The Anti-inflammatory Mechanism Of Resveratrol On LPS-induced Inflammation In RAW264.7 Cells

Resveratrol(3,4?,5-Trihydroxy-trans-stilbene,Res)is a natural polyphenolic substance with strong biological activity.Previous studies in our laboratory have shown that resveratrol can regulate the transcription and expression of related genes IKB?,P38,JNK,ERK1,ERK2 and ERK5 by affecting the signal cascade of TLR4/NF-?Bp65/MAPKs pathway in rat liver and lung tissues.Finally,it regulated the release and expression of inflammatory factors IL-1,IL-6,IL-8,COX2,etc to play the anti-inflammatory activities.This study intends to establish an inflammation model of RAW264.7 cells stimulated by lipopolysaccharide at the cellular level.The effects of resveratrol on signal transduction of TLR4-NF-?B/MAPKs/IRF3 pathway in LPS-induced inflammation of RAW264.7 cells were studied by WB,RT-PCR and other experimental techniques,and the pathway of resveratrol to play a role was determined.On this basis,we added the pathway-specific inhibitors which including SP600125,BAY11-7082 and SB203580 to detect the expression of important proteins in TLR4/NF-?B/MAPKs pathway,confirming the pathway of resveratrol action and the protective effect of inflammation.Finally,the transcription and secretion of related inflammatory factors IL-1?,IL-6,IL-8,IL-10 and TNF-? were detected to clarify the relationship between the anti-inflammatory effect of resveratrol and the secretion of inflammatory factors,so as to reveal the protective mechanism of resveratrol on LPS-induced inflammatory injury in RAW264.7 cells.The main research contents and results are as follows:1 Establishment of RAW264.7 cell inflammation model and screening of resveratrol drug concentrationRAW264.7 cells were cultured and the effects of different concentrations of LPS and resveratrol on the activity of RAW264.7 cells and the secretion of inflammatory mediators TNF-? and IL-6 were detected by CCK-8 and ELISA,respectively.The appropriate modeling method and the concentration of resveratrol were determined.The results showed that: there was not toxic to RAW264.7 cells when the concentration of LPS was less than or equal to 2.5 ug/ml.When the concentration of resveratrol was 12.5-200 umol/L,there was not toxic to RAW264.7 cells.LPS can promoted the secretion of TNF-? and IL-6 in a dose-dependent manner.When adding 6.25-50 ?mol/L,resveratrol can significantly reduced the secretion of inflammatory mediators TNF-alpha and IL-6 which was induced by LPS.The above results indicated that the cell inflammation model could be established by adding 500 ng/ml LPS to RAW264.7 cells and culture the cells for 12 hours;The range concentration of resveratrol which is 6.25-25?mol/L.2 Effects of resveratrol on TLR4-NF-?B/MAPKs/IRF3 signaling pathway induced by LPS in RAW264.7 cells.2.1 Effects of resveratrol on NF-?B/MAPKs/IRF3 signaling pathway induced by LPS in RAW264.7 cells: We studied the RAW264.7 inflammatory cells which stimulated by LPS.The expressions of NF-?B/MAPKs/IRF3 signaling pathway-related proteins I?B?,p65,P38,JNK,ERK1/2 and IRF3 were tested by using Western blot and RT-PCR after treated with different concentrations of resveratrol.The results showed that the relative expression of I?B?,p65,P38,JNK,ERK1/2 and IRF3 phosphorylated proteins was significantly increased after LPS stimulation,and resveratrol could decrease the expression of the phosphorylated proteins and inhibit NF-?B/MAPKs/IRF3 signaling pathway.Therefore,resveratrol plays an anti-inflammatory effect mainly through inhibiting NF-?B,MAPKs pathway signaling.2.2 Effect of resveratrol on the upstream MYD88-dependent/independent signaling pathway in LPS-induced RAW264.7 cell inflammation: Western blot and RT-PCR were used to detect the protein expression of TLR4,the transcription of genes TLR4,MyD88 and TRAM which are in the TLR4-MYD88 dependent/independent pathway after treated with different concentrations of resveratrol.The results showed that resveratrol can down-regulate the expression of TLR4 protein and inhibit the transcription of TLR4,MyD88 and TRAM mRNA.Therefore,resveratrol can mediate inflammation through TLR4 signaling pathway,influence the MyD88 and TRAM gene mRNA transcription,inhibit the MyD88-dependent and TRIF-dependent signaling pathways,and finally inhibit the activation of downstream NF-?B and MAPKs,IRF3 pathway3 The verification and inhibitory effect on pathway of resveratrol on LPS-induced RAW264.7 cells inflammatory injury.Cells were treated with NF-?B pathway-specific inhibitor(BAY11-7082),P38 pathway-specific inhibitor(SB203580),JNK pathway-specific inhibitor(SP600125),and 25 ?mol/L resveratrol respectively.Then Western Blot technique was used to detect the expression of related proteins I?B?,p65,P38 and JNK.The results showed that resveratrol was similar to NF-?B and MAPKs signaling pathway inhibitors which can inhibit the phosphorylation of I?B?,p38 and JNK in RAW264.7 inflammatory cells.Therefore,resveratrol mainly inhibited the cytotoxicity of NF-?B and MAPKs signaling pathways.Resveratrol had anti-inflammatory effects similar to those inhibitors.4 Effects of resveratrol and specific inhibitors on the transcription and secretion of inflammatory factors in raw264.7 cells induced by lps.RT-PCR and ELISA were used to detect the expression and secretion of related inflammatory factors of IL-8,IL-6,TNF-?,IFN-? and IL-10 in RAW264.7 cells after treated with resveratrol and specific inhibitors BAY11-7082,SB203580 and SP600125.The results showed that resveratrol was similar to NF-?B and MAPKs signaling pathway inhibitors,which significantly down-regulated the expression of inflammatory factors TNF-?,IL-6,IL-8 and IFN-? mRNA,and up-regulated the anti-inflammatory factor IL-10 mRNA;resveratrol and NF-?B,MAPKs signaling pathway inhibitors can significantly inhibit the secretion of inflammatory factors TNF-?,IL-6,IL-8,IFN-? which was induced by LPS,and significantly improve the anti-inflammatory factor IL-10 release.Therefore,resveratrol and the specifiic inhibitors could regulate the release of the inflammatory factors TNF-?,IL-6,IL-8,IFN-? and IL-10 to exert an anti-inflammatory effect through significantly down-regulating TNF-?,IL-6,IL-8,and IFN-? gene transcription in RAW264.7 inflammatory cells and up-regulating the transcription of the anti-inflammatory factor IL-10 gene.In summary,resveratrol can inhibit the activation of TLR4 signaling pathway by affecting TLR4,MyD88,TRAM mRNA transcription and TLR4 protein translation.Further through TLR4-NF-?B /MAPKs/IRF3 Signal cascade,inhibiting the phosphorylation levels of related proteins I?B?,p65,P38,JNK,ERK1/2,IRF3 in the pathway.Ultimately resveratrol exerts a protective effect on inflammatory damage through regulating the release of inflammatory factors TNF-?,IL-6,IL-8,IFN-? and IL-10.