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目的探讨磷脂酰肌醇3激酶(phosphoinositide-3-kinase,PI3K)/丝/苏氨酸激酶(serine thre-onine kinase,AKT)通路在基质金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinase-1,TIMP-1)抑制高糖诱导大鼠肾小球系膜细胞(rat mesangial cells,RMC)凋亡中的作用和机制。方法(1)脂质体法将正、反义人TIMP-1(hTIMP-1)及空载体转染到大鼠肾小球系膜细胞。(2)Annexin V/PI双染法流式检测系膜细胞凋亡率。(3)Caspase9/Mch6Colorimetric Assay试剂盒检测Caspase9活性。(4)Western blot检测BAD、phospho-BAD、AKT、phospho-AKT蛋白水平。结果与对照组(1·06±0·08)%相比,正义转染组细胞凋亡率降低[(4·51±0·50)%,P<0·05],反义转染组凋亡率显著提高[(24·97±3·64)%,P<0·05],LY294002的加入可部分抵消TIMP-1的抗凋亡作用。TIMP-1的高表达可下调Caspase9活性(0·111±0·064,P<0·05),低表达可上调Caspase9活性(0·461±0·012,P<0·05)。正义TIMP-1能提高细胞phos-pho-BAD、phospho-AKT的蛋白水平,而反义TIMP-1的两种蛋白水平明显减少,且该作用可被LY294002阻断。结论(1)PI3K/AKT通路参与TIMP-1抑制高糖诱导的大鼠肾小球系膜细胞凋亡过程。(2)BAD在其中起重要作用。(3)AKT对Caspase9的抑制亦起主要作用。
Objective To investigate the role of phosphoinositide 3-kinase (PI3K) / serine threonine kinase (AKT) pathway in tissue inhibitor of metalloproteinase-1 TIMP-1) to inhibit the apoptosis of rat mesangial cells (RMC) induced by high glucose. Methods (1) Positive and antisense human TIMP-1 (hTIMP-1) and empty vector were transfected into rat glomerular mesangial cells by liposome method. (2) Annexin V / PI double staining flow cytometry detection of apoptosis rate of mesangial cells. (3) Caspase9 / Mch6Colorimetric Assay assay Caspase9 activity. (4) Western blot detection of BAD, phospho-BAD, AKT, phospho-AKT protein levels. Results Compared with the control group (1.06 ± 0.08%), the apoptotic rate in the positive transfection group was significantly lower than that in the control group [(4.51 ± 0.50)%, P <0.05] Apoptosis rate was significantly increased [(24.97 ± 3.64)%, P <0.05]. The addition of LY294002 could partially counteract the anti-apoptotic effect of TIMP-1. High expression of TIMP-1 downregulated the activity of Caspase9 (0 · 111 ± 0 · 064, P <0 · 05), while down-regulated the activity of Caspase9 (0 · 461 ± 0 · 012, P <0 · 05). Sense TIMP-1 can increase the protein levels of phos-pho-BAD and phospho-AKT in cells, and the two protein levels of antisense TIMP-1 decrease obviously, and this effect can be blocked by LY294002. Conclusion (1) PI3K / AKT pathway is involved in TIMP-1 inhibition of high glucose-induced rat mesangial cell apoptosis process. (2) BAD plays an important role in it. (3) AKT also plays a major role in the inhibition of Caspase9.