Platelet rich plasma mitigates silver nanoparticles induced - pulmonary fibrosis in Wister rats via CASPASE -3 &amp;TWIST-1genes downregulation

doi:10.21203/rs.3.rs-3235996/v1

Download PDF

Article

Platelet rich plasma mitigates silver nanoparticles induced - pulmonary fibrosis in Wister rats via CASPASE -3 &TWIST-1genes downregulation

https://doi.org/10.21203/rs.3.rs-3235996/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Silver nanoparticles (AgNPs) are widely known for their anticancer, anti-inflammatory, and antimicrobial uses. Still, hazardous usage of AgNPs can damage various tissues, including the lung and is considered as environmental toxin. The ability of platelet rich plasma (PRP) to lessen the pulmonary damage caused by AgNps is not widely recognized. The aim of this study was to evaluate the efficacy of PRP, a natural yet harmless biological substance, in reducing lung fibrosis and damage caused by AgNps in rats. Forty-eight adults male Wistar albino rats were divided into six groups; Group 1, donors for PRP; Group 2, control rats; Group 3, received AgNPs (10 mg kg^-1 of body weight (BWt) once daily by intraperitoneal (i.p.) injection for4 successive weeks; Group 4, received AgNPs +PRP (0.5ml kg^-1. BWt i.p.) two days / week after AgNPs for for 3 weeks); Group 5, AgNPs+ dexamethasone (0.5 mg kg ^-1 i.p) for for 4 weeks after AgNPs treatment; Group 6, recovery group received Ag-NPs and then left for another 4 weeks without treatment. This study included the assessment of; BWt, hydroxyproline level in lung tissue. In addition, Caspase-3, and TWIST -1 gene expression in lung tissue as markers for apoptosis and fibrosis, respectively, was assessed using Rt-PCR. Also, histological (H/E &Siris red staining) and immunohistochemical studies were conducted to detect the expression of inducible nitric oxide synthase (iNOS) and cluster of differentiation 68 (CD68) in lung tissue associated with histomorphometry studies.

Significant increase in BWt was noted in AgNPs group when compared to AgNPs +PRP group (p<0.001). Hydroxyproline levels were markedly increased by AgNPs and decreased after PRP treatment (p<0.001). Caspase-3 and TWIST-1 genes were up-regulated in AgNPs group and significantly downregulated in AgNPs+PRP group. AgNPs induced deleterious changes in the lung tissues, including thickening of the interalveolar septa, collapse of the lung alveoli, diffuse lymphocytic infiltration and pulmonary fibrosis. This was also associated with increased alveolar macrophage CD68 expression and iNOS positivity in the cells lining the alveoli compared to AgNPs group. Treatment with PRP markedly improved histopathological and immunohistochemical picture. Surprisingly, the effect of PRP was comparable to dexamethasone. The herein study showed that treatment with PRP is a promising, natural, safe, and effective treatment against AgNPs induced lung fibrosis and injury by playing anti-apoptotic, anti-fibrotic, anti-inflammatory, and anti-oxidative stress roles.

Biological sciences/Drug discovery

Earth and environmental sciences/Natural hazards

Physical sciences/Nanoscience and technology

Silver nanoparticles

Platelet rich plasma

Lung fibrosis

TWIST-1

Caspase 3

A component of the so-called "fourth industrial revolution" is nanotechnology. The advancement of industry over the past two decades has depended heavily on this multidisciplinary field, which has given existing materials new proprieties¹. In the last ten years, nanotechnology has been employed in different applications across a variety of fields, including drug delivery, the food industry, hardware, diagnosis, cosmetics, sports, and medicine². One of the most prominent nanoparticles is silver nanoparticles (AgNPs). Textiles, food packaging, cosmetics, water purifiers, paints, and electronics are just a few examples of common consumer goods that include AgNPs which improve our quality of life ³. AgNPs can interact with biological targets of a comparable size thanks to their small size (1-100 nm), which enables them to cross the normal plasma membrane barrier⁴. Materials acquire antifungal and antimicrobial properties when AgNPs are added. These benefits help advance technology utilized in a variety of fields, including agriculture (biopesticides, food preservation) and biomedicine (wound dressings, catheters, dental materials, bone cement, medications, etc.) ⁵. AgNPs' larger surface area compared to silver in its solid state opens a wide range of potential biomedical uses⁶.

Unfortunately, these widespread applications inevitably raise the risk of unintentionally environmental release of these nanoparticles (NPs), which increases the exposure to people and other living organisms⁷. Smaller Ag NPs are more toxic and released fatster, and when coupled, they impair the immune responses⁵Silver ions (Ag⁺) released from Ag NPs ingested products into the blood stream and collected in different body organs revealed toxic effects ⁸. Current research has shown that AgNPs can enter food under a variety of usage scenarios from packaging⁹.AgNPs can also build up along ecological chains in aquatic environments after being released from urban and industrial effluents ¹⁰. As a result, ingesting AgNPs may be the most pertinent method of exposure for humans, with skin absorption and inhalation likely playing an additional substantial role¹¹.

There are many factors that determine the toxicities of AgNPs including physicochemical properties (e.g. source, size, dispersion state, concentration, and nanofabrication), and biological factors related to the organism such as species, age, gender, and size of the tested animals¹².

The first organ consistently exposed to nano particles is the lung¹³. More tissue damage, inflammation, and lung tumors have been linked to the insoluble NPs, according to reports¹⁴. Since the macrophage clearing capability of the insoluble NPs is greater than that of the soluble NPs, lung damage results from the dysfunctioning of the lungs' defense mechanisms.¹⁵. In addition to various experimental designs could have had an impact on the study's findings¹⁶ .AgNPs’ intrinsic characteristics such as concentration, coating nature, animal model, and several extrinsic factors including exposure time and cell type were crucial in the cellular response exerted by AgNPs in vitro and in vivo¹⁷.

One of the most frequently investigated aspects of AgNPs is time-dependent cytotoxicity. The pH, internalization, and dissolution of NPs into silver ions (Ag⁺) are only a few of the factors that are directly impacted by the various AgNPs exposure times¹⁸.The potential impact of lung exposure to AgNPs in industrial settings and AgNPs manufacturing facilities, where it has been claimed that the air's content of AgNPs doubled during production, has been supported by a large range of evidence¹⁹.

Pulmonary fibrosis (PF) is an incurable and progressive condition that affects the flexibility of the alveoli and is characterized by the infiltration of inflammatory substances that might result in tissue hypoxia. ²⁰. Furthermore, inhaled nanoparticles have been shown to be able to overcome the alveolar-capillary barrier, enter the bloodstream, and accumulate in secondary organs, with the liver being one of the most pertinent organs⁶. AgNPs were said to have a long-lasting negative impact on other organs because of translocated NPs accumulation in secondary organs or a leakage of inflammatory mediators and pro-oxidants from the lung into the plasma, which may subsequently injure distant organs¹⁹. Currently used drugs in the treatment regimens of lung fibrosis and damage can have undesirable side effects and disrupt the clinical trajectory of the disease. Opting for natural and safe alternative treatments is therefore a top priority.

Since the 1950s, the first line of treatment for pulmonary fibrosis has been the oral administration of glucocorticoids. To avoid the negative effects of glucocorticoids, which are often employed as anti-inflammatory and antifibrotic medication in similar situations, it is crucial to pinpoint an effective and safe treatment for lung injury and fibrosis caused by AgNPs. Utilizing the therapeutic potential of platelets and platelet-rich plasma (PRP) is a biologic approach that falls under the umbrella of regenerative medicine. To do this, a little of the patient's blood is drawn to obtain a plasma ²¹fraction with a concentration of platelets that is either equal to or greater than blood levels. In this plasma fraction, platelets that have been exogenously or internally activated release biomolecular components that will act on target tissues and cells²². The PRP is a popular treatment in many medical domains due to its biosafety, ease of collection, and application as a result of its autologous nature²³.

Despite encouraging results and a growing number of studies on PRP, more research is still required to fully understand the processes by which it functions. However, it appears to be well established that many of PRP's effects are brought about through altering cellular activity ²⁴. As a result, multiple studies have demonstrated that PRP increases cell proliferation, a crucial step in the treatment of diseases including degenerative lesions and healing processes that require cellular reconstruction of tissues. This was demonstrated by several cell types, including chondrocytes ²⁵, fibroblasts ²⁶, endometrial cells ²⁷, and corneal cells²⁸.

Thus, the present work was based on the hypothesis that PRP has an antifibrotic and regenerative abilities that can help in reducing AgNPs induced lung fibrosis and injury. Besides, little is referred to regarding the effect of PRP in treatment of lung injury caused by AgNPs.To our knowledge, this is the first research investigating the impact of PRP on AgNPs induced lung injury in experimental model. Currently and based on this knowledge gap, this work is aiming to prove the efficacy of PRP in treatment of AgNPs induced lung harmful effects.

I- Changes of body weight in different groups:

Treatment with Ag-NP 10 mg kg^-1 BWt once daily i.p for 4 weeks significantly(p<0.001) reduced body weight by 78% compared to the control group, according to Table 1. When compared to the AgNps group, PRP treatment caused a significant (p<0.001) increase in body weight of 71.04%, nearly reaching the weight of the control group. In contrast, Dexa therapy in the AgNPs+Dexa group significantly (p<0.001) increased body weight when compared to the AgNps group, yet it substantially (p<0.001) decreased body weight when compared to the control and AgNps+PRP groups. The recovery group's body weight was still and noticeably lower (p< 0.001) when compared to the AgNps+PRP and AgNps+dexa groups. However, there were no weigh differences noted between the recovery group and the AgNPs group. Also, the recovery group had significantly (p<0.001) less weight gain compared to the control group.

Table 1. Changes of body weight in different experimental groups. The results are presented as the means ± standard deviation (SD) The groups with different superscripts in the same row represent a statistical significance (p<0.05). a: control group, b: Ag-NP group, c: Ag-NP +PRP group, d: Ag-NP +Dexa group. n=number /group ; g=gram.

Parameters

Experimental group

F- value

P-value

Control

(n=8)

AgNPs

(n=8)

AgNPs +PRP

(n=8)

AgNPs +Dexa

(n=8)

Recovery

(n=8)

Initial body weight (g)

170± 6.32

171.66± 8.75

176.66± 8.75

177.5± 5.24

175.6± 3.8

1.37

<0.27

Final body weight (g)

286.66±5

224.16^a±4.91

292.5^b±16

255.66^a^,b,c±13.6

235.5 ^a,c,d ±3.01

47.7

<0.000

II- Levels of lung tissue hydroxyproline in different groups:

Based on Table. 2, the level of hydroxyproline increased significantly (p<0.001) after administrations of Ag-NP in comparison to the control group. The administration of PRP revealed a substantial (p<0.001) decrease in hydroxyproline level in contrast to the AgNPs group. Regarding the AgNPs treatment group and the AgNPs+PRP group, the hydroxyproline lung tissue level was significantly (p< 0.001) reduced after Dexa treatment. Additionally, rats in the recovery group shown substantial gains in hydroxyproline level when compared to the AgNps+PRP, AgNps+Dexa, and control groups, but non-significant changes were seen when compared to the AgNps group.

III- Relative expression of lung tissue Caspase-3 and Twist-1 in different groups:

In contrast to the control group, lung tissue Caspase-3 and Twist-1 gene expression was dramatically (p< 0.001) upregulated after AgNPs injection to rats. When compared to the AgNPs group, PRP treatment caused a significant (p< 0.001) downregulation of the expression of the Caspase-3 and Twist-1 genes. In contrary to the AgNPs group, the expression of Caspase-3 and Twist-1 decreased considerably (p< 0.001) in rats treated with Dexa; however, the alterations were not statistically significant when compared to the AgNPs+PRP group. Additionally, the recovery group did not noticeably differ from the AgNPs group, but it did significantly upregulate both gens when compared to the control, AgNps+PRP, and AgNps+Dexa groups (Table 2).

Table 2. Levels of lung tissue of Hydroxyproline and relative expression of Caspase -3and Twist- 1 in different groups. The results are presented as the means ± standard deviation (SD) The groups with different superscripts in the same row represent a statistical significance (p<0.05). a: control group, b: Ag-NP group, c: Ag-NP +PRP group, d: Ag-NP +Dexa group. n=number /group; g=gram

Parameters	Experimental groups									F value	P-value
	Control (n=8)		AgNPs (n=8)		AgNPs +PRP (n=8)		AgNPs +Dexa (n=8)	Recovery (n=8)
Hydroxyproline (µg/mg)	29.13± 2.48	125.81^a± 10.4		76.05 ^a,b±8.16		63.96 ^a,b,c± 1.4			121^a,c,d ± 3.16	256.61	<0.001
Caspase -3	1.02±0.01	5.73^a ±0.95		2.77^a,b±0.34		2.21 ^a,b ±0.26			5.78^a,c,d ±0.6	110.98	<0.001
Twist -1	1.01±0.4	6.55^a ±2.99		3.27^a,b±0.1.57		3.04 ^a,b ±0.03			6.55^a,c,d ±2.8	64.19	<0.001

VI: Histological studies

-Hematoxylin and eosin stain(H&E)

The results of the H&E stain used to examine the lung tissue in the control group are shown in Figure 1. In contrast, H&E-stained lung sections from the Ag-NPs group and the recovery group revealed loss of the normal architecture of the lung tissue in the form of numerous and enormous hemorrhage areas, destroyed and narrowed alveoli with noticeable thickening of the interalveolar septa, and extravasated red blood cells (RBCs) within the lung interstitium. Large alveolar macrophages with vacuolated acidophilic cytoplasm, congested dilated pulmonary blood vessels, extensive lymphocytic infiltration, and epithelial desquamation were observed in a few bronchioles. The ANPs+PRP group demonstrated a significant structural improvement compared to the Ag-NPs group, essentially restoring the typical lung architecture of the control group. Dexa therapy in the AgNPs +Dexa group appeared to lower the thickness of the interalveolar septa, there was less cellular infiltration, and the alveolar lumen was wider than in the AgNPs-injected group. However, there were still few congested blood vessels and interalveolar septal thickening in some regions.

-Sirius Red stain

Small strands of collagen fibers could be observed in the thin interalveolar septum, surrounding blood vessels, around pulmonary bronchioles, and around alveoli in Siris red stained slices from the control group. Collagen fiber distribution and density were clearly elevated in parts of the AgNPs and recovery groups. The thickened interalveolar septa, the area around the bronchioles, and the area around the clotted blood all showed signs of interstitial fibrosis as seen in Figure 2.In contrast, lung tissue sections of the AgNPs+PRP and AgNPs+Dexa groups revealed decreased deposition of collagen fibers around blood vessels, around pulmonary bronchioles, and around alveoli, alveolar sacs, and thin interalveolar septa, especially in the AgNPs +PRP group, which appeared analogous to that of the control group. These results validated our biochemical investigation of the down-regulated TWIST-1 gene and the lower quantity of hydroxyproline in lung tissues.

-Immunohistochemical assessment of iNOs

Figure3 showed the presence of few and sparse cytoplasmic immunoreactions of iNOS in the bronchioles and lung interstitium cells in the control group. Compared to the control group, the AgNPs group and the recovery groups both displayed strong positive dark brown iNOS immunoreactions that were clearly visible in the cytoplasm of the alveolar epithelial cells as well as in those of the interalveolar septa, the interstitial cells, and the bronchiolar epithelial lining, indicating apoptotic changes in lung tissue.

Similar to the control group, the AgNPs+PRP group displayed few and sparse cytoplasmic immunoreactions of iNOS among the cells of the lung interstitium and bronchioles. In the AgNPs+Dexa group, a significant decrease in iNOS expression was observed. AgNPs+PRP and AgNPs+Dexa groups revealed a weak positive brown cytoplasmic immunoreaction for iNOS in bronchiolar epithelial lining, pulmonary interstitial cells, and alveolar epithelial cells.

-Alveolar macrophage CD-68 immunohistochemical reaction

According to immunohistochemically stained CD68 sections, only a small percentage of the control group's interalveolar macrophages exhibited a slightly positive cytoplasmic immunoreaction. This was depicted in Figure 4. However, the interalveolar septa and the bronchial wall of the AgNPs and the recovery groups in contrast to the control group showed a significant number of cells with a positive cytoplasmic immunoreaction. The favorably stained CD-68 immunoreaction significantly decreased in the Ag-NPs+PRP group, but it was still roughly comparable to the control group. There was a noticeable decrease in the number of CD-68 immunoreactions that were positively stained when comparing the Ag-NPs group to the Ag-NPs+Dexa group. In cells of the inter alveolar septa, these immunoreactions manifested as slightly positive cytoplasmic immunoreactions.

-Morphometric analysis:

Table 3 showed a considerable increase in the mean thickness of the interalveolar septa in AgNPs group and in the recovery group compared to the control rats in H&E-stained sections (P ˂ 0.001).

However, there was a noticeable improvement in the AgNPs+PRP group and AgNPs+Dexa group, particularly in the Ag-NPs+PRP group, which resembled the control group in appearance.

Table 3: Mean±SD for morphometric measurements in different groups. The results are presented as the means ± standard deviation (SD) The groups with different superscripts in the same row represent a statistical significance (p<0.05). a: control group, b: Ag-NP group, c: Ag-NP +PRP group, d: Ag-NP +Dexa group. n=number /group

Parameters	Experimental groups					F -value	P-value
Parameters	Control (n=8)	AgNPs (n=8)	AgNPs +PRP (n=8)	AgNPs +Dexa (n=8)	Recovery (n=8)	F -value	P-value
Thickness of interalveolar septum (um)	0.72± 0.19	16.84^a ± 2.34	2.6^b± 1	3.44^b± 1.7	13.23^a,b,c,d ± 2.2	107.23	<0.001
Area percentage of collagen fibers	2.19±0.59	23.01^a±2.8	4.2^b± 1.41	5.87^b ±1.82	20.88^a,c,d ±6.6	50.42	<0.001
Area percentage of iNOS immunopositivity	0.04±0.19	39.68a±19	1.76^b±0.82	2.09b±0.87	34.8^a,c,d±16.3	68.14	<0.001
Mean number of CD68 alveolar macrophages	2.4±1.02	29.83a ±2.7	4.5^b ±2.5	6.83a,b±1.7	23.83a,b,c,d ±3.3	185.13	<0.001

Extensive uses of nanoparticles (NPs) in medical and industrial settings increase the chances of human exposure to a verity of NPs (Reference!). This could result in severe side effects and deleterious impacts on internal organs ². Silver nanoparticles are the most prevalent nanomaterials in the fast-growing field of nanotechnology, and they are employed in a variety of products, from antibacterial sprays to athletic apparel. Most products in modern technology are made with nanotechnology⁴⁴ .

The respiratory system is of particular concern because it interacts with NPs most extensively during medical treatments and through exposure to the environment⁴⁵. Inhalation is the most significant occupational health concern associated with exposure to silver nanoparticles in the industrial sectors⁴⁶.By persistent over time exposures, silver deposit will have a direct effect on respiratory functions. Acute and chronic toxicity cases resulting in argyria (grayish discoloration of the skin), hypoxia, tachycardia, and even lung failure which have been reported in severe cases are linked to daily exposure to silver ⁴⁷. In addition, longer circulation times of AgNPs in the lungs compared to Ag ions results in increased hazardous effects of AgNps¹⁷.

Body weight is frequently employed as a sensitive marker of chemical toxicity of materials⁴⁸.Furthermore,the body weight of rats is sensitive to AgNPs, according to a study conducted by Zhang et al⁴⁹. In the current study, administering 10mg Kg^− 1 bw of AgNPs intraperitoneally once daily for 4 consecutive weeks caused a significant (p < 0.001) who reached the same conclusions. Furthermore, our findings were in line with those of ⁵⁰who concluded the same results. Additionally, our results were consistent with ⁵¹who demonstrated that administering AgNPs at concentrations of 20 and 40 mg/kg caused body weight to decrease. Additionally, Yin et al .,2015⁵² validated the current results by stating that when the exposure dose was higher than 0.2 mg/kg/day, the average body weight growth in the AgNPs-treated rats was considerably reduced over the course of 7 weeks of treatment ⁵². Additionally, the results of Dziendzikowska et al. 2012 and Xia et al 2014 ⁵³⁵⁴complement the findings of the current study ^{53 ,54}.

However, the research on body weight reported contradictory results ⁵⁵.Some investigations have demonstrated that acute inhalation exposure (high-dose 3.08 106 particle/cm3, 750 mg/m3) and chronic oral treatment (1000 mg/kg/day) do not affect body weight ^56,57.Similarly to the latter reported results, no apparent shifts in body weight were seen by ⁵⁸ when rats were exposed to AgNPs (200 µL,1mg/mL) through a single intratracheal instillation.

In contrast, Adeyemi & Adewumi, 2014 have demonstrated that rats' body weight rose when exposed to AgNPs at doses of 100, 1000, and 5000 mg/kg per day for 7, 14, and 21 days⁵⁹. Additionally, AgNPs allegedly caused rats to gain more body weight ⁶⁰.

Our findings diverge from those published in other investigations for several reasons. First, compared to prior research, the current study's administration time was different. Second, the dose of Ag NPs used in our investigation is less hazardous (subchronic) than the lethal dose. Third, the utilization of an in vitro or in vivo study model.The treatment with PRP (0.5ml kg^− 1 BWt i.p.) significantly enhanced body weight in comparison to AgNPs only treated group and AgNPs + Dexa group. These results supported the findings of ⁶¹who determined that PRP reduced weight loss in diabetic rats. Additionally, our findings in mice lined with the results of Tong et al⁶².

The PRP plays an essential role in tissue proliferation and growth. The effectiveness of this therapy is associated with results from the local introduction of many growth factors and proteins that mimic and assist physiologic tissue reconstruction ⁶³.The PRP also affects cell cycle progression by promoting cyclin expression. Cyclins are substances that adhere to and activate cyclin-dependent kinases⁶⁴. Furthermore, PRP is claimed to have a proliferative effect on preadipocytes ⁶⁵.All the above information lends credence to our findings and provide an explanation for the weight gain that offset AgNP's negative effects.

Pulmonary fibrosis is the worst scenario in the story of AgNPs lung affection. So, in this study we investigated the indicators for lung fibrosis to assess PRP effect. The "gold standard" for determining collagen content in the study of organ fibrosis has traditionally been the biochemical assay of hydroxyproline. Levels of hydroxyproline can also be used to track the development of lung fibrosis and evaluate the efficacy of treatment ⁶⁶.

In the current study, hydroxyproline levels significantly increased in the AgNPs treated group compared to the control group. Hydroxyproline is a key biomarker in fibrotic illnesses that indicates the proportion of collagen deposition. An improvement in lung tissue architecture and a dramatic reduction in the distribution of collagen fibers were revealed by histological study of the lung tissues in PRP-treated rats. These results imply that PRP effectively reduced pulmonary fibrosis by reducing collagen buildup in the lungs. Dexa treatment reduced hydroxyproline more than PRP, but in a comparable manner.

The present results are in line with Salem et al.,2018 who detailed that PRP (i.p.) injection at dose (0.5 mlkg^− 1) two times weekly for three weeks improved clinical and pathological picture of lung fibrosis induced by amiodaron in rats³². Moreover, Salem and colleagues proved that PRP lessen hydroxyproline⁶⁸levels in liver tissue and mitigate liver fibrosis ^{68 ,67}.This was linked to the fact that platelet-rich plasma contains 5 to 10 times more growth factors than whole blood. These growth factors supports the body's own healing mechanisms.by supplying it with platelets to the injured location and draw stem cells as an initial response to the injury⁶⁹.

The PRP was introduced in the 1950s and is currently used in many branches of medicine ⁷⁰. The PRP is an autologous blood derivative with high platelets concentration in a small volume of plasma and considered an alternative treatment for several diseases as it is low-cost human by-product. It also decreases the chances of adverse effects and rejection ⁷¹.

Over the past ten years, the usage of biologic therapy in the treatment of numerous diseases has grown substantially. The PRP is a cutting-edge treatment method that is well-known all over the world. It offers researchers and medical professionals a large space because of its richness in growth factors. For instance, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), transforming growth factor (TGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF)^72,73 .

Platelets' effects on the pulmonary endothelium through releasing these growth factors include maintaining barrier integrity via cell proliferation, promoting endothelial cell development, and decreasing barrier permeability. When these concentrated growth factors interact with target cells' surface receptors, they activate intracellular signaling pathways that trigger the transcription of mRNA and proteins required for the regenerative process⁷⁴.PRP concentrates have the ability to induce the supraphysiological release of growth factors, which can speed up the healing process for both acute and chronic injuries ⁷⁵. Several cytokines, and locally acting regulators participate in most fundamental cell processes at all stages of tissue repair via endocrine, paracrine, autocrine, and intracrine system. However, the exact mechanism of how PRP regenerates tissue is yet unknown⁷⁶. PRP is frequently used in medical procedures including spinal surgery ⁷⁷ and oral and maxillofacial surgery⁷⁸. Later, PRP applications were expanded to include lung fibrosis in COVID 19 ⁷⁹.

Respiratory epithelial cells are efficient tissues that defend off the attack of dangerous chemicals, and their damage either directly or indirectly results in the toxicity of cells. Because of their small size, AgNPs can easily infiltrate the inside of cells, where they can hinder cell development and trigger programmed cell death (apoptosis) and necrosis ⁸⁰. According to several research, AgNPs induce cytotoxicity by causing apoptosis ^81,82,83.

The degree of lung damage brought on by subchronic exposure to AgNPs was also assessed at the molecular level by analyzing the activation of the Caspase-3 and TWIST-1 genes. The current study demonstrated that following AgNps i.p. injection, compared to the control group, Caspase-3 and TWIST-1 genes were downregulated (p < 0.001). These results was consistent with the findings of Blanco et al (2018) ¹who demonstrated that subchronic poisoning by AgNPs elevated caspase-3 considerably in rats due to impairment of DNA repair ¹. Surprisingly, PRP injection caused the Caspase-3 and TWIST-1 genes to be significantly upregulated, much like Dexa therapy. Our findings were consistent with those of Sekerci et al. (2017) ⁸⁴who claimed that PRP dramatically lowers Caspse-3 levels in rats ⁸⁴. The present histological analysis of lung tissues in this work confirmed that PRP somewhat has anti- apoptotic effect.

The anti-apoptotic protein caspase-3, which belongs to a specific class of aspartate-dependent proteases, can be activated both internally and externally during apoptosis. ⁸⁵.Moreover,caspase-3 overexpression is interpreted as an upsurge in apoptosis⁸⁴.According to Anand et al. (2023), exposure to NPs reduced mitochondrial activity⁸⁶. Consequently, apoptosis increased. The release of cytochrome c from mitochondria activates caspase 3, which causes the creation of an apoptosome⁸⁷. ⁸⁶

Caspases like caspase-3 may be activated by oxidative stress, which would enhance the death of cells ⁸⁸.According to a prior study, lung fibroblast exposed to AgNPs experience oxidative stress, cell morphological changes, reduced cell viability, and genotoxicity⁸⁹. The direct action of Ag⁺ released from AgNPs⁹⁰ and the resultant augmented formation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are responsible for causing oxidative and nitrosative damage, may contribute to AgNP-induced cytotoxicity. As a result, biological membranes become lipid peroxidized, which causes oxidative DNA damage as well as structural protein degradation^91,92.Moreover, Blanco et al. (2017) reported that daily exposure to low doses of AgNPs, or a single exposure to high doses of these nanoparticles for 72 h, increased caspase-3 with a high increase in ROS generation and DNA damage. If the cells are unable to trigger apoptosis due to a lack of caspase-3, necrosis may develop as a substitute for programmed cell death ⁹³. Almost all existing studies reported that caspase-3 increased after AgNPs exposure ^{94, 95}. And this was lined with our results. Also, Suliman et al. (2015) reported the same in human lung epithelial cells ⁹⁶.

Once inside the cell, AgNPs promote autophagy by gathering in autophagosomes and partially dissolve into silver ions (Ag⁺) by fusion of the autophagosomes and lysosomes⁹⁷. Lysosomal membrane integrity was compromised, which led to their permeabilization and the release of Ag⁺ and AgNPs into the cytosol¹. When they are released, they can interact directly with macromolecules and cell organelles, disrupting normal cell activity. AgNPs interact with mitochondria, preventing respiratory chain enzymes from functioning and obstructing the electron transport chain⁹⁸.Increased amounts of ROS and oxidative cell damage lead to apoptosis and ATP depletion⁹⁹. Additionally, AgNPs may cause apoptosis via the caspase-dependent mitochondrial pathway, which has been shown to alter cell dynamics, harm the cell membrane, inactivate ATPase activity to suppress Ca²⁺-ATPase, Na+/K+-ATPase and Ca²⁺/Mg²⁺⁻ATP as produce excessive amounts of ROS and MDA, sensitize cell signaling to the release of cytochrome C and pro-apoptotic protein into the cytoplasm, and activate the caspase cascade as reported by¹⁰⁰;¹⁰¹. Clearance of Ca²⁺and cell homeostasis maintenance are controlled by the calmodulin (CaM)-dependent enzyme Ca²⁺-ATPase, which can prevent cells from going through apoptosis ¹⁰².

According to literature, carbon nano particles upregulate the expression of the alveolar macrophage TWIST-1 protein which partially agree with the current findings¹⁰³ .A transcription factor, TWIST-1, contributes to the age and angiogenesis-related diseases, including pulmonary fibrosis ¹⁰⁴. TWIST-1 has a critical role as a regulator in the fibrotic lung ¹⁰⁵.Furthermore, TWIST-1 regulates vascular development and function¹⁰⁶ through multiple angiogenic signaling (e.g., Tie2, platelet-derived growth factor (PDGF), VEGFR2, and transforming growth factor beta receptor (TGFβR))¹⁰⁷ .Inhibition of TWIST-1 activity increases the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARα) that stimulates mitochondrial biogenesis and angiogenesis ¹⁰⁴.Also,Vlenzi et al (2022) ¹⁰⁵reported that TWIST-1 and other E-box transcription factor motifs were significantly upregulated in pulmonary fibrosis in bleomycin-injured mice ¹⁰⁵. This upregulation was also associated with increased collagen synthesis which was concurrent with our results.

One of the main causes of NPs' toxicity is their propensity to produce ROS like superoxide, singlet oxygen, hydrogen peroxide, and hydroxyl radicals ⁴⁴.It is noteworthy that that the antibacterial properties of AgNPs depend heavily on ROS production. Also, AgNPs it can exert antimicrobial properties by the release of free Ag + and the subsequent formation of ROS ¹⁰⁸.However if undesirable species' cells are damaged, this could be dangerous for humans and other organisms. Therefore, generation of ROS or the induction of inflammatory reaction can alter the cells’ membrane and harm organelles ^109,110.

The lack of safety issues has encouraged the use of PRP in the treatment of condition diseases with unmet medical needs including lung fibrosis where therapy with currently available medications is inadequate. Yet, the lack of standardization in PRP materials, doses, and treatment methods limits the advancement of PRP based therapies^111,75.

Regarding the histological results, the current study showed that administering AgNPs caused several histopathological abnormalities in the lung tissue. These alterations include alveolar collapse, thickening of the interalveolar septum, dilated congested blood vessels, numerous regions of hemorrhage, lymphocytic infiltration, and desquamation of bronchiolar epithelial cells inside its lumen. ^112,113 attributed these changes to the systemic and immunotoxic effect of AgNPs through the release of ROS as well as alveolar macrophages activation concomitant with the up regulation of pro-inflammatory and pro-neutrophilic cytokines which increase the membrane permeability to ions such as Ca^+ 2. Moreover, inactivation of structural proteins increased lipid peroxidation of the cellular membranes and depleted glutathione ¹¹⁴.Also, AgNPs potentiates elastase secretion from the inflammatory cells because of decreased α-antitrypsin1 activity. Then the α-antitrypsin1 breaks down elastin protein lining the air sacs and the pulmonary vessels by disrupting the alveolar capillary membranes¹¹⁵.Thus, it allows leakage of proteinaceous fluid into the pulmonary parenchyma causing alveolar epithelial injury with alterations of the alveolar capillary barrier and cytolysis, as well as increasing oxidative stress load.

Exposure to AgNPs changes oxygen metabolism, which in turn alters lung toxicity. The reduced lung function because of alveolar epithelial injury could be initiated by oxidative damage caused by AgNPs¹¹⁶.Our results were in agreement with Abd El-Maksoud et al.(2019) and Yousef et al (2019) who stated that the cytotoxicity caused by Ag-NPs could be partially caused by the direct action of dissolved Ag⁺ ions dissociated from Ag-NPs ^117,118. The AgNPs can penetrate cell membranes and other biological barriers leading to their subsequent bioaccumulation in the lung tissue. The same authors stated also that AgNPs bind to thiol groups in the cell which is implicated in the structural protein damage by desulphurization and enhancement of free radicals generation, intracellular ROS, and RNS. Flores-López et al. (2019) have mentioned that these activated substances are responsible for the induction of oxidative and nitrosative damage which may affect the lung epithelium increasing the permeability of the cellular membrane to Ag-NPs ¹¹⁹. Moreover, it potentiates the conceivable mechanisms including actuation of lysosomal acid phosphatase activity, interruption of the actin cytoskeleton, incitement of phagocytosis, eliciting oxidative DNA damage, and enhancing cellular necrosis and apoptosis. Hence, it increases the levels of the proinflammatory cytokines including TNF-α and IL-6, and cause inflammatory reactions.

Cellular infiltrations and extravasation were reported by some authors who stated that NPs may disturb the vascular endothelial cells cadherin (VE-cadherin) due to the direct combination of nanomaterials to VE-cadherin ^120,121. This attracts immune cells in the peripheral tissue due to alteration of endothelial cell-cell integrity and permeability of the cell membranes causing inflammatory reactions. Arpag et al. (2018) described that thickening of the interalveolar septa which was confirmed histomorphometrically in the present work could be attributed to the infiltration of inflammatory cells and the predominance of type II pneumocytes together with increased deposition of extracellular matrix proteins and endothelial hyper proliferation that may be linked to the ROS accumulation in lung ¹²².

Increased deposition of collagen fibers in AgNPs group aligns with the results of many studies who reported that pulmonary fibrosis is simply the consequence of inflammation that associated with increased levels of proinflammatory cytokines, C-reactive protein, tissue plasminogen activator (t-PA), and nuclear transcription factor– Kappa-B (NF-k_B) ^{123,124, 125}.These factors directly upregulate profibrotic transforming growth factor-beta 1 genes (TGF- β1) in the lung which then induce elevation of hydroxyproline content, fibronectin production and type I collagen by the activated fibroblasts, and promotes epithelial mesenchymal transformations into myofibroblasts, as well as inhibition of proteases that degrade the extracellular matrix contribute greatly to severe pulmonary fibrosis. These results were confirmed by morphometric measurement of the mean area percentage of collagen fibers within the lung interstitium in the Sirius red stained sections of AgNPs group that was significant compared to control group.

The current work revealed substantial increase in expression of iNOS in the lung sections of the Ag-NPs group. This occurred due to pro inflammatory, catalyze the decomposition of arginine and consequently the generation of large quantities of nitric oxide (NO). Therefore, strong oxidant (ONOO) will be formed by binding excess NO to O2 that will affect the permeability of the pulmonary vessels and the diffusion function of lung tissue resulting in destruction of the cytomembrane and DNA ^126,127.

This finding was in agreement with Alpert et al. (2017) ¹²⁸who stated that ONOO which will be formed by binding excess NO to O₂ will mediate mitochondrial dysfunction and will inhibit sirtuin 1 (SIRT1), thus inhibiting P53-dependent apoptosis pathway through a deacetylation pathway and ultimately promoting apoptosis and necrosis^127,129.This was proved by morphometrical measurement of area percentage of iNOS immunoreaction that revealed significant increase in the area percentage of iNOS positive immunoreaction within the lung interstitium in Ag-NPs group.

A significant increase in the mean number of CD-68 positive immunoreactive cells that was observed in the lung sections of the AgNPs group compared to control group. This result could be attributed to a compensatory mechanism to maintain normal lung function against the hazards of the inflammatory cascade which may affect oxidative metabolism of alveolar macrophages and undergoes activation as a response to foreign NPs stimuli ^{130, 127}.

In our study, remarkable improvement was observed in AgNPs + PRP group, that showed almost regain of the histological architecture of lung tissue when compared to AgNPs treated group. This might have occurred due to platelets stimulation of the mitogenic activity of human bone cells to increase the proliferation of stem cells, thus result in regeneration of lung tissues ¹³¹. This improvement may be attributed to PRP’s higher concentration in growth factors compared to whole blood. These growth factors enhance natural healing processes because of the first response of the body to tissue injury by delivering platelets to the injured area in situ and attracting stem cells to the site of the injury¹³².

Marked decrease in the deposition of collagen fibers in AgNPs + PRP group coincided with Shoeib et al.(2018) who reported that PRP treatment increased the level of cAMP which has antifibrotic effect ¹³³. This antifibrotic effect is mediated by Exchange proteins activated by cAMP (Epac) that adjust many cellular reactions through their capability to produce substitution of GTP for GDP on different G-proteins. Also, cAMP decreases proliferation of fibroblasts, inhibits extra cellular matrix protein synthesis and stimulates fibroblast apoptosis. They further added that PRP has anti-inflammatory role which is facilitated through its impact on macrophage inflammatory proteins (MIP-1α) levels in many diseases. Additionally, PRP suppresses the phosphorylation and translocation of NF-κB-p65 subunit to the nucleus which induce suppression of pro-inflammatory genes expression. Consequently, PRP inhibits the matrix metalloproteinase (MMPs) secretion and pro-inflammatory chemokines ¹³⁴.

Moreover, other researchers stated that PRP decreased the expression of mRNA of fibrosis associated genes including TGF-β, and inflammatory related gene as NF-kβ.Moreover, they reported a significant increase in the anti-apoptotic marker Bcl-2 after PRP treatment, confirming the role of PRP in tissue regeneration ¹³⁵.This could explain the significant decrease in the mean area percentage of iNOS immunopositivity in AgNPs + PRP group compared with that of Ag-NPs group. Additionally, this was consistent with the decrease in lung tissue levels of caspase-3 that was demonstrated in this study.

In the current study, administration of Dexa. after induction of lung injury by AgNPs injection caused great improvement in histological structure of lung compared to AgNPs group which was anticipated. Dexamethasone decreased the production of inflammatory cytokines, alleviated endothelial cell damage and alveolar epithelial cells. It also decreased pulmonary edema and improved the alveolar epithelium endothelial barrier function.

Dexamethasone has been shown to reduce the generation of NO, limit the expression of inflammatory markers, and scavenge oxygen free radicals to treat acute lung injury¹³⁶.

Histological finding in AgNPs + Dexa group agreed with Raish et al. (2018) ¹³⁷who state that Dexa reduced lung inflammation by reducing inflammatory cell migration and proliferation. Decreased deposition of collagen in lung tissue in AgNPs + Dexa group was confirmed statistically by significant decrease of area percentage of collagen fibers compared with Ag-NPs group ¹³⁷. The herein results are in line with Serrano-Mollar et al. (2003) ¹³⁸who reported that Dexa treatment directly inhibited lung fibrosis by direct suppression of fibroblasts and transcription of type1 procollagen mRNA in the fibroblast, thus suppressing collagen synthesis ¹³⁸.

According to another study, Dexa's restriction of NO generation lowers parasympathetic activity while sharply raising sympathetic vasoconstrictor tone. ¹³⁹¹³⁹. This may explain significant decrease in the mean area percentage of iNOS immunopositivity in AgNPs + Dexa. group compared with that of Ag-NPs group. Dexamethasone was reported to ameliorate lung fibrosis in many models as bleomycin induced fibrosis that was proved by Shi et al. (2014) ¹⁴⁰study¹⁴⁰. Additionally, there is currently a variety of effective treatments for pulmonary fibrosis, including pirfenidone, N-acetylcysteine, and prednisone. It has been demonstrated that corticosteroids suppress neutrophil recruitment, collagenase activity, and alveolar Type II pneumocyte proliferation ¹⁴¹. Due to corticosteroid's immunomodulatory effects, patients may be more susceptible to developing pneumonia or TB ¹⁴². Also, there is evidence that these lung infections increase the chance of developing lung cancer¹⁴³. Therefore, finding safe alternatives for lung fibrosis treatment is an actual necessity. In contrast to routinely used corticosteroids, PRP is an autologous substance with no known side effects ¹⁴⁴. This study showed that PRP improved lung damage and reduced lung fibrosis comparable to dexamethasone, which was extremely promising.

The recovery group in our study the lack of any improvement in histological findings compared to the control group was confirmed by non-significant difference between the recovery and the Ag-NPs groups in all histomorphometric and biochemical results.

In the present scenario, three lines of evidence were provided to support the claim that PRP helps in treating lung fibrosis and damage :1) improvement of histological and histomorphometry pictures of lung tissue with Sirius red staining which considered to be specifically suitable for the evaluation of very small fibrotic lesions due to its high sensitivity, 2) Hydroxyproline level reduction, and 3)TWIST-1 downregulation.

Given that there is no single standardized PRP protocol, most products follow the ordinary methods. Interestingly, PRP falls into a category of products that does not require abiding to the US Food and Drug Administration (FDA) traditional regulatory pathway of animal and clinical trial¹⁴⁵. As several PRP systems on the market do have FDA clearance, there are no clear regulations for PRP injectate composition, and currently PRP can vary greatly in platelet, white blood cells (WBCs) and red blood cells(RBCs) contamination, and growth factor concentrations depending on the spinning protocol and other preparation protocol¹⁴⁶.

This study has some limitations that should be addressed. First, this study was conducted on experimental animals and to apply PRP in clinical practice these results must be supported by clinical trials. Second, different doses and duration of treatment should be used to conclude the best optimum protocol of PRP treatment.

Despite these limitations, we suggested that PRP treatment can reduce lung fibrosis and damage caused by AgNPs and affords potential therapeutic alternative to several hazardous treatments. In Brief, the current work proved that PRP mitigates AgNPs induced lung injury.

This study showed that intraperitoneal PRR therapy could enhance histological and immunohistochemical study outcomes equivalent to dexamethasone by reducing the expression of the fibrosis and apoptotic markers TWIST-1 and Caspase-3 as well as hydroxyprolines. Our studies on the effects of PRP therapy on apoptosis, oxidative stress, and fibrosis point in new directions for the development of modern AgNPs lung fibrosis treatments. The PRP therapy may be utilized in conjunction with other regimens to decrease the harmful effects of AgNPs as a pulmonary hazardous substance.

Animals and husbandry

forty-eight adult male albino rats of local strain (weighing 160±30 g) were provided by Helwan breeding farm, Helwan, Egypt. Animals were kept in an animal care facility under controlled temperature(25±2 C), and humidity (54±5%) and on a 12 h/12 h light/dark cycle, with ad libitum access to water and a standard laboratory chow diet in conventional clean properly ventilated wire-mesh cages (25×30×25 cm^-2 rats per cage) at the animal house, Faculty of Medicine (Girls), Al-Azhar University, Cairo, Egypt. Rats were acclimated for one week for adaptation be for beginning the experiment. All the ethical protocols for the experimental use of laboratory animals were carried out after the approval of the Faculty of Medicine (Girls) Institutional Research Board, Al-Azhar University, Cairo, Egypt.

Every step was taken to reduce the number of animals (n = 8 per group) and their suffering.

Chemicals and reagents:

Silver nanoparticles (AgNPs): were obtained from NanoTech, Cairo, Egypt. They were used as received without further purification. Silver nanoparticles were given in a dose of 10 mg kg^-1bw by intraperitoneal (i.p.) injection once daily for 4 successive weeks. The AgNPs dose applied in this study was selected due to its potency in producing appreciable subchronic toxicity in albino rats according to Yousef et al. (2

012) ²⁹ .

Characterization:

A s colloidal solution of spherical nanoparticles with an average size of 45 ± 5 nm and dark yellow color at a concentration of 2000 ppm were purchased from NanoTech Egypt for Photo-Electronics. The consistency of AgNPs was ensured through transmission electron microscopy (TEM) and ultraviolet (UV) / Visible spectral analysis (Fig.5 &6).

PRP preparation:

Platelet rich plasma was prepared using the double centrifugation tube method according to Pazzini et al., 2016 ³⁰. Eight male Albino rats used as blood donors were anesthetized with ether and 3mL of blood was collected from the retro-orbital plexus into tubes containing 0.3 mL (3.2% sodium citrate) anticoagulant. Blood was centrifuged twice. First centrifugation was at 1600 rpm for 10 min. The portion just above the buffy coat was obtained without disturbing the buffy coat. The second centrifugation was at 2000 rpm for 10 min to yield two portions: the top consisting of platelet-poor plasma (PPP) and the bottom consisting of the platelet button. After discarding the PPP, the platelet button was resuspended in phosphate-buffered saline (PBS) (1: 1). Hematology analyzer (MICROS abc LC-152; Horiba Ltd., Kyoto, Japan; at least 1, 000, 000 cells/ μL) was used for platelets counting and then PRP was frozen at –80C until use ³¹.The PRP was activated immediately before use with CaCl2 (0.8mL PRP + 0.2mL 10% CaCl2) ³². The prepared PRP was administered intraperitoneally at a dose of (0.5ml/kg. BWt) two days a week for 3 weeks and 24 hours after the last dose of AgNPs³³.

Dexamethasone: Each injectable vial of dexamethasone contains 8 mg/2 mL (Pharm. Inco. Amryia, Cairo, Egypt).

Experimental Protocol:

According to our protocol, the rats were randomly divided in six groups. Group1 rats served as blood donors for the preparation of PRP. Group 2 were used as control (saline). Group 3 rats were treated by AgNPs in intraperitoneal (i.p.) injection 10 mgkg^-1 BWt/day for four weeks then sacrificed²⁹.Group 4-(AgNPs+ PRP group) AgNPs similar to group 3 were given to rats followed by PRP i.p. injection at dose of 0.5mlkg^-1 bw twice weekly for three weeks 24 hours after last dose of AgNPs. Group 5 (AgNPs+ Dexamethasone (Dexa): rats received AgNPs like group 3 followed by Dexa i.p. at (0.5 mgkg^-1BWt) for four weeks according to Chen et al.,2006. Group 6-(Recovery group), Rats were given AgNPs that were comparable to Group-3 and left unattended for three weeks until the end of the study. Because i.p. injection route of administration is significantly more precise, dependable, and consistent than other routes of potentially dangerous exposure, they were chosen as the route of administration for AgNPs in this study to achieve subchronic toxicity ³⁴ .Additionally, by using this method, rats will always receive the same dose of AgNPs.

A-Body weight:

Animals were weighed before and at the end of the investigations.

B-Biochemical Assay

At the end of the experiment rats were anaesthetized with a sodium pentobarbital injection (30 mg kg^-1), then they were sacrificed by decollation. Then, lungs were processed for biochemical study (lung tissue hydroxyproline activity), polymerase chain reaction (PCR) technique to detect Caspase III and Twist 1 gene expression in addition to histological and immunohistochemical studies.

1-Detection of hydroxyproline level in lung tissue

Lung tissue hydroxyproline activity was determined using Rat Hydroxyproline (HYP) ELISA Kit (Hangzhou East biopharm Co., Ltd., China). Lung tissue was homogenized and centrifuged for 20 minutes (min) at 4C, 1000 ×g. The supernatant was separated and stored on ice. Absorbance was read at 450 nm using a plate reader.

2-Detection of gene expression of Caspase-3 and Twist-1 in lung tissue by Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA was isolated using Qiagen tissue extraction kit (Qiagen, USA) according to instructions of manufacture. The total RNA was used for complementary DNA (cDNA) conversion using high capacity cDNA reverse transcription kit (Fermentas, USA). Moloney murine leukemia virus (MMLV) reverse transcriptase was used for the synthesis of cDNA from RNA. Human Placental Ribonuclease Inhibitor (HPRI) was used for the inhibition of RNase activity. Real-time qPCR amplification and analysis were performed using Applied Biosystem with software version 3.1 (StepOne™, USA). The reaction contained SYBR Green Master Mix (Applied Biosystems), gene-specific primer pairs which were shown in the table (4) and were designed with Gene Runner Software (Hasting Software, Inc., Hasting, NY) from RNA sequences from the gene bank. All the primer sets had a calculated annealing temperature of 60C. Amplification conditions were: 2 min at 50C, 10 min at 95C and 40 cycles of denaturation for 15 seconds and annealing/extension at 60C for 10 min. The relative expression of the studied genes was calculated according to Applied Bio system software using the comparative threshold cycle method.

Table 4: Primers for gene expression by RT-PCR.

Gene

Primer sequence

Caspase III

Forward primer 5′-GTGGAACTGACGATGATATGGC-3′

Reverse primer 5′-CGCAAAGTGACTGGATGAACC-3′

Twist 1

Forward primer 5′-CCGGAGACCTAGATGTCATTGT5-3′

Reverse primer 5′- CTGGGAATCTCTGTCCACCG-3′

C-Histological study

Lung specimens were fixed in a 10% formalin saline solution. After dehydration in different grades of ethyl alcohol (100%, 5 min; 95%, 2 min; 80%, 2 min; 70%, 2 min), cleaning in xylol, impregnation, and proper fixing, we embedded the specimens in paraffin wax (5 mm thick pieces). Then, specimens were cut and fixed on glass slides using a rotatory microtome (LEICA RM 2125 UK). Staining with hematoxylin and eosin (H/E) solutions (G1120, Solarbio, China) for 30 min at 55 C was used to explore the lung’s general histological structure under a light microscope³⁵.Collagen fibers were stained by using Sirius red stain ³⁶. Slides were examined under a light microscope (Primo star, ZEISS, China). The photos were taken using (Axiocam ERc 5s, ZEISS, China) camera, at Histology Department, faculty of Medicine for Girls, Al Azhar University.

D-Immunohistochemical study

1-Inducible nitric oxide synthetase (iNOS) and Cluster of differentiation 68 (CD 68)

Lung specimens were deparaffinized and rehydrate, then sections were microwaved for 15 min for antigen retrieval. After that, sections were treated with a blocking solution for 20 min then incubated overnight at 4C with primary antibody recognizing iNOS (rat monoclonal antibody, 1:500 dilution, Transduction Laboratories, San Diego, California, USA)³⁷, and CD-68 antibody (mouse monoclonal antibody, 1:200 dilution, code NCL-L-CD68; Leica Biosystems, Benton La, Newcastle Ltd, UK) in a humidified chamber according to the manufacturer’s instructions^38,39Later, secondary antibody was added for 10 minutes. Finally, one to two drops of diaminobenzidine (DAB) were added for 10 min. At last, lung sections were counterstained with Mayer’s hematoxylin, then dehydrated and cleared. A light microscope (Primo star, ZEISS, China) was used for section examination. By which iNOS expressed brownish coloration of the cytoplasm of the alveolar and bronchiolar epithelial cells. While CD68 stained the interstitial lung macrophages by brownish positive.

C. Morphometric study

Each part was studied and documented using Leica DM2500 optical microscopes and a Leica ICC50 W digital camera (Leica Microsystems, Nussloch, Germany), employing a computerized image analyzer Leica Q500 MC application and shown on the screen. (Leica Microsystems Ltd, Cambridge, UK). To change the measurement units (pixels) generated by the image analyzer program into actual micrometer units, the data were automatically calibrated. ⁴⁰. Ten distinct, non-overlapping fields from five distinct lung sections in each group were investigated to quantitatively assess the following:1• The thickness of the inter alveolar septa in H&E stained section at Xm 400..magnification⁴¹.2• The mean area percentage of the red stained collagen fibers /μm2 in the lung interstitium and around the pulmonary blood vessels in sirius red-stained sections at X 200 magnification ⁴¹.3• The mean area percentage of positive iNOS immunoreactivity in anti-iNOS immune-stained sections was assessed at X 400 magnification ^42, ³⁷4• The mean number of alveolar macrophages in anti-CD-68immunostained sections in at X 400 magnification was estimated ^{39 , 43.}

Statistical analysis

The normality of numerical data was investigated by examining the distribution of the data and employing normality tests (Kolmogorov–Smirnov and Shapiro–Wilk tests). The data is presented as mean±standard deviation (SD), and range values. To determine statistical differences, a one-way analysis of variance (ANOVA) test was performed, followed by a Bonferroni post hoc test. p ˂ 0.05 was considered as the level of significance. The statistical package SPSS for Windows (v 23.0; SPSS, IBM Corporation, Armonk, NY, USA) was used.

Author contributions: Conceptualization: Shaimaa R. Abdelmohsen, Ranya M. Abdelgalil^, Asmaa M. ElmaghrabyAmira M. Negm, Fatma El-zahraa Abd EL Hakam. Methodology and Formal analysis: Shaimaa R. Abdelmohsen, Ranya M. Abdelgalil^, Asmaa M. ElmaghrabyAmira M. Negm, Fatma El-zahraa Abd EL Hakam. All authors participated in the Writing original draft and revision of the manuscript.

Ethical declarations:

All experimental protocols in this study involving animals were conducted in accordance with the ethical standards and the international regulations regarding the usage and welfare of laboratory animals and were approved by the Research Ethics Committee of Al-Azhar University’s Faculty of Medicine for Girls (No2018122001) and carried out in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85–23, revised 1996. Additionally, this work is done in accordance with the ARRIVE Guidelines.

Funding:

No funds

Data availability: Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request .

Competing interests:

The authors declare no competing interest.

Blanco, J. et al. Oral exposure to silver nanoparticles increases oxidative stress markers in the liver of male rats and deregulates the insulin signalling pathway and p53 and cleaved caspase 3 protein expression. Food and Chemical Toxicology 115, 398–404 (2018).
Noga, M., Milan, J., Frydrych, A. & Jurowski, K. Toxicological Aspects, Safety Assessment, and Green Toxicology of Silver Nanoparticles (AgNPs)—Critical Review: State of the Art. Int J Mol Sci 24, 5133 (2023).
Shehzad, A. et al. Synthesis, characterization and antibacterial activity of silver nanoparticles.
He, Q. et al. Antiviral properties of silver nanoparticles against SARS-CoV-2: effects of surface coating and particle size. Nanomaterials 12, 990 (2022).
Altwaijry, N., El-Masry, T. A., Alotaibi, B., Tousson, E. & Saleh, A. Therapeutic effects of rocket seeds (Eruca sativa L.) against testicular toxicity and oxidative stress caused by silver nanoparticles injection in rats. Environ Toxicol 35, 952–960 (2020).
Nemmar, A. et al. Assessment of the Hepatotoxicity of Intratracheally Instilled Silver Nanoparticles in Hypertensive Mice. Hamdan Medical Journal 16, 39 (2023).
Burdușel, A., Gherasim, O., Nanomaterials, A. G.- & 2018, undefined. Biomedical applications of silver nanoparticles: an up-to-date overview. mdpi.com.
Cho, Y.-M. et al. Size-dependent acute toxicity of silver nanoparticles in mice. J Toxicol Pathol 31, 73–80 (2018).
Choi, J. I. et al. Potential silver nanoparticles migration from commercially available polymeric baby products into food simulants. Food Additives & Contaminants: Part A 35, 996–1005 (2018).
McGillicuddy, E. et al. Silver nanoparticles in the environment: Sources, detection and ecotoxicology. Science of the Total Environment 575, 231–246 (2017).
Motta, G. et al. Preliminary Toxicological Analysis in a Safe-by-Design and Adverse Outcome Pathway-Driven Approach on Different Silver Nanoparticles: Assessment of Acute Responses in A549 Cells. Toxics 11, 195 (2023).
Hou, J., Zhou, Y., Wang, C., Li, S. & Wang, X. Toxic effects and molecular mechanism of different types of silver nanoparticles to the aquatic crustacean Daphnia magna. Environ Sci Technol 51, 12868–12878 (2017).
González-Vega, J. G. et al. Lung Models to Evaluate Silver Nanoparticles’ Toxicity and Their Impact on Human Health. Nanomaterials 2022, Vol. 12, Page 2316 12, 2316 (2022).
Ferreira, A. J., Cemlyn-Jones, J. & Cordeiro, C. R. Nanoparticles, nanotechnology and pulmonary nanotoxicology. Revista Portuguesa de Pneumologia (English Edition) 19, 28–37 (2013).
Alqahtani, S., Xia, L. & Shannahan, J. H. Enhanced silver nanoparticle-induced pulmonary inflammation in a metabolic syndrome mouse model and resolvin D1 treatment. Part Fibre Toxicol 19, 1–18 (2022).
Zayerzadeh, E., Shabanian, M. & Koohi, M. K. Toxicopathological examinations after repeated intraperitoneal administration of silver nanoparticles in the Wistar rat model. Nanomedicine Research Journal 3, 51–57 (2018).
Ferdous, Z. & Nemmar, A. Health impact of silver nanoparticles: a review of the biodistribution and toxicity following various routes of exposure. Int J Mol Sci 21, 2375 (2020).
Molleman, B. & Hiemstra, T. Time, pH, and size dependency of silver nanoparticle dissolution: the road to equilibrium. Environ Sci Nano 4, 1314–1327 (2017).
Ferdous, Z. & Nemmar, A. Health impact of silver nanoparticles: a review of the biodistribution and toxicity following various routes of exposure. Int J Mol Sci 21, 2375 (2020).
Rezaee, R. & Hosseinzadeh, H. Pulmonary Fibrosis. Comprehensive Pharmacology 4, 803–811 (2022).
Chen, F. et al. Short courses of low dose dexamethasone delay bleomycin-induced lung fibrosis in rats. Eur J Pharmacol 536, 287–295 (2006).
Meznerics, F. A. et al. Platelet-Rich Plasma in Chronic Wound Management: A Systematic Review and Meta-Analysis of Randomized Clinical Trials. J Clin Med 11, 7532 (2022).
Zavan, B. et al. Action of Platelet-Rich Plasma on In Vitro Cellular Bioactivity: More than Platelets. International Journal of Molecular Sciences 2023, Vol. 24, Page 5367 24, 5367 (2023).
Sánchez, M. et al. Isolation, activation, and mechanism of action of platelet-rich plasma and its applications for joint repair. in Regenerative Medicine (IntechOpen, 2019).
Fice, M. P. et al. The role of platelet-rich plasma in cartilage pathology: an updated systematic review of the basic science evidence. Arthroscopy: The Journal of Arthroscopic & Related Surgery 35, 961–976 (2019).
Berndt, S., Turzi, A. & Modarressi, A. Production of autologous platelet-rich plasma for boosting in vitro human fibroblast expansion. J. Vis. Exp 168, e60816 (2021).
Wang, Y. et al. Knee effusion volume assessed by magnetic resonance imaging and progression of knee osteoarthritis: data from the Osteoarthritis Initiative. Rheumatology 58, 246–253 (2019).
Anitua, E. et al. Beneficial Effects of Plasma Rich in Growth Factors (PRGF) Versus Autologous Serum and Topical Insulin in Ocular Surface Cells. Curr Eye Res 48, 456–464 (2023).
Yousef, J. et al. Toxicity of silver nanoparticles after Injected intraperitoneally in rats. J Am Sci 8, 589–593 (2012).
Pazzini, J. M. et al. Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus). Pesquisa Veterinária Brasileira 36, 39–44 (2016).
Kazemnejad, S. et al. Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone marrow-derived mesenchymal stem cells to functional hepatocytes‐like cells. Vox Sang 95, 149–158 (2008).
Salem, N., Helmi, N. & Assaf, N. Renoprotective effect of platelet-rich plasma on cisplatin-induced nephrotoxicity in rats. Oxid Med Cell Longev 2018, (2018).
Hesami, Z. et al. Effect of platelet-rich plasma on CCl4-induced chronic liver injury in male rats. Int J Hepatol 2014, (2014).
Minigalieva, I. A. et al. In vivo toxicity of copper oxide, lead oxide and zinc oxide nanoparticles acting in different combinations and its attenuation with a complex of innocuous bio-protectors. Toxicology 380, 72–93 (2017).
Kiernan, J. Histological and histochemical methods. (Scion Publishing Ltd, 2015).
Bancroft, J. D. & Gamble, M. Theory and practice of histological techniques. (Elsevier health sciences, 2008).
Yang, R. et al. Effects of simvastatin on iNOS and caspase-3 levels and oxidative stress following smoke inhalation injury Corrigendum in/10.3892/mmr. 2020.11621. Mol Med Rep 22, 3405–3417 (2020).
Vrolyk, V., Schneberger, D., Le, K., Wobeser, B. K. & Singh, B. Mouse model to study pulmonary intravascular macrophage recruitment and lung inflammation in acute necrotizing pancreatitis. Cell Tissue Res 378, 97–111 (2019).
Ramos-Vara, J. A. et al. American association of veterinary laboratory diagnosticians subcommittee on standardization of immunohistochemistry suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories. J Vet Diagn Invest 20, 393–413 (2008).
Afsar, T., Razak, S. & Almajwal, A. Acacia hydaspica ethyl acetate extract protects against cisplatin-induced DNA damage, oxidative stress and testicular injuries in adult male rats. BMC Cancer 17, 1–14 (2017).
Jensen, E. C. Quantitative analysis of histological staining and fluorescence using ImageJ. Anat Rec 296, 378–381 (2013).
Abdel-Dayem, M. M., Hatem, M. M. & Elgendy, M. S. Histological and immunohistochemical study on nitric oxide synthase and effects of angiotensin receptor blockade in early phase of diabetes in rat kidney. Egyptian Journal of Histology 37, 248–257 (2014).
Nouno, T. et al. Elevation of pulmonary CD163 + and CD204 + macrophages is associated with the clinical course of idiopathic pulmonary fibrosis patients. J Thorac Dis 11, 4005 (2019).
Singh, K. et al. Safety of Nanoparticles: Emphasis on Antimicrobial Properties. Biomedical Applications and Toxicity of Nanomaterials 425–458 (2023).
Pacurari, M., Lowe, K., Tchounwou, P. B. & Kafoury, R. A review on the respiratory system toxicity of carbon nanoparticles. Int J Environ Res Public Health 13, 325 (2016).
Lee, P. et al. Biokinetics of subacutely co-inhaled same size gold and silver nanoparticles. Part Fibre Toxicol 20, 9 (2023).
Jaswal, T. & Gupta, J. A review on the toxicity of silver nanoparticles on human health. Mater Today Proc 81, 859–863 (2023).
Silva, A. V et al. Associations between clinical signs and pathological findings in toxicity testing. ALTEX-Alternatives to animal experimentation 38, 198–214 (2021).
Zhang, Y. et al. Toxicity and efficacy of carbon nanotubes and graphene: the utility of carbon-based nanoparticles in nanomedicine. Drug Metab Rev 46, 232–246 (2014).
El-Naggar, S., Khandil, R. & Salem, M. Phoenix dactylifera seed extract ameliorates the biochemical toxicity induced by silver nanoparticles in mice. International Journal of Cancer and Biomedical Research 5, 73–84 (2021).
Tiwari, D. K., Jin, T. & Behari, J. Dose-dependent in-vivo toxicity assessment of silver nanoparticle in Wistar rats. Toxicol Mech Methods 21, 13–24 (2011).
Yin, N., Yao, X., Zhou, Q., Faiola, F. & Jiang, G. Vitamin E attenuates silver nanoparticle-induced effects on body weight and neurotoxicity in rats. Biochem Biophys Res Commun 458, 405–410 (2015).
Dziendzikowska, K. et al. Time-dependent biodistribution and excretion of silver nanoparticles in male Wistar rats. Journal of Applied Toxicology 32, 920–928 (2012).
Xia, D.-L. et al. Influence of reducing agents on biosafety and biocompatibility of gold nanoparticles. Appl Biochem Biotechnol 174, 2458–2470 (2014).
Gromadzka-Ostrowska, J. et al. Silver nanoparticles effects on epididymal sperm in rats. Toxicol Lett 214, 251–258 (2012).
Sung, J. H. et al. Subchronic inhalation toxicity of silver nanoparticles. Toxicological sciences 108, 452–461 (2009).
Kim, J. S. et al. In vivo genotoxicity of silver nanoparticles after 90-day silver nanoparticle inhalation exposure. Saf Health Work 2, 34–38 (2011).
Ma, W. et al. Ameliorative Effect of Sodium Selenite on Silver Nanoparticles-Induced Myocardiocyte Structural Alterations in Rats. Int J Nanomedicine 8281–8292 (2020).
Adeyemi, O. S. & Adewumi, I. Biochemical evaluation of silver nanoparticles in wistar rats. Int Sch Res Notices 2014, (2014).
Moradi-Sardareh, H. et al. Toxicity of silver nanoparticles on different tissues of Balb/C mice. Life Sci 211, 81–90 (2018).
Xu, N. et al. Wound healing effects of a Curcuma zedoaria polysaccharide with platelet-rich plasma exosomes assembled on chitosan/silk hydrogel sponge in a diabetic rat model. Int J Biol Macromol 117, 102–107 (2018).
Tong, S., Yin, J. & Liu, J. Platelet-rich plasma has beneficial effects in mice with osteonecrosis of the femoral head by promoting angiogenesis. Exp Ther Med 15, 1781–1788 (2018).
Lai, F. et al. Platelet-rich plasma enhances the proliferation of human adipose stem cells through multiple signaling pathways. Stem Cell Res Ther 9, 1–10 (2018).
Roskoski Jr, R. Cyclin-dependent protein serine/threonine kinase inhibitors as anticancer drugs. Pharmacol Res 139, 471–488 (2019).
Fukaya, Y. et al. Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system. Exp Mol Med 44, 330–339 (2012).
Song, S. et al. Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis. European Respiratory Journal 59, (2022).
Salem, N. A., Hamza, A., Alnahdi, H. & Ayaz, N. Biochemical and molecular mechanisms of platelet-rich plasma in ameliorating liver fibrosis induced by dimethylnitrosurea. Cellular Physiology and Biochemistry 47, 2331–2339 (2018).
Maher, Z. M., Elsayied, A.-R. A. & El Din, M. M. M. Clinicopathological studies on the remodeling effect of platelet-rich plasma on lung fibrosis induced by amiodarone in albino mice. Veterinary Medicine & Public Health Journal 1, 108–114 (2020).
Lai, L. P. et al. Use of platelet-rich plasma in intra-articular knee injections for osteoarthritis: a systematic review. PM&R 7, 637–648 (2015).
Lana, J. F. et al. Leukocyte-rich PRP versus leukocyte-poor PRP-The role of monocyte/macrophage function in the healing cascade. J Clin Orthop Trauma 10, S7–S12 (2019).
Jain, N. K. & Gulati, M. Platelet-rich plasma: a healing virtuoso. Blood Res 51, 3–5 (2016).
Abd Elzaher, F., Moussa, M. H., Raafat, M. H. & Emara, M. M. Histological Effect of Platelet Rich Plasma on CCL4 Induced Liver Fibrosis in Adult Albino Rat. Egyptian Journal of Histology 44, 932–940 (2021).
Everts, P., Onishi, K., Jayaram, P., Lana, J. F. & Mautner, K. Platelet-rich plasma: new performance understandings and therapeutic considerations in 2020. Int J Mol Sci 21, 7794 (2020).
Everts, P., Onishi, K., Jayaram, P., Lana, J. F. & Mautner, K. Platelet-rich plasma: new performance understandings and therapeutic considerations in 2020. Int J Mol Sci 21, 7794 (2020).
Andia, I. & Maffulli, N. A contemporary view of platelet-rich plasma therapies: moving toward refined clinical protocols and precise indications. Regenerative Med 13, 717–728 (2018).
Knight, A. D. & Kacker, S. Platelet-Rich Plasma Treatment for Chronic Respiratory Disease. Cureus 15, (2023).
Cervelli, V., Gentile, P. & Grimaldi, M. Regenerative surgery: use of fat grafting combined with platelet-rich plasma for chronic lower-extremity ulcers. Aesthetic Plast Surg 33, 340–345 (2009).
Beilin, G. Regenerative medicine with platelet rich plasma and other blood derived products for dermatology, genitourinary disorders, musculoskeletal disorders and surgical procedures. Handbook of Anti-Aging Medicine (2023).
Karina, K. et al. The Effect of Intravenous Autologous Activated Platelet-Rich Plasma Therapy on “Profibrotic Cytokine” IL-1β Levels in Severe and Critical COVID-19 Patients: A Preliminary Study. Scientifica (Cairo) 2021, (2021).
Yin, N. et al. Silver nanoparticle exposure attenuates the viability of rat cerebellum granule cells through apoptosis coupled to oxidative stress. Small 9, 1831–1841 (2013).
Chen, Z.-Y. et al. The effect of the chorion on size-dependent acute toxicity and underlying mechanisms of amine-modified silver nanoparticles in zebrafish embryos. Int J Mol Sci 21, 2864 (2020).
Mao, B.-H., Chen, Z.-Y., Wang, Y.-J. & Yan, S.-J. Silver nanoparticles have lethal and sublethal adverse effects on development and longevity by inducing ROS-mediated stress responses. Sci Rep 8, 1–16 (2018).
Lee, Y.-L. et al. Toxic effects and mechanisms of silver and zinc oxide nanoparticles on zebrafish embryos in aquatic ecosystems. Nanomaterials 12, 717 (2022).
Sekerci, C. A. et al. Effects of platelet-rich plasma against experimental ischemia/reperfusion injury in rat testis. J Pediatr Urol 13, 317-e1 (2017).
Lee, J.-M. et al. Historical control data from 13-week repeated toxicity studies in Crj: CD (SD) rats. Lab Anim Res 28, 115–121 (2012).
Anand, A. S., Jain, K., Chauhan, A., Prasad, D. N. & Kohli, E. Zinc oxide nanoparticles trigger dysfunction of mitochondrial respiratory complexes and repair dynamics in human alveolar cells. Toxicol Ind Health 07482337231152956 (2023).
Li, L., Li, J., Ma, L., Shang, H. & Zou, Z. SAR-guided development of indole-matrine hybrids as potential anticancer agents via mitochondrial stress/cytochrome c/caspase 3 signaling pathway. Bioorg Chem 106341 (2023).
Farshori, N. N. et al. Copper oxide nanoparticles exhibit cell death through oxidative stress responses in human airway epithelial cells: a mechanistic study. Biol Trace Elem Res 200, 5042–5051 (2022).
Yousef, M. I., Abuzreda, A. A. & Kamel, M. A. Cardiotoxicity and lung toxicity in male rats induced by longterm exposure to iron oxide and silver nanoparticles. Exp Ther Med 18, 4329–4339 (2019).
Singh, R. P. & Ramarao, P. Cellular uptake, intracellular trafficking and cytotoxicity of silver nanoparticles. Toxicol Lett 213, 249–259 (2012).
Foldbjerg, R. et al. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Toxicol Lett 190, 156–162 (2009).
Kim, S.-H. et al. Silver nanoparticles induce apoptotic cell death in cultured cerebral cortical neurons. Mol Cell Toxicol 10, 173–179 (2014).
Blanco, J. et al. Polyvinyl pyrrolidone-coated silver nanoparticles in a human lung cancer cells: time-and dose-dependent influence over p53 and caspase-3 protein expression and epigenetic effects. Arch Toxicol 91, 651–666 (2017).
Piao, M. J. et al. Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis. Toxicol Lett 201, 92–100 (2011).
Satapathy, S. R. et al. Silver-based nanoparticles induce apoptosis in human colon cancer cells mediated through p53. Nanomedicine 8, 1307–1322 (2013).
Suliman Y, A. O. et al. Evaluation of cytotoxic, oxidative stress, proinflammatory and genotoxic effect of silver nanoparticles in human lung epithelial cells. Environ Toxicol 30, 149–160 (2015).
Qiao, D., Zhang, T. & Tang, M. Autophagy regulation by inorganic, organic, and organic/inorganic hybrid nanoparticles: Organelle damage, regulation factors, and potential pathways. J Biochem Mol Toxicol e23429 (2023).
Mello, D. F. et al. In vivo effects of silver nanoparticles on development, behavior, and mitochondrial function are altered by genetic defects in mitochondrial dynamics. Environ Sci Technol 56, 1113–1124 (2022).
Ahmed, L. B. et al. Impact of surface functionalization on the uptake mechanism and toxicity effects of silver nanoparticles in HepG2 cells. Food and Chemical Toxicology 107, 349–361 (2017).
Tang, J. et al. Enzymatic oxydate-triggered AgNPs etching: A novel signal-on photoelectrochemical immunosensing platform based on Ag@ AgCl nanocubes loaded RGO plasmonic heterostructure. Biosens Bioelectron 130, 125–131 (2019).
Jiang, L. et al. Oxidative damage and energy metabolism disorder contribute to the hemolytic effect of amorphous silica nanoparticles. Nanoscale Res Lett 11, 1–12 (2016).
Selstø, C. H. & Ruoff, P. A basic model of calcium homeostasis in non-excitable cells. bioRxiv 2012–2022 (2022).
Barna, B. P. et al. Carbon Nanotube-Induced Pulmonary Granulomatous Disease: Twist 1 and Alveolar Macrophage M1 Activation. Int J Mol Sci 14, 23858–23871 (2013).
Hunyenyiwa, T. et al. Obesity inhibits angiogenesis through TWIST1-SLIT2 signaling. Front Cell Dev Biol 9, 693410 (2021).
Valenzi, E. et al. Single-Nucleus Chromatin Accessibility Identifies a Critical Role for TWIST1 in Idiopathic Pulmonary Fibrosis Myofibroblast Activity. bioRxiv 2021–2022 (2022).
Li, J. et al. Endothelial TWIST1 promotes pathological ocular angiogenesis. Invest Ophthalmol Vis Sci 55, 8267–8277 (2014).
Mammoto, A., Hendee, K., Muyleart, M. & Mammoto, T. Endothelial Twist1-PDGFB signaling mediates hypoxia-induced proliferation and migration of αSMA-positive cells. Sci Rep 10, 7563 (2020).
pubs.rsc.org.
Elsevier.
De Matteis, V. & Rinaldi, R. Toxicity assessment in the nanoparticle era. Adv Exp Med Biol 1048, 1–19 (2018).
Harrison, P. et al. The use of platelets in regenerative medicine and proposal for a new classification system: guidance from the SSC of the ISTH. Journal of Thrombosis and Haemostasis 16, 1895–1900 (2018).
Cui, X. et al. Relationship between free and total malondialdehyde, a well-established marker of oxidative stress, in various types of human biospecimens. J Thorac Dis 10, 3088 (2018).
Dąbrowska-Bouta, B., Sulkowski, G., Strużyński, W. & Strużyńska, L. Prolonged exposure to silver nanoparticles results in oxidative stress in cerebral myelin. Neurotox Res 35, 495–504 (2019).
Zhang, B., Zhang, T., Hu, S. & Sun, L. Association of serum lipid peroxidation and glutathione peroxidase 4 levels with clinical outcomes and metabolic abnormalities among patients with gestational diabetes mellitus: A case-control study in the Chinese population. Frontiers in Bioscience-Landmark 27, 68 (2022).
Li, L. et al. Silver nanoparticles and silver ions cause inflammatory response through induction of cell necrosis and the release of mitochondria in vivo and in vitro. Cell Biol Toxicol 37, 177–191 (2021).
Garcés, M. et al. Alterations in oxygen metabolism are associated to lung toxicity triggered by silver nanoparticles exposure. Free Radic Biol Med 166, 324–336 (2021).
Abd El-Maksoud, E. M., Lebda, M. A., Hashem, A. E., Taha, N. M. & Kamel, M. A. Ginkgo biloba mitigates silver nanoparticles-induced hepatotoxicity in Wistar rats via improvement of mitochondrial biogenesis and antioxidant status. Environmental Science and Pollution Research 26, 25844–25854 (2019).
Yousef, M. I., Abuzreda, A. A. & Kamel, M. A. Cardiotoxicity and lung toxicity in male rats induced by longterm exposure to iron oxide and silver nanoparticles. Exp Ther Med 18, 4329–4339 (2019).
Flores-López, L. Z., Espinoza‐Gómez, H. & Somanathan, R. Silver nanoparticles: Electron transfer, reactive oxygen species, oxidative stress, beneficial and toxicological effects. Mini review. Journal of Applied Toxicology 39, 16–26 (2019).
Alarifi, S. et al. Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats. Int J Nanomedicine 3937–3943 (2013).
Setyawati, M. I. et al. Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE–cadherin. Nat Commun 4, 1673 (2013).
Arpag, H. et al. Protective effects of alpha-lipoic acid on methotrexate-induced oxidative lung injury in rats. Journal of investigative surgery 31, 107–113 (2018).
Mohamed Abd El-Galil, M. & Mohammad Farag Alkot, A. Impact of Crude Aloe Vera gel on Silver Nanoparticle-Induced Lung Cytotoxicity in Adult Male Albino Rats: Functional, Histological and Immunohistochemical Study. Al-Azhar Medical Journal 51, 563–604 (2022).
Asghari, M. H., Hobbenaghi, R., Nazarizadeh, A. & Mikaili, P. Hydro-alcoholic extract of Raphanus sativus L. var niger attenuates bleomycin-induced pulmonary fibrosis via decreasing transforming growth factor β1 level. Res Pharm Sci 10, 429 (2015).
Gamad, N. et al. Metformin alleviates bleomycin-induced pulmonary fibrosis in rats: pharmacological effects and molecular mechanisms. Biomedicine & Pharmacotherapy 97, 1544–1553 (2018).
Li, W.-C. et al. Effects of simvastatin on the expression of inducible NOS in acute lung injury in septic rats. Int J Clin Exp Pathol 8, 15106 (2015).
Ahmed, S. M., Abdelrahman, S. A. & Shalaby, S. M. Evaluating the effect of silver nanoparticles on testes of adult albino rats (histological, immunohistochemical and biochemical study). J Mol Histol 48, 9–27 (2017).
Albert, M. et al. Comparison of inhaled milrinone, nitric oxide and prostacyclin in acute respiratory distress syndrome. World J Crit Care Med 6, 74 (2017).
Liu, X., Jin, X., Yu, D. & Liu, G. Suppression of NLRP3 and NF-κB signaling pathways by α-Cyperone via activating SIRT1 contributes to attenuation of LPS-induced acute lung injury in mice. Int Immunopharmacol 76, 105886 (2019).
Anderson, D. S. et al. Persistence of silver nanoparticles in the rat lung: Influence of dose, size, and chemical composition. Nanotoxicology 9, 591–602 (2015).
Everts, P. A., Sadeghi, P. & Smith, D. Basic Science of Autologous Orthobiologics Part 1. Platelet-Rich Plasma. Physical Medicine and Rehabilitation Clinics (2023).
Mahera, Z. M., Abdel-rahman, A. E. & El-Dinc, M. M. M. Clinicopathological studies on the remodeling effect of Platelet-Rich plasma on lung fibrosis induced by amiodarone in Albino rats. (2020).
Shoeib, H. M., Keshk, W. A., Foda, A. M. & Abo El Noeman, S. E.-D. A. E. A study on the regenerative effect of platelet-rich plasma on experimentally induced hepatic damage in albino rats. Can J Physiol Pharmacol 96, 630–636 (2018).
Keshk, W. A. & Zahran, S. M. Mechanistic role of cAMP and hepatocyte growth factor signaling in thioacetamide-induced nephrotoxicity: Unraveling the role of platelet rich plasma. Biomedicine & Pharmacotherapy 109, 1078–1084 (2019).
Salem, N. A., Hamza, A., Alnahdi, H. & Ayaz, N. Biochemical and molecular mechanisms of platelet-rich plasma in ameliorating liver fibrosis induced by dimethylnitrosurea. Cellular Physiology and Biochemistry 47, 2331–2339 (2018).
Yu, Z., Ouyang, J.-P. & Li, Y.-P. Dexamethasone attenuated endotoxin-induced acute lung injury through inhibiting expression of inducible nitric oxide synthase. Clin Hemorheol Microcirc 41, 117–125 (2009).
Raish, M. et al. Sinapic acid ameliorates bleomycin-induced lung fibrosis in rats. Biomedicine & Pharmacotherapy 108, 224–231 (2018).
Serrano-Mollar, A. et al. In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats. Br J Pharmacol 138, 1037 (2003).
Kopincová, J., Púzserová, A. & Bernátová, I. L-NAME in the cardiovascular system—nitric oxide synthase activator? Pharmacological Reports 64, 511–520 (2012).
Shi, K. et al. Dexamethasone attenuates bleomycin-induced lung fibrosis in mice through TGF-β, Smad3 and JAK-STAT pathway. Int J Clin Exp Med 7, 2645 (2014).
Zhang, Z. et al. Inducible factors and interaction of pulmonary fibrosis induced by prenatal dexamethasone exposure in offspring rats. Toxicol Lett 359, 65–72 (2022).
Hendee, K. et al. Twist1 signaling in age-dependent decline in angiogenesis and lung regeneration. Aging (Albany NY) 13, 7781 (2021).
Patel, B. & Priefer, R. Impact of chronic obstructive pulmonary disease, lung infection, and/or inhaled corticosteroids use on potential risk of lung cancer. Life Sci 120374 (2022).
Puzzitiello, R. N. et al. Adverse impact of corticosteroids on rotator cuff tendon health and repair: a systematic review of basic science studies. Arthrosc Sports Med Rehabil 2, e161–e169 (2020).
Beitzel, K. et al. US definitions, current use, and FDA stance on use of platelet-rich plasma in sports medicine. J Knee Surg 28, 29–34 (2015).
Fadadu, P. P., Mazzola, A. J., Hunter, C. W. & Davis, T. T. Review of concentration yields in commercially available platelet-rich plasma (PRP) systems: a call for PRP standardization. Reg Anesth Pain Med 44, 652–659 (2019).

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Platelet rich plasma mitigates silver nanoparticles induced - pulmonary fibrosis in Wister rats via CASPASE -3 &TWIST-1genes downregulation

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Conclusion

Materials and Methods

Declarations

References

Additional Declarations

Status:

Version 1