基于HMGB1/NF-κB通路探讨温经通络汤对IL-1β诱导小鼠原代软骨细胞炎症的影响

Based on HMGB1/NF-κB Signal Pathway to Explore Inhibitory Effect of Wenjing Tongluo Decoction on IL-1β-Induced Inflammation in Primary Chondrocytes in Mice

  • 摘要:
      目的  观察温经通络汤对IL-1β诱导关节软骨细胞炎症的影响并探讨其作用机制。
      方法  从小鼠膝关节软骨中提取原代软骨细胞, 并用甲苯胺蓝染色法鉴定; IL-1β诱导软骨细胞炎症模型, 采用慢病毒RNAi-HMGB1转染软骨细胞, 干扰HMGB1的表达; 后用不同浓度的温经通络汤含药血清干预细胞; Western blot分析各组细胞中HMGB1、NF-κB信号通路蛋白的表达水平; qPCR检测HMGB1、NF-κB1、NF-κB2 mRNA的表达水平; ELISA检测细胞上清中炎症因子IL-6、PGE-2和TNF-α的表达水平。
      结果  与正常培养的细胞相比, IL-1β诱导软骨细胞炎症模型中HMGB1的表达水平显著上升(P < 0.01), 且p-IκBα/IκBα和p-P65/P65蛋白的相对表达量与NF-κB1、NF-κB2 mRNA的表达水平均有所提高(P < 0.01);与模型组比较, RNAi-HMGB1慢病毒转染可显著下调HMGB1的表达水平(P < 0.01), 并抑制p-IκBα/IκBα和p-P65/P65蛋白的相对表达量(P < 0.01)与NF-κB1、NF-κB2 mRNA的表达水平(P < 0.05);与模型组相比, 不同浓度的温经通络汤含药血清均表现出对HMGB1和NF-κB信号通路的抑制效应, 表现为HMGB1与p-IκBα/IκBα和p-P65/P65的蛋白表达水平下降(P < 0.05)。
      结论  温经通络汤能够改善IL-1β诱导软骨细胞炎症, 其作用机制可能与抑制HMGB1介导NF-κB信号通路的活化相关。

     

    Abstract:
      OBJECTIVE  To observe the effect of Wenjing Tongluo Decoction on IL-1β-induced inflammation of articular chondrocytes and explore its mechanism.
      METHODS  Primary chondrocytes were extracted from knee cartilage of mice and identified by toluidine blue staining. The chondrocyte inflammation model was induced by IL-1β. Lentiviral RNAi-HMGB1 was transfected into chondrocytes to interfere the expression of HMGB1. Then the cells were intervened with different concentrations of Wenjing Tongluo Decoction containing serum. Western Blot was used to analyze the protein expression of HMGB1 and NF-κB signaling pathway. The expression of HMGB1, NF-κB1, NF-κB2 mRNA was detected by qPCR. ELISA was applied to detect the expression of inflammatory factors IL-6, PGE-2 and TNF-α in cell supernatants.
      RESULTS  Compared with normal cultured cells, the expression of HMGB1 increased significantly (P < 0.01) in the IL-1β-induced chondrocyte inflammatory model, and the relative expression of p-IκBα/IκBα and p-P65/P65 proteins and the expression of NF-κB1 and NF-κB2 mRNA were increased (P < 0.01). Compared with the model group, lentiviral RNAi-HMGB1 transfection could significantly downregulate the expression of HMGB1 (P < 0.01), and inhibit the upregulation of the relative expression of p-IκBα/IκBα (P < 0.01) and p-P65/P65 protein and NF-κB1 and NF-κB2 mRNA expression (P < 0.05). Compared with the model group, different concentrations of Wenjing Tongluo Decoction showed inhibitory effects on HMGB1 and NF-κB signaling pathways, demonstrated by the decrease in the protein expression of HMGB1, p-IκBα/IκBα and p-P65/P65 (P < 0.05).
      CONCLUSION  Wenjing Tongluo Decoction can improve IL-1β-induced chondrocyte inflammation, and its mechanism may be related to inhibition of HMGB1-mediated activation of NF-κB signaling pathway.

     

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