Protective Effect Of Black Ginseng And Ginsenoside Rk1 On Acetaminophen-induced Liver Injury In Mice And Its Possible Mechanisms

Black ginseng(BG),as s product derives from Panax ginseng C.A.Meyer after nine steaming and drying cycles,has significant anti-inflammatory,anti-tumor and anti-diabetes effects.During the steaming process,the original ginsenosides in BG will transform into secondary saponins which are different from other secondary saponins.Not only are these saponins related to the hydrolysis of glycosyl,but include the C-20 dehydration isomerization,and known as "ginseng pyrolysis saponin".As a pyrolytic saponin in BG,Ginsenoside Rk1(G-Rk1)exerts strong pharmacological effects including anti-apoptosis,anti-cancer and anti-platelet aggregation.In this study,we investigated the hepatic protective effects of BG and G-Rk1 in acetaminophen(APAP,Acetaminophen)induced mouse model from the aspects of inflammation,oxidative/nitrative stress and apoptosis,and explored its potential molecular mechanism.1.Ameliorative effects of BG on APAP-induced liver injury.The mice were randomly divided into 4 groups(8 mice per group): normal group,APAP group(APAP,250mg/kg),APAP + BG(300mg/kg)and APAP + BG(600mg/kg).Mice in BG groups were treated with BG(300,600mg/kg)by oral gavage once per day for seven days.Mice in normal and APAP groups were given 0.9% saline in the same way.After the last dose,all mice in APAP and BG groups were treated with 250 mg/kg APAP intraperitoneally to establish a severe liver injury model.The mice were sacrificed and serum and liver tissues were collected for histopathological staining and determination of each index.Our results showed that pretreatment with BG significantly decreased the liver and spleen indexes,serum alanine aminotransferase(ALT)and aspartate transaminase(AST)levels compared with the APAP group.Meanwhile,an increase in hepatic antioxidant including glutathione(GSH)and a decrease in lipid peroxidation product malondialdehyde(MDA)were detected compared with the APAP group.In addition,BG also decreased the levels of cytochrome P450 El(CYP2E1),4-hydroxynonenal(4-HNE),3-nitrotyrosine(3-NT),Bax,iNOS,COX-2,increased the expression of Bcl-2 protein and reduced APAP-induced nucleus apoptosis.2.Ameliorative effects of G-Rk1 on APAP-induced liver injury.After adaptive breeding for 1 week,the mice were randomly divided into four groups(eight mice per group): normal group,APAP(250mg/kg)group,APAP+Rk1(10mg/kg)group and APAP+Rk1(20mg/kg)group.G-Rk1 was prepared by suspending in 0.05%(w/v)sodium carboxymethylcellulose and given to the mice in treatment groups by gavage for 7 consecutive days.Mice in the normal and APAP groups were treated with 0.9% saline in the same way.After final administration,mice in the APAP and APAP+Rk1 treatment groups were given a single intraperitoneal injection of 250mg/kg APAP to induce acute liver injury.Simultaneously,mice in the normal group were given 0.9% saline in the same way.All mice were executed after 24 h.Our results showed that pretreatment with G-Rk1 significantly decreased the levels of serum ALT,AST,tumor necrosis factor-?(TNF-?),and interleukin-1?(IL-1?)compared with the APAP group.Meanwhile,rises in hepatic antioxidants,GSH and superoxide dismutase(SOD),were detected compared with the APAP group.Moreover,a significant decrease of the lipid peroxidation product MDA was observed in the G-Rk1 treated groups compared with the APAP group.Immunofluorescence staining indicated that G-Rk1 supplementation downregulated the expression of CYP2E1?4-HNE and 3-NT proteins in liver tissues.Western Blot analysis and immunohistochemical staining revealed that G-Rk1 could suppress activation of apoptotic pathways by increasing anti-apoptosis Bcl-2 and decreasing pro-apoptosis Bax protein expression levels.Hoechst 33258 and TUNEL staining further showed that G-Rk1 could reduce APAP-induced nucleus apoptosis.Histopathological observation also revealed that G-Rk1 pretreatment significantly reversed APAP-induced hepatocyte apoptosis.In addition,immunohistochemical analysis also showed that G-Rk1 could reduce the expression of two inflammation markers,iNOS and COX-2,in liver tissue.In summary,the results of this study clearly demonstrate that BG and its major rare saponin G-Rk1 can prevent APAP-induced acute liver injury in mice by alleviating the oxidative/nitrative stress injury,inhibiting inflammation and apoptosis,which will provide theoretical basis for clinical treatment of APAP liver injury and development of hepatoprotective drugs.