Ameliorative Effects of Nilotinib on CCl4 Induced Liver Fibrosis Via Attenuation of RAGE/HMGB1 Gene Expression and Oxidative Stress in Rat

Abstract

Nilotinib as a tyrosine kinase inhibitor has been recently used to improve the liver fibrosis process, but the exact mechanisms still require further clarification. In this study, we investigated the anti-fibrotic effects of Nilotinib via RAGE/HMGB1axis and antioxidant mechanisms. This experimental study was performed in the Hamadan University of Medical Sciences, Iran, from May 2015 to December 2016. Liver fibrosis was induced in Wistar male rats by CCL₄. Rats were gavaged daily with Nilotinib (10 mg/kg). RAGE, HMGB1, TNF-α and TGF-β mRNA expression were evaluated by quantitative RT-PCR. TNF-α protein levels were measured using the immunoassay method. Thiol groups, carbonyl groups, nitric oxide levels and glutathione peroxidase activity were measured by spectrophotometric methods.The results showed that Nilotinib decreased TNF-α, TGF-β, RAGE and HMGB1 mRNA expression (p<0.001) in the liver tissues of the fibrosis group. Nilotinib also decreased carbonyl groups and nitric oxide levels and increased thiol groups and glutathione peroxidase activity in the fibrosis groups. The histopathological changes were found to be attenuated by Nilotinib. In conclusion, Nilotinib can improve liver fibrosis and open new mechanisms of the anti-fibrotic properties of Nilotinib.

Keywords: HMGB1 Protein; Liver Cirrhosis; Nitric Oxide; Rats.

FIG. 1. Effectsof Nilotinib on RAGE mRNA levels (A) and HMGB1 mRNA levels (B) in the liver tissues of rats with liver fibrosis. C: Control normal, O: Olive control group, CN: Nilotinib control group, F: Liver fibrosis group, FN: Liver fibrosis treated with Nilotinib (10 mg/kg body weight), the data represent mean±SD. ^*ap<0.001 versus groups C, O and CN.^*bp<0.001 versus group F.

FIG. 2. Effectsof Nilotinib on TNF-α protein levels (A), TGF-β1 mRNA levels (B) and TNF-α mRNA levels (C), in the liver tissues of rats with liver fibrosis. C: Control normal, O: Olive control group, CN: Nilotinib control group, F: Liver fibrosis group, FN: Liver fibrosis treated with Nilotinib (10 mg/kg body weight), the data represent mean±SD. ^ap<0.001 versus groups C, O and CN.^bp<0.001 versus group F.

FIG. 3. Effects of Nilotinib on oxidative stress stutus in the liver tissues of rats with liver fibrosis. Hepatic thiol group (A), Glutathion peroxidase (B) and carbonyl group (C) levels after 12 weeks in the different groups. C: Control normal, O: Olive control group, CN: Nilotinib control group, F: Liver fibrosis group, FN: Liver fibrosis treated with Nilotinib (10 mg/kg body weight), the data represent mean±SD. ^ap<0.001 versus groups C, O and CN.^*bp<0.001 versus group F. ^cp<0.05 versus group F.

FIG. 4. Effects of Nilotinib on nitric oxide (NO) levels in the liver tissues of rats with liver fibrosis. C: Control normal, O: Olive control group, CN: Nilotinib control group, F: Liver fibrosis group, FN: Liver fibrosis treated with Nilotinib (10 mg/kg body weight), the data represent mean±SD. ^ap<0.001 versus groups C, O and CN. ^bp<0.001 versus group F.

FIG. 5. Pathological features of rat liver tissue in different groups (HE staining; Bar=100 µm). (A) severe macrovesicular steatosis with many hepatocytes showing ballooning degeneration (stars) in CCl₄ group; (B) mild macrovesicular steatosis (star) in CCl₄+nilotinib group; (C) severe infiltration of mononuclear cells around the portal area (arrow) in CCl₄ group; (D) mild infiltration of mononuclear cells around the portal area (arrow) in CCl₄+nilotinib group; (E) CCl₄ group; severe focal necrosis in CCl₄ group; (F) mild focal necrosis in CCl₄+nilotinib group.

FIG. 6. Masson's trichrome staining of hepatic tissue for collagen in different groups. (A) control group; no fibrosis; (B) CCL₄ group; fibrosis expansion of the portal area with marked bridging as well as portal to central; (C) marked reduction of collagen deposition in CCl4+ nilotinib group (Bar=320 µm).