海风藤提取物保护白细胞介素-1β诱导的软骨细胞损伤机制研究  

The protective mechanism of Kadsura pepper stem extract against interleukin-1β-induced chondrocyte injury

作　　者：亓英国 刘长鹏 王石磊 房师强 谷增泉 谢学升 Qi Yingguo;Liu Changpeng;Wang Shilei;Fang Shiqiang;Gu Zengquan;Xie Xuesheng(Department of Spine and Joint Surgery,People’s Hospital Affiliated to Shandong First Medical University,Jinan 271199,China)

机构地区：[1]山东第一医科大学附属人民医院脊柱关节外科,济南271199

出　　处：《中华实验外科杂志》2022年第9期1717-1720,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨海风藤提取物(KPSE)对白细胞介素-1β(IL-1β)诱导的软骨细胞损伤的保护作用及其机制。方法采用IL-1β处理大鼠软骨细胞建立软骨细胞损伤模型。0.1、1.0、10.0 mg/ml浓度KPSE与10 ng/ml IL-1β处理细胞(分别记为KPSE-L+IL-1β组、KPSE-M+IL-1β组、KPSE-H+IL-1β组)。将anti-miR-con、anti-miR-499a、miR-con、miR-499a mimics转染至软骨细胞并加入10 ng/ml的IL-1β培养(分别记为IL-1β+anti-miR-con组、IL-1β+anti-miR-499a组、IL-1β+miR-con组、IL-1β+miR-499a组)。分别将anti-miR-NC、anti-miR-499a转染至软骨细胞后加入10 mg/ml的KPSE及10 ng/ml的IL-1β培养(分别标记为anti-miR-NC+KPSE-H+IL-1β组、anti-miR-499a+KPSE-H+IL-1β组)。噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞活力,酶联免疫吸附法检测肿瘤坏死因子-α(TNF-α)、IL-1β、IL-6表达,蛋白质印迹法检测裂解的半胱氨酰天冬氨酸特异性蛋白酶(cleaved-Caspase-3)表达,实时荧光定量聚合酶链反应(RT-qPCR)检测微小核糖核酸-499a(miR-499a)表达。两组间比较行t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果KPSE+L+IL-1β组、KPSE+M+IL-1β组、KPSE+H+IL-1β组细胞吸光度值显著高于IL-1β组(0.50±0.05、0.73±0.07、0.79±0.08比0.38±0.03),cleaved-Caspase-3表达(0.71±0.07、0.57±0.05、0.48±0.04比0.83±0.08)、细胞凋亡率[(22.43±2.17)%、(16.38±1.54)%、(9.85±0.91)%比(27.86±2.61)%]、TNF-α[(61.02±6.23)、(40.56±3.57)、(30.15±2.86)pg/ml比(86.37±8.12)pg/ml]、IL-1β[(516.76±41.05)、(362.45±32.87)、(214.59±20.54)pg/ml比(637.15±53.32)pg/ml]和IL-6表达[(531.52±51.68)、(418.36±40.53)、(246.28±21.08)pg/ml比(584.11±55.05)pg/ml]显著低于IL-1β组,差异有统计学意义(F=83.568、70.682、103.211、291.687、402.183、250.447,P<0.05)。KPSE+L+IL-1β组、KPSE+M+IL-1β组、KPSE+H+IL-1β组细胞miR-499a表达显著高于IL-1β组(0.53±0.05、0.78±0.07、0.93±0.09比0.34±0.03),差异有统计学意义(F=13Objective To study the protective mechanism of Kadsura pepper stem extract(KPSE)against interleukin-1β(IL-1β)-induced chondrocyte injury.Methods Rat chondrocytes were treated with IL-1βto establish a cell injury model.The cells were treated with different concentrations of 0.1,1.0,10.0 mg/ml KPSE and 10 ng/ml IL-1β,and served as KPSE-L+IL-1βgroup,KPSE-M+IL-1βgroup,KPSE-H+IL-1βgroup respectively.Anti-miR-con,anti-miR-499a,miR-con,and miR-499a mimics were transfected into chondrocytes and cultured by adding 10 ng/mll IL-1β(recorded as IL-1β+anti-miR-con group,IL-1β+anti-miR-499a group,IL-1β+miR-con group,and IL-1β+miR-499a group,respectively).Anti-miR-NC and anti-miR-499a were transfected into chondrocytes,respectively,and then cultured with 10 mg/ml KPSE and 10 ng/ml IL-1β(marked as anti-miR-NC+KPSE-H+IL-1βgroup and anti-miR-499a+KPSE-H+IL-1βgroup,respectively).The cell viability was detected by methyl thiazolyl tetrazolium(MTT)method,the expression of tumor necrosis factorα(TNF-α),IL-1β,and IL-6 was detected by enzyme-linked immunosorbent assay,cleaved cysteinyl aspartate-specific protease-3(cleaved-Caspase-3)expression was detected by Western blotting,and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the miR-499a expression.The t test was used for comparison between two groups,one-way ANOVA was used for comparison between multiple groups,and SNK-q test was used for pairwise comparison between groups.Results The absorbance of cells in KPSE+L+IL-1βgroup,KPSE+M+IL-1βgroup,KPSE+H+IL-1βgroup was significantly higher than that in IL-1βgroup(0.50±0.05,0.73±0.07,0.79±0.08 vs.0.38±0.03),while cleaved-Caspase-3 expression(0.71±0.07,0.57±0.05,0.48±0.04 vs.0.83±0.08),apoptosis rate[(22.43±2.17)%,(16.38±1.54)%,(9.85±0.91)%vs.(27.86±2.61)%],TNF-α[(61.02±6.23),(40.56±3.57),(30.15±2.86)pg/ml vs.(86.37±8.12)pg/ml],IL-1β[(516.76±41.05),(362.45±32.87),(214.59±20.54)pg/ml vs.(637.15±53.32)pg/ml]and IL-6[(531.52±51.68),(418.36±40.53),(246.28±21

关 键 词：海风藤提取物 微小RNA 白细胞介素-1Β 软骨细胞 损伤 

分 类 号：R285[医药卫生—中药学]