梓醇对IL-1β诱导软骨细胞损伤的保护机制研究  

Protective mechanism of catalpol on IL-1β-induced chondrocyte injury

作　　者：马玲[1] 钟利国[1] 崔裕如 刘彬 MA Ling;ZHONG Liguo;CUI Yuru;LIU Bin(The First People's Hospital of Jingzhou/The First Affiliated Hospital of Yangtze University,Jingzhou 434000,China)

机构地区：[1]荆州市第一人民医院,长江大学附属第一医院,434000

出　　处：《天津医药》2023年第7期718-723,共6页Tianjin Medical Journal

基　　金：荆州市科学技术局资助项目(2020HC23)。

摘　　要：目的探讨梓醇对白细胞介素-1β(IL-1β)诱导软骨细胞损伤的影响及其可能作用机制。方法分离培养人膝关节软骨细胞,分为对照(Con)组、IL-1β组(用10μg/L IL-1β处理48 h),IL-1β+梓醇-低组、IL-1β+梓醇-中组、IL-1β+梓醇-高组(分别用10、20及50 ng/L的梓醇处理软骨细胞后,用10μg/L IL-1β处理24 h),IL-1β+miR-NC组、IL-1β+miR-140-5p组(分别转染miR-NC和miR-140-5p mimics后,用10μg/L IL-1β处理24 h),IL-1β+梓醇+anti-miR-NC组、IL-1β+梓醇+anti-miR-140-5p组(分别转染anti-miR-NC和anti-miR-140-5p后,用50 ng/L梓醇和10μg/L IL-1β处理24 h)。细胞计数试剂盒-8(CCK-8)法检测细胞活力;酶联免疫吸附试验(ELISA)检测白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)的水平;流式细胞术检测细胞凋亡;采用实时荧光定量PCR检测miR-140-5p的表达水平;Western blot检测活化的胱天蛋白酶3(Cleaved-caspase 3)、Cleaved-caspase 9蛋白表达。结果与IL-1β组比较,IL-1β+梓醇-低组、IL-1β+梓醇-中组、IL-1β+梓醇-高组IL-6、TNF-α、IFN-γ水平,细胞凋亡率和Cleaved-caspase 3、Cleaved-caspase 9蛋白表达水平降低(P<0.05),细胞活力和miR-140-5p表达水平升高(P<0.05);与IL-1β+miR-NC组比较,IL-1β+miR-140-5p组IL-6、TNF-α、IFN-γ水平,细胞凋亡率和Cleavedcaspase 3、Cleaved-caspase 9蛋白表达水平降低(P<0.05),细胞活力和miR-140-5p表达水平升高(P<0.05);与IL-1β+梓醇+anti-miR-NC组比较,IL-1β+梓醇+anti-miR-140-5p组IL-6、TNF-α、IFN-γ水平,细胞凋亡率和Cleavedcaspase 3、Cleaved-caspase 9蛋白表达水平升高(P<0.05),细胞活力和miR-140-5p表达水平降低(P<0.05)。结论梓醇可能通过上调miR-140-5p表达,抑制细胞炎性因子释放及细胞凋亡,减轻IL-1β诱导的软骨细胞损伤。Objective To investigate the effect of catalpol on interleukin-1β(IL-1β)-induced chondrocyte injury and its possible mechanism.Methods Human knee joint chondrocytes were isolated,cultured and divided into the control(Con)group,the IL-1βgroup(treated with 10μg/L IL-1βfor 48 h),the IL-1β+catalpol-low group,the IL-1β+catalpol-medium group,the IL-1β+catalpol-high group(treated with 10,20 and 50 ng/L catalpol and then treated with 10μg/L IL-1βfor 24 h),the IL-1β+miR-NC group(transfected with miR-NC,treated with 10μg/L IL-1βfor 24 h),the IL-1β+miR-140-5p group(transfected with miR-140-5p mimics,treated with 10μg/L IL-1βfor 24 h),the IL-1β+catalpol+anti-miR-NC group(after transfection with anti-miR-NC,treated with 50 ng/L catalpol and 10μg/L IL-1βfor 24 h)and the IL-1β+catalpol+anti-miR-140-5p group(transfected with anti-miR-140-5p,treated with 50 ng/L catalpol and 10μg/L IL-1βfor 24 h).Cell viability was detected by cell count Kit 8(CCK-8 method).Levels of interleukin-6(IL-6),tumor necrosis factorα(TNF-α)and interferonγ(IFN-γ)were detected by enzyme-linked immunosorbent assay(ELISA).The apoptosis rate was detected by flow cytometry.The expression of miR-140-5p was detected by real-time quantitative fluorescence PCR(qPCR).Expression levels of cleaved aspartate specific cysteine proteinase(Cleaved-caspase)3 and Cleaved-caspase 9 were detected by Western blot assay.Results Compared with the IL-1βgroup,levels of IL-6,TNF-α,IFN-γ,cell apoptosis rate,Cleaved-caspase 3 and Cleaved-caspase 9 protein expression levels were decreased in the IL-1β+catalpol-low group,the IL-1β+catalpol-medium group and the IL-1β+catalpol-high group,but cell viability and miR-140-5p expression were increased(P<0.05).Compared with the IL-1β+miR-NC group,levels of IL-6,TNF-α,IFN-γ,cell apoptosis rate,Cleaved-caspase 3 and Cleaved-caspase 9 protein expression levels were decreased in the IL-1β+miR-140-5p group(P<0.05),and cell viability and miR-140-5p were increased(P<0.05).Compared with the IL-1β+catalpol+anti-miR

关 键 词：膝关节 软骨 关节 梓醇 细胞凋亡 miR-140-5p 炎性因子 

分 类 号：R684.76[医药卫生—骨科学]