羟氯喹对类风湿性关节炎成纤维样滑膜细胞基质金属蛋白酶-1表达的影响  

Effect of hydroxychloroquine on inhibiting matrix metalloproteinase-1 in rheumatoid arthritis fibroblast-like synoviocytes

作　　者：刘国钰 郑雅丹 戴嘉婧 张杰[2] 方征宇[1] LIU Guoyu;ZHENG Yadan;DAI Jiajing;ZHANG Jie;FANG Zhengyu(Medical Center,Shenzhen Peking University-The Hong Kong University of Science and Technology,Shenzhen 518036,China;Department of Dermatology,Peking University Shenzhen Hospital,Shenzhen 518036,China)

机构地区：[1]深圳北京大学香港科技大学医学中心,广东深圳518036 [2]北京大学深圳医院皮肤科,广东深圳518036

出　　处：《中华实用诊断与治疗杂志》2019年第12期1173-1176,共4页Journal of Chinese Practical Diagnosis and Therapy

基　　金：广东省自然科学基金(2016A030313754);深圳市基础研究计划(JCYJ20170306161757367)

摘　　要：目的探讨羟氯喹(hydroxychloroquine,HCQ)对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的类风湿性关节炎成纤维样滑膜细胞基质金属蛋白酶(matrix metalloproteinase,MMP)-1表达的调控作用及分子机制。方法对数生长期MH7A细胞分为空白组(细胞正常培养)、TNF-α组(细胞培养基中加入10μg/L TNF-α)、HCQ联合A组(细胞培养基中加入2.5μmol/L HCQ+10μg/L TNF-α)、HCQ联合B组(细胞培养基中加入5μmol/L HCQ+10μg/L TNF-α)、HCQ联合C组(细胞培养基中加入10μmol/L HCQ+10μg/L TNF-α)。5组细胞培养24h,采用ELISA法检测细胞培养上清液MMP-1水平,采用实时荧光定量PCR法检测细胞MMP-1mRNA相对表达量,采用Western blot法检测细胞c-Jun氨基末端激酶(Jun N-terminal kinase,JNK)、磷酸化JNK(p-JNK)蛋白相对表达量。将MH7A细胞分为对照组和茴香霉素组,对照组细胞按HCQ联合C组培养方法培养24h;茴香霉素组细胞培养基中先加入10μg/L茴香霉素培养2h后,再按HCQ联合C组的培养方法继续培养至24h;采用实时荧光定量PCR法检测2组MMP-1mRNA相对表达量。结果TNF-α组[(791.32±175.40)ng/L、6.89±1.20]、HCQ联合A组[(366.71±224.44)ng/L、4.12±0.71]、HCQ联合B组[(283.22±154.86)ng/L、2.99±0.18]、HCQ联合C组[(173.44±36.25)ng/L、2.01±0.80]、空白组[(133.13±62.19)ng/L、1.00±0.00]细胞MMP-1水平和MMP-1mRNA相对表达量均依次降低(P<0.05);TNF-α组(2.02±0.07)、HCQ联合A组(1.31±0.09)、HCQ联合B组(0.86±0.09)、HCQ联合C组(0.56±0.05)、空白组(0.43±0.03)细胞p-JNK蛋白相对表达量依次降低(P<0.05),而TNF-α组(2.30±0.16)、HCQ联合A组(2.16±0.13)、HCQ联合B组(2.03±0.05)、HCQ联合C组(2.16±0.16)、空白组(2.23±0.11)JNK蛋白相对表达量比较差异无统计学意义(P>0.05);茴香霉素组细胞MMP-1mRNA相对表达量(5.13±1.43)高于对照组(2.01±0.80)(P<0.05)。结论HCQ能通过JNK信号通路调控MH7A细胞MMP-1的表达。Objective To explore the role of hydroxychloroquine(HCQ)in regulating the expression of matrix metalloproteinase-1(MMP-1)in tumor necrosis factor-α(TNF-α)-induced rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)and its molecular mechanism.Methods MH7Acells in logarithmic growth phase were cultured with normal cells(blank group),10μg/L TNF-αin culture medium(TNF-αstimulation group),2.5μmol/L HCQ+10μg/L TNF-αin culture medium(HCQ joint group A),5μmol/L HCQ+10μg/L TNF-α(HCQ joint group B)and 10μmol/L HCQ+10μg/L TNF-αin culture medium(HCQ joint group C).After incubation for 24h,ELISA was used to detect the concentration of MMP-1in cell culture supernatant;Real-time quantitative PCR was used to detect MMP-1mRNA relative expression;Western blot was used to detect the relative expressions of Jun N-terminal kinase(JNK)and phosphorylated JNK(p-JNK).The MH7Acells were divided into control group and anisomycin group.Control group was cultured for 24haccording to the method of HCQ joint group C.Anisomycin group was added anisomycin in the culture medium and cultured with 10μg/L for 2h,and then was cultured according to the method of HCQ joint group C for 24h.The relative expression of MMP-1mRNA was detected by real-time fluorescent quantitative PCR.Results The relative expressions of MMP-1protein and MMP-1 mRNA decreased gradually in turn in TNF-αgroup((791.32±175.40)ng/L,6.89±1.20),HCQ joint group A((366.71±224.44)ng/L,4.12±0.71),HCQ joint group B((283.22±154.86)ng/L,2.99±0.18),HCQ joint group C((173.44±36.25)ng/L,2.01±0.80)and blank group((133.13±62.19)ng/L,1.00±0.00)(P<0.05).The relative expression of p-JNK decreased in turn in TNF-αgroup(2.02±0.07),HCQ joint group A(1.31±0.09),HCQ joint group B(0.86±0.09),HCQ joint group C(0.56±0.05)and blank group(0.43±0.03)(P<0.05),while the relative expression of JNK protein showed no significant difference in TNF-αgroup(2.30±0.16),HCQ joint group A(2.16±0.13),HCQ joint group B(2.03±0.05),HCQ joint group C(2.16±0.16)and blank group(2.23±0

关 键 词：类风湿性关节炎 羟氯喹 类风湿性关节炎成纤维样滑膜细胞 基质金属蛋白酶1 c-Jun氨基末端激酶信号通路 

分 类 号：R593.22[医药卫生—内科学]