| Objective: The aims of this study are to investigate the anti-inflammatory effect of combination therapy(curcumin and Erzhi pill group)on concanavalin A(ConA)-induced hepatitis in mice,and to elucidate its underlying molecular mechanisms.Methods: Female Balb/c mice were randomly divided into five groups: normal group,ConA model group,erzhi pill group(160mg/kg/day),curcumin group(30mg/kg/day),and erzhi pill curcumin(160mg/kg/day)+curcumin(30mg/kg/day)group.For the first 3 weeks,the mice in the normal group and the model group were given the same amount of 0.5% CMC-Na solution and the mice in the treated groups were given the corresponding medicines once a day.At the last day,one hour after intragastric administration,ConA(20 mg/kg)was injected into the caudal veins of mice,except for the normal group,which received 0.9% physiological saline(0.05 mL/10 g).The blood and liver tissues was collected at two points(10h,24h)after ConA injected.All samples are stored at-80℃ before measurement.Liver injury was evaluated by measuring serum transaminases and cytokines levels and assessing liver tissue histology.Hepatocyte apoptosis were identified by the TUNEL assay.We also measured hepatic expression of IL-6 and INF-γ mRNA level and the proteins of phosphorylated p65,cleaved caspase-3,nuclear factor(erythroid-derived 2)-like 2(Nrf2).Results: The acute hepatitis model was established successfully by intravenous administration of Con A(20 mg/kg)in male BALB/c mice.(1)Compared with ConA model group,the mice in curcumin and erzhi pill were able to significantly reduce the elevated plasma AST levels at 8h and 24 h time-points(P<0.05),while ALT level was decreased only at 24 h time-point(P<0.05).We also found that the necrosis of hepatocytes and the infiltration of inflammatory cells in the mice pretreated both erzhi pill and curcumin were significantly less than that of the ConA model group(P<0.05).(2)Elisa showed that the serum levels of inflammatory cytokines(IL-4,IL-6 and IFN-γ)in the erzhi pill + curcumin group were lower than ConA model group(P<0.05).(3)Realtime PCR showed that the treatment of mice with erzhi pill and curcumin were significantly inhibited the up-regulation of IL-6 and IFN-γ and the down-regulation of IL-10 in the liver after Con A injection compared with ConA model group(P<0.05).(4)In addition,erzhi pill and curcumin pretreatment dramatically suppressed the phosphorylation of p65,increased Nrf2 expression,and decreased the expression ratio of cleaved caspase-3/caspase-3 in liver tissues(P<0.05).Conclusion: These results suggest that erzhi pill and curcumin protects against ConA-induced hepatitis through suppressing inflammatory cytokine production,reducing hepatocyte necrosis,inhibiting NF-κB mediated inflammatory pathway and promoting Nrf2 mediated anti-oxidative stress signaling pathway. |
