Propolis versus Nigella sativa Oil challenging the apoptotic pathway in Propylthiouracil-induced Hypothyroidism on parotid gland

doi:10.21203/rs.3.rs-3462540/v1

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Propolis versus Nigella sativa Oil challenging the apoptotic pathway in Propylthiouracil-induced Hypothyroidism on parotid gland

https://doi.org/10.21203/rs.3.rs-3462540/v1

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Defending parotid gland against deleterious effects of hypothyroidism may be conquered by using herbal substances such as propolis or Nigella sativa oil.

Aim of work: This study was theorized to evaluate probolis versus NsO in amelioration of hazards of Propylthiouracil-induced Hypothyroidism on parotid gland; amending the influences of apoptosis and oxidative stress

Material and Methods: 30 male rats were utilized; group I (control), group II (sham control), group III (PTU-induced hypothyrodism), Group IV: (PTU + probolis), Group V: (PTU + NsO), Histo-pathological; H&E, masson trichrome, toluidine blue, α-SMA were performed. MDA and GPx, molecular studies for Bcl2, Bax, caspase and p53 were evaluated.

Results: severe pathological alterations in group III; irregular outline, shrunken acini with darkly stained pyknotic nuclei, interstitial exudate, inflammatory infiltrates, and extravasated blood were encountered. Marked increment in mean values of collagen, mast cells, SMA, P53, Bax and caspase. Surprisingly, Nso and probolis ameliorated these deleterious changes in an outstanding manner.

The parotid gland necessitates high energy expenditure and high blood flow to maintain its function (Rodriguez et al., 2009). The parotid gland is influenced by numerous conditions such as infection, tumors and hypothyroidism.

Hypothyroidism may be congenital, acquired or induced (Hayat et al., 2010). Propylthiouracil (PTU) is a thiouracil derived drug, used for induction of hypothyrodism through inhibition of thyroperoxidase enzyme so dropping T3 and T4 release and also interfering with conversion of T4 to T3 (Alkalby and Alzerjawi, 2013).

In hypothyroidism, the basal metabolic rate decreases and the liberation of reactive oxygen species (ROS) increases which result in lipid peroxidation and DNA impairment (Messarah et al., 2010). The later consequences apoptosis-induced tissue destruction (Zhou et al., 2009).

Tissue impairment starts with lipid radical release in the cell membrane, then the damage is accomplished by the formation of toxic products such as aldehyde, alkane and malondialdehyde (valko et al., 2006). With prolonged ROS production, there is progression of inflammation with recruitment of inflammatory cells such as macrophages and mast cells (Griffith et al., 2009).

P53 is a pro-apoptotic molecule. It is triggered by extreme ROS and DNA damage. It constrains the expression of Bcl-2 family proteins which play a crucial role in direction of apoptotic pathway. This family comprises pro-apoptotic and anti-apoptotic proteins. Bcl-2 and Bcl-xl are anti-apoptotic proteins; they prevent the release of cytochrome C and maintain mitochondrial integrity. On the other hand, Bcl-2 associated X protein (Bax) is pro-apoptotic protein which allows the release of cytochrome C and induction of apoptosis (Ghobrial et al., 2005).

Shielding of parotid gland against deleterious effects of hypothyroidism may be attained by using herbal substances such as propolis or Nigella sativa oil.

Propolis is a natural resinous mixture collected by hony bee. It is composed of several compounds such as flavonoids, terpenes and phenolics and their esters (Zullkiflee et al., 2022). It acts as an anti-inflammatory, antimicrobial and antioxidant agent (Araujo et al., 2012).

Nigella sativa is known as black seeds. The main constituent of its volatile oil is thymoquinone which has an antioxidant, anti-inflammatory and antitumor activity (Talib and Abukhader, 2013).

This study was carried out to compare probolis versus NsO in modulating the hazardous effects of Propylthiouracil-induced Hypothyroidism on parotid gland specially in ameliorating the impacts of apoptosis and oxidative insult

2.1-Chemicals:

2.1.1 Propylthiouracil (PTU): purchased in the form of 50mg tablets, commercially known as Thyrocil, manufactured by Amoun Pharmaceutical Company, Egypt.The tablets were crushed and dissolved in 5ml saline; each 1ml contained 10mg PTU.

2.1.2 Propolis: purchased in the form of 5g powder from Shana Honey Company, Egypt .The powder was dissolved in 50ml distilled water ; each 1ml contained 100mg propolis .

2.1.2Nigella sativa oil (NsO): purchased in the form of 450mg Baraka capsules (2ml), manufactured and packed by Pharco Pharmaceuticals Company, Egypt. The oil was extracted from each capsule by a graduated syringe then given to the rats by gastric gavage.

2.2- Animals:

The study was conducted on 40 adult male albino rats, weighing 150–200g, obtained from the Animal House, Faculty of Medicine, Cairo University and maintained according to the standard guidelines of Institutional Animal Care and Use Committee and after Institutional Review Board approval (approval number: CU-III-F-82-22).

The animals were acclimatized in the laboratory for a period of two weeks before carrying out the experiment. They were housed in cages, five rats/cage, under standard laboratory and environmental conditions and were given standard rodent food and water ad libitum. Twenty animals were randomly selected and used as normal and sham control. The other 20 rats were subjected to hypothyroidism induction.

2.3.1 Induction of hypothyroidism:

The allocated rats received PTU dissolved in saline at a daily dose of 10 mg/kg bw via gastric gavage till the determined date for scarification (Hwang et al., 2018). The induction of hypothyroidism was confirmed to be established 6 weeks after the onset of PTU administration. The PTU was given throughout the experiment to sustain the hypothyroid effect on the parotid; if PTU is withdrawn after 2 weeks, the rats will return to the euthyroid state (Massoud et al., 2012).

2.3.2 Animal groups:

The rats were divided into the following groups:

Group I (Normal control): consisted of 6 rats that received nothing and were sacrificed after 6weeks
Group II (Sham control): consisted of 6 rats that received 1ml/kg bw daily saline solution via gastric gavage and were sacrificed after 6weeks:
Group III: (PTU-induced hypothyrodism): consisted of 6 rats that were subjected to induction of hypothyroidism and were sacrificed after 6weeks.
Group IV: (PTU + propolis): consisted of 6 rats that were subjected to induction of hypothyroidism. After two weeks, propolis was given at a daily dose of 100 mg/kg BW via gastric gavage for 4 weeks then the animals were sacrificed (Samah et al., 2018). Therefore, sacrifice was performed 6 weeks after the onset of the experiment.
Group V: (PTU + NsO): consisted of 6 rats that were subjected to induction of hypothyroidism. After two weeks, NsO was given at a dialy dose of 400 mg /kg bw via gastric gavage for 4 weeks then the animals were sacrificed (Khalawi et al., 2013). Therefore, scarification was performed 6 weeks after the onset of the experiment.

2.3.4- Confirmation of hypothyriodism induction

The technique was carried out in the Biochemistry Department, Faculty of Medicine, Cairo University. Subsequent to six weeks (the allocated experimental period) a local anesthetic cream was applied on the animal ҆s tail. Blood samples were collected then transported to centrifuge tubes without anticoagulant. The serum was separated by low-speed centrifugation and stored at -20°C until the analysis time for evaluation of thyroid hormones level (El-Wakf et al., 2009). Serum T3, T4 and TSH (thyroid stimulating hormone) concentration were measured in each rat by radio-immunoassay in the serum samples using Amersham Test Kits No. 74 and 80 (Amersham International, Amersham, UK).

2.3.5- Sacrifice

At the end of the allocated experimental period for each animal, local anesthetic cream was applied on the abdominal skin, 30min before scarification, it was injected with phenobarbitone sodium at a lethal dose of 80µg/kgbw by intra-peritoneal injection (Shin et al., 2012). Intra cardiac perfusion was done with 1.5% glutaraldehyde buffered in 0.1M sodium cacodylate at pH 7.4, for partial fixation of the parotid gland. The parotid glands were extracted through an incision in the side of the neck. The right gland was prepared for light microscopic examination while the left was allocated for biochemical and molecular study.

2.4- Preparation of the parotid gland for light microscopy

Fixation: to preserve the cell structure and sub-cellular components, parotid specimens were immediately immersed in 10% buffered formalin for 3hours, then washed under running tap water.
Dehydration: was done in ascending grades of ethyl alcohol (50%, 70%, 90%) for 24hours, changed every six hours, then in absolute alcohol for another 24hours, changed every half an hour, then cleared in xylol for one day to remove the alcohol.
Embedding: in liquid paraffin wax overnight in an oven at 50 ⁰C then cooling to prepare paraffin blocks, then long axis of parotid sections, 5µm thick, were obtained by a steel knife on a microtome then mounted on glass slides (Bancroft and Gamble, 2008). The slides were used for both histological and immunohistochemical study.

2.5- Histological (Light microscopic) study

2.5.1 Hematoxylin and eosin stain (Avwioro,2010)

2.5.2 Masson’s trichromes stain (Ross and Michael, 2011)

2.5.3 Toluidine blue stain

Toluidine blue (TB) is a direct, rapid, highly effective and efficient antibody-independent method to identify mast cells in tissue sections. TB belongs to a set of dyes with metachromatic properties, conferred by its thiazine group which stain tissues with differing degrees of intensity of the same color (Sridharan and Shankar, 2012).

2.5.4-Immunohistochemical Study

The immunohistochemical staining was carried out using the streptavidin–biotin–peroxidase technique (Takahashi et al., 2004). Anti-alph-smooth muscle actin (α-SMA) rat monoclonal primary antibody (Dako Cytomation, Heverlee, Belgium) at a dilution 1/1000 was used as a marker for identification of myoepithelial cells. Deparaffinization in xylene for 30 minutes then re-hydrated. The sections were treated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS), pH 7.4 for 30 min to block endogenous peroxidase activity.The sections were incubated for 30 min with the monoclonal antibody: anti-α-SMA antibody, dried, covered by protein blocking reagent (normal rabbit serum) followed by covering with primary antibody. Sections were covered with biotin–caproyl secondary anti- mouse antibody then washed in PBS. Streptavidin and biotinylated horse-radish peroxidase were assorted at appropriate concentrations for at least 30 minutes at room temperature to form the complex. The pre-formed complex was then attached to the biotinylated antibody. The slides were dried then taken to distilled water, counter stained with Mayer’s haematoxylin, washed, dehydrated, left to dry, mounted, covered with glass cover then examined (Kiernan, 2017). Positive results were indicated by brown coloration in cytoplasm of myoepithelial cells around the acini and ducts of parotid gland.

2.5.6- Histomorphometric study

Quantitative data were obtained using Leica Qwin 500 Image Analyzer software system (National Institute of Mental Health, Bethesda, Maryland, USA). The slides were examined within the standard measuring frame of a known area equal to 11694 µm2. The mining of quantitative information from images was the final stage of analysis. The following parameters were examined at X40 objective lens in 10 non-overlaping fields for each gland in all animal groups:

Area percentage of collagen fibers in Masson’s trichrome-stained sections.
Number of mast cells in toliudine blue-stained sections.
Immune-reactivity of α-SMA-stained sections.

2.6 Tissue Biochemical Analysis of the Parotid Gland

The technique was done according to the manufacturer's instructions (LAB-SA system, Zymed Laboratories Inc, San Francisco, USA, 95-9643). Pieces of the left parotid gland tissue were homogenized in 1ml phosphate buffer saline, pH7.0 by a micro pestle in a micro tube. The supernatant was used to determine tissue level of malondialdehyde (MDA) and Glutathione peroxidase (GPx)

2.6.1 Oxidative stress marker (malondialdehyde)

Two and a half ml of 20% trichloroacetic acid and 1ml of 0.6% thiobarbituric acid (TBA) were added to 0.5 ml of tissue homogenate. The mixture was heated for 30 min in a boiling water bath followed by rapid cooling. The resulting chromogen was extracted with 4 ml of n-butyl alcohol .The absorbance of organic phase was determined at the wave length of 530 nm by spectrophotometer against butanol. The concentration of MDA was calculated using the standard curve (Onaolapo et al., 2017).

2.6.2 Anti-oxidative stress marker (Glutathione peroxidase)

The enzyme activity in tissue homogenate was measured by the inhibition of nitrobluetetrazolium reduction by O₂-generated by the xanthine/xanthine oxidase system. The glutathione peroxidase (GPx) activity unit was defined as the enzyme amount causing 50% inhibition in 1 ml reaction solution per gram tissue protein and the result was expressed in U/mg of tissue (Ahmed et al ., 2021).

2.7 Molecular Study

2.7.1 Real-time PCR for Bcl-2, Bax, Caspase-3 and P53 genes

The technique was done according to the manufacturer's instructions (LAB-SA system, Zymed Laboratories Inc, San Francisco, USA, 95-9643).The parotid tissue was processed for RNA extraction followed by cDNA synthesis by Reverse Transcriptase and quantitative real time PCR using SYBR Green I .

Total RNA extraction: Total RNA was extracted from parts of the parotid gland tissue using the TRIzol method according to the manufacturer’s protocol.RNA was extracted by homogenization in TRIzol reagent (Invitrogen, Life Technologies, USA).The homogenate was then incubated for 5min at room temperature.A 1:5 volume of chloroform was added, and the tube was vortexed and centrifuged at 12000g for 15min.The aqueous phase was isolated and the total RNA was precipitated with absolute ethanol. After centrifugation and washing, the total RNA was finally eluted in 20 µl of the RNase-free water. RNA concentration and purity were measured with an ultraviolet spectrophotometer.

Complementary DNA (cDNA) synthesis

The cDNA was synthesized from 1ug RNA using Superscript III First-Strand Synthesis;a system as described in the manufacturer’s protocol (Invitrogen, Life Technologies).A 1 ug of total RNA was mixed with 50 µM oligo deoxy thymidylate (DT) 20, 50 ng/µl random primers, and 10 mM deoxy nucleotide triphosphate (dNTP) mix in 10 µl of total volume .The mixture was incubated at 56°C for 5 min then placed on ice for 3 min.The reverse transcriptase master mix containing 2 µl of 10× RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT and 1 µl of SuperScript® III RT (200 U /µl) was added to the mixture and was incubated at 25°C for 10 min followed by 50 min at 50°C.

Real-time quantitative PCR

a- The relative abundance of mRNA species was assessed using the SYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems, Foster City, CA).PCR primers were designed with Gene Runner Software (Hasting Software, Inc., Hasting, NY) from RNA sequences from Gen Bank. All primer sets had a calculated annealing temperature of 60°.Quantitative RT- PCR was performed in a 25µl reaction volume consisting of 2X SYBR Green PCR. Gene primers sequances were as follows (Mitupatum et al., 2016).

i. Bcl-2 :

Forward primer: ( 5-ATGTGTGTGGAGACCGTCAA-3 ) Reverse primer: ( 5-GCCGTACAGTTCCACAAAGG-3 )

ii. Bax :

Forward primer: ( 5-ATGTTTTCTGACGGCAACTTC-3 )

Reverse primer: ( 5-AGTCCAATGTCCAGCCCAT-3 )

iii. Caspase-3 :

Forward primer: ( 5-TGTTTGTGTGCTTCTGAGCC-3 ) Reverse primer: ( 5-CACGCCATGTCATCATCAAC-3 )

iv. P53 :

Forward primer: ( 5-ATGTTTTGCCAACTGGCCAAG-3 )

Reverse primer: ( 5-TGAGCAGCGCTCATGGTG-3 )

b- Preparation of the Reaction Master Mix for Real Time- PCR: Master Mix (Applied Biosystems), 900 nM of each primer and 2–3µl of cDNA. Amplification conditions were 2min at 50°, 10min at 95° and 40 cycles of denaturation for 15s and annealing/ extension at 60° for 10 min.

c- Calculation of Relative Quantification (RQ) (relative expression): Data from real- time assays were calculated using the v1•7 Sequence Detection Software from PE Biosystems (Foster City, CA). Relative expression of studied gene mRNA was calculated using the comparative Ct method. All values were normalized to the beta actin gene and reported as fold change.

3 Statistical analysis

Numerical data of the histomorphometric measurements and the biochemical and molecular levels were recorded using Microsoft Excel worksheet, 2010. Statistical analysis was performed using statistical package for social sciences (SPSS) version 21.0 (IBM Corporaton, Somers, NY, USA) statistical software. The results were expressed as means ± standard deviation (SD). Statistical evaluation was done using one-way analysis of variance (ANOVA). Significance was considered when p value is ≤ 0.05 throughout the study.

3.1.1 Histological (Light microscopic) Study:

Sections of the parotid gland from groups I (normal) and II (sham) control presented almost identical features in haematoxylin and eosin, Masson ҆s trichrome and immune reaction stains and morphometry.

Haematoxylin and eosin-stained sections of the parotid gland in groups I and II revealed a thick fibrous capsule from which connective tissue septa extended between lobes and lobules. Each lobule was composed of serous acini and intralobular ducts. The acini were mostly spherical, closely packed, with regular outline and lined by pyramidal cells with pale eosinophilic cytoplasm and basal basophilic rounded nuclei.

The striated ducts were more frequent and easily identified. They exhibited a wide lumen and were lined by large number of simple columnar epithelial cells having eosinophilic cytoplasm and basal oval basophilic nuclei with occasional prominent nucleoli

The C.T.septa between the lobes and lobules were thin and featured excretory ducts and blood vessels. The excretory ducts were lined by simple columnar epithelium with eosinophilic cytoplasm and oval basophilic nuclei. Blood vessels lined by flat epithelial cells were seen around both striated and excretory ducts (Fig. 1a). Sections of the parotid gland in group III exposed serous acini widely separated and exhibited irregular outline. Some acini were shrunken with darkly stained pyknotic nuclei and cytoplasmic vacuoles. Within the lobules, interstitial exudate, inflammatory cellular infiltration, dilated striated and intercalated ducts and extravasated blood were encountered (Fig. 1b, c). sections of the parotid gland in group IV revealed an apparently normal architecture and thin C.T. septa between the lobes and lobules (Fig. 1d). sections of the parotid gland exhibited a thin fibrous capsule and lobes divided by connective tissue septa into lobules. Inside the lobules, most acini were closely packed. Some acini, the acini were mostly lined by pyramidal cells with basal rounded nuclei and occasional prominent nucleoli (Fig. 1e).

Masson ҆ s trichrome-stained sections of group I showed minimal amount of collagen fibers around the ducts (Fig. 2a). Sections in group III showed minimal amount of collagen fibers around the ducts (Fig. 2b). Masson ҆ s trichrome-stained sections in group IV and V featured moderate amount of collagen fibers around the ducts (Fig. 2c, d).

Toluidine blue-stained sections in group I revealed few numbers of mast cells in the connective tissue septa (Fig. 3a), Sections of group III revealed large number of mast cells in the connective tissue septa (Fig. 3b), group IV and V sections elucidated moderate number of mast cells in connective tissue septa (Fig. 3c, d).

Immune- histochemical; α-SMA-stained sections in group I exhibited weak positive immune-reactivity around the acini (Fig. 4a). Sections of group III exhibited strong positive immune-reactivity around the acini (Fig. 4b). α-SMA-stained sections revealed moderate positive immune-reactivity around the acini in group IV, V (Fig. 4c, d).

3.1.2 Histomorphometic study

The mean area percentage of collagen fibers and alpha SMA and the mean number of mast cells in group III revealed significant increment when compared to results of group I, while the mean values of group IV and V revealed a significant decrement when compared to the values of group III

3.2 Oxidative markers

The mean values of MDA in group III exposed significant raise when compared to that of group I, while the mean values of group IV and V bare a significant decrease when compared to the values of group III, while the mean levels of Gpx in group III showed significant decline when compared to that of group I, nevertheless, the mean values of group IV and V bare a significant increase when compared to the values of group III.

3.3 Gene expression of Bax by Real-Time Quantitative PCR:

The mean levels of Bax, caspase and P53 in group III pointed out a significant increment when compared to the mean values of group I, while the mean values of group IV and V revealed a significant decline when compared to the values of group III, however, the mean values of Bcl2 in group III showed a significant reduction when compared to the mean values of group I, while the mean values of group IV and V revealed a significant increase when compared to that of group III

In the present work, the hypothyroid status was achieved by administration of propylthiouracil (PTU) for 6 weeks and its establishment was confirmed by a decrease in serum level of T3 and T4 and an increase in TSH level (Faddladdeen et al., 2021).

After six weeks of hypothyroidism, haematoxylin and eosin-stained sections of the parotid gland in group III showed a thickened fibrous capsule and C.T. septa between the lobes and lobules. These findings are in line with Hayat et al. (2010) who reported an increase in the interacinar and interlobular connective tissue in hypothyroid rats. Masson trichrome stained-sections of parotid gland in the same group showed massive amount of collagen fibers deposition around the ducts compared to control groups. Hayat et al. (2016) andAbd Elazeem et al. (2016) emphasizedthat carbimazole-induced hypothyroidism had harmful changes on the parotid tissue which include excess deposition of collagenous fibers around the blood vessels and interlobular ducts. Khalawi et al. (2013) explained that signs of inflammation as cellular infiltration and fibrosis have the ability of oxidative stress to stimulate the expression of genes involved in collagen biosynthesis.

Group III in the present study featured also widely separated serous acini with irregular outlines. Several acini were shrunken with cytoplasmic vacuoles and pyknotic nuclei. Previous studies on PTU-induced hypothyroidism in rats agreed with the above stated findings. Hashem and Saad (2020) and Abd Elazeem et al. (2016) pointed outthat the acinar cells lost their normal arrangement and manifested large cytoplasmic vacuolations and pyknosis. Most of the lobules showed cellular infiltration.

El-bakry et al. (2010) and Hashem and Saad (2020) attributed cytoplasmic vacuoles in the brain of hypothyroid rats to lipid peroxidation and oxidative stress that leads to damage of cell membrane, increase in water content and swelling. Uzun et al. (2022) declared that the atrophy in serous acinar structure increases with the increase in the duration of hypothyroidism.

Furthermore, in the present study, interstitial exudate, inflammatory cellular infiltration, dilated intercalated and striated ducts and extravasated blood were encountered within the lobules. The C.T. septa exhibited dilated excretory duct with irregular outlines, inflammatory cells and congested blood vessels.

In accordance with the above stated changes, Treesh and Khair (2014) confirmed that congested blood vessels featured in the ovary of hypothyroid rats. Ibrahim et al. (2021) believed that the degenerative alterations which occurred in rat testis are linked to elevated blood endotoxins and oxidative stress, which resulted in capillary damage, exudation and apoptosis. Also, Proctor (2016) attributed the importance of the striated duct to its role in transporting electrolytes between the ductal lumina and the extracellular spaces for secretion and reabsorption.

Al-Refai et al. (2014) proposed that dilated salivary gland ducts may be due to affection of the myoepthelial cells (MECs). This in turn causes failure of expelling the salivary secretion leading to high ductal pressure.

In the present study, toluidine blue-stained sections of group III revealed high number of mast cells in the connective tissue septa group compared to control. In line with the present results, Oncu et al. (2004) reported that the number of mast cells increased significantly, six weeks after thyroidectomy in the sublingual gland of rats. This increase has been explained by Bischoff and Sellge (2002) who suggested that mast cells recruitment is the result of inflammation and fibrotic disorders through the production of histamine, heparin and tryptase.

In the present work, immune-stained sections of parotid gland from group III showed a strong positive immune-reactivity of myoepithelial cells (α -SMA) around acini and ducts compared to control. Histomorphometric analysis elucidated that in group III the mean area percentage of α-SMA immunoreaction significantly increased compared to control group, indicating proliferated or hypertrophied myoepithelial cells.

The above finding is in agreement with Cotroneo et al. (2008) who emphasized the increase in the size and number of the MECs in the degenerated acini and ducts of submandibular glands of rat to compensate this degeneration, a phagocytic or stem cells.

Kassab and Tawfik (2018) proposed that the MECs capacity is activated in case of parenchymal injury of submandibular gland aiming to preserve the secretory function of the cells.

Biochemical results of the present study showed a significant increase in tissue level of malondialdehyde (MDA) and a significant decrease in level of glutathione peroxidase (GPx) in parotid gland of group III compared to normal control group.

These data come in agreement with Ayuob et al. (2019) who reported a rise in the formation of plasma MDA and a reduction in the activity of plasma superoxide dismutase in hypothyroid status. These changes could lead to development of oxidative stress resulting in cell destruction.

de Jesus et al. (2015) suggested that the degenerative changes observed in the submandibular glands of hypothyroid rats are due to the free radicals resulting from hypothyroidism, which exert a damaging effect.

In the present work, P53 gene expression in rat parotid gland of group III elucidated a significant upregulation compared to control.The fore-stated results agree with the studies of Hassan et al. (2022) and Foster et al. (2012) who postulated that P53 level increased as a reaction to DNA damage during oxidative stress which guide the transcription and translation of the Bcl-2 family and participate in cell apoptosis .

Wolff (2008) emphasized that after exposure to stress, the cytoplasmic pool of P53 is the major source for P53 mitochondrial translocation of cytochrome C which is responsible for the activation of pro-caspase-3 and pro-caspase-9.

In group III in this study, Bcl-2 gene expression in rat parotid gland revealed a significant downregulation while Bax and Caspase-3 genes were upregulated significantly compared to control groups.

In accordance with these results, Mohammed et al. (2022) reported that the cytoplasm of parotid acinar cells in hypothyroid groups showed weak immune reaction for Bcl-2 compared to normal groups of rats. Furthermore, Ayuob (2016) elucidated an increased expression of caspase-3 in the salivary gland of hypothyroid rats compared to control animals, indicating an increase in cell apoptosis.

Kulsoom et al. (2018) hypothesized that Bax is activated by a process of DNA fragmentation while Bcl-2 is inhibited via P53 protein in case of tissue injury. Gokalp-Ozkorkmaz et al. (2018) concurred that Bcl-2 inhibits apoptosis while Bax induces it.

Administration of propolis as a protective measurement for parotid gland in group IV (PTU+propolis) produced an increase in the mean T3 and T4 level and a decrease in TSH mean level compared to group III. These data are in agreement with Elsheikh (2017) who reported that chlorpyrifos plus propolis produce a highly significant increase in the serum level of T3 and T4 compared to chlorpyrifos administration. The latter author attributed these results to the antioxidant effect of propolis

In this work, the ameliorating effect of propolis was confirmed by histopathological results. Haematoxylin and eosin-stained sections of the parotid in group IV showed thin C.T.septa between the lobes and lobules. Most acini were well packed and were lined with pyramidal cells. In accordance with these results, Hafez (2016) stressed that there was an improvement in the testicular histopathological and immunohistochemical alterations when the thyroidectomized rats were treated by propolis. Labah and Abd-Elmotelb (2016) reported that diabetic animals treated with propolis showed marked improvement in parotid gland architecture which appeared to be almost normal.

In the present study, Masson`s trichrome-stained sections of parotid gland in group IV presented moderate amount of collagen fibers deposition around the ducts compared to group III . In accordance, the mean area percentage of collagen fibers in parotid gland of group IV was significantly lower than that of group III.

The present finding come in agreement with Elsheikh (2017) whoemphasized thatchlorpyrifos plus propoils group sections showed highly significant reduction in collagen fibers percentage area. Furthermore, Badr et al. (2019) concluded that supplementation of propolis led to decrease in the collagen fibers surrounding the central vein and this was attributed to the reduction in chronic inflammation by the effect of propolis. Toluidine blue-stained sections of group IV revealed lower number of mast cells in the connective tissue septa compared to group III. Histo-morphometric results confirmed these findings as the mean number of mast cells in parotid gland of group IV was significantly lower than group IIIb.

In accordance with the present work, Araujo et al. (2012) and Korish and Arafa (2011) agreed that propolis acts as a potent anti-inflammatory agent in vitro and in vivo against both acute and chronic inflammation. The last authors explained the anti-inflammatory effect of propolis through preventing neutrophil penetration and deactivation of pro-inflammatory cytokines such as TNF-α.

Nader (2013) pointed out that caffeic acid phenethyl ester (CAPE) decreased histamine and leukotrienes release in vitro from isolated rat peritoneal mast cells. Chirumbolo (2012) suggested that propolis down-regulates type I allergy and inflammation by affecting mast cells.

Group IV in the present study showed moderately positive immune-reactivity of myoepithelial cells around acini and ducts compared to group III. Histomorphometric analysis proved that group IV presented a significant decrease in the mean area percentage of α-SMA immunoreaction compared to group III.

The forementioned results augment the work conducted by Badr et al. (2019) who emphasized thatCCl4-treated animals, orally supplemented with propolis, exhibited a significant downregulation of α-SMA in liver compared to that in CCl4-treated mice.

The present study spotted a significant decrease in the tissue level of MDA and significant increase in the level of GPx enzyme in parotid gland of group IV compared to Group III. These data confirm the antioxidant protective role of propolis.

In agreement with the stated results, Labah and Abd- Elmotelb (2016) and Jasprica et al. (2007) reported thatpropolis significantly reduced the MDA level and increased the activity of GPx. The latter authors attributed the propolis action to its scavenging of hydroxyl superoxide free radicals and lipid peroxides.

Several authors including Nirala et al. (2008) and El Sohaimy and Masry (2014) concluded thatpropolis has high a content of phenolics that have the ability to donate hydrogen ions which can attack free radicals to prevent oxidation reactions in the cell.

In the present study, the expression of P53, Bax and caspase-3 genes in rat parotid gland of group IV illustrated a significant downregulation while Bcl-2 gene revealed a significant upregulation, compared to group III.

The abovementioned results support the study of Badr et al. (2019) who found that supplementation of CCl4-treated animals with propolis increased the expression of Bcl-2 and downregulated the expression of Bax and cytochrome C. The expression level of P53 was significantly restored compared to CCl4-treated animals.

Alm-Eldeen et al. (2017) stated that the propolis extract (EPE) reduced pro-apoptotic P53 and increased anti-apoptotic Bcl-2 expression in aflatoxin B1 treated male mice. These findings confirm the ability of EPE to improve the oxidative stress and consequently the toxicity that induces hepatic dysfunction and apoptosis.

Tan et al. (2005) proved thatcaspase-3 activity in cardiac tissue was inhibited with administration of CAPE .In addition, Khan et al. (2007) stated that CAPE reduced caspase-3 and increased the expression of Bcl-xl in rat brain. These findings support its anti-apoptotic action.

The employment of Nigella sativa oil as a protective measurement for parotid gland in group V (PTU+NsO) was followed by an increase in the mean T3 and T4 level and a decrease in the mean TSH level compared to group III.

In the present experiment, haematoxylin and eosin-stained sections of the parotid gland in group V exhibited a thin fibrous capsule and C.T.septa. The acini were mostly well packed and lined by pyramidal cells with basal rounded nuclei. The protective effect of NsO in the present work supports the study of El-Din and Fattah (2020) who reported thatinthe hypothyroid plus NsO group the parotid gland tissues restored most of its characteristic acinar arrangement and the vacuolations of the acinar epithelial cells were highly minimized.

The present findings are in alignment with El-ghazouly (2017) whose study elucidated that Nigella sativa oil succeeded in protecting the kidney from the side action of the red bull-induced histopathological and biochemical alterations. The latter author reported that red bull plus NsO group revealed moderate amount of collagen fibers around basement membrane of the renal tubules and glomerular capillaries. Mohammed et al. (2014) stated that administration of NsO significantly decreased the toxic effect of cadmium administration also in the kidney tissues.

In the present work, toluidine blue-stained sections of group V presented a moderate number of mast cells in the connective tissue septa compared to group III. Morphometric results confirmed these findings; the mean number of mast cells in parotid gland of group V revealed a significant decrease compared to group III.

The previous data are in agreement with the study of Ayuob (2016) who reported that PTU plus TQ group illustrated a significant decrease in the mean number of mast cells compared to the PTU group. Kanter et al. (2006) pointed out that N. sativa and thymoquinone application by gavage decreased mast cell number and histamine level in gastric tissue which was damaged by absolute alcohol. Also, Kilinc et al. (2017) reportedthat N. sativa has several therapeutic properties such as analgesic, anti-inflammatory and antihistaminic.

In the present study, group V showed moderately positive immune-reactivity of the myoepithelial cells around acini and ducts. Histomorphometric analysis revealed a significant decrease in the mean area percentage of α-SMA immune-reaction compared to group IIIb.

In agreement with this result, Ayuob (2016) emphasized that PTU plus TQ group presented a significant decrease of α-SMA expression compared to the PTU group in rat parotid.

NsO administration in the present study reduced the tissue level of MDA and raised the level of GPx in parotid gland of group V compared to Group III. However, group V showed a statistically significant decrease in the level of MDA compared to group IV.

The fore-mentioned findings support Mohammed et al. (2014) who spotted that treatment with NsO resulted in an elevation in the level of GSH while MDA level was declined. Hosseinzadeh et al. (2007) concluded that pretreatment with TQ has a protective effect on lipid peroxidation. Furthermore, Ismail et al. (2010) hypothesized that TQ reduce oxidative stress through a direct antioxidant effect as well as through the induction of endogenous antioxidant enzymes.

With NsO administration in the present study, P53 gene expression was significantly downregulated compared to group III. This finding comes in alignment with Lu et al. (2018) who believed that NsO effect on P53 depends on its expression level in the target cells; if P53 expression is already low as in actively proliferating malignant cells, NsO act by increasing P53 expression thus increases their apoptosis rates and vice versa in cases of high P53 expression; as in case of injured non-malignant cells preventing its apoptosis. Hence, NsO has a dual contradictory effect regarding cell proliferation, either anti-proliferative or anti-apoptotic.

In the present study, Bax and caspase-3 gene expression in rat parotid gland of group V revealed a significant downregulation while Bcl-2 gene was significantly upregulated compared to group III.

In accordance with these findings, El-Din and Fattah (2020) reported thatBcl-2 immune-expression was significantly increased and Bax/Bcl-2 ratio significantly decreased in the hypothyroid plus NsO group compared to hypothyroid group.

Moreover, Fouad et al. (2016) stated that TQ decreased the cadmium-induced expression of caspase-3 which was an executioner of cell apoptosis in the kidney tissue. Faddladdeen et al. (2021) concurred that caspase-3 immunoexpression in pancreatic islet cells was significantly reduced in TQ-treated hypothyroid group compared to hypothyroid group.

The present study revealed different outcomes between the ameliorating effect of propolis or NsO in rat parotid gland in several parameters, though this difference was statistically non-significant. However, the MDA level in group V (PTU + NsO) was statistically lower than group IV (PTU + propolis), denoting that the antioxidant property of NsO is stronger than propolis.

Furthermore, in this work, the mean number of mast cells presented a non-significant increase in group IV (PTU + propolis) compared with control groups, indicating that the anti-inflammatory effect of propolis is more potent than NsO.

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Table ( ): Area percentage of collagen fibers, number of mast cells and area percentage of myoepithelial cells in all groups:

Group (n=5)	Area percentage of collagen fibres Mean ± SD	Number of mast cells Mean ± SD	Area percentage of myoepithelial cells Mean ± SD
I	5.78 ± 1.76	30.62 ± 7.33	4.69 ± 1.50
III	38.79 ± 7.66	227.2 ± 41.43	25.31 ± 2.27
IV	21.04 ± 4.40	75.80 ± 25.44	16.88 ± 2.64
V	20.79 ± 4.76	91.00 ± 20.65	15.57 ± 2.33

Statistically significant p-value ≤ 0.05.

Table ( ): Level of MDA and GPx in all groups:

Group (n=5)	MDA level (nmol/g/tissue) Mean ± SD	GPx level (U/g/protein) Mean ± SD
I	58.3 ± 3.90	125.1 ± 2.03
III	183.4 ± 1.57	44.9 ± 1.92
IV	91.4 ± 1.50	116.02 ± 2.12
V	84.3 ± 1.50	112.70 ± 1.88

Statistically significant p-value ≤ 0.05.

Table ( ): Level of Bcl-2, Bax, caspase-3 and P53 in all groups:

Group (n=5)	Bcl-2 gene expression level Mean ± SD	Bax gene expression level Mean ± SD	Caspase-3 gene expression level Mean ± SD	P53 gene expression level Mean ± SD
I	1.01 ± 0.02	1.005 ± 0.008	1 ± 0.01	1.007 ± 0.01
III	0.20 ± 0.01	5.5 ± 0.71	7.5 ± 0.20	5.18 ± .035
IV	0.79 ± 0.03	2.5 ± 0.15	3.30 ± 0.15	2.4 ± 0.35
V	0.85 ± 0.02	2.8 ± 0.21	3.3 ± 0.13	2.6 ± 0.42

No competing interests reported.

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Propolis versus Nigella sativa Oil challenging the apoptotic pathway in Propylthiouracil-induced Hypothyroidism on parotid gland

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1-Chemicals:

2.2- Animals:

2.3.1 Induction of hypothyroidism:

2.3.2 Animal groups:

The rats were divided into the following groups:

2.3.4- Confirmation of hypothyriodism induction

2.3.5- Sacrifice

2.4- Preparation of the parotid gland for light microscopy

2.5- Histological (Light microscopic) study

2.5.1 Hematoxylin and eosin stain (Avwioro,2010)

2.5.2 Masson’s trichromes stain (Ross and Michael, 2011)

2.5.4-Immunohistochemical Study

2.5.6- Histomorphometric study

2.6 Tissue Biochemical Analysis of the Parotid Gland

2.6.1 Oxidative stress marker (malondialdehyde)

2.6.2 Anti-oxidative stress marker (Glutathione peroxidase)

2.7 Molecular Study

2.7.1 Real-time PCR for Bcl-2, Bax, Caspase-3 and P53 genes

Complementary DNA (cDNA) synthesis

Real-time quantitative PCR

3 Statistical analysis

3. Results

3.1.1 Histological (Light microscopic) Study:

3.1.2 Histomorphometic study

3.2 Oxidative markers

3.3 Gene expression of Bax by Real-Time Quantitative PCR:

4. Discussion

References

Tables

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