抗炎合剂通过TLR4/MyD88/NF-κB通路抑制脂多糖诱导的过表达高迁移率蛋白-1转染小鼠巨噬细胞系RAW264.7细胞炎症反应的机制研究  

Mechanism research of Kangyan mixture through TLR4/MyD88/NF-κB signaling pathway on inhibiting inflammatory response of lipopolysaccharide-induced mouse macrophage cell line RAW264.7 with overexpression of high mobility group box 1 protein transfection

作　　者：闫国良[1] 李淑芳[2] 王馨璐[1] 孙雪 汪海慧[1] YAN Guoliang;LI Shufang;WANG Xinlu(Emergency Department,Shanghai Traditional Chinese Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200070;不详)

机构地区：[1]上海中医药大学附属市中医医院急诊科,上海200070 [2]上海中医药大学附属曙光医院感染ICU科,上海200021

出　　处：《河北中医》2020年第9期1362-1368,F0003,共8页Hebei Journal of Traditional Chinese Medicine

基　　金：上海中医药大学预算内项目(编号:2016YSN55)。

摘　　要：目的研究抗炎合剂对脂多糖(LPS)诱导的过表达高迁移率蛋白-1(HMGB1)慢病毒转染的小鼠巨噬细胞系RAW264. 7的炎症反应中Toll样受体4/髓样分化因子/核转录因子-κB(TLR4/MyD88/NF-κB)信号通路的调控作用。方法对10只SD雄性大鼠分别予抗炎合剂9. 9 g/kg灌胃7 d后,腹主动脉采血制备含药血清。将RAW264. 7细胞分为空白对照组、LPS组、LPS+中药血清组,将过表达HMGB1慢病毒转染的RAW264. 7细胞分为LPS+HMGB1组、LPS+HMGB1+中药血清组,将HMGB1 RNAi慢病毒转染的RAW264. 7细胞分为LPS+HMGB1 RNAi组、LPS+HMGB1 RNAi+中药血清组,以上7组分别进行相应的干预处理。观察干预前后RAW264. 7细胞、过表达HMGB1慢病毒转染的RAW264. 7细胞及HMGB1 RNAi慢病毒转染的RAW264. 7细胞的形态学变化。酶联免疫吸附(ELISA)法检测各组细胞上清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素10(IL-10)水平,实时荧光定量聚合酶链式反应(PCR)法检测各组细胞TRL4 mRNA、MyD88 mRNA、NF-κB mRNA表达量。结果干预后镜下可见,各不同干预组之间细胞分化区别较为明显,空白对照组细胞无分化,LPS组、LPS+HMGB1组细胞分化程度较高,LPS+中药血清组、LPS+HMGB1 RNAi组、LPS+HMGB1+中药血清组、LPS+HMGB1 RNAi+中药血清组细胞分化程度略低。与空白对照组比较,LPS组、LPS+HMGB1组、LPS+HMGB1+中药血清组中IL-6、IL-10、TNF-α水平及TLR4 mRNA、MyD88 mRNA、NF-κB mRNA表达量均明显升高(P <0. 05)。与LPS组比较,LPS+HMGB1组IL-6、IL-10、TNF-α水平及TLR4 mRNA、MyD88 mRNA、NF-κB mRNA表达量均升高(P <0. 05);LPS+HMGB1 RNAi组IL-6、IL-10、TNF-α水平及TLR4 mRNA、MyD88 mRNA、NF-κB mRNA表达量均降低(P <0. 05);LPS+中药血清组IL-6、TNF-α水平及TLR4 mRNA、MyD88 mRNA、NF-κB mRNA表达量均明显降低(P <0. 05),IL-10水平明显升高(P <0. 05)。与LPS+HMGB1组比较,LPS+HMGB1+中药血清组中IL-6、TNF-α水平及TLR4 mRNA、MyD88 mRNA、NF-κB mRNA表达�Objective To study the regulating effect of Kangyan mixture on the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB(TLR4/MyD88/NF-κB)signaling pathway in the inflammatory response of lipopolysaccharide-induced(LPS-induced)mouse macrophage cell line RAW264.7 with overexpression of high mobility group box 1 protein(HMGB1)lentiviral transfection.Methods Ten SD male rats were administered with Kangyan mixture for gavage for 7 days at a dosage of 9.9 g/kg.And the drug serum was prepared by using the abdominal aortic blood.RAW264.7 cells were assigned into the control group,the LPS group,and the LPS+drug serum group.RAW264.7 cells with overexpression of HMGB1 lentiviral transfection were divided into the LPS+HMGB1 group and the LPS+HMGB1+drug serum group.RAW264.7 cells with HMGB1 RNAi lentiviral transfection were divided into the LPS+HMGB1 RNAi group and the LPS+HMGB1 RNAi+drug serum group.And the corresponding intervention was performed for the above 7 groups.The morphological changes of RAW264.7 cells,cells with overexpression of HMGB1 lentiviral transfection,and cells with HMGB1 RNAi lentiviral transfection were observed.The enzyme linked immunosorbent assay(ELISA)was applied to test the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-10(IL-10)of cells in each group.And the expression indexes of TLR4 mRNA,MyD88 mRNA,and NF-κB mRNA of cells in each group was tested by the fluorescence-based real-time quantitative polymerase chain reaction.Results By the microscopical observation after the intervention,there was more obvious cell differentiation for cells in the groups with intervention,while no cell differentiation for the control group.Meanwhile,there was the well differentiation for cells in the LPS group and the LPS+HMGB1 group,while the poor differentiation in the LPS+drug serum group,the LPS+HMGB1 RNAi group,the LPS+HMGB1+drug serum group,and the LPS+HMGB1 RNAi+drug serum group.In the comparison of the results in the control group,the levels of IL-6,IL-10

关 键 词：脓毒症 RAW264.7细胞 炎症因子 抗炎合剂 体外研究 

分 类 号：R631.2[医药卫生—外科学] R9-33[医药卫生—临床医学]