Protective Effect Of Ginsenosides On Parkinson’s Disease In Vitro And Its Mechanism

Objective: The protective effect of ginseng on rotenone-induced Parkinson’s disease(PD)cell model was observed,and the highly active ginseng components with neuroprotective effect were screened,and the molecular mechanism was explored on the basis.Methods:(1)Use Rot to treat SH-SY5 Y cells to establish PD model in vitro,CCK-8 method to determine the optimal concentration of Rot and the best processing time;(2)CCK-8 method to detect total ginsenosides and different ginsenoside monomer pairs Rot induces protective effects of SH-SY5 Y cell damage,and screens out active components with strong protective effects;(3)LDH method is used to detect cell activity against screened highly active ginseng components;flow cytometry is used to detect cell cycle changes;Hoechst33342 Staining and Annexin V-FITC/PI were used to detect the degree of apoptosis;(4)Ginseng detected the mitochondrial-related apoptosis pathway of SH-SY5 Y cells induced by Rot: Caspase activity detection kit was used to detect Caspase activity;UV spectrophotometry was used to detect ATP Content,flow cytometry was used to detect changes in mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)production;mitochondrial protein was extracted from SH-SY5 Y cells by mitochondrial isolation kit,and cytochrome C(Cyt-C)was detected by western blotting.Mitochondria release to cytoplasm and Bax,Bcl-2 and Cleaved-Caspase3 protein expression levels;(5)Protective effect of ginseng on Rot-induced SH-SY5 Y cells oxidative damage: fine Nuclear separation kit was used to extract SH-SY5 Y nuclear protein,and immunoblot was used to detect the nuclear transfer of Nrf2 cells and the expression level of its downstream antioxidant protein HO-1;siRNA interfered with Nrf2 expression,and qRT-PCR was used to analyze the interference efficiency;CCK-8 method was used to examine the interference of Nrf2 Changes in viability of Rot-induced SH-SY5 Y cells after ginseng and changes in ROS levels detected by flow cytometry.Results:(1)The results of CCK-8 showed that the optimal action concentration of Rot was 0.3 μM,and the optimal action time was 24 h.(2)Ginsenoside Re is most effective in protecting Rot-induced SH-SY5 Y cytotoxicity,and the optimal concentration is 5 μM(p<0.01);(3)Ginsenoside Recan significantly inhibit Rot-induced LDH release rate The increase(p<0.01),relieved the G1 phase arrest of SH-SY5 Y cells induced by Rot,and significantly inhibited the level of apoptosis induced by Rot(p<0.001).(4)Ginsenoside Re resists Rot-induced cytotoxicity by inhibiting mitochondrial apoptotic pathway: 5 μM ginsenoside Re significantly inhibits Rot-induced increase in Caspase-3 and Caspase-9 activity(p<0.001,p<0.01),No significant effect on Caspase-8 activity;ginsenoside Re significantly inhibited Rot-induced reduction of MMP in SH-SY5 Y cells(p<0.01),relieved the decrease in ATP production(p<0.01),and inhibited Rot-induced intracellular ROS Increase in production(p<0.01).Compared with Rot-induced SH-SY5 Y cell model,ginsenoside Re inhibits Bax protein and induces Bcl-2protein expression,reduces Bax/Bcl-2 protein expression ratio(p<0.01);prevents Cyt-C from mitochondria to cytoplasm.Release,thereby inhibiting Cleaved-Caspase3 protein expression(p<0.01).(5)Ginsenoside Re slows Rot-induced oxidative stress by activating the Nrf2 antioxidant pathway: Ginsenoside Re treatment can significantly increase the expression level of Nrf2 protein in the nucleus,accompanied by a decrease in cytoplasmic expression level(p<0.05);induce Nrf2 The expression level of downstream antioxidant protein HO-1(p<0.05).Compared with the PD model group,after the interference of Nrf2 expression,the cell survival rate and ROS content of the ginsenoside Re intervention group were not significantly different(p>0.05).Conclusion: In vitro PD model was successfully established by treating SH-SY5 Y cells with Rot,and it is preferred that ginsenoside Re has the most significant protective effect on Ro-induced SH-SY5 Y cell injury.Ginsenoside Re can antagonize Rot-induced oxidative stress by activating the Nrf2 antioxidant signaling pathway,relieve Rot-induced mitochondrial dysfunction,and exert its protective effect by inhibiting mitochondrial-mediated apoptosis pathways.