摘要
从LXRs/NF-κB信号通路调控膝骨关节炎(knee osteoarthritis,KOA)滑膜组织炎症角度,探讨壮骨健膝方减轻KOA滑膜组织炎症的作用机制。收集5名正常人滑膜组织及30例KOA滑膜炎患者滑膜组织进行体外培养,将正常人滑膜组织设为正常组,KOA滑膜炎患者滑膜组织随机分为滑膜炎症组、壮骨健膝方组、LXRα抑制剂组、N-CoR抑制剂组,分别用10%空白血清、10%空白血清、10%含药血清、10%含药血清+LXRα抑制剂、10%含药血清+N-CoR抑制剂干预7 d。干预后收集各组滑膜组织,采用HE染色观察滑膜组织形态并进行病理学评分;免疫荧光染色检测滑膜组织中成纤维细胞标志性蛋白α-平滑肌肌动蛋白(α-SMA)的表达情况;ELISA检测滑膜组织匀浆上清液中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶-3(MMP-3)及基质金属蛋白酶-13(MMP-13)的含量;RT-qPCR、Western blot检测滑膜组织LXRα、N-CoR、P50、P65 mRNA及蛋白表达。结果显示,与正常组比较,滑膜炎症组滑膜内衬层细胞增生、炎性细胞浸润明显,且滑膜细胞排列紊乱,形态学评分升高(P<0.05),滑膜成纤维细胞α-SMA荧光染色表达增强,细胞数量增多(P<0.05),滑膜组织中IL-1β、TNF-α、MMP-3、MMP-13含量均升高(P<0.05),LXRα、N-CoR mRNA及蛋白表达降低,P50、P65 mRNA及蛋白表达升高(P<0.05);与滑膜炎症组比较,壮骨健膝方组滑膜组织病理改变及形态学评分降低(P<0.05),滑膜成纤维细胞α-SMA荧光染色表达减弱,细胞数量减少(P<0.05),滑膜组织中IL-1β、TNF-α、MMP-3、MMP-13含量均降低(P<0.05),LXRα、N-CoR mRNA及蛋白表达升高,P50、P65 mRNA及蛋白表达降低(P<0.05);与壮骨健膝方组比较,LXRα抑制剂组、N-CoR抑制剂组滑膜组织病理改变及形态学评分升高(P<0.05),滑膜成纤维细胞α-SMA荧光染色表达增强,细胞数量增多(P<0.05),IL-1β、TNF-α、MMP-3、MMP-13含量均升高(P<0.05),LXRα、N-CoR mRNA及蛋白表达下降,P50、P65 mRNA及蛋白表达升高(P<0.05)。以上结果说明壮骨健膝方可上调KOA滑膜炎患者滑膜组织中LXRs/NF-κB通路上LXRα、N-CoR mRNA及蛋白表达,从而下调P50、P65 mRNA及蛋白表达,降低NF-κB通路的活性,发挥抑制KOA滑膜炎患者滑膜组织炎症的作用。
This study amis to observe the effect of Zhuanggu Jianxi Decoction on synovial tissue inflammation in human knee osteoarthritis(KOA)and explore its mechanism through LXRs/NF-κB signaling pathway.The synovial tissues of 5 normal subjects and 30 KOA synovitis patients were collected for culture in vitro,and the normal subjects were set as normal group.The synovitis tissues of KOA synovitis patients were randomly divided into synovitis group,Zhua nggu Jianxi Decoction group,LXRαinhibitor group and N-CoR inhibitor group.The synovitis tissues of the 5 groups were treated with 10%blank serum,10%blank serum,10%drug-containing serum,10%drug-containing serum+LXRαinhibitor,and 10%drug-containing serum+N-CoR inhibitor respectively for 7 days.After intervention,the synovial tissues of each group were collected,the morphology of synovial tissues was observed by HE staining and the pathological scoring was conducted.The expression intensity of fibroblast specific markerα-smooth musle actin(α-SMA)in synovial tissue was observed by immunofluorescence staining.The contents of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-3(MMP-3)and matrix metalloproteinase-13(MMP-13)in the supernatant of synovium homogenate were determined by ELISA.The mRNA and protein expression of LXRα,N-CoR,P50 and P65 in synovial tissue were detected by RT-qPCR and western blot.The results showed that compared with the normal group,there were obvious synovial lining cell proliferation,inflammatory cell infiltration,synovial cell arrangement disorder and other pathological changes in synovial inflammation group,the morphological score increased(P<0.05),and theα-SMA fluorescence staining expression of synovial fibroblasts enhanced,cell number increased(P<0.05).The contents of IL-1β,TNF-α,MMP-3 and MMP-13 in synovial tissue increased(P<0.05),and the mRNA and protein expression of LXRαand N-CoR decreased,P50 and P65 increased.Compared with the synovial inflammation group,the pathological changes and morphological scores of synovial tissue decreased(P<0.05),theα-SMA fluorescence staining expression of synovial fibroblasts in Zhuanggu Jianxi Decoction group decreased,the number of cells decreased(P<0.05).The contents of IL-1β,TNF-α,MMP-3 and MMP-13 decreased(P<0.05),and the mRNA and protein expression of LXRαand N-CoR in synovial tissue increased,P50 and P65 decreased(P<0.05).Compared with Zhuanggu Jianxi Decoction group,the pathological changes and morphological scores of synovial tissue in LXRαinhibitor group and N-CoR inhibitor group increased(P<0.05),theα-SMA fluorescence expression of synovial fibroblasts enhanced,the number of cells increased(P<0.05).The contents of IL-1β,TNF-α,MMP-3 and MMP-13 increased(P<0.05),and the mRNA and protein expression of LXRαand N-CoR decreased,P50 and P65 increased(P<0.05).These results above shows Zhuanggu Jianxi Decoction could alleviate the synovial inflammation in KOA patient and its mechanism may be related to upregulating the expression of LXRαand N-CoR mRNA and proteins on the LXRs/NF-κB pathway in synovial tissue,thereby downregulating P50 and P65 and reducing the activity of the NF-κB pathway.
作者
肖艳
刘俊
陈鹏
王美玲
郑卓铭
张英杰
张婷
翁绳健
苏友新
郭洁梅
XIAO Yan;LIU Jun;CHEN Peng;WANG Mei-ling;ZHENG Zhuo-ming;ZHANG Ying-jie;ZHANG Ting;WENG Sheng-jian;SU You-xin;GUO Jie-mei(College of Rehabilitation Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;Key Laboratory of Orthopedics&Traumatology of Traditional Chinese Medicine and Rehabilitation,Ministry of Education,Fuzhou 350122,China;First Clinical Medicine College,Fujian University of Traditional Chinese Medicine,Fuzhou 350004,China;FuZhou Second General Hospital,Fuzhou 350007,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第23期6481-6489,共9页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82274349)
福建省自然科学基金项目(2021J011320)
国家中医药管理局2021年岐黄学者支持项目(国中医药人教函[2022]No.6)
福建中医药大学苏友新岐黄学者传承工作室项目(闽中医[2023]No.56)。
