Study Of Anti-Inflammatory Effect Of Low Toxicity Polyphenols And Its Molecular Mechanism Based On The LPS-Induced RAW264.7 Cell Inflammation Model

When the body is exposed to external damage,an inflammatory response is triggered to protect the body from foreign pathogens.However,if the inflammatory response continues,it will adversely affect the body and cause various diseases such as non-alcoholic fatty liver,Alzheimer’s disease,ulcerative colitis,etc.Natural polyphenolic compounds are of interest for their ability to modulate the immune system and reduce the inflammatory response.This project focuses on the anti-inflammatory effects of polyphenolic compounds by studying the network pharmacology,anti-inflammatory effects,and anti-inflammatory mechanisms.The main research of the thesis is as follows:1.Screening of polyphenolic compounds with anti-inflammatory effects:Preliminary screening of the collected polyphenolic compounds for anti-inflammatory effects by network pharmacology.Ranked according to the degree value,and the top 10 compounds were selected for subsequent experiments.And the CCK-8 assay,lactate dehydrogenase(LDH)release assay and morphological observation were performed on the above 10 compounds to screen out the compounds with lower cytotoxicity.2.Anti-inflammatory effect of polyphenolic compounds:After treating RAW264.7 cells with the compounds for certain time(6 h.12 h,24 h)using the compounds,the cells were treated with lipopolysaccharide(LPS)for 12 h.The changes of NO content in the supernatant were detected by using nitric oxide detection kit,and the changes of inflammatory factors(TNF-α,IL6,IL-1β)in the cells were detected by using real-time fluorescence quantitative PCR(RT-qPCR)to determine the anti-inflammatory effect of polyphenolic compounds.3.Anti-inflammatory mechanism of polyphenolic compounds:The anti-inflammatory targets and pathways of target polyphenolic compounds were analyzed by network pharmacology,and the changes of mRNA expression and protein expression of key targets on each pathway were detected by RT-qPCR and Western Blot assay to reveal the anti-inflammatory mechanism of polyphenolic compounds.The main findings of the paper are as follows:1.Screening of polyphenolic compounds with anti-inflammatory effects:Network pharmacological screening yielded the following polyphenolic compounds with antiinflammatory effects:resveratrol,butein,gallic acid,curcumin,pterostilbene,paeonol,gentisic acid,protocatechuic acid,ellagic acid and epicatechin.According to the results of CCK-8 assay,lactate dehydrogenase(LDH)release assay and morphological observation assay,the cytotoxic effects of protocatechuic acid,gentisic acid and paeonol were small.Combining the results of toxicity experiments,protocatechuic acid,gentisic acid and paeonol were selected for antiinflammatory effect study.2.Anti-inflammatory effect of polyphenolic compounds:Protocatechuic acid exerted antiinflammatory effects by decreasing the expression of TNF-α,IL-1β,IL-6 mRNA and NO secretion;Gentisic acid exerted anti-inflammatory effects by decreasing the expression of TNFα,IL-1β and NO secretion,but had no significant effect on IL-6 expression;Paeonol exertedanti-inflammatory effects by decreasing the secretion of NO and the expression of TNF-α,IL1β,IL-6 mRNA The anti-inflammatory effect was exerted by reducing NO secretion and expression of TNF-α,IL-1β and IL-6 mRNA.All three compounds had anti-inflammatory effects,but there were differences.3.Anti-inflammatory mechanism of polyphenolic compounds:It was demonstrated by network pharmacology and experimental validation that protocatechuic acid could reduce the expression of TNFR1 and TRAF2 mRNA and decreased the expression of IKKα,IKKβ mRNA and protein,the key targets of downstream NF-κB signaling pathway,and reduced the phosphorylation of NF-κB/p65,protocatechuic acid could exert anti-inflammatory effects by regulating the TNF signaling pathway;Gentisic acid could significantly reduce IL-17A mRNA expression,reduce TRAF6,IKKα,IKKβ mRNA and protein expression,and significantly NFκB/p65 phosphorylation,and gentisic acid can exert anti-inflammatory effects by regulating the IL-17 signaling pathway;Paeonol can significantly reduce LCK,zap70,IKKα,IKKβ mRNA and protein expression,and significantly The anti-inflammatory effect of paeonol can be exerted by regulating the T cell receptor signaling pathway.The experimental results showed that the targets of the three polyphenolic compounds exerting anti-inflammatory effects may be different,but all of them inhibit the activation of NF-κB signaling pathway by blocking the upstream pathway of NF-κB signaling pathway to achieve anti-inflammatory effects.The above results indicate that polyphenolic compounds have anti-inflammatory effects and inhibit the activation of NF-κB signaling pathway mainly by blocking the upstream pathway of NF-κB signaling pathway,thus achieving anti-inflammatory effects.