摘要
目的探讨环磷酰胺(CTX)联合三氟拉嗪(TFP)阻滞系膜细胞周期于G0/G1期对狼疮性肾炎(LN)小鼠的保护作用。方法将6只C57BL/6小鼠作为对照组(N组),24只同周龄的MRL/LPR小鼠随机分为LN组、LN+TFP组、LN+CTX组、LN+1/2TFP+1/2CTX组。LN+TFP组小鼠每天灌胃30mg/kg的TFP;LN+CTX组每周腹腔注射60mg/kg的CTX;LN+1/2TFP+1/2CTX组的小鼠则每天灌胃15mg/kg的TFP,每两周腹腔注射60mg/kg的CTX。治疗12周后结束实验获取标本。对肾脏形态学检测,使用苏木精-伊红染色(HE)和糖原染色(PAS);生化比色法检测各组小鼠的肝、肾功能指标;蛋白质印迹法(WB法)检测增殖细胞核抗原(PCNA)、Ki67蛋白的表达。体外实验:肾小球系膜细胞分为:N组,20%FBS组、20%FBS+TFP组、20%FBS+CTX组、20%FBS+1/2TFP+1/2CTX组。流式细胞术检测各组系膜细胞细胞周期的情况,二苯基四氮唑溴盐(MTT)法检测各组系膜细胞的增殖情况;蛋白质印迹法(WB法)检测各组系膜细胞细胞周期蛋白(CyclinD1)、丝氨酸/苏氨酸激酶(AKT)、周期蛋白激酶抑制酶(p21)蛋白的表达。结果体内实验结果表明,CTX和TFP联合减量用药后系膜细胞增殖情况、血清尿素氮和血清肌酐的水平均低于LN组(P<0.05),且低于单独用药的LN+CTX组和LN+TFP组(P<0.05);LN+1/2TFP+1/2CTX组小鼠肾组织PCNA,Ki67蛋白表达均低于LN组且低于单独用药的LN+CTX组和LN+TFP组(F_(Ki67)=36.08,F_(PCNA=)10.04,P<0.01)。体外实验MTT法显示各组细胞间、12 h,24 h,36 h及组间与时间的交互效应的差异均有统计学意义(F_(组间)=337.83,F_(时间)=55.96,F_(交互)=7.73,P<0.001);流式细胞术结果显示相较于单独用药,20%FBS+1/2CTX+1/2TFP组G0/G1期细胞比例明显增加,S、G2期细胞比例明显降低;WB结果显示20%FBS+1/2CTX+1/2TFP组细胞p21蛋白的表达高于20%FBS组且高于单独用药的20%FBS+CTX组和20%FBS+TFP组(F_(p21)=12.78,P<0.01),20%FBS+1/2CTX+1/2TFP组的CyclinD1、p-AKT/AKT蛋白的表达均低于20%FBS组且低于单独用药的20%FBS+CTX组和20%FBS+TFP组(F_(p-AKT/AKT)=32.88,F_(CyclinD1)=13.59,P<0.01)。结论环磷酰胺联合三氟拉嗪减量使用可以将系膜细胞周期阻滞于G0/G1期,减轻系膜细胞的增殖,从而保护LN小鼠。
Objective To explore the safeguarding effect of trifluoperazine(TFP)when amalgamated with cyclophosphamide(CTX)on arresting the mesangial cell cycle at the G0/G1 phase in lupus nephritis(LN)-afflicted mice.Methods Six C57BL/6 mice were used as the control group(N),and 24 MRL/LPR mice of the same week were randomly divided into LN,LN+TFP,LN+CTX,and LN+1/2TFP+1/2CTX.Mice in the LN+TFP were given 30 mg/kg of TFP per day.In the LN+CTX group,mice were injected with 60mg/kg of CTX intraperitoneally once a week,while in the LN+1/2TFP+1/2CTX group,mice were given 15mg/kg of TFP orally every day and injected with 60mg/kg of CTX intraperitoneally every two weeks.After 12 weeks of treatment,the experiment was completed and specimens were obtained.HE staining and PAS staining were used to the morphological detection of kidneys.The biochemical spectrophotometry were used to the liver and kidney function indicators of each group of mice.WB method was used to the expression of PCNA and Ki67 protein.Glomerular mesangial cells were divided into N,20%FBS,20%FBS+TFP,20%FBS+CTX,20%FBS+1/2TFP+1/2CTX.Flow cytometry was used to detect the cell cycle of mesangial cells in each group,and the proliferation of mesangial cells in each group was detected by diphenyltetrazolium bromide salt(MTT)method.WB was used to the expressions of AKT,CyclinD1 and p21 in mesangial cells.Results The in vivo experiment results show that the pathological damage to mesangial cells,BUN and SCR levels were all lower in the CTX+TFP group than in the LN group(P<0.05),and also lower than in the LN+CTX group and the LN+TFP group treated with single drugs(p<0.05).The expression of PCNA and Ki67 protein in kidney tissue of mice in the LN 1/2TFP 1/2CTX group was lower than that in the LN group and lower than that in the LN CTX group and LN TFP group(F_(Ki67)=36.08,F_(PCNA)=10.04,P<0.01).In vitro experiments The MTT experiments conducted in vitro revealed striking disparities in the combined effects of different groups over time(F_(group)=337.83,F_(time)=55.96,F_(interaction)=7.73,P<0.001).Furthermore,flow cytometry analysis demonstrated a notable increase in the percentage of cells in the G0/G1 phase within the 20%FBS 1/2CTX 1/2TFP group,accompanied by a significant decrease in cells in the S and G2 phases when compared to treatment with the drug alone.The WB results showed that the expression of p21 protein in the 20%FBS 1/2CTX 1/2TFP group was higher than that in the 20%FBS group and higher than that in the 20%FBS CTX group and the 20%FBS TFP group with single medication(F_(p21)=12.78,P<0.01),in the group with 20%FBS+1/2 CTX+1/2 TFP,the expression of CyclinD1 and p-AKT/AKT protein was lower than the group with 20%FBS and lower than the groups with 20%FBS+CTX and 20%FBS+TFP taken alone(F_(p-AKT/AKT)=32.88,F CyclinD1=13.59,P<0.01).Conclusion The reduction of cyclophosphamide combined with trifluoperazine can arrest the mesangial cell cycle at the G0/G1 phase,so as to inhibit the proliferation of mesangial cells,thereby protecting lupus nephritis mice.
作者
何娟
严盘
成瑶
田艳
李荣山
王宝栋
HE Juan;YAN Pan;CHENG Yao;TIAN Yan;LI Rongshan;WANG Baodong(The Third Clinical College of Shanxi University of Traditional Chinese Medicine,Jinzhong 030619,Shanxi,China;Department of Nephrology,Shanxi Provincial People's Hospital,Taiyuan 030012,China;Department of Nephrology,The Fifth Clinical Medical College,Shanxi Medical University,Taiyuan 030012,China)
出处
《西部医学》
2025年第9期1293-1299,共7页
Medical Journal of West China
基金
山西省大学生创新创业项目(20240422)。
关键词
狼疮性肾炎
三氟拉嗪
环磷酰胺
细胞周期
细胞增殖
Lupus nephritis
Trifluoperazine
Cyclophosphamide
Cell cycle
Cell proliferation
