miR-224-3p调控JAK/STAT信号通路对LPS诱导的椎间盘退变髓核细胞增殖、凋亡和炎症反应的影响OA
Effects of miR-224-3p regulating JAK/STAT signaling pathway on proliferation,apoptosis,and inflammatory response of LPS-induced nucleus pulposus cells in intervertebral disc degeneration
目的 探讨miR-224-3p调控Janus激酶/信号转导和转录激活因子(JAK/STAT)信号通路对椎间盘髓核细胞增殖、凋亡和炎症反应的影响.方法 将髓核细胞分为对照(Control)组、模型(Model,以200 μg/mL脂多糖干预)组、miR-224-3p阴性对照(miR-NC)组、miR-224-3p模拟物(miR-224-3p mimics)组、miR-224-3p mimics+JAK1过表达空载体(miR-224-3p mimics+pc-DNA-NC)组、miR-224-3p mimics+JAK1过表达质粒(miR-224-3p mimics+JAK1)组.采用实时定量PCR检测细胞miR-224-3p、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、可聚蛋白多糖(aggrecan)、胶原蛋白Ⅱ(CollagenⅡ)、JAK1、STAT1 mRNA表达水平;采用CCK-8法检测细胞增殖情况;采用流式细胞术法检测细胞凋亡情况;采用ELISA试剂盒法检测炎性细胞因子水平;采用Western blotting检测JAK1、STAT3、BCL2 相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)、胱天蛋白酶-3(caspase-3)、aggrecan和CollagenⅡ蛋白表达;双萤光素酶报告基因实验验证miR-224-3p与JAK1之间的靶向关系.结果 与Control组相比,Model组髓核细胞miR-224-3p表达下调,JAK1和STAT1表达上调(P<0.05);过表达miR-224-3p可提高髓核细胞活性、aggrecan和Collagen Ⅱ表达水平、Bcl-2蛋白表达量,降低细胞凋亡率、TNF-α、IL-6和IL-8水平及p-JAK1/JAK1、p-STAT1/STAT1、caspase-3和Bax蛋白表达量(P<0.05);同时过表达miR-224-3p和JAK1可降低髓核细胞活性、aggrecan和CollagenⅡ表达水平、Bcl 2蛋白表达量,提高细胞凋亡率、TNF-α、IL-6和IL-8水平及p-JAK1/JAK1、p-STAT1/STAT1、caspase-3和Bax蛋白表达量(P<0.05).结论 椎间盘退变髓核细胞中miR-224-3p的表达水平显著下调,上调miR-224-3p可通过靶向抑制JAK/STAT信号通路,提高脂多糖诱导的椎间盘髓核细胞活性,抑制细胞凋亡、炎症反应和细胞外基质降解.
Objective To investigate the effects of miR-224-3p regulation of the Janus kinase/signal transducer and activator of tran-scription(JAK/STAT)signaling pathway on the proliferation,apoptosis,and inflammatory response of nucleus pulposus cells in interverte-bral discs.Methods Nucleus pulposus cells were divided into the following groups:Control,Model(intervened with 200 μg/mL lipopoly-saccharide),miR-224-3p negative control(miR-NC),miR-224-3p mimics,miR-224-3p mimics+JAK1 overexpression empty vector(miR-224-3p mimics+pcDNA-NC),and miR-224-3p mimics+JAK1 overexpression plasmid(miR-224-3p mimics+JAK1).miR-224-3p,tumor necrosis factor α(TNF-α),interleukin-6(IL-6),aggrecan,CollagenⅡ,JAK1,and STAT1 mRNA expression levels in cells were determined using real-time quantitative PCR.Cell proliferation was assessed using the Cell Counting Kit-8 assay,and apoptosis was mea-sured using flow cytometry.Inflammatory cytokine levels were determined using enzyme-linked immunosorbent assay kits.JAK1,STAT3,BCL2-associated X protein(Bax),B-cell lymphoma-2(Bcl-2),caspase-3,aggrecan,and CollagenⅡ were analyzed using Western blotting.The targeting relationship between miR-224-3p and JAK1 was verified using a dual-luciferase reporter assay.Results Compared with the Control group,the Model group showed significantly downregulated miR-224-3p expression and upregulated JAK1 and STAT1 expressions in nucleus pulposus cells(P<0.05).miR-224-3p overexpression significantly enhanced cell viability,increased aggrecan,CollagenⅡ,and Bcl-2 protein expression,whereas it reduced the apoptosis rate;TNF-α,IL-6,and IL-8 levels;and the p-JAK1/JAK1 and p-STAT1/STAT1 ratios,as well as caspase-3 and Bax protein expressions(P<0.05).Simultaneous miR-224-3p and JAK1 overexpression reduced cell viability,aggrecan and CollagenⅡ,and Bcl-2 protein expressions,while increasing the apoptosis rate;TNF-α,IL-6,and IL-8 levels,and p-JAK1/JAK1,p-STAT1/STAT1,caspase-3,and Bax protein expressions(P<0.05).Conclusion miR-224-3p expression is signi-ficantly downregulated in nucleus pulposus cells of degenerative intervertebral discs.Notably,miR-224-3p upregulation can enhance the viability of lipopolysaccharide-induced nucleus pulposus cells and inhibit cell apoptosis,inflammatory response,and extracellular matrix degradation by targeting the JAK/STAT signaling pathway.
熊和然;赵欣;王松;向超;万骐;程思玲;何生华
武汉市中医医院推拿科,武汉 430000武汉市中医医院推拿科,武汉 430000武汉市中医医院推拿科,武汉 430000武汉市中医医院推拿科,武汉 430000武汉市中医医院推拿科,武汉 430000武汉市中医医院推拿科,武汉 430000武汉市中医医院推拿科,武汉 430000
医药卫生
miR-224-3pJanus激酶/信号转导和转录激活因子椎间盘髓核细胞增殖凋亡炎症反应
miR-224-3pJanus kinase/signal transducer and activator of transcription pathwayintervertebral disc nucleus pulposus cellsproliferationapoptosisinflammatory response
《中国医科大学学报》 2026 (3)
265-271,7
湖北省中医药管理局中医药科研项目(ZY2023M026)
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