独活寄生汤含药血清对大鼠退变软骨细胞TLR4/NF-κB信号通路的影响

Effects of Medicated Serum of Duhuo Jisheng Decoction on TLR4/NF-κB Signaling Pathway in Rat′s Degenerated Chondrocytes

目的:观察独活寄生汤含药血清对大鼠退变软骨细胞TLR4/NF-κB信号通路相关调节因子的影响,探讨独活寄生汤治疗骨关节炎的作用机制。方法:建立体外退变软骨细胞模型,分别采用独活寄生汤含药血清(治疗组)和空白血清(模型组),干预24 h后,收集软骨细胞,采用ELISA法检测诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子α(TNF-α)和白介素6(IL-6)含量变化;Real-time PCR法检测TLR4、MyD88、TRAF6和NF-κB p65基因表达;Western blot法检测TLR4、MyD88、TRAF6和NF-κB p65蛋白表达。结果:与模型组相比,ELISA结果表明独活寄生汤含药血清可有效降低iNOS、TNF-α和IL-6含量(P<0.01);Real-time PCR结果表明独活寄生汤含药血清能抑制TLR4、MyD88、TRAF6和NF-κB p65基因表达(P<0.05);Western blot结果与Real-time PCR结果趋势一致,即独活寄生汤含药血清能抑制TLR4、MyD88、TRAF6和NF-κB p65蛋白表达(P<0.05)。结论:独活寄生汤可通过调控TLR4/NF-κB信号通路,抑制骨关节炎炎症反应,从而起到治疗骨关节炎作用。

Objective:To observe the effect of serum medicated with Duhuo Jisheng Decoction(DHJSD)on the related regulatory factors in TLR4/NF-κB signaling pathway in rat′s degenerated chondrocytes,so as to explore its mechanism on osteoarthritis treatment.Methods:The degenerated chondrocytes models in vitro were established,which were intervened by the serum medicated with Duhuo Jisheng Decoction(treatment group)and the blank serum(model group)for 24 h,then the chondrocytes were collected.ELISA method was used to measure the content of iNOS、TNF-αand IL-6.Real-time PCR method was used to detect the mRNA expression of TLR4、MyD88、TRAF6 and NF-κB p65.Western blot method was used to measure the protein levels of TLR4、MyD88、TRAF6 and NF-κB p65.Results:ELISA results showed that serum medicated with DHJSD could effectively reduce the content of iNOS、TNF-αand IL-6 compared with the model group(P<0.01).Real-time PCR results suggested that serum medicated with DHJSD could inhibit the mRNA expression of TLR4、MyD88、TRAF6 and NF-κB p65 compared with the model group(P<0.05).The results of Western blot were in agreement with the results of Real-time PCR.As serum medicated with DHJSD could inhibit the protein expression of TLR4、MyD88、TRAF6 and NF-κB p65.Conclusion:DHJSD could inhibit the inflammatory reaction in osteoarthritis by regulating TLR4/NF-κB signaling pathway,which may,in part,explain its clinical efficacy in the treatment of osteoarthritis.