多不饱和脂肪酸对骨关节病软骨细胞自噬的影响及机制研究  

Effect of polyunsaturated fatty acid on autography of human osteoarthritis chondrocytes and its mechanism

作　　者：马立峰[1] 郭艾[1] 王国栋[2] 于浩淼[1] 张良[1] 尹合勇 曹瑞祺 MA Li-feng;GUO Ai;WANG Guo-dong(Department of Orthopaedics,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;Department of Orthopaedics,Beijing Tongren Hospital,Capital Medical University,Beijing 100176,China)

机构地区：[1]首都医科大学附属北京友谊医院骨科,北京100050 [2]首都医科大学附属北京同仁医院骨科,北京100176

出　　处：《临床和实验医学杂志》2020年第10期1062-1066,共5页Journal of Clinical and Experimental Medicine

基　　金：北京市自然科学基金(编号:7192045)。

摘　　要：目的探讨多不饱和脂肪酸对人骨关节病(OA)软骨细胞自噬的影响及其促进自噬的作用机制。方法纳入2019年5在首都医科大学附属北京友谊医院骨科接受髋关节置换的1例患者,体外分离培养人股骨头表面关节软骨细胞,使用100 pg/ml白介素-1(Interleukin-1,IL-1)建立OA软骨细胞模型。按照单纯随机抽样法,将细胞随机分为4组:对照组、白介素1(IL-1)组、IL-1+DHA组和IL-1+DHA+3-MA组。对照组加入生理盐水;IL-1组加入100 pg/ml IL-1;IL-1+DHA组加入100 pg/ml IL-1和50μg/ml DHA;IL-1+DHA+3-MA组加入100 pg/ml IL-1和50μg/ml DHA,以及5 mmol/L自噬抑制剂3-甲基腺嘌呤(3-MA)。所有细胞培养24 h后,采用Western blotting法检测哺乳动物雷帕霉素靶蛋白(mTOR)、Beclin-1、B淋巴细胞瘤-2基因(Bcl-2)和轻链3-Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ)蛋白的表达,RT-PCR法检测相关信号通路mRNA的表达(JNK、pERK、IΚBα、PI3K、P38)。结果与对照组相比,IL-1组中的细胞自噬活性降低。与IL-1组相比,DHA减少了mTOR通路蛋白的表达,并增加了Beclin-1和LC3-Ⅰ/Ⅱ蛋白的表达,差异具有统计学意义(P<0.01),增加了细胞自噬活性。在IL-1+DHA+3-MA组,使用自噬抑制剂3-MA干预后mTOR表达增加,而Beclin-1和LC3-Ⅰ/Ⅱ蛋白的表达减少。IL-1组的JNK、pERK、pERK和P38 mRNA表达量升高,差异具有统计学意义(P<0.01);在IL-1+DHA组,使用DHA干预后JNK和P38的表达量较IL-1组下降,差异具有统计学意义(P<0.01)。在IL-1+DHA+3-MA组,使用自噬抑制剂3-MA干预后,JNK和P38表达量明显上升,差异具有统计学意义(P<0.01)。结论DHA可促进OA软骨细胞的自噬活性,其作用是通过抑制JNK和P38信号通路的蛋白表达而实现的。自噬抑制剂3-MA可以阻断DHA对OA软骨细胞自噬的促进作用。Objective To observe the effect of polyunsaturated fatty acid on autophagy of human osteoarthritis(OA)chondrocytes,and investigate its mechanism in promoting autophagy.Methods A patient who underwent hip arthroplasty in the Department of Orthopaedics,Beingjing Friendship Hospital,Capital Medical University in May 2019 was recruited.Human articular chondrocytes on the surface of the femoral head were isolated and cultured in vitro,and OA chondrocyte model was established with 100 pg/ml interleukin-1(IL-1).The OA chondrocytes were divided into four groups randomly:control group,IL-1 group,IL-1+DHA group and IL-1+DHA+3-MA group.The chondrocytes in the control group were treated with 0.9%saline;the chondrocytes in the IL-1 group were treated with 100 pg/ml IL-1;the chondrocytes in the IL-1+DHA group were treated with 100 pg/ml IL-1 and 50 g/ml DHA;the chondrocytes in the IL-1+DHA+3-MA group were treated with 100 pg/ml IL-1,50μg/ml DHA and autophagy inhibitor 3-methyladenie(3-MA),respectively.All the chondrocytes were cultured for 24 hours,then western blotting was used to detect the expression of mammalian target of rapamycin(mTOR),Beclin-1,B cell lymphoma-2(Bcl-2)and Light chain-Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ),while RT-PCR was used to detect the expression of the related signal pathway mRNA(JNK,pERK,IΚBα,PI3K,P38).Results Compared with the control group,cell autophagic activity decreased in the IL-1 group.Compared with the IL-1 group,the expression of mTOR decreased and the expression of Beclin-1 and LC3-Ⅰ/Ⅱincreased in the IL-1+DHA group,the differences were statistically significant(P<0.01),and DHA increased the autophagic activity.In the IL-1+DHA+3-MA group,the expression of mTOR increased after treating with 3-MA,while the expression of Beclin-1 and LC3-Ⅰ/Ⅱdecreased.In the IL-1 group,the expression of JNK,ERK,GRP78,pERK and P38 mRNA increased significantly(P<0.01).In the IL-1+DHA group,the expression of JNK and P38 decreased significant after treating with DHA(P<0.01).However,in the IL-1+DHA+3-MA group,JNK and P

关 键 词：骨关节病 软骨细胞 多不饱和脂肪酸 自噬 

分 类 号：R684[医药卫生—骨科学]