三七皂苷通过PPARγ调控SIRT1介导的狼疮肾炎小鼠脾淋巴细胞激素耐药及脂代谢影响的研究

PNS Regulate SIRT1 on Steroid Resistance and Lipid Metabolism in Spleen Lymphocyte of LN Mice by Acting on PPAR Gamma

[目的]观察三七皂苷(Panax notoginseng saponin,PNS)对激素耐药狼疮肾炎(lupus nephritis,LN)小鼠脾淋巴细胞过氧化物酶增殖体活化受体γ(peroxidase proliferator activated receptor gamma,PPARγ)、沉默信息调节因子相关酶1(silent information regulation factor related enzyme 1,SIRT1)和三磷酸腺苷结合盒转运体A1(adenosine triphosphate binding cassette transporter A1,ABCA1)表达的影响,探索PNS改善激素耐药和脂代谢紊乱的双重作用,为运用活血化瘀中药提高难治性LN临床疗效提供科学依据。[方法]采用密度梯度法分离NZB/WF1小鼠的脾淋巴细胞,用小剂量甲基强的松龙诱导细胞产生激素耐药,将诱导后的细胞分为模型组、PPARγ质粒转染组、PPARγsi RNA+PNS组和PNS组,并以未诱导耐药的NZB/WF1小鼠脾淋巴细胞作为对照组,各组细胞培养72h后以Real-time PCR检测各组淋巴细胞PPARγ和SIRT1 m RNA表达,Western blot检测PPARγ和SIRT1蛋白表达,荧光分光光度法检测细胞内胆固醇酯含量,流式细胞术检测ABCA1蛋白表达。[结果](1)上调PPARγ能抑制SIRT1高表达,模型组PPARγm RNA和蛋白表达水平均低于对照组,SIRT1 m RNA和蛋白表达水平均高于对照组(P<0.05);通过质粒转染上调PPARγ水平后,PPARγ质粒转染组的SIRT1 m RNA和蛋白表达水平均低于模型组(P<0.05);(2)PNS组PPARγ表达高于模型组,SIRT1表达则减低(P<0.05)。尽管PNS组PPARγm RNA表达水平低于PPARγ质粒转染组,但两组SIRT1 m RNA表达水平无统计学差异(P>0.05);与模型组比较,PPARγsi RNA+PNS组的SIRT1 m RNA和蛋白表达水平均下降(P<0.05)。(3)PNS具有调节细胞内脂代谢紊乱作用,模型组细胞内胆固醇酯含量低于对照组(P<0.05);PNS组细胞内胆固醇酯含量高于对照组,ABCA1蛋白表达则低于对照组(P<0.05)。[结论](1)激素耐药的LN小鼠脾淋巴细胞内存在脂代谢紊乱。(2)PNS具有逆转激素耐药和调节细胞内脂代谢紊乱的双重效应。

[Objective] To observe the effect of Pozu~ rtotoginseng saponin（PNS） on steroid resistance in spleen lymphocyte of lupus nephritis（LN） mice for peroxisome proliferator activated receptor gamma（PPAR~/）, silent information regulation faction related enzyme I（SIRT1） and ATP binding cassette transporter A1 （ABCA1） expression, and explore the dual role of steroid resistance and lipid metabolism disorder, providing a scientific basis for the use of Chinese herbal medicine to improve the clinical effect of refractory LN. [Methods]Splenic lymphocyte of NZB,rWF1 lupus mice is separated by density gradient method and induced steroid resistance by small dose of methylprednisolone. It is the establishment of LN mice spleen lymphocyte steroid resistance model, and divided into model group, PPAR gamma plasmid transfection group, PPAR gamma siRNA by PNS intervention group, and PNS intervention group. Meanwhile, it is used to NZB/WF1 LN mice spleen lymphocyte as control group. After 72h culture, PPAR gamma and SIRT1 mRNA expression is detected by Real-time fluorescence quantitative PCR; PPAR gamma and SIRT1 protein expression is detected by Western blot; cholesterol ester is detected by fluorescence spectrophotometer;, expression of ABCA1 protein is detected by flow eytometry. [Results] （1） The up-regulation of PPAR gamma can inhibit the high expression of SIRT1. The expression level of PPAR gamma mRNA and protein in model group is significantly lower than that in control group, and the expression level of SIRT1 mRNA and protein increased significantly（P〈0.05）. SIRT1 expression is decreased after upregulated PPAR gamma level compared with model group（P〈0.05）; （2）PNS has the dual effects of upregulated PPAR gamma and inhibited SIRT1. PPAR gamma is significantly higher in PNS group than that in model group, and expression of SIRT1 is decreased significantly（P〈0.05）. Although the level of PPAR gamma mRNA in PNS group is significantly lower than that in plasmid transfection group, the inhibition level of SIRT1 is similar in two groups（P〉O.05）. Compared with model group, expression of SIRT1 mRNA and protein are still decreased in PPAR siRNA by PNS intervention group（P〈0.05）. （3） PNS has the effect of regulating lipid metabolism disorder. The expression of cholesterol ester in the model group is lower than that in the control group（P〈0.05）, indicating that there is a lipid metabolic disorder in LN mice spleen lymphocyte steroid resistance model（P〈0.05）. PNS can improve the content of cholesterol ester in the model group（P〈 0.05）, and decrease the expression of ABCA1 protein in spleen lymphocyte steroid resistance model of lupus nephritis miee（P〈0.05）.[Conelusion] （1）There is a disorder of lipid metabolism in the steroid resistance of splenic lymphocyte of NZB/WF1 LN miee.（2）PNS has a dual role in reversing steroid resistance and improving lipid metabolism disorder.