基于TRPV1探讨黄五甘附膏治疗膝骨关节炎外周炎性痛敏的作用机制  

Mechanism of Huangwu Ganfu Ointment in Treatment of Peripheral Inflammatory Hyperalgesia of Knee Osteoarthritis Based on TRPV1

作　　者：姜楚洋 王兆南 姜洪亮 赵子苇 赵乐 马艳苗 李艳彦 JIANG Chuyang;WANG Zhaonan;JIANG Hongliang;ZHAO Ziwei;ZHAO Le;MA Yanmiao;LI Yanyan(Shanxi University of Traditional Chinese Medicine(TCM),Jinzhong 030619,China;Affiliated Hospital of Shanxi University of TCM,Taiyuan 030024,China)

机构地区：[1]山西中医药大学,山西晋中030619 [2]山西中医药大学附属医院,太原030024

出　　处：《中国实验方剂学杂志》2024年第14期97-106,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82104731);山西省中医药管理局中医药创新团队建设计划项目(zyytd2024010);山西省基础研究计划项目(202203021221204);山西省研究生教育创新项目(2022Y718);山西中医药大学研究生创业项目(2022CX020)。

摘　　要：目的:观察黄五甘附膏对阳虚寒凝证膝骨关节炎(KOA)大鼠滑膜组织瞬时受体电位锚蛋白1(TRPV1)受体表达、巨噬细胞极化及p38丝裂原活化蛋白激酶(p38 MAPK)信号通路调控作用,探讨其缓解KOA外周炎性痛敏的作用机制。方法:48只雄性SD大鼠随机分为空白组、模型组、黄五甘附膏高、中、低剂量组(9.3、4.65、2.325 g·kg^(-1))、塞来昔布组(208.2 mg·kg^(-1)),采用气候箱加游泳2周联合膝关节腔注射碘乙酸钠(MIA)建立阳虚寒凝证KOA大鼠模型,连续给药4周,观察各组大鼠一般情况,记录机械刺激缩足痛阈值(PWT)和关节直径,酶联免疫吸附测定法(ELISA)检测大鼠血清白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、神经生长因子(NGF)和降钙素基因相关肽(CGRP)炎性因子表达;苏木素-伊红(HE)染色观察膝关节滑膜组织病理学改变;蛋白免疫印迹法(Western blot)检测大鼠膝关节滑膜组织TRPV1、p38 MAPK、p-p38 MAPK蛋白表达水平;免疫荧光(IF)测量评估M1/M2巨噬细胞极化情况。结果:与空白组比较,模型组大鼠整体精神状态较差,自主活动减少,体质量偏低,关节直径增加,X射线提示关节边缘骨赘增生,关节面明显粗糙,关节腔间隙明显变窄,大鼠PWT显著降低(P<0.01),大鼠血清IL-1β、TNF-α、CGRP、NGF含量均显著升高(P<0.01),滑膜Krenn评分显著升高(P<0.01),TRPV1、p-p38 MAPK/p38 MAPK蛋白表达显著升高(P<0.01),M1巨噬细胞占比和M1/M2显著升高(P<0.01),M2巨噬细胞占比显著降低(P<0.01);与模型组比较,黄五甘附膏低、中、高剂量体质量有不同程度上升(P<0.05,P<0.01),黄五甘附膏高剂量组和塞来昔布组大鼠膝关节直径减少(P<0.01),黄五甘附膏高、中剂量组PWT恢复较为明显(P<0.05),各给药组大鼠血清IL-1β和CGRP的含量都显著降低(P<0.01),塞来昔布组和黄五甘附膏高剂量组大鼠血清TNF-α含量明显降低(P<0.05),黄五甘附膏中剂量组大鼠血清NGF含量明显降低(Objective:To observe the regulatory effect of Huangwu Ganfu ointment on transient receptor potential anchor protein 1(TRPV1)receptor expression,macrophage polarization,and p38 mitogenactivated protein kinase(p38 MAPK)signaling pathway in synovial tissue of knee osteoarthritis(KOA)rats with yang deficiency and cold coagulation syndrome and explore the mechanism of relieving peripheral inflammatory hyperalgesia of KOA.Method:Forty-eight male SD rats were randomly divided into blank group,model group,high-dose,middle-dose,and low-dose groups of Huangwu Ganfu ointment(9.3,4.65,2.325 g·kg^(-1)),and celecoxib group(20.82 mg·kg^(-1)).The KOA rat model of yang deficiency and cold coagulation syndrome was established through climate box and swimming for two weeks combined with an injection of sodium iodoacetate(MIA)in the articular cavity.After continuous administration for four weeks,the general condition of rats in each group was observed,and the pain withdrawal threshold(PWT)and joint diameter induced by mechanical stimulation were recorded.The expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),nerve growth factor(NGF),and calcitonin gene-related peptide(CGRP)inflammatory factor were detected by enzyme-linked immunosorbent assay(ELISA),and the histopathological changes of synovial tissue of the knee joint were observed by hematoxylin-eosin(HE)staining.Western blot was used to detect the protein expression of TRPV1,p38 MAPK,and p-p38 MAPK in synovial tissue of the knee joint,and immunofluorescence(IF)was used to evaluate the polarization of M1/M2 macrophages.Result:Compared with that in the blank group,the overall mental state of the model group was worse,and the autonomous activity was decreased.The body mass was lower,and the joint diameter was increased.The X-ray showed that the osteophyte at the edge of the joint proliferated,and the articular surface was obviously rough.The articular cavity was significantly narrowed,and the PWT was significantly decreased(P<0.01).The contents of IL-1β

关 键 词：黄五甘附膏 膝骨关节炎 瞬时受体电位锚蛋白1(TRPV1) 巨噬细胞极化 黄芪桂枝五物汤 甘草附子汤 

分 类 号：R2-0[医药卫生—中医学] R33R289R684.3