壮骨健膝方含药血清对人退变关节软骨细胞caveolin-p38MAPK信号通路的影响

Effects of serum containing Zhuanggu Jianxi Decoction on caveolin-p38MAPK signaling pathway in human degenerative articular chondrocytes

目的:观察壮骨健膝方含药血清对人退变关节软骨细胞caveolin-p38MAPK信号通路关键信号分子及下游效应产物的影响,阐明该方含药血清保护人退变关节软骨细胞的机制。方法:取正常人及膝骨关节炎(KOA)患者软骨标本体外分离软骨细胞并培养至第3代。将正常人软骨细胞设为正常组,KOA患者软骨细胞随机分为KOA组、阻滞剂组、含药血清组及含药血清+阻滞剂组。分别用20%空白血清、20%空白血清、20%空白血清+20μmol/L SB203580、20%含药血清、20%含药血清+20μmol/L SB203580进行干预;48h后,收集细胞;采用Western Blot检测细胞中caveolin-1、p-p38蛋白表达,RT-PCR检测细胞中TNF-α、IL-1β、MMP-3、MMP-13 mRNA表达。结果:与正常组比较,KOA组软骨细胞中caveolin-1、p-p38蛋白及TNF-α、IL-1β、MMP-3、MMP-13 mRNA表达显著升高(P<0.01,P<0.05);与KOA组比较,阻滞剂组、含药血清组、含药血清+阻滞剂组软骨细胞中caveolin-1、p-p38蛋白及TNF-α、IL-1β、MMP-3、MMP-13 mRNA表达显著降低(P<0.01);与阻滞剂组比较,含药血清组软骨细胞中caveolin-1蛋白及MMP-13 mRNA表达显著降低(P<0.01,P<0.05),p-p38蛋白及TNF-α、MMP-3 mRNA表达显著升高(P<0.01,P<0.05);含药血清+阻滞剂组软骨细胞中caveolin-1、p-p38蛋白及TNF-α、IL-1β、MMP-3、MMP-13 mRNA表达显著降低(P<0.01,P<0.05);与含药血清组比较,含药血清+阻滞剂组软骨细胞中caveolin-1、p-p38蛋白及TNF-α、IL-1β、MMP-3、MMP-13 mRNA表达显著降低(P<0.01)。结论:壮骨健膝方含药血清保护人退变关节软骨细胞的机制可能与其抑制caveolin-p38MAPK信号通路中关键信号分子caveolin-1、p-p38蛋白表达,降低下游效应产物TNF-α、IL-1β、MMP-3、MMP-13 mRNA表达有关。

Objective: To observe the effects of serum containing Zhuanggu Jianxi Decoction on the key signaling molecules and downstream effect products of the caveolin-p38 MAPK signaling pathway in human degenerative articular chondrocytes, and clarify the mechanism of medicated serum to protect human degenerative articular chondrocytes. Methods: Cartilage specimens from normal human and knee osteoarthritis(KOA) patients were isolated in vitro and cultured to the third passage. Normal human chondrocytes were set as normal group, and KOA patients’ chondrocytes were randomly divided into KOA group, blocker group, medicated serum group and medicated serum group + blocker group. Five groups were intervened respectively with 20% blank serum, 20% blank serum, 20% blank serum+20μmol/L SB203580, 20% medicated serum, 20% medicated serum+20μmol/L SB203580. After 48 hours, all chondrocytes were collected. Western Blot was used to detect the expression of caveolin-1 and p-p38 proteins;RT-PCR was used to detect the expression of TNF-α, IL-1β, MMP-3, MMP-13 mRNA. Results: Compared with the normal group, the expression of caveolin-1, p-p38 proteins and TNF-α、IL-1β、MMP-3、MMP-13 mRNA in chondrocytes of the KOA group were significantly increased(P<0.01, P<0.05). Compared with the KOA group, the expression of caveolin-1, p-p38 protein, TNF-α, IL-1β, MMP-3, MMP-13 mRNA in chondrocytes of the blocker group, medicated serum group, medicated serum + blocker group were significantly decreased(P<0.01);Compared with the blocker group, the expression of caveolin-1 protein and MMP-13 mRNA in chondrocytes of medicated serum group were significantly decreased(P<0.01, P<0.05), but p-p38 protein and TNF-α, MMP-3 mRNA in chondrocytes of the medicated serum group were significantly increased(P<0.01, P<0.05), the expression of caveolin-1, p-p38 protein and TNF-α, IL-1β, MMP-3, MMP-13 mRNA in chondrocytes of the medicated serum+blocker group were decreased(P<0.01, P<0.05). Compared with the medicated serum group, the expression of caveolin-1, p-p38 protein, TNF-α, IL-1β, MMP-3, MMP-13 mRNA in chondrocytes of the medicated serum+blocker group were significantly decreased(P<0.01). Conclusion: The mechanism of Zhuanggu Jianxi Decoction medicated serum to protect human degenerative articular chondrocytes may be related to its inhibition of the expression of caveolin-1 and p-p38 proteins in caveolin-p38 MAPK signaling pathway, and the reduction of the expression of TNF-α, IL-1β, MMP-3, MMP-13 mRNA which are the pathway downstream effect products.