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基于TLR4/MyD88/NF-кB信号通路研究十一方药酒对兔膝骨关节炎的保护作用
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| 篇名： | 基于TLR4/MyD88/NF-кB信号通路研究十一方药酒对兔膝骨关节炎的保护作用 |
| TITLE： | Study on protective effects of Shiyifang medicinal wine on knee osteoarthritis in rabbits based on TLR 4/MyD88/ NF-кB signaling pathway |
| 摘要： | 目的 研究十一方药酒（以下简称“SYF”）对木瓜蛋白酶诱导的兔膝骨关节炎（KOA）的保护作用及机制。方法将35只兔随机分为空白组、模型组、阳性组（双氯芬酸二乙胺乳胶剂200mg/kg）、SYF高剂量组（386mg/kg）、SYF低剂量组（97mg/kg），每组7只（均为雄性4只、雌性3只）。除空白组外，其余各组兔于第1、4、7天从右后肢膝关节腔注入木瓜蛋白酶混合液（含2%木瓜蛋白酶与0.03mol/LL-半胱氨酸）0.5mL复制KOA模型，空白组注射等体积生理盐水。从第15天开始于各组兔右后肢膝关节涂抹药物，每天给药2次，连续20d，同时在第0、8、14、35天，用游标卡尺测量兔右后肢膝关节直径计算其肿胀度。实验结束后，采用酶联免疫吸附测定法检测滑膜组织白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、前列腺素E（2PGE2）的水平；制作滑膜组织苏木精-伊红（HE）染色切片，观测病理学形态变化；采用实时荧光定量聚合酶链反应法检测滑膜组织Toll样受体4（TLR4）/髓样分化因子88（MyD88）/细胞核因子（NF-кB）信号通路中TLR4、MyD88、NF-кBp65mRNA相对表达水平；采用Westernblot法检测滑膜组织TLR4、MyD88、NF-кBp65、磷酸化NF-кBp65（p-NF-кBp65）蛋白相对表达水平。结果与空白组比较，模型组兔膝关节肿胀度升高，滑膜组织病理性损伤严重，滑膜组织中IL-1β、TNF-α、PGE2水平均显著升高（P＜0.05），TLR4、MyD88、NF-кBp65mRNA和TLR4、MyD88、p-NF-кBp65蛋白相对表达水平均显著升高（P＜0.05）。与模型组比较，各给药组兔右后肢膝关节肿胀度与滑膜组织病理损伤程度均有显著改善，滑膜组织中IL-1β、TNF-α、PGE2水平均显著降低（P＜0.05），TLR4、MyD88、NF-кBp65mRNA相对表达水平和TLR4、MyD88（SYF低剂量组除外）、p-NF-кBp65蛋白相对表达水平均显著降低（P＜0.05）。结论SYF对木瓜蛋白酶诱导的兔KOA有保护作用，其作用机制与降低炎症因子IL-1β、TNF-α、PGE2水平及下调TLR4/MyD88/NF-кB信号通路相关。 |
| ABSTRACT： | OBJECTIVE To study the protective effect and mechanism of Shiyifang medicinal wine （SYF） on knee osteoarthritis（KOA）of rabbit induced by papain . METHODS Thirty-five rabbits were randomly divided into blank group ，model group，positive group （Diclofenac diethylamine emulsion 200 mg/kg），SYF high -dose group （386 mg/kg）and SYF low -dose group（97 mg/kg），with 7 rabbits in each group （all had 4 males and 3 females）. Except for the blank group ，the other groups ’ rabbits were injected 0.5 mL papain mixture （containing 2% papain and 0.03 mol/L L -cysteine）into the right knee cavity on day 1， 4 and 7，to replicate KOA model . Blank group was given constant volume of normal saline . From the 15th day ，drugs were applied to right hind knee joints of rabbits in each group ，twice a day for 20 days. At the same time ，the diameter of right knee joints of rabbits was measured by vernier calipers at day 0，8，14 and 35 to calculate swelling degree . After the experiment ，the levels of IL-1β，TNF-α and PGE 2 in synovial tissue were determined by enzyme -linked immunosorbent assay . Hematoxylin-eosin（HE） staining was used to prepare the sections of synovial tissue ，and the pathological changes were observed . The relative mRNA expressions of TLR 4，MyD88 and NF - кB p 65 in the TLR 4/MyD88/NF- кB signaling pathway were detected by real -time quantitative polymerase chain reaction . The relative protein expressions of TLR 4，MyD88，NF-кB p 65 and p -NF-кB p 65 were detect by Western blot . RESULTS Compared with blank group，the degree of knee swelling could be increased in model group ，the pathological damage of synovial tissue was more serious ，and the levels of IL -1β，TNF-α and PGE 2 were increased significantly in synovial tissue （P＜0.05）. The relative expression levels of TLR 4，MyD88，NF-кB p 65 mRNAs and TLR 4，MyD88，p-NF-кB p 65 proteins were significantly increased（P＜0.05）. Compared with model group ，swelling degree of right hind knee and the pathological injury degree of synovial tissue were significantly improved in each treatment group ，while the levels of IL -1β，TNF-α and PGE 2 in synovial tissue were significantly decreased （P＜0.05）. The relative mRNA expressions of TLR 4，MyD88 and NF -кB p 65 and relative protein expressions of TLR 4，MyD88（except for SYF low -dose group ）and p -NF-кB p 65 were all significantly decreased （P＜0.05）. CONCLUSIONS SYF shows protective effect on KOA induced by papain ，the mechanism of which is associated with decreasing the levels of IL -1β，TNF-α and PGE 2 and down -regulating TLR 4/MyD88/NF-кB signaling pathway . |
| 期刊： | 2022年第33卷第18期 |
| 作者： | 张文涛，周玲梅，黄宏海，鲍慧慧，毛德文，田慧，刘舒凌 |
| AUTHORS： | ZHANG Wentao ，ZHOU Lingmei ，HUANG Honghai ，BAO Huihui ，MAO Dewen ，TIAN Hui，LIU Shuling |
| 关键字： | 十一方药酒；膝骨关节炎；炎症因子；TLR4/MyD88/NF-кB信号通路 |
| KEYWORDS： | Shiyifang medicinal wine ；knee osteoarth ritis；inflammatory factor ；TLR4/MyD88/NF-кB signaling pathway |
| 阅读数： | 133 次 |
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