白星 1高正超 1张凯 1康旭鹏 1段大鹏 1段亮1
作者信息
- 1. 陕西省人民医院骨科,陕西 西安 710068
- 折叠
摘要
Abstract
Objective To investigate the mechanism by which overexpression of optic atrophy 1(OPA1)inhibits in-terleukin-1β(IL-1β)-induced human chondrocyte injury,focusing on mitochondrial autophagy.Methods Human chon-drocytes(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/propidi-um 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 colo-rimetric assays.Mitochondrial dysfunction indicators[adenosine triphosphate(ATP),peroxisome proliferator-activated re-ceptor 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 pro-tein 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-reg-ulated 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.关键词
骨关节炎/视神经萎缩蛋白1/线粒体自噬/白细胞介素1β/人软骨细胞/细胞凋亡/细胞外基质/作用机制Key words
osteoarthritis/optic atrophy 1/mitochondrial autophagy/interleukin-1β/human chondrocytes/apoptosis/extracellular matrix/mechanism of action分类
医药卫生
