脂肪量和肥胖相关基因对骨关节炎软骨细胞凋亡和炎症反应的影响

Effects of the fat mass and obesity-associated gene on apoptosis and the inflammatory response of chondrocytes in osteoarthritis

目的探究脂肪量和肥胖相关基因(FTO)对骨关节炎(OA)软骨细胞凋亡和炎症反应的影响。方法分析人正常软骨组织样本与OA软骨组织样本中FTO的表达差异,分离并培养原代OA软骨细胞,并构建OA大鼠模型,从临床、动物和细胞水平检测FTO表达。利用FTO敲低慢病毒(sh-FTO)和m^(6)A甲基化抑制剂(cycloleucine)处理细胞,检测细胞中m^(6)A含量和炎症因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的表达水平;流式细胞术检测OA软骨细胞凋亡情况,蛋白质免疫印迹(Western blot)检测凋亡蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达水平。结果与正常对照比较,人OA软骨组织、OA大鼠软骨组织及OA软骨细胞中FTO的mRNA和蛋白表达均显著上升(均P<0.05)。敲低FTO后,原代OA软骨细胞中m^(6)A水平上升,IL-6、TNF-α水平显著降低,细胞凋亡率降低,凋亡蛋白Bax表达显著下调,Bcl-2表达显著上调;而cycloleucine干预能显著降低OA软骨细胞中m^(6)A水平,增加IL-6、TNF-α的丰度,促进细胞凋亡和凋亡相关蛋白表达,逆转FTO敲低慢病毒产生的干预效果(均P<0.05)。结论FTO可能介导m^(6)A机制促进OA软骨细胞凋亡和炎症反应,进而加速OA的进展。

Objective To explore the effects of the fat mass and obesity-associated gene(FTO)on apoptosis and the inflammatory response of chondrocytes in osteoarthritis(OA).MethodsDifferences in FTO expression between normal human cartilage tissue samples and OA cartilage tissue samples were examined.Primary OA chondrocytes were isolated and cultured,and a rat OA model was constructed.The expression of FTO was detected in clinical,animal and cellular samples.Cells were treated with an FTO knockdown lentivirus vector(sh-FTO)and an m^(6)A methylation inhibitor(cycloleucine).The amount of m^(6)A and the expression levels of inflammatory cytokines,interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α),were detected.Flow cytometry was used to detect apoptosis in OA chondrocytes,and Western blot was used to detect the expression levels of B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax).ResultsCompared with the normal control group,FTO mRNA and protein expression in human OA cartilage tissue,rat OA cartilage tissue and OA chondrocytes was significantly increased(all P<0.05).After FTO knockdown,the level of m^(6)A increased,the levels of IL-6 and TNF-αdecreased considerably,the apoptosis rate decreased,the expression of the proapoptotic protein Bax decreased considerably,and the expression of Bcl-2 increased considerably in primary OA chondrocytes.However,cycloleucine intervention clearly reduced the level of m6A,increased the levels of IL-6 and TNF-α,promoted cell apoptosis and the expression of apoptosis-related proteins,and reversed the effect induced by the FTO knockdown lentivirus in OA chondrocytes(all P<0.05).ConclusionsFTO may be involved in mechanisms related to the action of m^(6)A to promote OA chondrocyte apoptosis and the inflammatory response,thus accelerating the progression of OA.