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目的观察穿山龙总皂苷含药血清对IL-17和TNF-α联合诱导的大鼠滑膜细胞株RSC-364 NF-κB p65活性、STAT3表达及VEGF mRNA表达水平的影响,探讨穿山龙总皂苷抑制类风湿性关节炎血管新生的作用机制。方法制备穿山龙总皂苷和雷公藤(阳性对照)含药血清;取大鼠滑膜细胞株RSC-364经IL-17(10μg/L)和TNF-α(10μg/L)、穿山龙总皂苷含药血清、雷公藤多甙片含药血清单独或联合应用孵育,孵育24 h,提取各组细胞核蛋白用于TransAMTMNF-κB p65活性检测试剂盒检测NF-κB p65的DNA结合活性;提取各组细胞总蛋白后应用Western blot方法观察STAT3蛋白的表达;采用实时荧光定量PCR方法检测各组细胞VEGF mRNA的表达情况。结果 IL-17+TNF-α诱导的细胞模型组中NF-κB p65的DNA结合活性、STAT3蛋白表达及VEGFmRNA表达水平均显著高于空白对照组(P<0.01,P<0.01,P<0.05);与细胞模型组相比,雷公藤含药血清组、穿山龙总皂苷含药血清组的NF-κB p65 DNA结合活性、STAT3蛋白表达及VEGF mRNA表达水平均显著降低(P<0.01,P<0.01,P<0.05),且2组之间比较无显著性差异(P>0.05)。结论本研究证实穿山龙总皂苷含药血清可以抑制NF-κB p65的DNA结合活性及STAT3蛋白的表达,考虑穿山龙总皂苷通过上述信号转导途径来调控血管新生关键因子VEGF的产生,进而抑制RA血管新生。
Objective To observe the effects of total saponins of Asiastia medicinalum on the activity of RSC-364 NF-κB p65, the expression of STAT3 and the expression of VEGF mRNA in rat synovial cell line induced by IL-17 and TNF-α, Rheumatoid arthritis angiogenesis mechanism of action. Methods The total serum of Radix Scutellariae and Tripterygium wilfordii (positive control) were prepared. The synovial cell line RSC-364 was induced by IL-17 (10μg / L) and TNF- Serum and tripterygium glycosides tablets containing drug serum alone or in combination incubated for 24 h, each group of nuclear protein was extracted for detection of DNA binding activity of NF-κB p65 by TransAMTMNF-κB p65 activity detection kit; Western blot was used to observe the expression of STAT3 protein. The expression of VEGF mRNA in each group was detected by real-time fluorescence quantitative PCR. Results The DNA binding activity, STAT3 protein and VEGF mRNA expression of NF-κB p65 in the model group induced by IL-17 + TNF-α were significantly higher than those in the blank control group (P <0.01, P <0.01, P <0.05) ; Compared with the cell model group, the content of NF-κB p65 DNA binding, the expression of STAT3 protein and the level of VEGF mRNA in tripterygium wilfordii containing drug serum group and PNS group were significantly decreased (P <0.01, P <0.01 , P <0.05), and there was no significant difference between the two groups (P> 0.05). Conclusions This study demonstrated that S. argenosus serum can inhibit DNA-binding activity and STAT3 protein expression of NF-κB p65, and that S. pilosicoli regulates the production of VEGF, a key factor of angiogenesis, through these signal transduction pathways, thereby inhibiting the growth of RA blood vessels newborn.