Protective Effect of Co Q10 and Candesartan on Bleomyycin Induced Lung Fibrosis in Rats

AJPS (2021) 16 Protective Effect of Co Q10 and Candesartan on Bleomyycin Induced Lung Fibrosis in Rats Fatima Mohammad Saeed Mahdi *, Mustafa Ghazi Alabbassi **, Muthanna Ibrahim Al-Ezzi **. *Department of Pharmacology and Toxicology, College of Pharmacy, Mustansiriyah University, M.Sc. program, Iraq. **Department of Pharmacology and Toxicology, College of Pharmacy, Alkafeel university, Iraq. ***Department of Clinical Laboratory Sciences, College of Pharmacy, Mustansiriyah University, Iraq. ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ــــــــــــــــــــــــــــــــــــــــــــ ــــــــــــــــــــــ   Abstract:


Introduction
Fibrosis of the lungs (PF) is the final phase of many lung illnesses. Its characterized by excessive matrix production leading to the lung architecture destruction and eventually death [1] . Pulmonary fibrosis occurs when there is damage to the pulmonary tissue and a fibrotic reaction ensues that leads to thickening of the alveolar walls and obliteration of the alveolar air spaces. Its etiology could be not known or could be one of the [2] various chemical toxins and other injuries well known to cause pulmonary fibrosis [3] . The principle histological marks of pulmonary fibrosis are continuing and nonrepaired epithelial injury, proliferation and buildup of myofibroblasts and fibroblasts, and collagen deposition increase [4] .
Pulmonary fibrosis is considered as a disease caused by repeated subclinical injury leading to epithelial injury and subsequent destruction of the alveolarcapillary basement membrane [5] . This process promotes the infiltration of fibrotic cells and the activation of (myo)fibroblasts. The usual clearance of inflammatory and mesenchymal cells by phagocytosis and apoptosis is dysregulated in pulmonary fibrosis [6] . This leads to the damage to the architecture of the normal lung and function loss. Transforming growth factor β (TGF-β) is considered as the most potent profibrotic mediator described until this date which induce pulmonary fibrosis via the recruitment and activation of fibroblasts and monocytes, and promotes ECM production. Lung fibroblast proliferation occurs indirectly by TGF-β1 through induction of fibroblast growth factor-2 and consequently activating the mitogenactivated protein kinase (MAPK) pathway [7] .
additionally, TGF-β promotes fibroblasts to differentiation to myofibroblasts. TGF-β induces ECM formation by induction of ECM gene transcription, including fibronectin, collagen and proteoglycans and by inhibiting the actions of matrix metalloproteinases, elastases and plasminogen activators which leads to the inhibition of degradation of collagen (1).
Bleomycin is a member of a group of antimicrobial and antineoplastic glycopeptides. The agent is widely used to treat a variety of malignant tumors Although bleomycin generally lacks toxicity in normal hematopoietic cells, a serious adverse reaction associated with the drug is pulmonary toxicity [8] . Bleomycin has been invested to induce pulmonary fibrosis in animal studies. The bleomycin toxicity is instigated by oxidative damage directly to the pulmonary tissue, which later advances to inflammatory and fibrotic response. The damage is caused by generating extracellular and intracellular reactive oxygen species (ROS) [9] . BLM can cause DNA strand scission in the attendance of Fe and O2 elements, leading to double or single strand breaks and resulting in more ROS and reactive nitrogen species (RNS) formation, which explains the antineoplastic effects of bleomycin [10] . Co enzyme Q10 (CoQ10) is a lipid soluble molecule available in the cell membrane of every mammalian cell type [11] . Since CoQ10 can reversibly accept or give 2 electrons to form hydroquinone or benzoquinone, respectively, it is a key component in the mitochondrial electron transport chain playing an important role in the formation of cellular adenosine triphosphate (ATP) [12], CoQ10 is also an essential constituent of membrane oxidoreductase System It is an endogenously synthesized intracellular antioxidant enzyme in the human body. CoQ10 protects cellular components from free radical-induced oxidative cell destruction [13] . Candesartan is an angiotensin II type 1 receptor (AT1) selective antagonist and is widely employed in treating hypertension [14] . Studies indicate that angiotensin II has close involvement in damage to tissues and fibrogen formation in the organs of circulation. It's been reported that Angiotensin II promotes proliferation and synthesis of collagen from cardiac fibroblasts this action was inhibited by AT1 antagonists [15]. The fibrosis progression has close relation to a complex network compromised of several chemical mediators, cytokines, growth factors (GF), and biophysical peptides derived from inflammatory immune cells, endothelial cells and alveolar type II cells. Since Ag II is a member of this network, an angiotensin II pathway has been reported to be involved with PF [16] . A high level of ACE has been reported in bronchoalveolar lavage (BAL) fluid from individuals with idiopathic pulmonary fibrosis [17] . Radiation induced lung fibrosis in animal model, revealed that the levels of ACE and angiotensin II were elevated in pulmonary tissue homogenates [16] . These investigations show that the process of lung fibrosis is induced by a local reninangiotensin system in the pulmonary tissue. proof that AT1 is situated not just in the cardiac and renal tissues but also in the pulmonary tissue supports this theory. Human pulmonary fibroblasts were elevated by induction of angiotensin II with AT1 [18] . Giving AT1 antagonist greatly reduced radiation caused lung fibrosis. All in all, the proof from the following investigations suggest that Ag II is a key factor in the induction of lung fibrosis, and AT1 is an essential receptor for angiotensin II signaling transmission [19] . Moreover, angiotensin II is closely involved with TGF-β1 formation in fibrosis of the heart and kidney which indicates that Ag II promotes fibrosis in pulmonary fibrosis models by inducing TGF-β1 production via AT1. Promoting TGF-β1 synthesis are mediated by the (AT1) receptor in vivo and in vitro [20]. This study aimed to assess the potential protective role of Co Q10 and candesartan as antifibrotic agent against pulmonary fibrosis induced by bleomycin. Materials and Methods: Study Design: Forty-eight rats were divided randomly into four groups, each consisting of 12 male rats: Group I: (Control group), the rats in this group received single injection of 0.2 ml normal saline via intratracheal route. Group II: (The BLM group), received BLM (8.3 U/kg) as sulfate salt dissolved in 0.1 ml of normal saline as a single dose via the intratracheal rout. Group III: (BLM+CoQ10 group) rats receive BLM (8.3 U/kg) as sulfate salt dissolved in 0.1 ml of normal saline via the intratracheal instillation concomitant with 100 mg/kg co Q 10 per day orally for 5 days before and 10 days after BLM injection. Group IV: (BLM+ candesartan group) rats receive BLM (8.3 U/kg) as sulfate salt dissolved in 0.1 ml of normal saline via the intratracheal instillation concomitant with (10 mg/kg) candesartan per day orally for 5 days before and 10 days after BLM injection. Induction: Acute Pulmonary fibrosis was induced by intratracheal instillation of BLM (8.3 U/kg) as sulfate salt dissolved in 0.1 ml of normal saline to a healthy male albino rat (21). Rats anesthetized using ketamine (45mg/kg) and xylazine (8mg/kg) I.P.) [22] . A midline incision made in the neck, the trachea exposed and BLM instilled. Rats should be kept in vertical position and rotated several times to ensure uniform distribution of BLM within the lung tissues. The incision should be surgically sutured and povidone iodine applied topically to the wound [21] . Sample Collection and Preparation: A blood sample collected by using heart puncture, put in plain gel tube and centrifugation at 3000x for fifteen minutes. The serum was stored at -80°C in preparation for ELISA analysis where glutathione and ALOX5 were measured in serum according to manufacturer instruction (Mybiosource -USA), then sacrificed the rats by using ketamine and xylazine for anesthesia, then removed the lungs and washed by distilled water, then the lung tissue kept in 10%formaline to prep for trichrome stain. Statistical Analysis: The statistical analysis was done via utilizing the 16.0 version of SPSS. Every single result was described as mean ± standard deviation (SD). To differentiate between group results, the One-way ANOVA test was utilized, and a post hoc LSD test followed. For the data when the P<0.05 the Statistically significant differences were confirmed, when P <0.01 high significant difference and when the P <0.001 very high significant differences was considered. Results: Glutathione: The descriptive statistics for serum GSH conc. which is described as mean ± SD (table 1). BLM group (78.06±8.89, μg /ml) showed nonsignificant difference when compared with control group and the (BLM+ CoQ10) group (83.15±5.47,82.31±3.81 μg/ml, respectively) (P >0.05). The serum GSH levels showed a significant elevation in (BLM+ Candesartan) group (86.91±3.52 μg/ml; P >0.05) when comparing it with the levels in the BLM group (78.06±8.89μg/ml; P >0.05). serum GSH concentration showed non-significant differences (P >0.05) in both (BLM +CoQ10) and (BLM+ Candesartan) group in compare with the control group. as shown in figure (1).  0.39±0.08 Values are expressed as mean ±SD. The statistical analysis done by applying oneway ANOVA followed by LSD test. * significant difference (p < 0.05) when comparing BLM with control group. # high significant difference (p < 0.01) when comparing (BLM + CoQ10) and (BLM + Candesartan) group with BLM group.

Figure (2):
Effect of CoQ10 and Candesartan on serum ALOX5 level in BLM induced lung toxicity all values are expressed as mean ±SD. The statistical analysis done by applying one-way ANOVA followed by LSD test. * significant difference (p < 0.05) # high significant difference (p < 0.01) Group Ⅰ (Control), Group Ⅱ (BLM), Group Ⅲ (BLM+ CoQ10), Group Ⅳ (BLM+ Candesartan) Histological changes CoQ10 and Candesartan both showed reduce in the collagenous fibers production in BLM pulmonary toxicity. All groups results are shown below: A. Control group: the lung tissue in control group appear normal with no collagen fibers as in figure (3-A). B. BLM group: the lung tissue in BLM group showed a moderate to severe inflammation and moderate peribronchial fibrosis (3-B). C. (BLM+CoQ10) group: the lung tissue in this group showed a moderate to severe inflammation and moderate peribronchial fibrosis the cardiac tissue in this group showed only mild inflammation and a mild peribronchial fibrosis as in figure (3-C). D. (BLM +Candesartan) group: the lung tissue in this group appear to have a moderate to severe inflammation and moderate peribronchial fibrosis as shown in figure (3-D).

Discussion:
Glutathione is part of the thiol redox system. Redox systems keep the reducing state of the cell by electron transfer. glutathione is commonly referred to as the main cellular antioxidant, however the role of GSH is more than just an antioxidant, it also has many other essential cellular functions, including xenobiotic detoxification, cell proliferation modulation, storage and transport of cysteine and redox status maintenance [23] . GSH levels is used as the main indication of cellular redox state since the GSH pool is roughly three to four folds more abundant than other major redox couples [24,25] . This study revealed that BLM decrease the level of the antioxidant GSH and that both CoQ10 and candesartan caused significant increase (P<0.05) in plasma GSH level.Chen et al and Mcmillan et al (2016), showed that treatment with bleomycin causes a decrease in the GSH levels in the blood since bleomycin induces injury by increasing oxidative stress [26,27] [29][30][31] . Arachidonate 5-lipoxygenase is a member of the lipoxygenase family of enzymes. It forms leukotrienes [32] substrates as well as forming various other biologically active products from essential fatty acids. ALOX -5 plays an essential part in asthma and inflammation. This enzyme promotes bronchiole constriction in response to cysteinyl leukotrienes which leads asthma. It promotes neutrophilic inflammation as well [33] . Leukotrienes are arachidonic acid metabolites and they are important regulators of lung fibrosis. The production of LTB4 and cysteinyl LT (cysLT) are catalyzed by ALOX-5. LTB4 plays a great part in the host defense system against invasion by foreign bodies and infection. LTB4 is regarded as a cause of various inflammatory disorders, and it is synthesized by alveolar cells in patients with idiopathic pulmonary fibrosis. This enzyme is responsible for converting arachidonic acid into the leukotrienes LTB4 and LTC4. These leukotrienes have inflammatory and fibrotic properties and are upregulated in the lungs of IPF patients [34] .This study revealed that BLM increased the level of the inflammatory mediator ALOX-5 and that both CoQ10 and candesartan caused significant reduction (P<0.05) in plasma ALOX-5 level. BLM was shown to increase oxidative stress and thus cause an increase in the levels of inflammatory mediators like ALOX-5. This was confirmed by Wiley et al (2019) [35] . Tsai et al (2011), showed significant reduction of ALOX-1 level by oral CoQ10 supplementation, they support that CoQ10 has significant antioxidant and protective effects on cell injury [36] . to our knowledge this is the first study on the effect of Co Q10 and candesartan on ALOX-5 levels. The pulmonary tissue in the control group of this study is normal showing the absence of inflammatory cell infiltration and no fibrosis. the BLM group showed moderate to severe infiltration of inflammatory cells and fibrotic lesions. On the other hand, both (BLM + CoQ10) and (BLM + Candesartan) groups showed only mild inflammatory cell infiltration and mild fibrosis. Swaisgood et al (2000), showed that BLM causes changes on normal morphology of pulmonary cells and mononuclear cells infiltration due to the action of oxidative stress that considered as an indication for pulmonary injury and dysfunction [37] . zaghloul et al (2017), showed that BLM 5mg /kg single dose causes the deposition of fibrotic tissue in the lings [21] . In this study both BLM + CoQ10 and BLM + Candesartan groups showed little to no fibrosis and very mild inflammatory cell infiltration. These finding are in agreement with Mohamed et al (2019), that reported therapy of coQ10 removed histopathological changes produced by methotrexate so exerting an antifibrotic effect [38] .Oto et al (2013), showed that coQ10 treatment had a preventive role on BLM induced pulmonary fibrosis and alterations [39] .

Conclusion:
this study showed that both CoQ10 and candesartan have a good pulmonary protective and antifibrotic effect against bleomycin pulmonary toxicity by different mechanisms.

Acknowledgments:
the investigator thanks Al-Mustansiriyah University (www.uomustansiryiah.edu.iq) for the supporting this study.