| [Objective]To study the effects of 20(R)-ginsenoside Rg3 on autophagy induced by cerebral ischemia reperfusion injury(CIRI)in rats,and to explore the regulation of 20(R)-ginsenoside Rg3 on autophagic signaling pathway of PI3K/Akt.This study provides theoretical and experimental basis for further analysis of the effect of 20(R)-ginsenoside Rg3 on improving CIRI.[Methods]1.Animal experimental study:the effect of 20(R)-ginsenoside Rg3 on CIRI-induced autophagy in rats.Male specific-pathogen free(SPF)SD rats were randomly divided into sham operation group,MCAO/R model group,20(R)-ginsenoside Rg3 group(5,10,20mg/kg)and nimodipine group(1mg/kg).Except for sham operation group,MCAO/R model was replicated in the other groups.After 2 h ischemia and reperfusion for 24 h,the open field test was performed in each group.Then the rats were sacrificed and the brain tissues were taken out for HE staining and nissl body staining.The number of autophagosomes was observed by transmission electron microscopy.The expressions of autophagy-related proteins Beclinl,P62 and LC3-II/I were detected by immunofluorescence and Western blot,respectively.The protein expressions and phosphorylation levels of PI3K,Akt and mTOR in PI3K signaling pathway in rat cerebral cortex were detected by Western blot.2.CCK8 method was used to explore the optimal time and concentration of 20(R)-ginsenoside Rg3 on PC 12 cells.PC 12 cells were aseptically cultured,and the cells with good growth were selected and randomly divided into:Normal group,OGD/R model group,20(R)-ginsenoside Rg3 groups with different concentrations(1 μmol/L,5 μmol/L,25 μmol/L,125μmol/L,and 250 μmol/L).After 2 h OGD treatment,each group was incubated for 6,12,24 h,respectively.Cell viability was determined by CCK8 assay.3.Cell experiment:the effect of 20(R)-ginsenoside Rg3 on autophagy of PC 12 cells injured by OGD/R.The experimental groups were as follows:normal group,OGD/R group,20(R)-ginsenoside Rg3 groups with different concentrations(5 μmol/L,25 μmol/L,125 μmol/L),and 10 μmol/L nimodipine group.The expressions of Beclinl,P62 and LC3-Ⅱ/Ⅰ were detected by Western blot.The effect of 20(R)-ginsenoside Rg3 on autophagy signal pathway of PI3K/Akt in PC 12 cells injured by OGD/R was further studied.The cells were randomly divided into normal group,OGD/R group and 20(R)-ginsenoside Rg3 group(125 μmol/L).The expressions of PI3K,p-Akt and p-mTOR were detected by Western blot.4.The following experiments were designed to confirm whether 20(R)-ginsenoside Rg3 could protect PC 12 cells from OGD/R-induced injury mediated by PI3K/Akt pathway.After preconditioning of PI3K inhibitor LY294002(20 μmol/L),the effects of 20(R)-ginsenoside Rg3 on autophagy and PI3K/Akt/mTOR signaling pathway in OGD/R-injured PC 12 cells were observed.The experimental groups were normal group,OGD/R group,normal control group+LY294002,20(R)-ginsenoside Rg3 group,and LY294002+20(R)-ginsenoside Rg3 group,respectively.The protein expressions of Beclinl,P62,PI3K,Akt and mTOR were detected by Western blot.[Results]1.Open field test showed that compared with control group,the average speed of rats in MCAO/R group decreased significantly.The pathological examination showed disorder of neuron arrangement in the cortex,intercellular broadening,the cell body shrinkage,nuclear pyknosis,and nucleolus disappeared,and a large number of nuclei of neurons abnormity and chromatin hyperchromatic,and the number of damaged neurons increased significantly,while the nissl body was dissolved.Compared with the model group,the average velocity of the rats in 20 mg/kg 20(R)-ginsenoside Rg3 group was significantly increased,the number of neuron damage was significantly decreased,and the dissolution of nissl bodies was significantly decreased.Electron microscopy results showed that the number of autophagosomes in the model group was increased compared with that in the sham group,and the number of autophagosomes in the 20 mg/kg 20(R)-ginsenoside Rg3 group was significantly decreased compared with that in the model group.Immunofluorescence results showed that beclin1 expression in model group was increased compared with sham group(P<0.01),and beclin1 expression in 20(R)-ginsenoside Rg3 group was significantly decreased compared with model group(P<0.01).Western blot results showed that beclin1 and LC3-Ⅱ/Ⅰ expressions in model group were significantly increased compared with sham group(P<0.05).The expression levels of P62,p-PI3K,p-AKT and p-mTOR were significantly decreased(P<0.05).Compared with model group,the expressions of Beclinl and LC3-Ⅱ/Ⅰ in 20 mg/kg 20(R)-ginsenoside Rg3 group were significantly decreased(P<0.05),and the expressions of P62,p-PI3K,p-Akt and p-mTOR were significantly increased(P<0.05).2.Compared with normal control group,the cell survival rate decreased significantly after 2 h of glucose deficiency and 12 and 24 h of reoxygenation(P<0.01).Compared with OGD/R group,the survival rate of the cells in 25,125 and 250 μmol/L 20(R)-Rg3 groups had no significant changes.Compared with OGD/R group,125 and 250 μmol/L 20(R)-ginsenoside Rg3 significantly increased cell survival rate after 12 h glucose and oxygen reoxygenation(P<0.05);compared with OGD/R group,25,125 and 250 μmol/L 20(R)-ginsenoside Rg3 significantly increased the cell survival rate(P<0.05,P<0.01),indicating that 125 μmol/L 20(R)-ginsenoside Rg3 intervention for 24 h had the best effect on the cell viability of PC 12 treated by OGD/R.Therefore,125 μmol/L 20(R)-ginsenoside Rg3 was selected for 24 h for subsequent experiments.3.The expression of autophagy-related proteins in OGD/R-injured PC 12 cells pretreated with 20(R)-ginsenoside Rg3 was detected by Western blot.Compared with normal control group,the expressions of beclin1 and LC3 in OGD/R group were significantly increased,and the expressions of P62,p-PI3K,p-AKT and p-mTOR were significantly decreased(P<0.01,P<0.05);compared with OGD/R,125 μmol/L 20(R)-ginsenoside Rg3 significantly decreased the expression of beclin1 and LC3,and increased the expression of P62(P<0.01),and significantly increased the expression of p-PI3K,p-AKT and p-mTOR(P<0.01,P<0.05).4.After LY294002 pretreatment,the protein expressions of beclinl,LC3-Ⅱ/Ⅰ,P62,PI3K,Akt and mTOR in OGD/R injured PC 12 cells were detected by Western Blot,and the results showed that:compared with the normal control group,the expression of beclin1 was significantly increased in LY294002 and OGD/R groups,and the expression of P62,p-PI3K,p-Akt and p-mTOR was significantly decreased(P<0.01,P<0.05).Compared with OGD/R group,the expression of beclin1 in 20(R)-ginsenoside Rg3 group was decreased,and the expression of P62,p-PI3K,p-Akt and p-mTOR were increased(P<0.01,P<0.05).Compared with 20(R)-ginsenoside Rg3,the expression of Beclinl in LY294002+Rg3 group was increased,and the expression of P62,p-PI3K,p-Akt and p-mTOR was decreased(P<0.01,P<0.05).[Conclusion]1,20(R)-ginsenoside Rg3 can improve the motor dysfunction and reduce neuronal damage in MCAO/R rats.2,20(R)-ginsenoside Rg3 inhibits autophagy,down-regulats the expression of autophagy-related proteins beclin1 and LC3-Ⅱ/Ⅰ,and up-regulats the expression of P62.3,20(R)-ginsenoside Rg3 inhibits autophagy and plays a neuroprotective effect,which may be related to its up-regulation of the expression of PI3K/Akt signaling pathway. |
