膝痹通络方通过调节MAPK-MEK信号通路延缓对白细胞介素1β诱导的关节软骨细胞退变  

Xibi Tongluo Recipe Delays IL-1β-Induced Degeneration of Articular Chondrocytes though Regulating MAPK-MEK Signaling Pathway

作　　者：宋奕 丁道芳[2,3] 朱寅 王星华 SONG Yi;DING Dao-Fang;ZHU Yin;WANG Xing-Hua(Suzhou Hospital of Integrated Chinese and Western Medicine,Suzhou 215000 Jiangsu,China;Shi’s Traumatology Medicine Center of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Institute of Orthopedics and Traumatology of Shanghai Research Institute of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]苏州市中西医结合医院,江苏苏州215000 [2]上海中医药大学附属曙光医院石氏伤科医学中心,上海201203 [3]上海市中医药研究院骨伤科研究所,上海201203

出　　处：《广州中医药大学学报》2021年第1期135-141,共7页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：苏州市科技局科技示范工程(编号:SS201815);苏州市科技局指导性项目(编号:SYSD2016051);江苏省中医药管理局科技项目(编号:YB201834)。

摘　　要：【目的】观察膝痹通络方对白细胞介素1β(IL-1β)诱导的软骨细胞退变的影响,并从丝裂原活化蛋白激酶(MAPK)-MAPK激酶(MEK)信号通路探讨其机制。【方法】提取新生SD大鼠四肢关节软骨细胞,选择P1代细胞进行实验,共设5组:正常对照组,IL-1β组,高、中、低浓度中药组。正常对照组软骨细胞加入正常大鼠血清培养;IL-1β组软骨细胞加入正常大鼠血清、20 ng/mL IL-1β培养;高、中、低浓度中药组软骨细胞分别加入对应浓度的膝痹通络方含药血清进行培养,并加入20 ng/mL IL-1β。24 h后倒置显微镜下观察软骨细胞大小、密度、形态,采用实时定量聚合酶链反应(qPCR)检测软骨细胞基质金属蛋白酶3(MMP3)、聚蛋白多糖酶4/5(ADAMTS4/5)、蛋白多糖(A-CAN)、性别决定区Y框蛋白(SOX9)等软骨退变相关基因的表达,蛋白免疫印迹(Western Blot)法检测软骨细胞SOX9、基质金属蛋白酶13(MMP13)、磷酸化原癌基因丝苏氨酸蛋白激酶(P-Raf1)、Raf-1、磷酸化丝裂原活化蛋白激酶激酶(P-MEK1)、MEK1的蛋白表达。【结果】倒置显微镜下可见,经IL-1β炎性因子诱导后的软骨细胞出现退变形态;q PCR结果显示,与IL-1β组比较,各浓度中药组MMP3、ADAMTS4/5的mRNA表达水平降低(P<0.05),A-CAN的mRNA表达水平升高(P<0.05),高浓度中药组SOX9的mRNA表达水平升高(P<0.05);Western Blot结果显示,与IL-1β组比较,各浓度中药组软骨细胞MMP13蛋白表达,Raf1、MEK1磷酸化水平降低(P<0.05),SOX9蛋白表达水平升高(P<0.05)。【结论】膝痹通络方可延缓IL-1β诱导的软骨细胞退变,其机制可能与调节MAPK-MEK信号通路相关蛋白及基因表达有关。Objective To observe the effect of Xibi Tongluo Recipe(XTR)on the degeneration of chondrocytes induced by interleukin 1β(IL-1β), and to explore its mechanism based on mitogen-activated protein kinase(MAPK)-MAPK kinase(MEK) signaling pathway. Methods The articular chondrocytes were isolated from newborn SD rats,and then the cells in first generation were selected and divided into 5 groups,namely normal control group,IL-1β group,and high-,middle-and low-concentration XTR groups. The chondrocytes in the normal control group were cultured with rat normal serum,those in IL-1β group were cultured with rat normal serum and 20 ng/mL of IL-1β, and those in the high-, middle-and low-concentration XTR groups were cultured with high,middle and low concentrations of XTR-containing serum,respectively,and 20 ng/mL of IL-1β. After 24 hours,the size,density and shape of chondrocytes were observed under inverted microscope,the levels of cartilage degeneration-related genes of matrix metalloproteinase 3(MMP3),A disintegrin-like and metallopteinase with thrombospondin type 1 motifs 4/5(ADAMTS4/5),Aggrecan(A-CAN)and Sry related HMG box 9(SOX9)were detected by quantitative polymerase chain reaction(qPCR),and the protein expression levels of SOX9, matrix metalloproteinase 13(MMP13), phosphorylated rapidly accelerated fibrosarcoma(P-Raf1),Raf-1,phosphorylated mitogen-activated protein kinase 1(P-MEK1),MEK1 were detected by Western blotting assay. Results The changes in degeneration of chondrocytes were observed under inverted microscope. The qPCR results showed that compared with the IL-1β group,the mRNA expression levels of MMP3 and ADAMTS4/5 were decreased,and A-CAN mRNA expression level was increased in the various concentrations of XTR groups(P<0.05),and SOX9 mRNA expression level was increased in high-concentration XTR group(P<0.05). Western blotting results showed that compared with the IL-1β group, the protein expression level of MMP13, and phosphrylation levels of Raf1 and MEK1 in chondrocytes were decreased,and SOX9 pro

关 键 词：膝痹通络方 膝关节骨性关节炎 MAPK-MEK信号通路 白细胞介素1Β 软骨细胞 

分 类 号：R285.5[医药卫生—中药学]