Study On The Protective Mechanism Of Ginseng Root Extract And Its Fermented Products On Macrophage RAW264.7 Oxidative Damage

Objective:In this study,we established an in vitro LPS-induced RAW264.7 cell oxidative damage model to study the protective effects of ginseng root extract and its fermentation products on oxidative damage.On this basis,explore the anti-oxidative damage mechanism of ginseng root extract.Provide data support for further research on the antioxidant effect of ginseng.Methods:1.Lactobacillus plantarum was used to study the fermentation process,and the optimal fermentation process was screened by orthogonal test method by investigating the three factors of pH,inoculation amount and fermentation time,and the antioxidant activity of ginseng root extracts before and after fermentation was tested.2.Fluorescence probe detection method and flow cytometry were used to detect the changes of intracellular ROS,O2-,mitochondrial number and mitochondrial membrane potential in each group.3.Study on the Effect of Ginseng Root Extract on the Expression of Antioxidant Related Pathway ProteinsWestern blot was used to detect the protein expression changes of Nrf2,Keap1,HO-1,GCLC and GCLM,and to study the changes in intracellular ROS content and related protein expression before and after the silence of p62,a key factor of p62-Keap1-Nrf2 pathway.4.Effect of Ginseng Root Extract on Autophagy Level of Macrophage RAW264.7 CellsThe AO staining method was used to detect the number of acidic vesicles in each administration group,and the protein expression changes of autophagy-related proteins p62,LC3 B,p-AKT and p-mTOR were detected by Western blotting.5.The role of autophagy in the reduction of oxidative damage by ginseng root extract.Ginseng root extract and autophagy regulators work together.Western blot was used to detect the expression of p62,Keap1 and Nrf2 nuclear proteins in the antioxidant pathway.Result:1.The optimal fermentation process of ginseng root extract was screened by orthogonal test as fermentation pH5.5,inoculation amount 3.5%,fermentation time 24 h.The antioxidant activity before and after fermentation was compared,and the results showed that the samples before and after fermentation had significant antioxidant activity,Ginseng root extract before fermentation is more effective.2.Compared with the LPS control group,as the concentration of ginseng root extract increased,the content of ROS and O2-in the cells decreased,and the mitochondrial dysfunction caused by oxidative damage of cells was inhibited.3.Ginseng root extract can promote phosphorylation of p62,reduce the protein expression of Keap1,promote Nrf2 to enter the nucleus,and increase the expression of antioxidant proteins such as HO-1,GCLC and GCLM;silencing p62 can significantly inhibit ginseng root extraction Antioxidant activity of the substance.4.Ginseng root extract can increase the protein expression of LC3 B,Beclin1 and Atg7,and reduce the phosphorylation level of AKT and mTOR.The autophagy inducer Rapamycin can significantly promote the p62-Keap1-Nrf2 signaling pathway,and the addition of the autophagy inhibitor 3-MA inhibits the expression of Nrf2 into the nucleus.Conclusion: Ginseng root extract can mediate p62 phosphorylation to increase the antioxidant activity of Nrf2,increase the expression of antioxidant factors,reduce the levels of ROS and O2-in cells,protect mitochondrial function,thereby reducing LPS-induced cell oxidative damage.Gene silencing experiments proved that p62 played an important role in this process.At the same time,ginseng root extract can increase the autophagy level of RAW264.7 cells,so as to better play the role of reducing oxidative damage.