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目的 基于线粒体自噬探讨过表达视神经萎缩蛋白1(OPA1)抑制白细胞介素1β(IL-1β)诱导人软骨细胞损伤的作用机制。方法 取对数生长期人软骨细胞（C28/I2细胞），将其随机分为对照组、IL-1β组、IL-1β+空质粒腺病毒（Ad-null）组、IL-1β+OPA1腺病毒（Ad-OPA1）组。除对照组外，其他各组加入10 ng/mL的IL-1β诱导细胞损伤，24 h后取IL-1β+Ad-OPA1组、IL-1β+Ad-null组细胞，分别转染Ad-OPA1（过表达）、Ad-null，用实时荧光定量反转录聚合酶链式反应（RT-qPCR）验证转染效率。用Annexin V/碘化丙啶双染色法检测细胞凋亡率，用Western blotting法检测细胞凋亡相关蛋白[半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3)、B细胞淋巴瘤2(Bcl-2)、Bcl-2关联X蛋白（BAX）]、细胞外基质（ECM）降解相关蛋白[Ⅱ型胶原蛋白（ColⅡ）、聚集蛋白聚糖（AGC）、基质金属蛋白酶13(MMP-13)]表达，用比色法检测细胞中氧化应激指标[活性氧（ROS）、谷胱甘肽（GSH）、超氧化物歧化酶（SOD）]，分别用紫外-可见分光光度计、RT-qPCR测定细胞线粒体功能障碍指标[三磷酸腺苷（ATP）及过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)、线粒体融合蛋白1(MFN1)、动力蛋白相关蛋白1(Drp1)] mRNA表达，Western blotting法检测细胞中线粒体自噬相关蛋白[p62、自噬相关蛋白1(Beclin-1)、溶酶体相关膜蛋白1(LAMP1)、抑癌基因诱导激酶1(PINK1)、帕金森病蛋白2(Parkin)、泛素特异性蛋白酶30(USP30)]表达。结果 IL-1β+Ad-OPA1组OPA1 mRNA相对表达量高于IL-1β+Ad-null组（P均<0.05）。与对照组比较，IL-1β组、IL-1β+Ad-null组细胞凋亡率高，Caspase-3、BAX蛋白相对表达量高而Bcl-2蛋白相对表达量低（P均<0.05）,ColⅡ、AGC蛋白相对表达量低而MMP-13蛋白相对表达量高（P均<0.05）,ROS水平高而GSH、SOD水平低（P均<0.05）,ATP及PGC-1α、MFN1mRNA相对表达量低而Drp1 mRNA相对表达量高（P均<0.05）,p62、USP30蛋白相对表达量高而Beclin-1、LAMP1、PINK1、Parkin蛋白相对表达量低（P均<0.05）。与IL-1β+Ad-null组比较，IL-1β+Ad-OPA1组以上指标变化均逆转（P均<0.05）。结论 OPA1过表达可能通过激活线粒体自噬以减轻线粒体功能障碍和氧化应激，从而抑制IL-1β诱导的人软骨细胞凋亡及ECM降解。
Abstract：Objective To investigate the mechanism by which overexpression of optic atrophy 1(OPA1) inhibits interleukin-1β(IL-1β)-induced human chondrocyte injury, focusing on mitochondrial autophagy. Methods Human chondrocytes(C28/I2 cells) in the logarithmic growth phase were randomly divided into four groups: control, IL-1β, IL-1β +adenovirus empty vector(Ad-null), and IL-1β + OPA1 adenovirus(Ad-OPA1) groups. Except for the control group, all groups were treated with 10 ng/mL IL-1β to induce cellular injury. After 24 h, cells in the IL-1β + Ad-OPA1 and IL-1β +Ad-null groups were transfected with Ad-OPA1 and Ad-null, respectively. Real-time reverse transcription-quantitative PCR(RT-qPCR) was used to verify the transfection efficiency. Apoptosis rates were measured using Annexin V/propidium iodide(PI) double staining. Western blotting was performed to detect apoptosis-related proteins [cleaved caspase-3, B-cell lymphoma 2(Bcl-2), and Bcl-2-associated X protein(BAX)] and extracellular matrix(ECM) degradation-related proteins [type Ⅱ collagen(Col Ⅱ), aggrecan(AGC), and matrix metalloproteinase 13(MMP-13)]. Oxidative stress markers [reactive oxygen species(ROS), glutathione(GSH), and superoxide dismutase(SOD)] were quantified via colorimetric assays. Mitochondrial dysfunction indicators [adenosine triphosphate(ATP), peroxisome proliferator-activated receptor γ coactivator 1α(PGC-1α), mitofusin 1(MFN1), and dynamin-related protein 1(Drp1)] were analyzed using UV-Vis spectrophotometry(for ATP) and RT-qPCR(for mRNA expression). Additionally, Western blotting was utilized to assess the expression levels of mitochondrial autophagy-related proteins [p62, Beclin-1, lysosome-associated membrane protein 1(LAMP1), PTEN-induced kinase 1(PINK1), Parkin, and ubiquitin-specific protease 30(USP30)]. Results The relative expression level of OPA1 mRNA in the IL-1β+Ad-OPA1 group was higher than that in the IL-1β+Ad-null group(P<0. 05). Compared with the control group, the IL-1β and IL-1β + Ad-null groups exhibited significantly increased apoptosis rates, elevated levels of cleaved caspase-3 and BAX, reduced Bcl-2 expression(all P<0. 05), decreased Col Ⅱand AGC, increased MMP-13(P<0. 05), higher ROS levels, lower GSH and SOD activity(all P<0. 05), reduced ATP and PGC-1α/MFN1 mRNA expression, elevated Drp1 mRNA(all P<0. 05), up-regulated p62 and USP30, and down-regulated Beclin-1, LAMP1, PINK1, and Parkin(all P<0. 05). These effects were reversed in the IL-1β + Ad-OPA1 group compared to the IL-1β + Ad-null group(all P<0. 05). Conclusion Overexpression of OPA1 may alleviate mitochondrial dysfunction and oxidative stress by activating mitochondrial autophagy, thereby inhibiting IL-1β-induced chondrocyte apoptosis and ECM degradation.
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中图分类号:R684.3
引用信息:
[1]白星,高正超,张凯等.基于线粒体自噬探讨OPA1过表达抑制IL-1β诱导人软骨细胞损伤的作用机制[J].山东医药,2025,65(05):18-23.
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