Study On Mechanism Of Ginseng Root Extract And Its Fractions Related To The Relieving Effect On Macrophage Inflammation Induced By LPS

Objective: By establishing an LPS-induced inflammation model of RAW264.7 cells,we carried out a study on the preparation of ginseng root extract extraction and its effect on inflammatory response.On this basis,the mechanism of action of the active ingredients in ginseng root extracts on anti-inflammation was further explored to provide data support for further studies on the anti-inflammatory effects of ginseng.By establishing an LPS-induced inflammation model;The active ingredients in the ginseng root extract were treated with RAW264.7 cellMethods:1.In this paper,ginseng root extracts were first extracted and prepared,and inflammation models were established using RAW264.7 cells and LPS.The levels of intracellular nitric oxide were measured by nitrate reductase assay,the secretion levels of inflammatory cytokines were measured by ELISA,the changes in m RNA levels were measured by RT-PCR,and protein blotting was used to measure the protein expression levels in the cytoplasm and nucleus,and the possible target of action studies of ginseng root extract were verified by exogenous addition of inhibitors.2.The active ingredients in the ginseng root extract were treated with RAW264.7 cells and LPS to establish an inflammation model.To determine the optimal active ingredient,intracellular nitric oxide levels were measured by nitrate reductase assay,changes in m RNA levels were measured by RT-PCR,and target inflammatory proteins were measured by protein blotting.3.To further explore the anti-inflammatory mechanism of the active ingredients in ginseng root extract,the intracellular nitric oxide level was detected by nitrate reductase assay,inflammatory cytokine secretion was detected by ELISA,m RNA level changes were detected by RT-PCR,intracellular reactive oxygen species and superoxide anion levels in each group were detected by fluorescent probes,autophagosome formation was detected by acridine orange staining,protein expression levels in the cytoplasm and nucleus were determined by protein blotting,and the possible targets of the action of the active ingredients in ginseng root extract were investigated by adding inhibitors and the expression of the corresponding target proteins were detected.Results:1.The yield of ginseng root extract was 21.58 % after extraction.The experimental results demonstrated that ginseng root extract significantly decreased the levels of nitric oxide,TNF-α and IL-6 secreted by LPS-treated cells.q PCR results also showed that ginseng root extract up-regulated IL-10 transcript levels and decreased IL-6 and IL-1β transcript levels in LPS-treated cells.Protein blotting results showed that ginseng root extract inhibited the protein expression of NF-κB,COX2 and decreased the phosphorylation levels of MAPKs(JNK,ERK1/2 and p38).the expression levels of NF-κB and phosphorylation levels of MAPKs(JNK,ERK1/2 and p38)showed a decreasing trend after NF-κB and JNK inhibitor interventions,which consistent with ginseng root extract.2.The results of the analysis of the active ingredients of ginseng root extract showed a protein content of 7.32 %,polysaccharide content of 8.7 % and total saponin content of9.5 %.After functional screening for anti-inflammatory and antioxidant activity,the activity of total saponins was superior to those of protein and polysaccharide.The results showed that total saponins significantly reduced the level of nitric oxide secreted by cells,significantly inhibited the expression of IL-1β and IL-6 at the m RNA level,and reduced the protein expression of NF-κB and COX2 compared to proteins and polysaccharides.3.A series of results,including protein immunoblotting and q PCR techniques,confirmed that,compared with the LPS model group,with increasing concentrations of total saponin,cellular NO levels and TNF-α secretion decreased,IL-10 m RNA levels in LPS-treated cells were upregulated,IL-6 and IL-1β m RNA levels were decreased,intracellular ROS and superoxide anion levels were reduced,total saponin increased the the number of acidic autophagosomes in macrophages,and total saponins increased the protein expression of Nrf2,HO-1,GCLC,GCLM,Beclin1,ATG7 and LC3,while decreasing the protein expression of COX-2,NF-кB and phosphorylation of MAPKs(JNK,ERK1/2 and p38).Conclusion: In this paper,we demonstrated that in RAW264.7 macrophages,ginseng root extract exerted anti-inflammatory functions through MAPK/NF-κB pathway,and the anti-inflammatory effects of total saponins in the active ingredients of ginseng root extract were superior to those of proteins and polysaccharides,and total saponins could reduce LPS-induced inflammatory responses and oxidative damage through NF-кB and Nrf2/ARE pathways as well as by increasing autophagy levels.