Study On Anti-inflammatory Activity Of Resveratrol Derivatives

Non-steroidal anti-inflammatory drugs are currently the most widely used anti-inflammatory drugs in the world,and long-term use in large quantities can cause side effects such as indigestion or gastrointestinal toxicity.Therefore,it is of great significance to find a low-toxic,high-efficiency natural product with anti-inflammatory effects.In recent years,scholars have confirmed that resveratrol has excellent anti-inflammatory effects,but because the hydroxyl group on the benzene ring in the resveratrol structure is easily oxidized,resulting in structural and activity instability,the clinical efficacy is not satisfactory.Therefore,it has been found that resveratrol derivatives with similar activity become one of the research hotspots.This paper selected three resveratrol methylated derivatives as the research object to determine whether it has anti-inflammatory activity.The three compounds are pterostilbene,pinostilbene,and 4'-methoxyresverarol.Since the hydroxy group on the benzene ring is substituted with methoxy group,the lipophilicity is enhanced,which is more conducive to cell entry and functions.It will increase its bioavailability.The first chapter mainly introduces the general situation of inflammation,inflammatory mediators and inflammation-related signal transduction pathways,the research status of resveratrol and its biological activity,and the general situation of resveratrol derivatives and their biological activities.The second chapter mainly uses LPS to induce macrophage RAW264.7 to establish in vitro cell inflammation model.The cells were stimulated with LPS at 50,100,and 150 ng/mL for 24 hours,and RNA was extracted with TRizol reagent.The mRNA expression levels of inflammatory factors?MCP-1,IL-6,IL-1band TNF-a?produced by the cells were detected by reverse transcription and RT-qPCR to determine the intensity of inflammation.The results showed a dose-dependent increase in the expression of inflammatory cytokines.When the concentration reached 100 ng/mL,the expression of inflammatory cytokines reached a very significant effect.Therefore,100ng/mL LPS was selected for follow-up tests.The third chapter is mainly to screen out compounds with good anti-inflammatory activity.First,crystal violet staining was used to examine the effects of pterostilbene,pinostilbene,and 4'-methoxyresverarol on cell viability.Pterostilbene and pinostilbene had a significant reduction in cell viability at more than 10?M.4'-methoxyresverarol had a significant decrease in cell viability at more than 30?M.Therefore,the final selected drug concentration was 5?M.Based on the inflammatory model of RAW264.7cells induced by LPS at 100 ng/mL,three compounds were intervened,and LPS and compounds were simultaneously added and cultured together for 24 hours.TRizol reagent was used to extract cellular RNA,and mRNA levels of MCP-1,IL-6,IL-1band TNF-awere detected by reverse transcription and qRT-PCR.The results showed that Pterostilbene and 4'-methoxyresverarol were able to inhibit the expression of inflammatory factors to varying degrees and exhibited good anti-inflammatory activity,while pinostilbene did not show anti-inflammatory effects.Therefore,the follow-up experiment selected pterostilbene and 4'-methoxyresverarol for anti-inflammatory mechanism analysis.The fourth chapter mainly analyzes the anti-inflammatory mechanism of pterostilbene and 4'-methoxyresverarol.First,the anti-inflammatory effects of pterostilbene and 4'-methoxyresverarol were verified and supplemented.The mRNA expression levels of inflammatory factors MCP-1,IL-6,IL-1b,TNF-a,eNOS,iNOS and COX-1,COX-2,mPGEs-1 were detected by q-PCR.The amount of NO released from the culture medium was detected by nitrate reductase assay.The concentration of PGE₂ in the culture medium was used Enzyme-linked immunosorbent assay.The results showed that both Pterostilbene and4'-methoxyresverarol significantly inhibited the expression of four inflammatory factors mRNA.In addition,Pterostilbene could inhibit the release of NO?P<0.001?by down-regulating the mRNA expression level of iNOS?P<0.001?,and could significantly inhibit the release of PGE₂?P<0.01?,while 4'-methoxyresverarol ether could inhibit the release of NO?P<0.05?by down-regulating the mRNA expression of iNOS and eNOS?P<0.01?,but it has no obvious effect on PGE₂ and its related COXs,suggesting that 4'-methoxyresverarol ether exerts anti-inflammatory effects and does not pass the COX pathway.Next,the gene levels of c-jun and c-Fos were detected by PCR.The effect of pterostilbene and 4'-methoxyresverarol on LPS-induced protein expression of c-jun,P-JNK/JNK,P-ERK/ERK,p-p38/p38 and p-p65/p65 were detected by Western blot.The results showed that pterostilbene significantly inhibited the phosphorylation of ERK,p38 and p65,and may play an anti-inflammatory role by blocking the transduction of MAPK and NF-kB signaling pathways.4'-methoxyresverarol significantly inhibited the expression of c-jun gene and protein expression,and also inhibited the phosphorylation of JNK,p38 and p65,thus 4'-methoxyresverarol plays anti-inflammatory role by blocking the signal transduction of AP-1,MAPK and NF-kB.