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七叶皂苷A在小鼠软骨细胞及骨关节炎中的作用及相关分子机制
王淼,万锐杰,刘伟,彭科
0
重庆市沙坪坝区中医院骨伤一科重庆400030;重庆市中医院康复科重庆400021;重庆医科大学附属第三医院骨科重庆401120;重庆医科大学基础医学院重庆400016
摘要:
目的:探讨七叶皂苷A(EsA)在小鼠骨关节炎(OA)中的作用及相关分子机制。方法:分离培养C57BL/6小鼠原代软骨细胞,将细胞用10 mg/L白细胞介素(IL)-1β及不同浓度(10、50、100 μmol/L)EsA进行处理后,CCK-8检测细胞活性,ELISA法检测细胞培养液上清中IL-6和肿瘤坏死因子-α(TNF-α)含量,实时定量PCR检测基质金属蛋白酶-13(MMP-13)和聚蛋白多糖酶(ADAMTS5)mRNA表达水平,Western blotting检测II型胶原蛋白(Collagen II)、基质金属蛋白酶-9(MMP-9)及缺氧诱导因子(HIF)-2α和核因子-κB(NF-κB)途径相关蛋白表达,免疫荧光染色检测p65表达,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及丙二醛(MDA)试剂盒检测氧化活性水平。将60只C57BL/6雄性小鼠随机分为假手术组、OA组、OA+低剂量EsA组和OA+高剂量EsA组,每组15只,建立了小鼠OA模型后,OA+低剂量EsA组、OA+高剂量EsA组小鼠分别按照5 mg/kg、10 mg/kg进行腹膜注射EsA,每隔一天注射1次,持续6周后处死小鼠,骨密度测量仪检测小鼠股骨近端骨密度,HE染色和番红O固绿染色检测骨组织病理形态学改变。结果:EsA能够抑制IL-1β刺激后引起的细胞存活率下降与TNF-α、IL-6含量升高,使MMP-13和ADAMTS-5 mRNA表达下降,Collagen II蛋白表达升高且MMP-9蛋白表达降低,并抑制了IL-1β诱导的HIF-2α与NF-κB信号通路的激活,使MDA水平下降、SOD与CAT活性升高,差异均有统计学意义(P<0.05)。OA小鼠经低、高剂量EsA治疗后,小鼠股骨骨密度值均升高(P<0.05),骨组织病变现象得到改善。结论:EsA能够抑制小鼠骨关节炎,其机制可能与HIF-2α和NF-κB途径及其发挥抗氧化活性有关。
关键词:  骨关节炎  七叶皂苷A  缺氧诱导因子-2α  核因子-κB  抗氧化中图分类号:R684,R977 文献标识码:A 文章编号:1007-6948(2021)02-0189-09
DOI:10.3969/j.issn.1007-6948.2021.02.005
投稿时间:2020-08-02
基金项目:重庆市技术创新与应用发展专项面上项目(cstc2019jscx-msxmX0853)
Escin AInhibits the Progression of Osteoarthritis by Down-regulating the HIF-2α, NF-κB Pathways and Their Antioxidant Activity
WANG Miao,WAN Rui-jie,LIU Wei
Abstract:
Objective To explore the role and related molecular mechanism of Escin A (Escin A, EsA) in mouse osteoarthritis (OA). Methods Isolate and culture the primary chondrocytes of C57BL/6 mice, and treat the cells with 10 mg/L IL-1β and different concentrations (10, 50, 100 μmol/L) of Escin A. CCK-8 detects cell activity.The Enzyme-linked immunosorbent assay was used to detect the content of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant of cell culture fluid,real-time quantitative PCR to detect the mRNA expression levels of matrix metalloproteinase-13 (MMP-13) and proteoglycanase (ADAMTS5), western blot detection of collagen type II (Collagen II), matrix metalloproteinase-9 (MMP-9), hypoxia-inducible factor-2 (HIF-2α) and nuclear transcription factor-Kappa B (NF-κB) pathway related protein expression, immunofluorescence staining to detect p65 expression, superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) kits were used to detect the level of oxidative activity. The 60 C 57 BL/ 6 male mice were randomly divided into sham operation group, OA group, OA+low-dose EsA group and OA+high-dose EsA group, which 15 mice in each group.After establishing the mouse OA model, mice in the OA+low-dose EsA group and OA+high-dose EsA group were injected with EsA intraperitoneally at 5 mg/kg and 10 mg/kg respectively, every other day, and the mice were sacri?ced after 6 weeks. Bone density measuring instrument detects the bone density of the proximal femur in mice,HE staining and safranine O fast green staining were used to detect the pathological morphological changes of bone tissue. Results Under the action of Escin A, it can inhibit the decrease of cell survival rate and the increase of TNF-α and IL-6 content caused by IL-1β stimulation (P<0.05). At the same time, it can make MMP-13 and ADAMTS-5 mRNA expression decreased (P<0.05), Collagen II protein expression increased and MMP-9 protein expression decreased (P<0.05), inhibited IL-1β-induced activation of HIF-2α and NF-κB signaling pathways, and make MDA level drop, SOD and CAT activity increase (P<0.05).After OA mice were treated with Escin A at low and high doses, the bone density of the femur of the mice increased (P<0.05), the bone tissue lesions were improved. Conclusion Escin A can inhibit osteoarthritis in mice, and its mechanism may be related to the HIF-2α and NF-κB pathways and their antioxidant activity.
Key words:  Osteoarthritis  Escin A  HIF-2α  NF-κB pathway  antioxidant

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