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目的探讨细胞外信号调节激酶(ERK1/2)通路是否参与基质金属蛋白酶抑制剂(TIMP-1)抑制大鼠肾小球系膜细胞的凋亡。方法选择大连医科大学附属第一医院2005年8月至2006年2月应用人正、反义TIMP-1转染大鼠肾小球系膜细胞,分别用高糖(25mmol/L)和MEK1特异性抑制剂PD98059(50μmol/L)刺激24h,采用流式细胞仪检测细胞凋亡情况;逆转录聚合酶链式扩增(RT-PCR)观察细胞外信号调节激酶信使RNA(ERK1 messenger RNA)的表达;酶联免疫吸附实验(ELISA)方法检测胞浆中p-ERK1/2的表达情况。结果(1)未加PD98059的未转染组、空载体组、正义转染组及反义转染组各组细胞的凋亡率分别为(12.10±2.21)%、(11.90±3.34)%、(5.50±0.50)%和(20.70±3.41)%;正义转染组的凋亡率明显低于未转染组(P<0.01);反义转染组凋亡率高于未转染组(P<0.05)。加入PD98059后各组细胞的凋亡率明显增加。(2)加入PD98059后,各组细胞ERK1mRNA的表达较相应未加PD98059各组明显上调;以正义转染组上调最为明显。(3)ELISA结果显示:加入PD98059后,p-ERK1/2较相应未加PD98059各组明显下调(P<0.05),以正义组下调最为明显(P<0.01)。结论ERK1/2信号传导通路是TIMP-1抑制高糖诱导的大鼠肾小球系膜细胞凋亡的主要途径之一。
Objective To investigate whether extracellular signal-regulated kinase (ERK1 / 2) pathway is involved in the inhibition of rat mesangial cell apoptosis by matrix metalloproteinase inhibitor (TIMP-1). Methods The rat mesangial cells were transfected with human positive and negative antisense TIMP-1 from the first affiliated hospital of Dalian Medical University from August 2005 to February 2006, respectively. The mesangial cells were treated with high glucose (25mmol / L) and MEK1 The cell apoptosis was detected by flow cytometry after PD98059 (50μmol / L) for 24 hours. The expression of ERK1 messenger RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) The expression of p-ERK1 / 2 in cytoplasm was detected by enzyme-linked immunosorbent assay (ELISA). Results (1) The apoptotic rates of untransfected group, empty vector group, positive transfected group and antisense transfected group without PD98059 were (12.10 ± 2.21)%, (11.90 ± 3.34)%, (5.50 ± 0.50)% and (20.70 ± 3.41)%, respectively. The apoptosis rate in sense transfection group was significantly lower than that in untransfected group (P <0.01). The apoptosis rate in antisense transfected group was higher than that in untransfected group P <0.05). After adding PD98059, the apoptosis rate of each group increased obviously. (2) After adding PD98059, the expression of ERK1mRNA in each group was significantly up-regulated compared with the corresponding group without PD98059; the up-regulation was the most obvious in the positive transfection group. (3) The result of ELISA showed that the expression of p-ERK1 / 2 was significantly down-regulated in PD98059 group (P <0.05) after PD98059 was added compared with that in PD98059 group. Conclusion ERK1 / 2 signal transduction pathway is one of the main pathways that TIMP-1 inhibits high glucose-induced rat mesangial cell apoptosis.