Fasudil Ameliorates Methotrexate-Induced Hepatotoxicity by Modulation of Redox-Sensitive Signals

Abstract

Methotrexate (MTX) is one of the most widely used cytotoxic chemotherapeutic agents, and it is used in the treatment of different autoimmune disorders. However, the clinical applications of MTX are limited by its hepatic toxicity. Hence, the present study was conducted to evaluate the efficacy of fasudil (Rho-Kinase inhibitor) in the amelioration of MTX hepatotoxicity and the possible underlying mechanisms. Experimentally, 32 male Sprague Dawley rats were used and divided into four groups: control, MTX (20 mg/kg, i.p., single dose), fasudil (10 mg/kg/day i.p.) for one week, and fasudil plus MTX. It was found that MTX significantly induced hepatitis and hepatocellular damage, as shown by abnormal histological findings and liver dysfunction (ALT and AST), with up-regulation of the inflammatory mediators NF-κB-p65 and IL-1β. Moreover, MTX remarkably disrupted oxidant/antioxidant status, as evidenced by malondialdehyde (MDA) up-regulation associated with the depletion of superoxide dismutase (SOD), catalase, and reduced glutathione (GSH) levels. Moreover, MTX reduced the hepatic expression of B-cell lymphoma 2 (Bcl-2). On the contrary, the i.p. administration of fasudil significantly ameliorated MTX hepatotoxicity by histopathological improvement, restoring oxidant/antioxidant balance, preventing hepatic inflammation, and improving the hepatic anti-apoptotic capability. Furthermore, fasudil hepatic concentration was determined for the first time using the validated RP-HPLC method. In conclusion, the present study revealed that fasudil has a reliable hepatoprotective effect against MTX hepatotoxicity with underlying antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. It also introduced a new method for the determination of fasudil hepatic tissue concentration using the RP-HPLC technique.

Keywords: MTX hepatotoxicity; RP-HPLC technique; hepatic concentration; rho-kinase inhibitor.

Figure 1

Representative RP-HPLC chromatogram of fasudil (2.12 min, 3.0 μg/mL) under optimized conditions.

Figure 2

Effect of fasudil on hepatic enzymes and lipid peroxidation after MTX challenge: (A) serum ALT; (B) serum AST; (C) hepatic content of MDA. Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX control group (p < 0.05). **** p < 0.0001. NS, non-significant.

Figure 3

Fasudil ameliorated MTX-induced hepatic histopathological aberrations. Routine staining (hematoxylin and eosin (H&E) stain, ×400) was conducted to examine control, MTX, fasudil, and MTX + fasudil groups. Arrows mark hepatic changes, including hepatocyte degeneration and congestion with inflammatory cell infiltration. Scale bar = 50 μm.

Figure 4

Effect of fasudil on the hepatic GSH content as well as antioxidant enzyme activity: (A) hepatic GSH content; (B) hepatic SOD activity; (C) hepatic catalase activity. Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). **** p < 0.0001. NS, non-significant.

Figure 5

Effect of fasudil on IL-1β expression. Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). **** p < 0.0001. NS, non-significant.

Figure 6

Effect of fasudil on NF-κB-p65 immunostaining protein expression after MTX-challenge (IHC ×400). Arrows indicate the localization and expression of NF-κB-p65 in hepatic sections. Scale bar = 200 μm. The bar graph represents the quantitative determination of NF-κB-p65 protein expression in different groups (n = 6); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). *** p < 0.001. **** p< 0.0001. NS, non-significant.

Figure 7

Effect of fasudil on Bcl-2 immunostaining protein expression after MTX-challenge (IHC X 400). Arrows indicate the localization and expression of Bcl-2 in hepatic sections. Scale bar = 200 μm. The bar graph represents the quantitative determination of Bcl-2 protein expression in different groups (n = 6); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). ** p < 0.05. **** p < 0.0001. NS, non-significant.