沉默HMGB1通过抑制p38 MAPK信号通路减少LPS或IL-17A诱导的中耳腔上皮细胞的炎症与凋亡

doi:10.3969/j.issn.1006-6233.2024.05.05

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摘要 目的: 探讨高迁移率族蛋白B1(High Mobility Group Protein 1,HMGB1)对脂多糖(lipopolysaccharide,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。

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	阿不拉江·托合提
	阿布利克木·依
	吾买尔·亚生
	韩志国

关键词 ：高迁移率族蛋白B1, p38-丝裂原激活的蛋白激酶, 人中耳腔上皮细胞, 细胞凋亡

Abstract：Objective: To investigate the regulatory effects and mechanisms of high-mobility group protein 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 lymphoma 2 protein (Bcl-2), and Bcl-2 associated X protein (Bax). Flow cytometry was used to detect cell apoptosis. 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 increased (all P<0.05). Conclusion: Silent HMGB1 alleviates LPS or IL-17A-induced inflammation and apoptosis of HMEECs via inhibiting p38 MAPK signaling pathway.

Key words： High mobility group protein 1 p38 mitogen-activated protein kinase Human middle ear epithelial cells Cell apoptosis

基金资助:新疆维吾尔自治区自然科学基金项目,(编号:2022D01C135);新疆维吾尔自治区人民医院院内项目,(编号:20200116)

通讯作者: 韩志国

引用本文:

阿不拉江·托合提, 阿布利克木·依, 吾买尔·亚生, 韩志国. 沉默HMGB1通过抑制p38 MAPK信号通路减少LPS或IL-17A诱导的中耳腔上皮细胞的炎症与凋亡[J]. 河北医学, 2024, 30(5): 724-730.
ABULAJIANG Tuoheti, ABULIKEMU Yi, UUMAIER Yasheng, et al. Silent HMGB1 Alleviates LPS or IL-17A Induced Inflammation and Apoptosis of Human Middle Ear Epithelial Cells via Inhibiting p38 MAPK Signaling Pathway. HeBei Med, 2024, 30(5): 724-730.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2024.05.05 或 http://www.hbyxzzs.cn/CN/Y2024/V30/I5/724

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