三氟拉嗪上调叉头框蛋白O1表达对MRL/lpr狼疮肾炎小鼠的保护作用

Protective effect of trifluoperazine on MRL/LPR mice by upregulating FoxO1 expression

目的探讨三氟拉嗪通过叉头框蛋白O1(FoxO1)的表达对系膜细胞增殖的影响及对狼疮肾炎(LN)小鼠的保护作用。方法体外实验:利用二苯四氮唑溴盐(MTT)法检测不同浓度三氟拉嗪对系膜细胞的增殖情况的影响;流式细胞术检测不同浓度三氟拉嗪对系膜细胞周期的影响;蛋白质印迹法(WB法)检测不同浓度三氟拉嗪对系膜细胞FoxO1、细胞周期蛋白(cyclinD1)表达的影响,及增强或抑制FoxO1表达后,三氟拉嗪对系膜细胞、cyclinD1,P21的表达;体内实验:通过苏木精-伊红(HE)染色、免疫组织化学、WB法检测三氟拉嗪对MRL/lpr小鼠肾小球系膜细胞增殖情况,ELISA方法检测三氟拉嗪对LN小鼠肾功能的影响,WB法检测三氟拉嗪对系膜细胞所分泌基质纤维连接蛋白(FN),Ⅰ型胶原蛋白(Col1)表达水平。计量资料组间比较采用重复测量方差分析和单因素方差分析,进一步两两比较采用Bonferroni法。结果三氟拉嗪抑制系膜细胞的增殖,组间、时间及组间×时间的交互效应均有统计学意义(F组别=162.58,F时间=50.84,F交互=19.12,P均<0.001),呈现出浓度依赖性;流式细胞术结果显示,不同浓度的三氟拉嗪处理系膜细胞后,G0/G1期的细胞百分比逐渐增加,而S期的细胞逐渐减少,这种效应具有剂量依赖性,差异具有统计学意义(P<0.05);WB法结果证明,三氟拉嗪抑制cyclinD1的表达(2.17±0.34,1.49±0.20,1.11±0.27,0.15±1.55,F=33.60,P<0.001),上调FoxO1的表达(0.81±0.45,2.31±0.81,3.51±0.52,5.13±10.07,F=35.63,P<0.001),且具有剂量依赖性;体内实验结果表明:肾脏病理提示三氟拉嗪可以抑制LN小鼠系膜细胞的增殖,差异具有统计学意义(F=8.47,P=0.007),ELISA法表明三氟拉嗪并对肾功能具有一定的保护作用[血肌酐:正常组(144±23)μmol/L,LN组(237±14)μmol/L,LN+三氟拉嗪组(211±36)μmol/L,F=20.47,P<0.001,尿素氮:正常组(22.84±0.56)μmol/L,LN组(19.99±0.92),LN+三氟拉嗪组(13.57±0.25)μmol/L,F=331.96,P<0.001],且通过WB法证明三氟拉嗪可抑制FN、Col1的表达,差异具有统计学意义(FFN=1 312.83,FCol1=171.16,P<0.001)。结论三氟拉嗪通过增加FoxO1的表达阻滞系膜细胞周期于G0/G1期,抑制细胞增殖,可能对LN具有一定治疗作用。

Objective To investigate the effect of trifluoperazine(TFP)on the proliferation of mesangial cells through FoxO1 pathway and its protective effect on lupus nephritis(LN)mice.Methods In vitro,MTT assay was used to detect the effect of different concentrations of trifluoperazine on the proliferation of mesangial cells.Flow cytometry was used to detect the effect of different concentrations of TFP on the mesangial cell cycle.Western blotting method(WB method)was used to detect the effect of different concentrations of TFP on the expression of FoxO1 and cyclinD1 proteins in mesangial cells,and the expression of TFP on mesangial cells,cyclinD1 and P21 after enhancing or inhibiting the expression of FoxO1.The effect of TFP on the proliferation of MRL/LPR mouse mesangial cells and the expression of FoxO1 was detected by HE staining,immunohistochemistry and WB method,and the effect of TFP on renal function in LN mice was detected by ELISA method in vivo.WB method was used to detect the effect of TFP on mesangial cell fibrosis,that is,the protein expression levels of FN and Col1.Repeated measures analysis of variance and One-way analysis of variance were used to compare measurement data between groups,and further pairwise comparisons were made using the Bonferroni method.Results Trifluoperazine inhibits the proliferation of mesangial cells,and the interaction effects was concentration dependence and were statistically signifiant between groups,time(Fgroup=162.58,Ftime=50.84,Finteraction=19.12,P<0.001).Flow cytometry results showed that after mesangial cells were treated with trifluoperazine at different concentrations,the percentage of cells in the G0/G1 phase gradually increased,while the cells in the S phase gradually decreased.This effect was dose-dependent,the difference was statistically significant(P<0.05).Results of WB test proved that trifluoperazine inhibited the expression of cyclinD1 protein(2.17±0.34,1.49±0.20,1.11±0.27,0.15±1.55,F=33.60,P<0.001)and up-regulated the expression of FoxO1(0.81±0.45,2.31±0.81,3.51±0.52,5.13±10.07,F=35.63,P<0.001),and also in a dose-dependent patten.In vivo experimental results showed that trifluoperazine could inhibit the proliferation of mesangial cells and promote the expression of FoxO1 in mice with lupus nephritis,and the difference wsa statistically significant(F=8.47,P=0.007).ELISA test results showed that trifluoperazine had a protective effect on renal function[serum creatinine:normalgroup(144±23)μmol/L,LNgroup(237±14)μmol/L,LN+TFP(211±36)μmol/L,Fvalue=20.47,P<0.001,urea nitrogen:normal group(22.84±0.56)μmol/L,LN group(19.99±0.92)μmol/L,LN+TFP(13.57±0.25)μmol/L,F=331.96,P<0.001]and it was proved by WB method that trifluoperazine could inhibit Fn and Col1 expression,the difference was statistically significant(FFN=1312.83,FCol1=171.16,P<0.001).Conclusion Trifluoperazine blocks the mesangial cell cycle in G0/G1 phase by increasing the expression of FoxO1 and inhibits cell proliferation,which may have a therapeutic effect on lupus nephritis nephritis.