JAK/STAT通路在金属蛋白酶1组织抑制剂抑制肾小球系膜细胞凋亡中的作用

目的 探讨金属蛋白酶1组织抑制剂（TIMP-1）抑制大鼠肾小球系膜细胞（RMC）凋亡与Janus激酶/信号转导和转录激活子（JAK/STAT）通路的关系。 方法 用无血清培养基体外培养pcDNA3空载体、人正义、反义TIMP-1基因重组真核表达载体转染RMC。根据是否加JAK2特异性抑制剂AG490刺激24 h,将细胞分为未转染组、未转染＋AG490组、空载体组、空载体＋AG490组、正义组、正义＋AG490组、反义组和反义＋AG490组。另外设正常培养条件下的RMC作为正常对照组。应用流式细胞技术检测各组RMC的凋亡率。RT-PCR检测TIMP-1、bcl-xl、cyclin D1、p27kip1和JAK2 mRNA的表达。Western印迹检测胞质中JAK2、STAT3、STAT5及其相应磷酸化蛋白(p-JAK2、p-STAT3、p-STAT5)的表达。 结果 未转染组、正义组及反义组RMC凋亡率分别为（10.59±0.96）％、（7.08±0.43）％和（21.91±0.25）％，各组间差异有统计学意义(P < 0.05或P < 0.01)。在未加AG490的各组RMC中,bcl-xl和cyclin D1 mRNA在正义组中表达最高，反义组中最低，p27kip1 mRNA在反义组中表达最高。加入AG490后，各组细胞的凋亡率均显著增加（P < 0.01）；TIMP-1、bcl-xl和cyclin D1 mRNA表达均减少；p27kip1 mRNA表达均增加。在未加AG490的各组细胞中，p-JAK2、p-STAT3和p-STAT5在正义组中表达最高，反义组中最低。加入AG490后上述蛋白表达均减少，且正义＋AG490组最高，反义＋AG490组最低。 结论 TIMP-1表达受JAK/STAT信号通路调控，后者可通过上调前者的表达抑制RMC凋亡；TIMP-1通过JAK/STAT信号通路抑制RMC凋亡。 bcl-xl、cyclin D1和p27kip1参与了上述过程。

Effect of JAK/STAT pathway on the apoptosis of rat mesangial cells inhibited by TIMP-1

Objective To investigate whether JAK/STAT signaling pathway is associated with the tissue inhibitor of metallproteinase-1(TIMP-1)-induced apoptosis in rat mesangial cells(RMC). Methods The human sense and antisense TIMP-1 recombinant plasmids were transfected into RMC through liposome. After RMC was stimulated under the condition of serum deprivation and AG490(JAK2 specific inhibitor) for 24 hours in vitro, the apoptosis rate was measured by flow cytometry. The expression of TIMP-1, bcl-xl, cyclin D1, p27kip1 and JAK2 mRNA was assayed by RT-PCR. The expression of JAK2, STAT3, STAT5, p-JAK2, p-STAT3 and p-STAT5 protein was analyzed by Western blot. Results The apoptosis rates of the non-transfected group, sense TIMP-1 group and antisense TIMP-1 group in the serum-deprived culture medium without serum and AG490 were （10.59±0.96）％, （7.08±0.43）％ and （21.91±0.25）％,respectively. There were statistical differences among those apoptosis rates. The expression of bcl-xl and cyclin D1 mRNA was the highest in sense group, while it was the lowest in antisense one. The expression of p27kip1 mRNA was the highest in antisense group. AG490 treatment enhanced the apoptosis rates of RMC significantly(P<0.01). AG490 decreased the expression of TIMP-1 mRNA and bcl-xl, as well as cyclin D1 mRNA, and increased the expression of p27kip1 mRNA. Before RMC was stimulated with AG490, the expression of p-JAK2, p-STAT3 and p-STAT5 was the highest in sense TIMP-1 group, and it was the lowest in antisense one. AG490 treatment significantly suppressed the expression of the phosphorylation proteins in all groups. Conclusions The expression of TIMP-1 can be regulated by JAK/STAT signaling pathway. The JAK/STAT can inhibit RMC apoptosis through up-regulating the expression of the TIMP-1. TIMP-1 inhibits the apoptosis of RMC induced by serum deprivation through JAK/STAT signal pathway. Bcl-xl, cyclin D1 and p27kip1 are involved in the above-mentioned courses