大鼠肾小球系膜细胞活性氧簇JAK2/STAT5通路与TIMP-1之间的关系及对其凋亡的影响

The Relationship Among ROS,JAK2/STAT5 Signal Pathway and TIMP-1 and the Effect of them on Apoptosis in Rat Mesangial Cells

目的:探讨高糖(HG)条件下大鼠肾小球系膜细胞(RMC)活性氧簇(ROS)、JAK2/STAT5信号通路与金属蛋白酶1组织抑制剂(TI MP-1)之间的关系及对其凋亡的影响。方法:在HG培养条件下的RMC中,根据是否使用DPI(NADPH氧化酶特异性抑制剂,可抑制ROS产生)和AG490(JAK2特异性抑制剂)刺激24 h,将RMC分为HG组、HG+AG490组、HG+DPI组和HG+AG490+DPI组四组。采用流式细胞技术检测各组细胞的凋亡率,RT-PCR检测各组RMC Bcl-xl、Cyclin D1、P27kip1、JAK2和TI MP-1 mRNAs的表达,Western blot检测胞浆中JAK2、STAT5及相应磷酸化蛋白的表达。结果:HG组、HG+AG490组、HG+DPI组和HG+AG490+DPI组的细胞总凋亡率分别为:(10.69±0.26)%、(20.52±0.51)%、(24.56±0.36)%和(26.01±0.28)%;加入AG490和(或)DPI后RMC总凋亡率明显升高(P<0.01),Bcl-xl、Cyclin D1、JAK2和TI MP-1 mRNAs表达显著减少,P27kip1 mRNA表达增加。AG490和(或)DPI可使各组RMC P-JAK2和P-STAT5的表达显著减少,尤其在加入DPI后减少更加明显。结论:HG条件下JAK2/STAT5信号通路受ROS调控;阻断ROS生成和(或)JAK2/STAT5信号通路,可使RMC TI MP-1 mRNA表达减少,凋亡增加。

Objective：To investigate the relationship among Reactive Oxygen Species（ROS）,JAK2/STAT5 signal pathway and TIMP-1 and the effect of them on apoptosis in rat mesangial cells under the condition of high glucose.Methods：After RMC was stimulated with HG,DPI（specific inhibitor of NADPH oxidase） and AG490（specific inhibitor of JAK2） for 24 hours,the total apoptosis rate of RMC was measured by flow cytometry.The expression of Bcl-xl,Cyclin D1,P27kip1,JAK2 and TIMP-1 mRNAs was assayed by RT-PCR.The expression of JAK2,STAT5,P-JAK2 and P-STAT5 proteins was analysed by Western blot.Results：The total apoptosis rates of HG,G＋AG490,HG＋DPI and HG＋AG490＋DPI groups were（10.69±0.26）%,（20.52±0.51）%,（24.56±0.36）% and（26.01±0.28）%,respectively.AG490 and（or） DPI treatment enhanced the apoptosis（P0.01） and the expression of P27kip1 mRNA,however,it obviously decreased the expression of Bcl-xl,Cyclin D1,JAK2,TIMP-1 mRNAs and the expression of P-JAK2 and P-STAT5 proteins in RMC.Conclusion：Under the condition of high glucose,JAK2/STAT5 signal pathway could be regulated by ROS;the inhibition of ROS and（or）JAK2/STAT5 signal pathway decreased the expression of TIMP-1 mRNA and increased apoptosis rate in RMC.