Study On The Anti-inflammatory Effect And Mechanism Of BLA On LPS-induced Macrophages

Objective:To investigate the inhibitory effect of BLA on the inflammatory response of LPS-induced RAW264.7 cells;to analyze the potential molecular mechanism of the anti-inflammatory effect of BLA on the LPS-induced RAW264.7 cells inflammation model.Methods: RAW264.7 cells were treated with different concentrations(0,25,50,100,150,200,400,600,800,1000,1500 ?g/mL)of BLA for 24 h,Cell viability was measured using CCK-8 assay for screening the safe cellular dose of BLA.ELISA: RAW264.7 cells were randomLy divided into normal control group(NC group),LPS group and BLA + LPS group.NC group: cells without any other treatment;LPS group: cells were stimulated with LPS of 500 ng/mL for 12 h on the basis of normal culture;BLA + LPS group: add the safe range concentration of BLA,and then add 500 ng / mL LPS to stimulate cells.After reaching a certain drug action time(0.5,1,2,3,4,5,6,8,12,16,18,20,24,30,36 and 48 h,respectively),stop culturing and collect the supernatant.After 48 h,the NC group and LPS group stopped culturing and collected the supernatant.The contents of inflammatory factors IL-6,IL-1?,TNF-? and macrophage chemotactic protein-1(MCP-1)in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Western blot:RAW264.7 cells were randomLy divided into normal control group(NC group),LPS group and BLA + LPS groups.The treatment of NC group and LPS group is the same as above.BLA + LPS groups: add the safe range concentration of BLA and then add 500ng/mL LPS to stimulate cells.After reaching a certain drug action time(0.5?1?2?3?4?5?6?8?12?16?18?20?24?30?36?48hrespectively),stop culturing and extract protein.NC group and LPS group stop culturing and extract protein after 24 h.Western blot method was used to detect the expression level of iNOS,COX2 and TLR4,effect of NF-?B,MAPK signal pathway activation.Results:1.The results of CCK-8 showed that the IC50 of BLA was 197.4?g/mL.Therefore,the concentration of BLA was 150?g/mL in the subsequent experiments.2.The ELISA results showed that compared with the NC group,the levels of TNF-?,IL-1?,IL-6 and MCP-1 in RAW264.7 cells were significantly increased in the LPS group(P<0.05);Compared with the LPS group,BLA can effectively reduce the levels of inflammatory factors TNF-? and IL-1? in RAW264.7 cells at all time points(P<0.05),among them,the inhibitory effect of BLA on TNF-? was most obvious at 30h(P<0.05);Compared with the LPS group,BLA can effectively reduce the levels of inflammatory factors MCP-1 and IL-6 in RAW264.7 cells at the most time points(P<0.05).3.The WB results showed that compared with the NC group,the expression levels of iNOS,COX2,TLR4,p-p65,p-p38,p-ERKand p-JNK in RAW264.7 cells were significantly increasedwere significantly increased in the LPS group(P<0.05).Compared with the LPS group,the expression levels of iNOS,COX2,TLR4,p-p38 and p-JNK were significantly reduced in the RAW264.7 cells at 2h,6h,12 h,18h,and 24hgroups(P<0.05);The expression of p-p65 was significantly reduced in RAW264.7 cells at 6h,12 h 18h and 24 h groups(P<0.05);The expression of p-ERK was significantly decreased in RAW264.7 cells at 12 h,18h and 24 h groups(P<0.05).Among them,compared with other groups,the inhibitory effect was most obvious at 24 hgroups(P<0.05).Conclusion: 1.BLA can effectively inhibit the inflammatory response of RAW264.7 cells induced by LPS.2.BLA can effectively inhibit the expression of inflammatory factors TNF-?,IL-1?,IL-6,MCP-1 and inflammatory proteins iNOS and COX2.3.BLA may play an anti-inflammatory role by inhibiting TLR4 and activating NF-?B and MAPK signals.