三七皂苷对高糖诱导的大鼠肾小球系膜细胞保护作用及其机制研究

Protective Effects and Mechanisms of Panax Nologinoside on Rat Mesangial Cells Induced by High Glucose

[目的]从氧化应激及炎症角度观察三七皂苷(panax nologinoside,PNS)对高糖诱导的大鼠肾小球系膜细胞(rat mesangial cell,RMC)保护作用及从SIRT1/NF-κB信号通路探讨其机制。[方法]培养RMC,并将其分为4组:正常对照组、高糖组、PNS组及SIRT1RNAi+PNS组;采用MTT及Annexin V-FITC分别检测细胞活力及凋亡情况;采用Real-time PCR技术检测RMC SIRT1、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)的m RNA表达;Western Blot技术检测SIRT1和乙酰化NF-κB P65蛋白的表达;采用经典的氮蓝四唑(NBT)显色法检测超氧化物歧化酶(superoxide dismutase,SOD)活性;ELISA检测细胞培养上清液中TNF-α、TGF-β1、丙二醛(malondialdehyde,MDA)等的浓度。[结果]与正常对照组比较,高糖刺激使RMC活力降低,细胞凋亡增加,SOD活性降低,MDA含量增加,SIRT1基因及蛋白表达降低,NF-κB P65蛋白乙酰化水平增高,TNF-α、TGF-β1的m RNA和蛋白水平增高。PNS干预可逆转高糖引起的上述变化,但沉默SIRT1基因后,PNS干预作用不明显。[结论]PNS对高糖诱导的RMC具有保护作用,其机制可能通过增加SIRT1表达及活性,降低NF-κB P65蛋白乙酰化水平,使其转录活性降低,进而抑制炎症及氧化应激。

[Objective]To explore the effect and mechanism of antioxidant and antiflammation of Panax Nologinoside（PNS） on high glucose cultured rat mesangial cells（RMC） from the SIRT1/NF-κB singal pathway. [Methods] RMC was cultured in DMEM medium supplemented with 10% FBS and was divided into control group, high glucose group, PNS group and SIRT1 RNAi ＋PNS group. The cell viability and apoptosis were analyzed by MTT and Annexin V-FITC respectively. The m RNA expression of SIRT1, TNF-α and TGF-β1was analyzed by Real-time quantitative PCR. The protein expression of SIRT1 and the acetylation of NF-κB P65 subunit were determined by Western blot. The activity of superoxide dismutase（SOD） was detected by using the classical method of nitroblue tetrazolium（NBT）.The protein concentration of TNF-α, TGF-β1and MDA was detected by ELISA. [Results]Compared with the control group, the cell viability, SOD activity, the expression of SIRT1 m RNA and protein levels were decreased by high glucose treatment. The cell apoptosis, the acetylation of NF-κB P65 subunit were significantly increased after interfered with high glucose, which resulted in the increases of TNF-α and TGF-β1secretions. PNS intervention can reverse the changes induced by high glucose. However, after silencing SIRT1 gene, PNS cannot significantly decrease the level of acetylation of NF-κB P65 subunit and the expression of TNF-α, TGF-β1by high glucose-induced. [Conclusion]PNS remarkably inhibits oxidant stress injury and the inflammatory reactions. The mechanisms may be involved in decreasing the level of acetylation of NF-κB P65 subunit via regulating activity of SIRT1.