鹰嘴豆素A对骨关节炎的体外作用机制研究  

Study on mechanism of action of biochanin A on osteoarthritis in vitro

作　　者：何鹏 李秀成 何争珍 周显臣 HE Peng;LI Xiu-cheng;HE Zheng-zhen;ZHOU Xian-chen(Department of Orthopedics,China Three Gorges University Traditional Chinese Medicine Hospital/Yichang Hospital of Traditional Chinese Medicine,Yichang Hubei 443000,China)

机构地区：[1]三峡大学中医医院/宜昌市中医医院骨伤科,湖北宜昌443000

出　　处：《局解手术学杂志》2024年第9期753-758,共6页Journal of Regional Anatomy and Operative Surgery

基　　金：2023年度宜昌市医疗卫生研究项目(A23-1-064)。

摘　　要：目的通过体外实验探讨鹰嘴豆素A(BCA)对骨关节炎的作用机制。方法提取大鼠原代软骨细胞进行培养,随后将其分为对照组(不加诱导剂及干扰剂)、模型组[10 ng/mL白细胞介素-1β(IL-1β)诱导]和BCA组(10 ng/mL IL-1β诱导+0、3、6、12、24、48µmol/L BCA干预)。采用四甲基偶氮唑蓝(MTT)法进行细胞活性评估,衰老β-半乳糖苷酶(SA-β-gal)染色观察细胞衰老情况,酶联免疫吸附(ELISA)法检测细胞内炎症因子和氧化应激指标,蛋白免疫印迹法检测相关蛋白水平,运用分子对接技术验证BCA与Nrf2之间的亲和力。结果MTT法筛选BCA浓度后,选择6、12、24µmol/L BCA进行后续实验。与对照组相比,模型组软骨细胞活性及过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性明显减弱(P<0.05),SA-β-gal阳性细胞数显著增加,肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、丙二醛(MDA)表达水平明显升高(P<0.05),细胞内Ⅱ型胶原蛋白(COL2A1)、蛋白多糖(ACAN)表达水平明显降低(P<0.05)。与模型组相比,BCA组上述情况均出现明显逆转(P<0.05)。此外,BCA可以显著减弱核因子κB抑制因子α(IκB-α)的降解和NF-κBp65的核转位,并促进Nrf2在细胞核中的表达和人血红素氧化酶1(HO-1)在全细胞中的表达(P<0.05)。分子对接研究结果则进一步证实BCA与Nrf2具有极高的亲和力。结论BCA可通过调控Nrf2/NF-κB信号通路体外抑制骨关节炎软骨细胞氧化应激及炎症反应,缓解关节损伤。Objective To investigate the mechanism of action of biochanin A(BCA)on osteoarthritis through in vitro experiments.Methods Primary rat chondrocytes were extracted and cultured,and then divided into the control group(without inducer or interferant),the model group[10 ng/mL interleukin-1β(IL-1β)induction],and the BCA group(10 ng/mL IL-1βinduction+0,3,6,12,24,48µmol/L BCA intervention).Cell activity was detected by methyl thiazolyl tetrazolium(MTT)method.Senescence-associatedβ-galactosidase(SA-β-gal)staining was used to observe cell senescence.Intracellular inflammatory factors and oxidative stress indexes were detected by enzyme-linked immunosorbent assay(ELISA),the levels of related proteins were detected by Western blot,and the affinity between BCA and Nrf2 was verified by molecular docking technology.Results After screening the concentration of BCA with MTT,6,12 and 24µmol/L BCA were selected for subsequent experiments.Compared with the control group,the chondrocyte activity and the activities of catalase(CAT)and superoxide dismutase(SOD)in the model group were significantly decreased(P<0.05).The number of SA-β-gal positive cells and expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and malondialdehyde(MDA)increased significantly(P<0.05).The expression levels of type II collagen(COL2A1)and aggrecan(ACAN)were significantly decreased(P<0.05).Compared with the model group,the above conditions were significantly reversed in the BCA group(P<0.05).In addition,BCA could significantly reduce the degradation of inhibitor kappa B alpha(IκB-α)and nuclear translocation of NF-κBp65,and promote the expression of Nrf2 in the nucleus and the expression of human heme oxygenase-1(HO-1)in whole cells(P<0.05).The results of molecular docking further confirmed that BCA had the highest affinity with Nrf2.Conclusion BCA can inhibit oxidative stress and inflammation of osteoarthritis chondrocytes by regulating Nrf2/NF-κB signaling pathway in vitro,and alleviate joint injury.

关 键 词：鹰嘴豆素A 核因子-E2相关因子2 NF-ΚB信号通路 氧化应激 炎症反应 

分 类 号：R684.3[医药卫生—骨科学]