〔Abstract〕 Objective To investigate the protective effect of Paeoniflorin-6′-o-benzene sulfonate(CP-25) on methotrexate(MTX)-induced liver injury in rats.Methods 40 SD rats were randomly divided into normal group, model group, CP-25 treatment group, CP-25 prevention group and resveratrol prevention group.A rat model of liver injury was established by single intraperitoneal injection of MTX(20 mg/kg), the levels of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), superoxide dismutase(SOD), glutathione(GSH) and malondialdehyde(MDA) were determined by ELISA, the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), B-lymphocyte tumor-2(Bcl-2), Bcl-2-associated X(Bax),cytochrome C(Cyt-C), cleaved cysteinyl aspartate specific proteinase8(cleaved-cas8), cleaved cysteinyl aspartate specific pro-teinase9(cleaved-cas9) and reduced nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4) containing cysteine were assessed by Western blot.Results Compared with the normal group,the levels of ALT and AST in MTX-induced liver injury model rats increased(F=70.23,P<0.01; F=11.09,P<0.05),and the liver histopathology showed extensive inflammatory cell infiltration,hepatic lobular structure disorder and hepatocyte swelling.Compared with MTX model group,the levels of ALT and AST in CP-25 prevention group and treatment group were lower(F=62.32,86.86,P<0.01; F=16.35,7.14,P<0.01),and the pathological score of liver tissue was lower(F=18.33,P<0.01; F=15.47,P<0.05).Further studies showed that compared with MTX model group,both CP-25 prevention group and treatment group could significantly down-regulate the expression of pro-apoptotic proteins Bax(F=21.37,P<0.01; F=19.35,P<0.05),Cyt-C(F(CP-25 prevention group)= 7.21,P<0.01),cleavedcas8(F(CP-25 prevention group)= 12.32,P<0.05) and cleaved-cas9(F=8.41,P<0.01; F=6.91,P<0.05),and upregulate the expression of anti-apoptotic protein Bcl-2(F=13.11,P<0.01; F=9.93,P<0.05).At the same time,compared with MTX model group,CP-25 prevention group and treatment group decreased the levels of IL-1β,IL-6 and TNF-α(F(CP-25 prevention group)= 160.7,46.55,28.89,P<0.01); F(CP-25 treatment group)= 127.4,53.70,26.46,P<0.01), down-regulated the levels of MDA and NOX4(F(CP-25 prevention group)= 31.71,38.45, P<0.01;F(CP-25 treatment group)=27.89,31.27,P<0.01),and up-regulated the level of GSH(F=39.65,29.04,P<0.01).Conclusion CP-25 has a good therapeutic and protective effect on hepatotoxicity induced by MTX,and this effect may be related to its inhibition of oxidative stress,inflammatory response,and reduction of apoptosis in liver tissue.