HIF-1α通过炎性小体ASC结构域调控膝骨关节炎的分子机制研究

doi:10.3969/j.issn.1007-3205.2023.06.014

摘要/Abstract

摘要： 目的探究下调缺氧诱导因子1(hypoxia-inducible factor 1,HIF-1）对膝骨关节炎模型大鼠的干预效果及对炎性小体凋亡相关斑点样蛋白(apoptosis-related dot-like protein ASC ，ASC) 通路及信号通路磷酸化修饰作用机制。
方法将40只SPF级SD大鼠随机分为对照组（n=10）、模型组（n=10）、阳性对照（negative control，NC）组（n=10）和si-HIF-1α组（n=10）。模型组、NC组与si-HIF-1α组建立大鼠膝关节炎模型，NC组和si-HIF-1α组分别尾静脉注射5 nmol NC质粒和si-HIF-1α质粒，对照组和模型组给与等量生理盐水，HE染色对大鼠膝关节进行HE染色分析并进行病理评分，通过酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）检测大鼠关节组织白细胞介素1β（interleukin-1β，IL-1β），白细胞介素6（interleukin-6，IL-6），肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）水平，qPCR检测各组大鼠膝关节组织HIF-1α和血管紧张素转化酶（angiotensin converting enzyme，ACE）表达水平，Western blot检测大鼠膝关节组织炎性小体JUN和ASC蛋白表达水平以及信号通路磷酸化水平。
结果与对照组相比，模型组、NC组、si-HIF-1α组大鼠膝关节损伤及病理评分均显著高于对照组，si-HIF-1α组显著低于模型组和NC组，ELISA结果表明与对照组相比，模型组、NC组、si-HIF-1α组关节组织IL-1、IL-6、TNF-α显著升高（P＜0.01），si-HIF-1α组显著低于模型组和NC组（P＜0.01）；qPCR结果表明与对照组相比，模型组和NC组JUN和ASC显著上调，si-HIF-1α组显著下调（P＜0.01）。Western blot 结果表明与对照组相比，模型组和NC组JUN和ASC显著升高，而si-HIF-1α组显著降低，并且其信号通路磷酸化修饰水平显著降低（P＜0.01）。
结论下调HIF-1α可以抑制膝关节炎模型大鼠组织炎症因子释放，降低组织损伤，发挥骨保护作用，其机制可能与炎性小体ASC结构域的磷酸化修饰调控相关。

关键词: 骨关节炎, 膝, 缺氧诱导因子1, 炎性小体

Abstract: Objective To explore the intervention effect of down-regulation of HIF-1α on knee osteoarthritis (KOA) model rats and the mechanism of phosphorylation modification of inflammasome ASC pathway and signaling pathway.
Methods Forty SPF SD rats were randomly divided into control group (n=10), model group (n=10), negative control (NC) group (n=10) and si-HIF-1α group (n=10). The model group, NC group and si-HIF-1α group were used to establish a rat model of KOA. The NC group and the si-HIF-1α group were injected with 5 nmol of NC plasmid and si-HIF-1α plasmid respectively through the tail vein, and the control group and the model group were given the same amount of normal saline.The rat knee joints were analyzed by HE staining and pathological scoring. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α （TNF-α） in the joint tissues of the rats were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of HIF-1α and angiotensin I-converting enzyme (ACE) in knee joint tissue of rats, and the protein expression levels of JUN and ASC and the phosphorylation level of signaling pathway in inflammasome of knee joint tissue of rats were detected by Western blot.
Results Compared with the control group, the knee joint injury and pathological scores in the model group, NC group and si-HIF-1α group were significantly higher than those in the control group, which were lower in the si-HIF-1α group than in the model group and NC group. ELISA results showed that, compared with the control group, IL-1, IL-6, IL-8 and TNF-α in joint tissues of model group, NC group and si-HIF-1α group were significantly increased (P＜0.01), which were significantly lower in the si-HIF-1 group than in the model group and NC group (P＜0.01). qPCR results showed that compared with the control group, JUN and ASC were significantly up-regulated in the model group and NC group, and significantly down-regulated in the si-HIF-1α group (P＜0.01). Western blot results showed that compared with the control group, JUN and ASC in the model group and NC group were significantly increased, while those in the si-HIF-1α group were significantly decreased, and the phosphorylation level of their signaling pathways was significantly decreased (P＜0.01).
Conclusion Down-regulation of HIF-1α can inhibit the release of inflammatory factors in KOA model rats, reduce tissue damage, and exert bone protective effect. The mechanism may be related to the regulation of phosphorylation modification of the ASC domain of the inflammasome.

Key words: osteoarthritis, knee, hypoxia-inducible factor 1, inflammasome

杨学钰, 郭文帆, 郑雪君, 卢吉高, 杨勇, 李会东. HIF-1α通过炎性小体ASC结构域调控膝骨关节炎的分子机制研究 [J]. 河北医科大学学报, 2023, 44(6): 692-696,706.

YANG Xue-yu, GUO Wen-fan, ZHENG Xue-jun, LU Ji-gao, YANG Yong, LI Hui-dong. Molecular mechanism of HIF-1α regulating knee osteoarthritis through inflammasome ASC domain[J]. Journal of Hebei Medical University, 2023, 44(6): 692-696,706.

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HIF-1α通过炎性小体ASC结构域调控膝骨关节炎的分子机制研究

Molecular mechanism of HIF-1α regulating knee osteoarthritis through inflammasome ASC domain

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