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(P<0.05),黄五甘附膏高剂量组滑膜Krenn评分明显降低(P<0.05),黄五甘附膏各剂量组大鼠滑膜组织中TRPV1和p-p38 MAPK/p38 MAPK蛋白表达都显著下降(P<0.01),塞来昔布组和黄五甘附膏各剂量组滑膜组织中M1巨噬细胞占比均降低(P<0.01),黄五甘附膏高剂量组M2巨噬细胞占比升高(P<0.05),黄五甘附膏中、高剂量M1/M2降低(P<0.05)。结论:黄五甘附膏可通过调控巨噬细胞极化减轻KOA炎症反应,减少炎性疼痛介质释放,同时还降低了TRPV1蛋白表达,其机制可能与p38 MAPK信号通路有关,从而达到改善KOA外周痛敏的效应。
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,TNF-α,CGRP,and NGF in serum and synovium Krenn score increased significantly(P<0.01).The protein expression of TRPV1 and p-p38 MAPK/p38 MAPK increased significantly(P<0.01),and the proportion of M1 macrophages and M1/M2 increased(P<0.01),while the proportion of M2 macrophages decreased(P<0.01).Compared with model group,the body mass in the low,middle,and high dose groups of Huangwu Ganfu ointment increased to different degrees(P<0.05,P<0.01).The diameter of the knee joint in the high dose group of Huangwu Ganfu ointment and celecoxib group decreased(P<0.01).The recovery of PWT in the high and middle dose groups of Huangwu Ganfu ointment groups was more obvious(P<0.05).The contents of IL-1βand CGRP in the serum of rats in each administration group were significantly decreased(P<0.01),and the content of serum TNF-αin the celecoxib group and high dose group of Huangwu Ganfu ointment decreased significantly(P<0.05).The content of serum NGF in the middle dose group of Huangwu Ganfu ointment decreased significantly(P<0.05),and the synovium Krenn score decreased in the high dose group of Huangwu Ganfu ointment(P<0.05).In addition,the protein expression of TRPV1 and p-p38 MAPK/p38 MAPK in synovial tissue decreased significantly in all groups of Huangwu Ganfu ointment(P<0.01).The proportion of M1 macrophages in synovial tissue in the celecoxib group and all groups of Huangwu Ganfu ointment decreased(P<0.01),and the proportion of M2 macrophages in the high dose group of Huangwu Ganfu ointment increased(P<0.05).The M1/M2 in the middle and high dose groups of Huangwu Ganfu ointment decreased(P<0.05).Conclusion:Huangwu Ganfu ointment can mediate the polarization of macrophages to reduce the inflammatory reaction of KOA,alleviate the release of inflammatory pain mediators,and lower the protein expression of TRPV1.The mechanism may be related to the p38 MAPK signaling pathway,so as to improve the peripheral hyperalgesia of KOA.
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)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
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)。
