Betulin, a Compound Isolated from Crinum asiaticum Bulbs Exerted Anti-Silicosis and Pulmonoprotective Effects Through the Inhibition of NF-κB activation in Rat model

doi:10.21203/rs.3.rs-4717683/v1

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Betulin, a Compound Isolated from Crinum asiaticum Bulbs Exerted Anti-Silicosis and Pulmonoprotective Effects Through the Inhibition of NF-κB activation in Rat model

https://doi.org/10.21203/rs.3.rs-4717683/v1

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Backgroung

Silicosis is an interstitial lung disease that causes shortness of breath, cough, fever, and blue skin and causes a heavy burden to patients both physically and mentally. Silicosis at the moment has no effective treatment, and patients depend on bronchodilators, antitussives, and mucolytic medicines for survival. After all other treatment alternatives have been exhausted, lung transplantation may be the sole choice. Betulin (BET) isolated from Crinum asiaticum bulbs (CAE) has exhibited a multitude of powerful pharmacological properties ranging from antitumor, anti-inflammatory, anti-parasitic, anti-microbial, and anti-viral activities. This work sought to investigate the anti-silicosis and pulmonoprotective effects of betulin and CAE as well as elucidating the possible mechanism in mitigating pulmonary silicosis in crystalline silica induced silicosis in rat model.

Method

In this study, anti-silicosis and pulmonoprotective effects of BET and CAE were investigated after rat models were subjected to lung injury through an intratracheal administration of crystalline silica. Rats were critically observed for morphological changes and were sacrificed. Lungs were harvested for biochemical and histological analysis.

Results

The results showed that CAE and BET reduced significantly (****p < 0.0001) the levels of NF-κB, TNF-α, IL-1β, IL-6, hydroxyproline, collagen types I and III when compared with the negative control group. On broncho alveoli lavage fluid (BALF) biomarkers such as macrophages, lymphocytes, monocytes, and neutrophils, CAE and BET were able to reduce their levels significantly (****p < 0.0001). The CAE and BET were investigated for their anti-oxidant activity and were shown to increase the levels of catalase (CAT) and superoxide dismutase (SOD) while lowering the level of malondialdehyde (MDA). There was also an improvement in lung function when lung tissues were examined histologically.

Conclusion

In conclusion, CAE and BET possessed anti-silicosis effect and occurred through the down regulation of NF-κB and some pro-inflammatory cytokines and also acted as protective mechanisms, facilitating the preservation of the lung’s physiological integrity. The outcome of this study could for serve as leads in drug discovery in the production of anti-silicosis agents

Silicosis

Betulin

Crinum asiaticum

Pulmonoprotective effect

Anti-silicosis effect

Silicosis is a lung disease that progresses over time, causing symptoms such as shortness of breath, cough, fever, and bluish skin. [1–3]. Silicosis causes acute lung injury (ALI) by damaging alveolar epithelial and capillary endothelial cells, leading to diffused pulmonary interstitial edema, alveolar edema, and acute hypoxic respiratory insufficiency [4–7]. A progressive hypoxemia or acute respiratory distress syndromes (ARDS) are clinical manifestation of silicosis [8, 9]. ARDS is characterized by hypoxia, tachypnea, loss of pulmonary compliance, and diffuse alveolar infiltration [10, 11]. The disease significantly impacts patients physically and mentally[12, 13] and induces acute lung injury (ALI) by destroying alveolar epithelial and capillary endothelial cells, which results in diffused pulmonary interstitial edema, alveolar edema, and acute hypoxic respiratory insufficiency [14, 15]. Silicosis is a harmful and severe occupational disease that occurs to people specifically in silica contaminated geographical regions around the world [16].

Individuals with silicosis stand on major risk factors such as cumulative lifetime exposure, total amount of inhaled crystalline silica dust, and individual susceptibility [17–19]. When crystalline silica is inhaled, particles reach the lower respiratory tract and alveolar spaces where gaseous exchange occurs. The inhaled silica interacts with lung tissue ultimately developing fibrotic nodules and scarring around the trapped silica particles which is driven by macrophages activation [20–22].

In acute lung injury, reactive oxygen species (ROS) play an important role [23–25]. Oxygenation is essential for life, and within the lungs, the cells that line the airways and alveolar surface are constantly in contact with oxygen and do not appear to suffer. The partial reduction of oxygen to ROS occurs during cellular metabolism and during cellular injury of any type [26, 27]. Pulmonary defense cells such as neutrophils, monocytes, and macrophages are particularly involved in converting molecular oxygen to ROS [28–30]. Reactive oxygen species balance with the antioxidants produced by the liver under normal physiological condition but a number of agents such as silica and drugs, have been recognizable factors in causing an increase in oxidative stress [31–33]. An oxidative stress is a phenomenon when endogenous antioxidants are not able to prevent the actions of ROS and therefore manifests clinically. Agents known to cause pulmonary injuries play major role in ensuring oxidant–antioxidant imbalance [34, 35].

Numerous variables contribute to the complex pathophysiology of acute lung injury (ALI), which induces the accumulation and activation of inflammatory cells in the lung [36, 37]. Inflammatory cascade is promoted when inflammatory cells during lung injury release pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α), interleukins (ILs). Pro-inflammatory cells such as TNF-α induce oxidative stress [38, 39] and is one of the main factors contributing to alveolar epithelial cell dysfunction and pulmonary vascular endothelial cell damage [40].

The alterations brought on by inhaling silica are closely linked to inflammation and oxidative stress caused by silica. These include the deposition of collagen fibers, thickening of the pulmonary interstitium mass, formation of hyalinized fibrotic nodules, and accumulation of lymphocytes and alveolar macrophages in the interstitium [41]. One of the clinical consequences of silicosis is TB, often known as silicotuberculosis, which continues to be a significant public health issue in low- and middle-income nations [42]. Persistent silica exposure exacerbates pre-existing pulmonary tuberculosis and raises the likelihood of tuberculosis infection in workers [42, 43].

At present, there's no effective treatment for silicosis, and patients are solely treated symptomatically with bronchodilators, antitusic, and mucolytic medicines [44]. After exhausting all other therapeutic alternatives, lung transplantation may be the sole choice [45, 46]. However, there is a poor prognosis for long-term survival following lung transplantation [47]. Much importantly the prevention of the additional exposures to silica and curing the respiratory infections is a key [48]. Novel experimental methods suppress inflammasome activation through antioxidants, autophagy-lysosomal system restoration, or blocking pro-inflammatory cytokines or pro-fibrotic factors [49–54].

Over the years natural products have been used as therapeutic medicines for millions of people globally which Ghana is one of the epicenters [55; 56–58]. Natural scavengers of free radicals and antioxidants can be used to treat acute lung injury and more so providing a protective mechanism to lungs when there is toxicity or injury [59, 60].

Natural products have a huge role to play in drug research and development and recent studies have shown that herbal medicines and some isolated compounds can be of benefit in the treatment of pulmonary silicosis as they can reduce changes related with silica-induced inflammation, fibrosis, and oxidative stress [2, 61–63]. Certain plant-based compounds have the ability to affect one or more of the silicosis pathomechanisms, which can help to lessen the inflammation and/or fibrosis caused by silica. This work was aimed at investigating the anti-silicosis effect and pulmonoprotective effects of Crinum asiaticum L. bulbs extract (CAE) and its active compound betulin (BET) in crystalline silica-induced silicosis in rat model as well as investigation the possible mechanism associated with this pharmacological activities.

Crinum asiaticum L. bulb (CAE) is a bulbous plant and it is widely distributed in Ghana and other part in Africa (56). It is used by the local folks in southern Ghana for several ethno-medicinal uses such as wound healing, skin infection and severe cough. In other part of the world the plant is used to treat skin conditions, mumps, tonsillitis, fever, earache, boils, hernia, rheumatism, urinary issues, bone fracture, edema, and poison antidote [64, 65]. This plant exhibits several biological and pharmacological effect and recently the anti-tuberculosis effects of the CAE in an aerosol induced mice model was reported [57]. The toxicity profile of the bulb extract of the plant was reported and was known to possess no known toxicity with the LD ₅₀ greater than 5000 mg/kg [66]. Pharmacological effects reported on the plant includes: the antimicrobial, anti-inflammatory, anti-pyretic, anti-analgesic, anti-cancer effects [58].

Triterpenes are one of the class of natural compounds having a lot of biological and pharmacological effects [67, 68]. Although betulin is widely isolated from the outer bark of birches and sycamore trees, it can be found in the bulbs of Crinum asiaticum L [69]. A triterpene of the lupine class, betulina exhibits a range of pharmacological and biological properties, including anticancer properties [70–73], anti-inflammatory [74, 75], antimicrobial [76, 77], anti-liver fibrosis effects [78] and anti pulmonary fibrosis effect through downregulation of pro-inflammatory and pro-fibrotic cytokines [79]. The betulin toxicity profile has been documented, and the doses chosen for this study were less than the lethal dose in a study carried out by Makarova in 2011 [80].

Materials

Crinum asiaticum L. bulbs, rotary evaporator (Buchi Labotechnik Rotavap R-210), autoclave (Sano clav), oral gavage, falcon tubes.

Chemicals and reagents

Crystalline silica, Betulin (purity > 98% Chem Cruz, USA) Prednisolone (PD) (Cipla Pharma, India), Interleukin-6 ELISA KIT (R and D Systems, UK), Rat TNF-alpha ELISA KIT (R and D Systems, UK), Rat interleukin-1β (Cusabio, China), Rat hydroxyproline kit (Cusabio, China), Rat collagen I and III (Cusabio, China), NF-κB (Cusabio, China), chloroform (BDH Prolabo).

Laboratory animals

Male specific pathogen-free wistar rats (140 ± 25 g, 8–10 weeks old) were purchased from Noguchi Memorial Institute, Ghana and were kept in the animal house in the Department of Pharmacology, KNUST. The rats were kept in sterilized cages in rooms at 60–70% humidity and 24–26°C with a 12/12 h dark–light cycle and given fresh water and food each week. The study was approved by the Institutional Animal Care and Use Committee of Kwame Nkrumah University of Science and Technology with an ethical code (FFPS-AEC/CA05/18) and experiment was conducted according to the national and institutional rules regarding animal experiments.

Groupings and modeling

Rats were divided into eight groups (group I-VIII) with each group having 7 rats (n = 7). In detail, the rats were placed on a platform and then anesthetized with intraperitoneal injection of 2% pentobarbital (0.2 mL/100 g). Next, the trachea of rat was separated and silica suspension (50 mg/mL, 1 mL) was slowly added to the trachea with a syringe. Intratracheal instillation of silica (50 mg/mL, 1 mL) was used to induce silicosis in rats in groups II-VIII. The control/naïve group (group I) was given equal amount of normal saline. After 24 hours of silicosis induction treatment began in groups III-VIII. Group II was the negative control group hence no treatment was carried out. Group III, 1V and V were treated with 100, 500 and 1000 mg/kg CAE respectively, also group VI and VII were treated with 25 and 50 mg/kg respectively for 28 days after silicosis induction. Group VIII was designated as the positive control group and was treated with 10 mg/kg of prednisolone (Pred). Assessment and recording of body weight of rats was conducted weekly during the experimental period.

At the end of the experimental period, all animals were weighed and sacrificed by an overdose of ether. Their lungs were harvested and washed in chilled PBS (pH 7.4). The left lungs were used for the determination of biochemical markers such as inflammatory mediators like IL-6, IL-1β and TNF-alpha and the assessment of anti-oxidant markers such as superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) activities. The right lungs were also fixed in 10% formalin for histological assessment.

Biochemical markers

Anti-oxidants levels

Anti-oxidant assay was performed on the lung homogenates for anti-oxidant markers.

Total protein content determination

The methodology of this assay has been reported [81]. A reference, bovine serum albumin (BSA) used and measurement taken at 660 nm. The viability of the test was maintained in a basic environment of pH between 10 and 10.5. The BSA calibration graph was used in the estimation of the total protein content in the sample.

Determination of superoxide dismutase (SOD) Levels

Superoxide dismutase activity was investigated and the procedures reported by [82]. In this assay, an amount of homogenate (500 µL) was added to an iced cold chloroform (150 µL) and 750 µL ethanol (96% v/v). It was followed by 60s vortexing and was centrifuged at 2000 rpm for 20 min, the supernatant was separated from the pellets. One mL carbonate buffer (0.1 M; pH 10.2) and 0.5 mL EDTA (0.6 mM) were used to treat 500 µL of supernatant. Adrenochrome formation was then initiated by the addition of adrenaline (0.05 mL of 1.3 mM). Absorbance was measured at 480 nm. The amount of SOD needed to inhibit adrenaline oxidation was expressed as follows:

% inhibition =\(\:\:\frac{\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{t}\text{e}\text{s}\text{t}\:--\:\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\text{o}\text{f}\:\:\text{b}\text{l}\text{a}\text{n}\text{k}}{absorbance\:of\:test}\) x 100

SOD activity was expressed in units per mg protein, where 1 unit is the enzyme quantity needed to show 50% dismutation of the superoxide radical at 25°C calculated using the following formula:

Unites of SOD activity/mg protein = \(\:\frac{\text{%}\:\text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:}{50\:x\:wt\:of\:protein}\)

Determination of catalase (CAT) Levels

The method for catalase levels assay was done as reported earlier by [83]. (0.1 mL) of homogenate was added to a mixture of 0.4 mL H₂O₂ (1.18 M) and 1 mL phosphate buffer (0.01 M; pH 7.0). Resulting mixture was incubated for 5 min at 25°C. The reaction was terminated with an amount of 2 mL dichromate-acetic acid reagent constituting (3:1 parts of glacial acetic acid and 5% w/v potassium dichromate respectively). The catalase activity was expressed as units per mg protein and absorbance read at 620 nm.

mUnit of CAT action/mg = \(\:\frac{absorbance\:}{39.4\:x\:wt\:of\:protein}\) x 100

Effects of CAE and BET on Malondialdehyde Levels

An amount of 3 mL (0.5% TBA in 3 ml 20% TCA ) was added to a small amount of homogenate of about 1 ml in a test tube. Mixture was heated at 95°C for 30 min, then cooled, and centrifuged at 5000 rpm for 10 min. Absorbance was read at 532 nm and then read again at 600 nm [84].

Nmol MDA/mg protein = \(\:\frac{absorbance\left(532nm\right)-absorbance\:\left(600nm\right)\:}{155\:x\:total\:protein}\) x 10⁶

Histological studies

The right lungs were produced as 5 mm thick slices, paraffin embedded, fixed in 10% neutral buffered formalin, and stained with hematoxylin and eosin (H&E) for examination under a light microscope. A semi-quantitative approach was used to quantify three factors: pneumocyte hyperplasia, infiltration by inflammatory cells, and thickening of the alveolar septa. The degree of damage was measured using a graded system from 0 to 4, where 0 represents no harm, 1 represents extremely low damage, 2 represents mild damage, 3 represents moderate damage, and 4 represents severe damage. Fields were randomly viewed by experts with no bias with a camera attached to a microscope at x100.

Statistical analysis

Results expressed as mean ± standard error of mean (SEM). Utilizing Graph Pad Software Prism 9.0, statistical analysis and graphing were performed (Graph pad Software Inc.,San Diego, CA, USA). Significant differences between groups were determined using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons.

Ethical approval

Ethical clearance from the animal ethical committee at the Faculty of Pharmacy and Pharmaceutical Sciences, KNUST was issued with ethical code FFPS-AEC/CA05/18.

Effect of CAE and BET on body weight of rats during silica-induced silicosis

As shown in table 1, the effects of CAE and BET on body weight of rats were assessed during the experimental period. Weight analysis on 500, 1000 mg/kg CAE and 25, 50 mg/kg BET groups showed significant gradual increase in body weight of rats when compared with the negative control group. The 100 mg/kg CAE poorly maintained body weight of rats since there was a reduction in body weight.

Table 1. Effect of Crinum asiaticum bulbs extract and betulin on the body weight of silica-induced silicosis in rats

Doses

Duration

Mean weight ± SEM

Significance

Naïve

Week one

Week two

Final week

141.0 ± 9.81

146.3 ± 4.21

151.7 ± 8.65

0.1433

Negative control

Week one

Week two

Final week

162.0 ± 3.21

158.4 ± 4.65

154.3 ± 2.40

0.2408

10 mg/kg PD

Week one

Week two

Final week

156.0 ± 5.78

157.8 ± 3.71

159.3 ± 6.98

0.6816

100 mg/kg CAE

Week one

Week two

Final week

147.0 ± 5.46

145.8 ± 2.54

142.7 ± 5.36

0.5309

500 mg/kg CAE

Week one

Week two

Final week

153.0 ± 11.60

153.8 ± 5.25

154.7 ± 9.03

0.8945

1000 mg/kg CAE

Week one

Week two

Final week

165.0 ± 6.60

167.3 ± 5.71

170.0 ± 6.66

0.4642

25 mg/kg BET

Week one

Week two

Final week

154.5 ± 4.21

155.5 ± 3.67

157.9 ± 5.11

0.3671

50 mg/kg BET

Week one

Week two

Final week

162.0 ± 6.19

162.9 ± 4.88

164.1 ± 7.22

0.4127

Data were expressed as mean ± SEM with Dunnett’s multiple comparison test. (N = 7), silicosis control group – Negative, Crinum asiaticum bulbs extract – CAE, Betulin – BET and Pred – Prednisolone.

Effect of CAE and BET on hydroxyproline content in silica-induced silicosis in rats

The significant increase in hydroxyproline level in the silica induced group (negative control) (####ρ ≤ 0.0001 negative control vrs naïve) was markedly reduced in the CAE and BET treatment groups as shown in Figure 1. The level of hydroxyproline increased in the 100 mg/kg treatment group.

Effect of CAE and BET on collagen type I and III level in silica-induced silicosis in rats

The administration of CAE and BET significantly reduced the levels of collagen I and III in the silicosis induced models when compared with the disease control group which saw high levels of collagen I and III contents in Figure 2.

Effects of CAE and BET on total protein, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels

The administration of CAE and BET in the silica-induced treatment groups significantly reduced the level of total protein content and malondialdehyde when compared to the negative control group (***ρ < 0.0001). The levels of CAT and SOD significantly elevated when compared with the silicosis-induced group (****ρ < 0.0001) as shown in Figure 3.

Effects of CAE and BET on pro-inflammatory cytokines

Pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 1β (IL-1β) and nuclear factor ƙappa-β (NF-ƙβ) were measured in silica induced silicosis in rats models. It was found out that CAE and BET significantly (ρ ≤ 0.005) reduced the levels of the cytokines measured when compared with the disease control group which saw increased in their levels.

Effect of CAE on broncho-alveolar lavage fluid (BALF) markers on silica-induced silicosis in rats.

Broncho-alveolar fluid lavage biomarkers such as neutrophils, lymphocytes, monocytes and macrophages were measured in the assessment of their levels. The outcome as shown in Figure 5 explicitly depicts that CAE and BET significantly (ρ ≤ 0.005) reduced BALF markers produced. On the other hand the 100 mg/kg CAE showed little effect in terms of reducing the BALF markers.

Effect of CAE and BET on lung tissue histology

Lungs of rats were stained with hematoxylin and eosin and tissues were examined histologically. Lungs harvested in silica-induced silicosis group stained with H & E showed extensive lung tissue damage compared to the CAE and BET treatment groups as shown in Figure 6. On the other hand the H & E stained sections of CAE and BET treatment groups presented with an improved lung function with several viable alveolar spaces and other indicators such as terminal bronchi, lung parenchyma were affected least as shown. Graphs in Figure 6 (a-d) showed statistical analysis of the various pathological indications that were observed such as the thickness of alveolar septum, severity of inflammation, infiltration of inflammatory cells and necrosis respectively.

Acute lung injury (ALI) damages the alveolar epithelial and capillary endothelial cells with a manifestation of diffused pulmonary interstitial edema, alveolar edema and an acute hypoxic respiratory insufficiency [36,85]. Silicosis is a progressive occupational lung disease caused by retention of crystalline-silica dust inhaled and deposited on the lung alveolar spaces [62,86]. It is a common life threatening lung disease and reactive oxygen species (ROS) play an important role in its pathogenicity [87,88]. There are several therapeutic strategies mapped by research scientists in mitigating pulmonary silicosis giving room for the increasing number of experimental studies to assess the potentials of medicinal plants and their related phytochemicals in the treatment of lung silicosis [89–93]. Some of these plant-based compounds can influence one or even several of the pathomechanisms of silicosis and thereby they can alleviate the silica-induced silicosis [61,94]. In the pathogenesis of silicosis, there are several grey areas that have not been elucidated fully therefore giving room for fewer treatment options [95]. Cytokines such as TNF-α, IL-1β, IL-6 and TGF-β1 are induced by some inflammatory cells such as NF-κB when silica is deposited in the lungs [96,97]. The inflammatory cytokines like IL-6, TNF-α and IL-1β produced up-regulate the proliferation, the recruitment and maturation of fibroblast in the lungs [96,97].

In this work, there was an oral treatment of Betulin (BET) and Crinum asiaticum bulbs extract (CAE) in crystalline silica-induced silicosis in rat model. The outcome of this experiment showed BET and CAE ameliorated crystalline silica-induced silicosis by the reduction of hydroxyproline levels, collagen type I and III levels and more so the levels of pro-inflammatory cytokines were reduced. It also decreased oxidative stress by elevating markedly the levels of anti-oxidants markers.

Weight loss is associated with silicosis because of the suppression of plasma leptin by pro-inflammatory cytokines like IL-6, TNF-α and IL-1β [42,98,99]. Plasma leptin is known for its role in regulating appetite [100]. The body weight of rats were assessed throughout the experimental duration (Table 1) and was shown that in the BET and CAE treatment groups there was gradual increase in the body weight of rats compared to a declined body weight in the silica-induced silicosis model (negative control group). This significantly suggested that BET and CAE were able to manage the disease condition well therefore preventing loss of body weight.

It is reported that in silicosis, there is a marked increase in hydroxyproline content in the lungs [62,63]. In silicosis there is lung fibrosis which is attributed to the deposition of collagen fibers produced by the fibroblasts. Hydroxyproline enhances the stability of collagen fibers on the lungs, therefore the higher the collagen deposition, the higher the hydroxyproline content [101–103]. Both collagen and hydroxyproline content are significant features within the plasma of patients with silicosis [3,104]. One of the therapeutic targets to treat pulmonary silicosis is to degrade collagens and to reduce the level of hydroxyproline content [105]. In this work, the levels of hydroxyproline content, collagen I and collagen III were assessed Figure 1 and Figure 2 respectively. In the negative control group, there were higher levels of hydroxyproline content, collagen I and III which saw a significant increase (****ρ < 0.0001). The various treatment doses of BET and CAE showed significant decrease in collagen I, III and hydroxyproline levels (****ρ < 0.0001) as compared with the negative control group. This confirmed what had been reported in regards to agents with anti-silicosis effects, which reduce some notable biomarkers like hydroxyproline content and collagen type I and III since these markers are crucial in lung damage [94,97,106,107].

One of the therapeutic strategies in treating silicosis is the demand for a therapeutic regimen that has a pulmonary protective effect and this could serve as an adjunct therapy and more so enhance therapeutic effectiveness. Over the years natural products have been used as a therapeutic medicine for millions of people globally. Natural scavengers of free radicals and antioxidants can be used to treat acute lung injury and more so providing a protective mechanism to lungs when there is toxicity or injury [108,109].

The instillation of crystalline silica in rats exhibited elevated levels of free radicals, which led to a marked increased in oxidative stress. The damaging effect was ameliorated by the administration of doses of CAE and BET. The result in Figure 3 showed that CAE and BET treatment groups significantly reduced total protein content (***ρ < 0.005). Reactive oxygen species (ROS) can damage different cellular components, including lipids and protein, and decline anti-oxidant enzymes. A decrease in total protein content signifies a reduced or controlled oxidative stress [110]. Oxidative stress is the imbalance between anti-oxidants and free oxygen, hydrogen radicals and oxidants [111]. This happens due to excessive production of ROS and the failure of the anti-oxidants to neutralize them. Lipid peroxidation is the process in which lipids are oxidized to liberate lipid peroxide as a primary product [112]. End product of lipid peroxidation is the elevation of serum malondialdehyde (MDA) which is a common biomarker used to assess the degrading nature of lipids when there is oxidative stress [113,114]. Agent with antioxidant effect decreases oxidative stress and that matter reduce lipid peroxidation and finally lowering MDA level.

In this study, CAE and BET significantly reduced the level of MDA (****ρ < 0.0001) as shown in Figure 3. The silica-induced silicosis (negative control) group in Figure 3 presented high level of MDA when compared to the naïve control group. It is reported that increase MDA increases permeability of Ca²⁺ across cell membrane which destroys capillary membrane of alveolar, membrane proteins and enzymes [115–118]. This leads to fluid leakage inside the pulmonary tissue contributing to cellular destruction and also growing oxidative burden [115].

Among the various antioxidants, the most studied and reported anti-oxidant metalloenzyme is the superoxide dismutase (SOD) (119,120). SOD plays a very significant role in anti-oxidant defense against free oxygen and hydrogen radicals [119]. In minimizing oxidative stress by SOD, superoxide anions (free radicals) are converted to hydrogen peroxide and oxygen molecule in which hydrogen peroxide is subsequently converted to water. This conversion is well executed and catalyzed by SOD because hydrogen peroxide is regarded as essential sensor in redox metabolism and when increased will lead to oxidative stress [121,122]. Oxidative stress and apoptosis are implicated in silica-induced lung injury as reported and this was confirmed in this study when the negative control group saw a significant decrease in SOD (****ρ < 0.0001) as shown in Figure 3. CAE and BET suppressed oxidative stress by enhancing anti-oxidant defense through SOD elevation. Another biomarker of anti-oxidant is the catalase (CAT). Same as the SOD, catalase catalyzes the neutralization of free oxygen and hydrogen radicals thus reducing oxidative stress from infection or inflammation. It was reported that increased lung catalase confers a protection on the lungs against infections and other degenerative lung diseases (123,124). Moreover catalase elevation had shown to down regulate inflammatory mediators (125,126). In this study, CAT level in the negative control group markedly reduced whereas on the other hand in the CAE and BET treatment groups showed significant increase (****ρ < 0.0001) in CAT level in Figure 3. The anti-oxidant defense exhibited by CAE and BET suggested these agents could offer a protective effect on the lungs during injury.

Oxidative stress has the tendency to induce inflammatory cytokines such as IL-1β, TNF-α, IL-6 and etc in the lungs and cytokines are known to enhance the progression of lung disease [127–130]. These mediators are known to up-regulate collagen fibers deposition and extracellular matrix remodeling which further cause scaring of lungs tissue with a period of time [131,132].

Nuclear factor ƙappa-β (NF-ƙβ) is a family of transcription factor that function as dimers and regulate genes involved in immunity, inflammation and cell survival [133–136]. Giuseppe et al reported that systemic inhibition of NF-κB activation protects individual from silicosis and it is a pivotal transcription factor in crystalline-silica induced silicosis [133]. This makes NF-κB a potential treatment target for silica induced lung injury. In a centrifuged lung tissue homogenates harvested from the various control and treatments groups, the level of NF-κB was measured and was found out that the level of NF-κB was significantly reduced (****ρ < 0.0001) in the BET and CAE treatment groups (Figure 4) as compared with the control group which there was high increased in NF-κB. The reduction of NF-κB by CAE and BET suggested the potential role of the these agents in ensuring the treatment of silicosis since agents that down regulate NF-κB level are better candidates in the treatment of silica-induced silicosis since they could mitigate the severity and prevent lung fibrosis.

Immune cells activation due to persistent deposition of silica in the lung leads to the activation of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6 and other associated cytokines and bioactive substances [137–139]. In this work, the levels of the inflammatory cytokines such as TNF-α, IL-1β, IL-6 were measured in lung tissue homogenates (Figure 4). It was found out that in the negative control group, there was increased levels of the cytokines measured which was in agreement with what was reported by other authors [41,51,89,93,94]. The BET and CAE treatment groups showed significant (****ρ < 0.0001) decrease in the levels of TNF-α and IL-1β as well as IL-6. The significant decrease in inflammatory cytokines by any potential anti-silicosis agent makes it a better choice in the management of silica-induced silicosis since these cytokines are linked to extracellular matrix deposition and lung remodeling [139–141].

In this study, the relative counts of leukocytes were provided to show a complex dynamic of the individual types of cells that may have a different rate of migration into the tissue. In the broncho alveoli lavage fluid (BALF) of silica induced silicosis control group (negative control), the levels of macrophages, lymphocytes, monocytes and neutrophils were measured and found out to be elevated when compared with the naïve controls (Figure 5). On the other hand, leucocytes counts measured in the BALF of the CAE and BET treated groups showed significant (****ρ < 0.0001) reduction in the levels of macrophages, lymphocytes, monocytes and neutrophils. Similar trend in the increase of leukocytes count particularly macrophages, neutrophils in BALF of silica-induced silicosis models have been reported by some authors [142–145].

Histological examination of hematoxylin and eosin (H and E) stained section of lung tissue showed an improved lung function and it was justified by the microscopic presentation of several focal points on the lung tissue as such the alveoli, alveolar septum, terminal bronchi, blood vessel congestion, infiltration of inflammatory cells, severity and architecture of lung parenchyma. CAE and BET treatment groups showed there were several viable alveoli, little or no blood vessel congestion, bronchioles were not infiltrated with inflammatory cells and the lung architecture was maintained when compared with the negative control group (Figure 6). Prednisolone control group also showed an improved lung function when lungs were assessed for pathological alterations. Stained lung tissue section of the negative control group unlike CAE and BET presented with necrosis, terminal bronchi infiltrated with inflammatory cells and blood vessel congestion. Level of inflammation was assessed, analyzed, quantified and presented in graphics (Figure 6 a-d). This was observed in multiple focal areas under the microscope and was based on parameters such as severity, thickness of alveolar septum, infiltration of inflammatory cells and necrosis. In assessing the thickness of interalveolar septum (Figure 6a), CAE and BET significantly (****ρ < 0.0001) maintained the thickness of various alveolar septum compared with an increased in thickness in the negative control group. Alveolar septal thickness is increased during inflammation as a result of congestion and infiltration of inflammatory cells which migrate across alveolar spaces [146–148]. In the assessment of the severity (Figure 6b), infiltration of inflammatory cells (Figure 6c) and necrosis (Figure 6d), CAE and BET attenuated the level of inflammation significantly (****ρ < 0.0001) which therefore contributed to the restoration of the physiological function of the lung function. The histological findings confirmed what were reported earlier [149–151].

In conclusion, this study demonstrated that BET and CAE attenuated silica-induced lung injury through the exertion of a protective effect on the lungs by exhibiting antioxidant defense, the reduction of tissue damage, decreased inflammatory cytokines, reduced hydroxyproline and collagen I and III concentration. There was also an improved lung function when lungs were observed histologically. The effects produced by BET and CAE could be attributed to the downregulation of NF-κB. The effects produced suggested that CAE and BET could be leads in the discovery of promising agents to treat pulmonary silicosis.

PD Prednisolone

BALF Broncho alveolar lavage fluid

IL-6 Interleukin 6

TNF-alpha Tumor necrosis factor - alpha

TGF Transforming growth factor

CAE Crinum asiaticum bulbs extract

BET Betulin

SOD Super oxide dismuthase

CAT Catalase

MDA Malondialdehyde

LD₅₀Lethal dose 50

NF-κB Nuclear factor – kappa B

ELISA Enzyme linked immunosorbent assay

ARDS Acute respiratory distress syndromes

ROS Reactive oxygen species

BSA Bovine serum albumin

Acknowledgment

Not applicable

Authors’ Contributions

Conceptualization of this study was done by M.O and C.A.D. Investigation was by M.O, C.A.D, R.O.A and Prince Gyan. Data curation was by M.O and J.A. Formal analysis by F.S and M.O. Methodology was conceived by A.A.T and W.A.A. Resources for the research provided by M.O, C.A.D and J.A. C.A.D supervised this work and was validated by M.O. Writing of original draft manuscript by M.O and review, editing were done by C.A.D, F.S and A.A.T

Funding

Not applicable

Availability of data and materials

Data sets formed part of a PhD thesis and is made available in Kwame Nkrumah University of Science and Technology repository unit. https://ir.knust.edu.gh and also published at figshare. https://doi.org/10.6084/m9.figshare.22970378 . Data would also be made available upon request from the corresponding author.

Ethics approval and consent to participate

All procedures and experiments involving animals were approved by the animal ethical committee at the Faculty of Pharmacy and Pharmaceutical Sciences, KNUST was issued with ethical code FFPS-AEC/CA05/18.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Table 1

Effect of Crinum asiaticum bulbs extract and betulin on the body weight of silica-induced silicosis in rats
Doses	Duration	Mean weight ± SEM	Significance
Naïve	Week one Week two Final week	141.0 ± 9.81 146.3 ± 4.21 151.7 ± 8.65	0.1433
Negative control	Week one Week two Final week	162.0 ± 3.21 158.4 ± 4.65 154.3 ± 2.40	0.2408
10 mg/kg PD	Week one Week two Final week	156.0 ± 5.78 157.8 ± 3.71 159.3 ± 6.98	0.6816
100 mg/kg CAE	Week one Week two Final week	147.0 ± 5.46 145.8 ± 2.54 142.7 ± 5.36	0.5309
500 mg/kg CAE	Week one Week two Final week	153.0 ± 11.60 153.8 ± 5.25 154.7 ± 9.03	0.8945
1000 mg/kg CAE	Week one Week two Final week	165.0 ± 6.60 167.3 ± 5.71 170.0 ± 6.66	0.4642
25 mg/kg BET	Week one Week two Final week	154.5 ± 4.21 155.5 ± 3.67 157.9 ± 5.11	0.3671
50 mg/kg BET	Week one Week two Final week	162.0 ± 6.19 162.9 ± 4.88 164.1 ± 7.22	0.4127
Data were expressed as mean ± SEM with Dunnett’s multiple comparison test. (N = 7), silicosis control group – Negative, Crinum asiaticum bulbs extract – CAE, Betulin – BET and Pred – Prednisolone.

No competing interests reported.

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Editorial decision: Revision requested
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Betulin, a Compound Isolated from Crinum asiaticum Bulbs Exerted Anti-Silicosis and Pulmonoprotective Effects Through the Inhibition of NF-κB activation in Rat model

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