To Explore The Mechanism Of Resveratrol Inhibiting Macrophage Polarization To M1 Based On TLR4/NF-?B/HIF-1? Pathway

ObjectiveA RAW264.7-derived macrophage M1 phenotypic polarization model was established to evaluate the effect and mechanism of resveratrol?Res?on inhibiting macrophages from polarizing to M1,and to provide experimental evidence for clinical research.Method1.Establish LPS+IFN-?induced RAW264.7-derived macrophages to M1phenotypic polarization model,and evaluate the efficacy of ResLPS+IFN-?was used to induce RAW264.7-derived macrophages to polarize into M1phenotype macrophage model,and the morphology of the control group and model group was observed;flow cytometry was used to detect the control group and model group The expression of M1 macrophage phenotype marker CD11c+was identified by model.Res underwent drug intervention under the premise of successful modeling.CCK8 and LDH efflux experiments to evaluate drug dose toxicity;morphological observation of the cell morphology of each experimental group;flow cytometry to detect the expression of M11 macrophage phenotypic marker CD11c+;RT-q PCR to detect the pro-inflammatory secretion of M1 phenotype macrophages Sex factor?IL-1?,TNF-?,IL-6?m RNA expression;ELISA detection of pro-inflammatory factors IL-1?,TNF-?,IL-6 secreted by M1 phenotype macrophages in each group of cell supernatants The NO kit was used to detect the content of NO secreted by M1phenotype macrophages in the cell supernatant of each group.2.Mechanism analysis and verificationUsing RT-q PCR and Western Blot to detect the expression mechanism of m RNA and protein expression of related factors on the TLR4/NF-?B/HIF-1?pathway;add HIF-1?agonist?Co Cl₂?,repeat the test of the index in the first experiment,comparative analysis Difference between Res group and Res+Co Cl₂group.It is clear that Res inhibits the polarization of macrophages towards the M1 phenotype and is related to HIF-1?.Result1 Evaluation of Res on LPS+IFN-?-induced polarization of RAW264.7-derived macrophages to M1 phenotype?1?Observe the morphological changes of RAW264.7-derived macrophages under a light microscope:unactivated macrophages are small and round.Compared with the control group,most cells in the model group are still round,but the volume is increased.Fullness,increased cytoplasm,consistent with the characteristics of M1phenotype macrophages,indicating that the modeling method used in this experiment is feasible;the Res intervention group has a larger number of small and round macrophages than the model group;?2?The expression level of M1 macrophage phenotype marker CD11c+in the model group was significantly increased compared with the control group?P<0.01?,indicating that the modeling method selected in this experiment was feasible;after intervention with different concentrations of Res,the M1 macrophage phenotype Compared with the model group,the expression of the marker CD11c+decreased to varying degrees,showing a certain concentration dependence of the drug,and the high concentration group Res had the best effect?P<0.01?;?3?The levels of IL-1?,IL-6,and TNF-?in the cell supernatant of the model group were significantly increased compared with the control group?P<0.01?;after intervention with different concentrations of Res,the levels of IL-1?and IL-6 in the cell supernatant were significantly increased.The levels of TNF-?were down-regulated compared with the model group,showing a drug concentration-dependent trend,and the effect was highest in the high concentration Res group?P<0.01?;?4?The m RNA expression levels of IL-1?,IL-6,and TNF-?in the model group were significantly up-regulated compared with the control group?P<0.01?;after intervention with different concentrations of Res,the m RNA expressions of IL-1?,IL-6,and TNF-?were significantly increased.Compared with the model group,the doses were all down-regulated,and the differences between the groups were statistically significant?P<0.05?,showing a drug concentration dependence.The effect was best in the high concentration Res group?P<0.01?;?5?The content of NO in the cell supernatant of the model group was significantly increased compared with the control group?P<0.01?;after intervention with different concentrations of Res,the content of NO in the cell supernatant was decreased compared with the model group,showing a drug concentration-dependent trend.The concentration group had the best effect?P<0.01?.2 Explore the mechanism of Res on LPS+IFN-?-induced phenotype polarization of RAW264.7-derived macrophages based on TLR4/NF-?B/HIF-1?pathway?1?The expression levels of TLR4 and HIF-1?m RNA in the model group were significantly increased compared with the control group?P<0.01?.After intervention with different concentrations of Res,the expression levels of TLR4 and HIF-1?m RNA were all down-regulated compared with the model group.The differences between groups were statistically significant.The significance was statistically significant?P<0.01?,showing drug concentration dependence,and the effect was highest in the high concentration Res group?P<0.01?.The expression levels of TLR4,p-NF-?B p65,and HIF-1?protein in the model group were significantly increased compared with the control group?P<0.01?.After intervention with different concentrations of Res,the expressions of TLR4,p-NF-?B p65,and HIF-1?protein were significantly increased.The level was lower than that of the model group.The effect was highest in the high concentration Res group?P<0.01?.Combining RT-q PCR and Western Blot results showed that Res could effectively down-regulate m RNA and protein expression in the TLR4/NF-?B/HIF-1?pathway.The effect was best in the high-Res group.?2?It is clear that Res inhibits the polarization of RAW264.7-derived macrophages to the M1 phenotype and is indeed related to HIF-1?:Combined with flow cytometry to detect the expression of M1 macrophage phenotype marker CD11c+;RT-q PCR was used to detect the expression of pro-inflammatory factors?IL-1?,TNF-?,IL-6?;ELISA was used to detect the contents of IL-1?,TNF-?,and IL-6 in the cell supernatant;The content of NO in the cell supernatant of the group,the experimental results show that Res can effectively inhibit the macrophages from polarizing to the M1 phenotype,and Co Cl₂can promote the macrophages from polarizing to the M1phenotype.Comparing the Res+Co Cl₂group with the Res intervention group In the presence of Co Cl₂,the effect of Res on inhibiting the macrophage polarization to M1phenotype was offset.It is suggested that Res inhibits the macrophage polarization to M1 phenotype and it is related to HIF-1?.Conclusion1.Res can inhibit macrophages from polarizing to M1 phenotype,and it shows a drug concentration-dependent trend.The effect of Res high concentration group is the most significant.2.Res can down-regulate the expression of m RNA and protein on the TLR4/NF-?B/HIF-1?pathway.After intervention with HIF-1?agonists,the efficacy is significantly inhibited,suggesting that Res inhibits RAW264.7-derived macrophages to M1Phenotypic polarization is related to the TLR4/NF-?B/HIF-1?pathway.