穿山龙总皂苷含药血清对IL-17和TNF-α诱导的大鼠滑膜细胞株RSC-364NF-κB p65活性、STAT3及VEGF mRNA表达的影响

Effects of Rhizoma Dioscoreae Nipponicae total saponin -medicated serum on NF-κB p65 activity, STAT3 protein expression,and VEGF mRNA expression in IL-17- and TNF-α-induced rat synovial cell strain RSC-364

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。

To evaluate the effects of Rhizoma Dioscreae Nipponicae（RDN） total saponin on angiogenesis of rheumatoid arthritis,we observed the effects of RDN total saponin-medicated serum on NF-κB p65 activity,STAT3 protein expression and VEGF mRNA expression in IL-17 and TNF-α-induced rat synovial cell strain RSC-364.Medicated serum of total saponin from RDN and Tripterygium（positive control group） were prepared.Then rat synovial cell strain RSC-364 was stimulated by IL-17（10 μg/L） ＋ TNF-α（10 μg/L） in the presence or absence of RDN total saponin-medicated serum or Tripterygium-medicated serum.After twenty-four hours incubation,the NF-κB p65 DNA-binding activity in nuclear extract of rat synovial cell strain RSC-364 was detected by TransAM TM NF-κB p65 Transcription Factor Assay Kit.Western blot was used to observe the expression of STAT3 protein in each cells group,while VEGF mRNA expression in rat synovial cell strain RSC-364 in each group was detected by PrimeScript TM real-time quantitative PCR detection kit.Compared with the control group,the NF-κB p65 DNA-binding activity and the expressions of STAT3 protein and VEGF mRNA were all increased remarkable in rat synovial cell strain RSC-364 induced by IL-17＋TNF-α model group（P〈0.01,P〈0.01,P〈0.05）.While the NF-κB p65 DNA-binding activity and the expressions of STAT3 protein and VEGF mRNA in Tripterygium medicated serum group and RDN total saponin-medicated serum group were remarkably lower than those of IL-17＋ TNF-α model group（P〈0.01,P〈0.01,P〈0.05）.There was no significant difference between the two medicated serum groups（P〉0.05）.In conclusion,RDN total saponin-medicated serum could remarkably lower NF-κB p65 DNA-binding activity and STAT3 expression,thus regulate VEGF secretion and restrain angiogenesis of rheumatoid arthritis.