中国中西医结合杂志2025，Vol.45Issue(4)：429-439,11.DOI:10.7661/j.cjim.20241220.300
痹痿同治同调控NF-κB、p38MAPK通路延缓滑膜—软骨细胞共培养体系软骨细胞退变
The Co-regulation of NF-κB and p38MAPK Pathways by Arthralgia-Flaccidity Simultaneous Treatment Delayed the Degeneration of Chondrocytes in Synovial-Chondrocyte Co-culture System
摘要
Abstract
Objective To investigate the interrelationship between synoviocyte inflammation and chondrocyte degeneration based on a cell co-culture system,to study the effect and mechanism of Zhuanggu Jianxi Decoction(ZGJXD)containing serum on inflammatory synoviocytes and normal chondrocytes through synoviocyte-chondrocyte interaction,and further to elucidate the scientific connotation of"arthralgia-flaccidity simultaneous treatment"of ZGJXD.Methods(1)Transwell was employed to establish a co-culture system of synoviocytes and chondrocytes,which was divided into six groups,i.e.Group A(normal chondrocytes),Group B(normal synoviocytes),Group C(normal synoviocytes+normal chondrocytes),and Group D(inflammatory synoviocytes+normal chondrocytes),Group E(normal synoviocytes+degenerated chondrocytes),Group F(inflammatory synoviocytes+degenerated chondrocytes).Following a 48-h cultivation chondrocyte viability was detected by CCK-8 assay.The expressions of collagen type Ⅱ alpha 1(COL2A1)and matrix metalloproteinase-13(MMP-13)mRNA in chondrocytes were determined by RT-qPCR.MMP-13,interleukin-1 beta(IL-1β),and tumor necrosis factor alpha(TNF-α)in chondrocytes,as well as IL-1 β and TNF-α contents in synoviocytes were detected by enzyme-linked immunosorbent assay(ELISA).Western Blot assay was used to detect COL2A1,p38 mitogen-activated protein kinase(p38MAPK),and phosphorylated p38 mitogen-activated protein kinase(p-p38MAPK)in chondrocytes,as well as nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB p65)and IκB alpha protein expressions in synoviocytes.(2)Further the rabbit synovial inflammatory cell model was constructed and a co-culture system of rabbit synovial cells and chondrocytes established,which were divided into the normal group(normal synovial cells+normal chondrocytes),the model group(inflammatory synovial cells+normal chondrocytes),and Zhuanggu Jianxi Decoction(ZGJXD)group(inflammatory synovial cells+normal chondrocytes).Following a 48 h intervention period with 10％blank serum,10％blank serum,and 10％ZGJXD containing serum,respectively.RT-qPCR was used to assess the expressions of IL-1 β,TNF-α,IL-6,and proliferating cell nuclear antigen(PCNA)in the synoviocytes of various groups,and the mRNA expressions of MMP-13,COL2A1,and aggrecan in the chondrocytes.Western Blot was used to detect PCNA,IκBα,and intranuclear NF-κB p65 protein expressions in synoviocytes,and COL2A1,MMP-13,p38MAPK,and p-p38MAPK protein expressions in chondrocytes.Results(1)A comparison of the corresponding cells in group C with those in groups D,E,and F demonstrated a significant decline in cell viability,COL2A1 mRNA and protein expressions in chondrocytes(P＜0.05).mRNA expression of MMP-13,and the contents of MMP-13,IL-1β,and TNF-α were significantly elevated(P＜0.05).Similarly,the p-p38MAPK protein expression and the p-p38MAPK/p38MAPK ratio significantly increased(P＜0.05).Furthermore,IL-1β and TNF-α mRNA expressions and contents,and intranuclear NF-κB p65 protein expression in synoviocytes significantly increased(P＜0.05),while IκBα protein expression significantly decreased(P＜0.05).Compared with the corresponding cells in group D,the expressions and contents of IL-1 β and TNF-α mRNA,and the expression of NF-κB p65 protein in the nucleus were significantly higher(P＜0.05)in synoviocytes of group F.In contrast,the expression of IκB-α protein was significantly lower(P＜0.05).A comparison of the corresponding cells in group E with those in group F revealed a significant reduction in cell viability,COL2A1 mRNA and protein expressions in the chondrocytes of group F(P＜0.05).In contrast,the chondrocytes of group F demonstrated significantly elevated mRNA expression of MMP-13,accompanied by elevated contents of MMP-13,TNF-α,and IL-1β,p-p38MAPK protein expressions,and a higher p-p38MAPK/p38MAPK ratio(P＜0.05).(2)The content and mRNA expression of COL2A1 in chondrocytes were the lowest.and the mRNA expression of MMP-13 was the highest in the co-culture of inflammatory synoviocytes and chondrocytes of rabbits for 48 h,which was used as the cell culture condition of the model group for subsequent experiments.Compared with the normal control group,the levels of IL-1 β,TNF-α,IL-6,and PCNA mRNA were significantly elevated(P＜0.05),while IκBα protein expression was significantly reduced(P＜0.05),and the levels of PCNA and intranuclear NF-κB p65 protein were significantly elevated(P＜0.05)in synoviocytes of the model group.Additionally,the mRNA expression of MMP-13 in chondrocytes was significantly elevated(P＜0.05),while the mRNA expression of COL2A1 and Aggrecan was significantly reduced(P＜0.05),and the protein expression of COL2A1 was significantly reduced(P＜0.05),and the p-p38MAPK protein expression and p-p38MAPK/p38MAPK ratio were significantly elevated(P＜0.05).Compared with the model group,ZGJXD group showed a notable reduction of IL-1β,TNF-α,IL-6,and PCNA mRNA expressions in synoviocytes(P＜0.05),an increase in IκBα protein expression(P＜0.05),and a decrease in PCNA and intranuclear NF-κB p65 protein expressions(P＜0.05).The mRNA expression of MMP-13 in chondrocytes was significantly reduced(P＜0.05),while the mRNA expression of COL2A1 and Aggrecan was significantly elevated(P＜0.05).Additionally,the protein expression of COL2A1 was significantly elevated(P＜0.05),protein expression of MMP-13,p-p38MAPK and p-p38MAPK/p38MAPK ratio significantly decreased(P＜0.05).Conclusions Inflammatory synoviocytes can cause and exacerbate chondrocyte degeneration,and degenerated chondrocytes can cause and exacerbate synoviocyte inflammation as a result of the interaction between the two.Meanwhile,ZGJXD was demonstrated to inhibit the inflammatory reaction and over-proliferation of synovial fibroblasts,as well as to attenuate the degeneration of chondrocytes caused by inflammatory synovial fibroblasts.Its mechanism of action may be related to inhibiting the activion of NF-κB in synovial fibroblasts and the p38MAPK signaling pathway in chondrocytes.关键词
膝骨关节炎/壮骨健膝方/痹痿同治/核因子-κB通路/p38丝裂原活化蛋白激酶通路/中药复方Key words
knee osteoarthritis/Zhuanggu Jianxi Decoction/arthralgia-flaccidity simultaneous treatment/nuclear factor-κB pathway/p38 mitogen-activated protein kinase pathway/Chinese herbal compound引用本文复制引用
郭洁梅,苏友新,王美玲,杨瑞芳,张鹏,张英杰,郑卓铭,邱灵,肖艳,陈鹏..痹痿同治同调控NF-κB、p38MAPK通路延缓滑膜—软骨细胞共培养体系软骨细胞退变[J].中国中西医结合杂志,2025,45(4):429-439,11.基金项目
国家自然科学基金项目(No.82274349) （No.82274349）
国家中医药管理局2021年岐黄学者支持项目(No.国中医药人教函[2022]No.6) （No.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(No.闽中医[2023]No.56) （No.闽中医[2023]No.56）
