Pharmacological study on the potential protective effects of a NADPH oxidase inhibitor against Concanavalin A-induced liver injury in rats

Abstract

This study was directed to explore the possible role of NOX enzymes and the potential antifibrotic effect of the NOX inhibitor, apocynin, in ConA-induced liver fibrosis, as a murine model of autoimmune hepatitis. Also, the current study aimed to investigate whether the antioxidant α-LA could potentiate the antifibrotic effect of apocynin and if so, what are the possible mechanisms? First, determination of the appropriate hepatoprotective dose was achieved by constructing a dose response study using different doses of apocynin and α-LA for one week. Rats were randomly assigned into nine groups; Group (I) served as control group, where it received i.v. injection of PBS (vehicle of ConA) and i.p. injection of DMSO (vehicle of apocynin and α-LA). Group (II) was given a single i.v. injection of ConA of 20 mg/kg. Groups (III), (IV), (V) and (VI) received a single i.v. injection of ConA of 20 mg/kg as well as i.p. injection of apocynin at doses of 15, 25, 50 and 75 mg/kg, respectively. Groups (VII), (VIII) and (IX) received a single i.v. injection of ConA of 20 mg/kg followed by i.p. injection of α-LA at doses of 50, 75 and 100 mg/kg, respectively. At the end of the week, blood samples were collected for the determination of ALT and AST serum levels, then animals of all groups were sacrificed, where liver tissues were dissected out for histopathological examination. Meanwhile, ConA-intoxication have significantly increased ALT and AST serum levels by 232 and 200%, respectively. On the other hand, co-treatment with 50 mg/kg of apocynin significantly decreased ALT and AST levels by 72 and 50%, respectively when compared to ConA-intoxicated group. While co-treatment with 50 mg/kg of α-LA revealed a significant decrease of ALT and AST levels by about 61 and 25%, respectively as compared to ConA group. Accordingly, the dose of 50 mg/kg of both agents was selected for further mechanistic study. These results were further confirmed by histopathological examination of the liver tissues of different groups. Second, based on data obtained from the previous step, the optimal dose of apocynin and α-LA was selected for further assessment of the potential antifibrotic effects using ConA-induced liver fibrosis model in rats. Animals were randomly divided into seven groups and treated for six consecutive weeks. Group (I) served as a control group and received i.v. injection of PBS once/week and i.p. injection of DMSO three times/week. Groups (II) received 50 mg/kg i.p. injection of apocynin three times/week. Group (III) received 50 mg/kg i.p. injection of α-LA three times/week. Group (IV) received 20 mg/kg ConA through i.v. injection once/week. Group (V) received 20 mg/kg i.v. injection of ConA once/week as well as 50 mg/kg i.p. injection of apocynin three times/week. Group (VI) received 20 mg/kg i.v. injection of ConA once/week as well as 50 mg/kg i.p. injection of α-LA three times/week. Group (VII) received 20 mg/kg i.v. injection of ConA as well as 50 mg/kg i.p. injection of both apocynin and α-LA three times/week. Afterwards, blood samples were collected, then animals of all groups were sacrificed and liver tissues were dissected out for further analysis. In the mechanistic study, it was found that ConA administration once per week for six consecutive weeks has significantly increased the serum levels of ALT and AST by 161 and 140%, respectively. In contrast, co-treatment with both apocynin and α-LA significantly lowered the levels of ALT and AST by 58 and 52%, respectively as compared to ConA group. Regarding albumin, total bilirubin, TC and TG, ConA-injected group showed a significant decrease in albumin levels by 34% as well as an increase in the serum levels of total bilirubin, TC and TG by 269, 88 and 174%, respectively. Interestingly, the combination group showed a more pronounced increase in albumin levels by 44% and a decrease in total bilirubin, TC and TG serum levels accounting for 68, 43 and 55%, respectively as compared to ConA-intoxicated group. Apparently, the administration of ConA significantly increased liver index by 38%. Notably, the group co-treated with both apocynin and α-LA showed a marked decrease in the liver index by 27% when compared to ConA group. These data confirm that the hepatic damage was reversed by the co-treatment with both agents.