摘要
目的:观察芍药内酯苷(AF)对白细胞介素(IL)-1β诱导软骨细胞损伤的保护作用,并探讨机制是否与调控微小RNA(miR)-149-5p/Wnt1诱导信号通路蛋白1(WISP1)通路有关。方法:分离培养远交群(SD)大鼠膝关节软骨细胞,采用10μg/L的IL-1β建立软骨细胞损伤模型。根据处理方法不同将软骨细胞分为对照(Con)、IL-1β、IL-1β+AF-L、IL-1β+AF-M、IL-1β+AF-H、IL-1β+miR-NC、IL-1β+miR-149-5p、IL-1β+si-NC、IL-1β+si-WISP1、IL-1β+AF+anti-miR-NC、IL-1β+AF+anti-miR-149-5p组。酶联免疫吸附实验(ELISA)检测细胞上清中IL-6、干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)含量;流式细胞术和蛋白质印记(Western blot)用于分析细胞凋亡和WISP1蛋白表达;实时定量聚合酶链反应(RT-qPCR)检测miR-149-5p和WISP1 mRNA表达。双荧光素酶报告实验和Western blot验证miR-149-5p对WISP1的靶向作用。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析和SNK-q检验。结果:IL-1β组软骨细胞上清液中IL-6(6.11±0.61比2.54±0.25,q=26.072,P<0.05)、IFN-γ(53.62±5.33比8.14±0.82,q=44.989,P<0.05)和TNF-α含量(29.54±2.71比4.21±0.42,q=44.463,P<0.05)高于Con组,细胞凋亡率(28.65±2.71比8.23±0.81,q=33.622,P<0.05)、WISP1表达(0.92±0.08比0.45±0.04,q=22.873,P<0.05)高于Con组,miR-149-5p表达(0.44±0.04比1.00±0.05,q=30.571,P<0.05)低于Con组。IL-1β+AF-L、IL-1β+AF-M、IL-1β+AF-H组软骨细胞上清液中IL-6(5.03±0.49、3.95±0.31、2.86±0.27比6.11±0.61,q=7.887、14.775、23.735,P<0.05)、IFN-γ(37.19±3.21、24.58±2.44、13.54±1.32比53.62±5.33,q=16.063、28.391、39.184,P<0.05)和TNF-α(21.65±2.17、12.64±1.28、6.95±0.63,q=14.014、30.017、40.123,P<0.05)含量低于IL-1β组,细胞凋亡率(22.47±2.23、16.98±1.54、10.54±1.12比28.65±2.71,q=10.175、19.215、29.818,P<0.05)、WISP1表达(0.78±0.07、0.66±0.06、0.53±0.05比0.92±0.08,q=6.813、12.653、18.980,P<0.05)低于IL-1β组,miR-149-5p表达高于IL-1β组。IL-1β+miR-149-
Objective To observe the protective effect of Albiflorin(AF)on interleukin-1β(IL-1β)-induced chondrocyte injury,and to explore whether the mechanism is related to the regulation of microRNA(miR)-149-5p/Wnt1-induced signaling pathway protein 1(WISP1)signaling pathway.Methods Chondrocytes from knee joints of Sprague dawley(SD)rats were isolated and cultured,and chondrocyte injury model was established by using IL-1βat 10μg/L.According to different treatment methods,chondrocytes were divided into control(Con),groups of IL-1β,IL-1β+AF-L,IL-1β+AF-M,IL-1β+AF-H,IL-1β+miR-NC,IL-1β+miR-149-5p,IL-1β+si-NC,IL-1β+si-WISP1,IL-1β+AF+anti-miR-NC,IL-1β+AF+anti-miR-149-5p.Enzyme-linked immunosorbent assay(ELISA)was used to detect IL-6,interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)levels in cell supernatants.The cell apoptosis and WISP1 protein expression were detected by flow cytometry and Western blotting.The expression of miR-149-5p and WISP1 mRNA was detected by Real-time quantitative polymerase chain reaction(RT-qPCR).The dual luciferase reporting experiment and Western blotting verified the targeting effect of miR-149-5p on WISP1.The independent-sample t test was used to estimate differences between two groups,and one-way ANOVA and SNK-q test were used to estimate differences between multiple groups.Results The levels of IL-6(6.11±0.61 vs.2.54±0.25,q=26.072,P<0.05),IFN-γ(53.62±5.33 vs.8.14±0.82,q=44.989,P<0.05)and TNF-α(29.54±2.71 vs.4.21±0.42,q=44.463,P<0.05)in the chondrocyte supernatant of the IL-1βgroup were significantly higher,the apoptosis rate(28.65±2.71 vs.8.23±0.81,q=33.622,P<0.05)and WISP1 expression(0.92±0.08 vs.0.45±0.04,q=22.873,P<0.05)were significantly higher,and the miR-149-5p expression(0.44±0.04 vs.1.00±0.05,q=30.571,P<0.05)was significantly lower than those in Con group.The levels of IL-6(5.03±0.49,3.95±0.31,2.86±0.27 vs.6.11±0.61,q=7.887,14.775,23.735,P<0.05),IFN-γ(37.19±3.21,24.58±2.44,13.54±1.32 vs.53.62±5.33,q=16.063,28.391,39.184,P<0.05)and TNF-α(21.6
作者
段贤斌
张强
刘弼
卢小虎
李孝静
Duan Xianbin;Zhang Qiang;Liu Bi;Lu Xiaohu;Li Xiaojing(Department of Orthopedics,Shenzhen People′s Hospital,Shenzhen 518000,China;School of Materials Science and Engineering,South China University of Technology,Guangzhou 510641,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第7期1221-1225,共5页
Chinese Journal of Experimental Surgery
基金
深圳市科技计划项目(JCYJ20170307100726777)。
