摘要
目的:探讨高迁移率族蛋白B1(High Mobility Group Protein 1,HMGB1)对脂多糖(lipopo-lysaccharide,LPS)或白细胞介素-17A(interleukin-17A,IL-17A)诱导人中耳腔上皮细胞(human middle ear epithelial cells,HMEEC)的炎症反应与凋亡的调控作用与机制.方法:培养 HMEEC 细胞系.将HMEEC分为对照组、LPS组、LPS联合短发夹RNA(shRNA)沉默质粒阴性对照处理组(LPS+shNC 组)、LPS联合shRNA沉默HMGB1 处理组(LPS+shHMGB1 组)、LPS 联合 p38-丝裂原激活的蛋白激酶(p38 MAPK)抑制剂SB202190 处理组(LPS+SB202190 组)、IL-17A 组、IL-17A+shNC 组、IL-17A+shHMGB1组、IL-17A+SB202190 组.酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测TNF-α、IL-1β和IL-6 的水平变化.用Western blot检测粘蛋白 5AC(mucoprotein 5AC,MUC5AC)、粘蛋白 8(mucoprotein 8,MUC8)、p38 MAPK、磷酸化的 p38 MAPK(p-p38 MAPK)、E26 样蛋白 1(E-twenty-six like 1 protein,ELK1)、B细胞淋巴瘤2 蛋白(B-cell lymphoma 2 protein,Bcl-2),以及Bcl-2 相关X蛋白(Bcl-2 associated X protein,Bax)的表达.流式细胞术检测细胞的凋亡.结果:与对照组比,LPS组或IL-17A组的 HMEEC 的凋亡率都增加,且 HMGB1、MUC5AC、MUC8、TNF-α、IL-1β、IL-6、p38、p-p38、ELK1、Bax的表达水平升高,而 Bcl-2 的表达水平降低(均 P<0.05).与 LPS 组或 IL-17A 组比,LPS+shHMGB1 组或IL-17A+shHMGB1 组的凋亡率都降低,且 HMGB1、MUC5AC、MUC8、TNF-α、IL-1β、IL-6、p38、p-p38、ELK1、Bax的表达水平都减少,而Bcl-2 的表达水平升高(均P<0.05).与LPS 组或IL-17A组比,LPS+SB202190 组或 IL-17A+SB202190 组的的凋亡率都降低,MUC5AC、MUC8、TNF-α、IL-1β、IL-6、p38、p-p38、ELK1、Bax的表达水平都降低,而 Bcl-2 的表达水平升高(均 P<0.05)(均 P<0.05).结论:沉默HMGB1 通过抑制p38 MAPK信号通路减少 LPS 或 IL-17A 诱导的 HMEEC 的炎症与凋亡.
Abstract
Objective:To investigate the regulatory effects and mechanisms of high-mobility group pro-tein B1(HMGB1)on the inflammatory response and apoptosis of human middle ear epithelial cells(HMEECs)induced by lipopolysaccharide(LPS)or interleukin-17A(IL-17A).Methods:HMEEC cell line was cultured.HMEECs were divided into the control group,LPS group,LPS+shRNA negative control group(LPS+shNC group),LPS+shRNA-mediated HMGB1 knockdown group(LPS+shHMGB1 group),LPS+p38 mitogen-activated protein kinase(p38 MAPK)inhibitor SB202190 group(LPS+SB202190 group),IL-17A group,IL-17A+shNC group,IL-17A+shHMGB1 group,and IL-17A+SB202190 group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the changes in the levels of TNF-α,IL-1β and IL-6.Western blot was used to detect the expression of mucin 5AC(MUC5AC),mucin 8(MUC8),p38 MAPK,phosphorylated p38 MAPK(p-p38 MAPK),E-twenty-six like 1 protein(ELK1),B-cell lympho-ma 2 protein(Bcl-2),and Bcl-2 associated X protein(Bax).Flow cytometry was used to detect cell apop-tosis.Results:Compared with the control group,the apoptosis rate of HMEECs in the LPS group or IL-17A group was increased,and the expression levels of HMGB1,MUC5AC,MUC8,TNF-α,IL-1β,IL-6,p38,p-p38,ELK1,and Bax were increased,while the expression level of Bcl-2 was decreased(all P<0.05).Compared with the LPS group or IL-17A group,the apoptosis rate was decreased in the LPS+shHMGB1 group or IL-17A+shHMGB1 group,and the expression levels of HMGB1,MUC5AC,MUC8,TNF-α,IL-1β,IL-6,p38,p-p38,ELK1,and Bax were all decreased,while the expression level of Bcl-2 was increased(all P<0.05).Compared with the LPS group or IL-17A group,the apoptosis rate was decreased in the LPS+SB202190 group or IL-17A+SB202190 group,and the expression levels of MUC5AC,MUC8,TNF-α,IL-1β,IL-6,p38,p-p38,ELK1,and Bax were all decreased,while the expression level of Bcl-2 was in-creased(all P<0.05).Conclusion:Silent HMGB1 alleviates LPS or IL-17A-induced inflammation and ap-optosis of HMEECs via inhibiting p38 MAPK signaling pathway.
基金项目
新疆维吾尔自治区自然科学基金(2022D01C135)
新疆维吾尔自治区人民医院院内项目(20200116)
维普
万方数据