| Part 1:Effect of ginsenoside Rb1 on hepatic ischemia-reperfusion injuryObjective:To investigate the effect and related mechanisms of ginsenoside Rb1 on liver function and hepatic tissues in the liver ischemia- reperfusion injury.Methods:Healthy male SD rats were randomized into 3 groups (n=18):sham-operated group(A), model control group(B) and ginsenoside Rb1 treated group(C).10 minutes before Ischemia, treatment group rats were intravenously injected ginsenoside Rb1 40mg/kg. Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle lobe, were established. Rats in the control group were administered with saline in the same volume. After 90 mins of ischemia, 6 rats of each group were sacrificed at 2h,6h and 24h after residual liver reperfusion respectively. Blood and I/R liver tissue samples were collected from each group. ALT and AST in serum were measured, part of the liver tissues were made into paraffin-embedded specimens to detect rat liver histological change and grade of hepatic IRI (Suzuki's score), the activity of SOD and MDA expression in liver tissues were detected by ELISA.Results:Compared with the control group, serum ALT and AST in the ginsenoside Rb1 treated group were significantly decreased (P<0.05). Hepatic pathology performance was improved and Suzuki's scores decrease significantly in the ginsenoside Rb1 treatment group (P<0.05). The MDA expression in I/R liver tissues were significantly decreased in the ginsenoside Rb1 treatment group comparing with the I/R control group(P<0.05), and the activity of SOD were significantly increased in the ginsenoside Rb1 treatment group (P<0.05).Conclusions:(1) Treatment with ginsenoside Rb1 can effectively improve the rat liver function and reduce the liver damage induced by liver ischemia-reperfusion injury;(2)Intervention with ginsenoside Rb1 can inhibit the productions of oxygen free radicals, and reduce the hepatic damage induced by lipid peroxidation.Part 2:Effect of ginsenoside Rb1 on ischemia-reperfusion-induced hepatocytes apoptosis and related protein expressionObjective:To study the molecular mechanisms of the effect of ginsenoside Rb1 inhibiting the ischemia-reperfusion-induced hepatocytes apoptosis and related protein expression in the rat hepatic ischemia-reperfusion injury.Methods:Healthy male SD rats were randomized into 3 groups (n=6):sham-operated group(A), control group(B), ginsenoside Rb1 treatment group(C).10 minutes before Ischemia, treatment group rats were intravenously injected ginsenoside Rbi 40mg/kg.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatic lobe, were established. Control group were administered with saline in the same volume. After 90 min ischemia,6 rats of each group were sacrificed at 6h after residual liver reperfusion respectively. Liver tissue samples were collected from each group. Hepatocytes apoptosis was determined by TUNEL staining. The activities of caspase-9, 8 and 3 were analyzed by Caspase Colorimetric Assay Kit. The expression of Bcl-2 and Bax was detected by immunohistochemical method and and Western blot, the level of Bcl-2 and Bax mRNA were measured by RT-PCR.Results:Compared with the model control group, the apoptosis indexes(AI) in the ginsenoside Rb1 treatment group were significantly decreased(P<0.05), and the activities of caspase-9,8 and 3 in the ginsenoside Rb1 treatment group were significantly decreased(P<0.05); Compared with the control group, the level of Bc1-2 expression in ginsenoside Rb1 treatment group were significantly increased(p<0.05), the level of Bax expression were significantly decreased(P<0.05); compared with the control group, the level of Bc1-2 mRNA in ginsenoside Rb, treatment group were significantly increased(p<0.05), the level of Bax mRNA were significantly decreased(P<0.05).Conclusions:(1)Treatment with ginsenoside Rb1 could attenuate reperfusion-induced hepatocytes apoptosis;(2)Ginsenoside Rb1 may play a protective role agains reperfusion-induced hepatocytes apoptosis, which may be realized by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax in the hepatic tissues.Part 3:Study of the relationship between the anti-apoptosis of ginsenoside Rb1 with PI3K/Akt pathway in hepatic ischemia-reperfusion injury in ratsObjective:To study the molecular mechanisms of the effect of ginsenoside Rb1 on the PI3K/Akt pathway in the rat hepatic ischemia-reperfusion injury.Methods:Healthy male SD rats were randomized into 4 groups (n=6):sham-operated group(A), control group(B), ginsenoside Rb1 treatment group (C) and LY294002+ ginsenoside Rb1 treatment group(D). For the rats in group C,20 minutes before ischemia, the same volume of DMSO were injected, then with the ginsenoside Rb1 40mg/kg 10 minutes before ischemia; for the rats in group D, we intravenously injected LY294002 0.3mg/kg which was diluted in 0.02% DMSO, then with the ginsenoside Rb1 40mg/kg 10 minutes before ischemia. Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.Control group and were administered with the same volume of DMSO and saline. After 90 mins ischemia,6 rats of each group were sacrificed at 6h after residual liver reperfusion respectively. Liver tissue samples were collected from the each group. Hepatocytes apoptosis was determined TUNEL staining.The expression of protein of PI3K, Akt and p-Akt were measured by immunohistochemical method and Western blot; the level of PI3K and Akt mRNA were measured by RT-PCR.Results:Compared with the control group, the apoptosis indexes(Al) in the treatment group of ginsenoside Rb1(C) were significantly decreased(P<0.05), pretreatment of LY294002 abrogated the anti-apoptosis effect of ginsenoside Rb1 significantly(P>0.05); the levels of PI3K and Akt in all groups have no significant difference (P>0.05); compared with the control group(B), the expression of p-Akt in ginsenoside Rb1 treatment group(C) were significantly increased(p<0.05). There was no significant difference in the expression of p-Akt in hepatic tissues between group A,B and D (P>0.05); the levels of PI3K mRNA and Akt mRNA in all groups have no significant difference (P>0.05).Conclusion:Ginsenoside Rb1 may play a protective role agains reperfusion-induced hepatocytes apoptosis by enhancing PI3K/Akt signal pathway activation.
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