Klotho蛋白对高糖作用下人脐静脉血管内皮细胞凋亡和衰老的作用

Klotho protein inhibits apoptosis and senescence of human umbilical vein endothelial cell induced by high glucose

目的研究抗衰老Klotho蛋白对高糖作用下人脐静脉血管内皮细胞(HUVECs)凋亡和衰老的抑制作用及其机制。方法培养HUVECs细胞,设置C组(加同体积PBS培养基)、L组(加含低浓度5.5 mmol/L葡萄糖组培养基)、H组(加含高浓度33.3 mmol/L葡萄糖组培养基)、K1组(加含0.1μmol/L Klotho+33.3 mmol/L葡萄糖组培养基)、K2组(加含1μmol/L Klotho+33.3 mmol/L葡萄糖组培养基)、K3组(加含10μmol/L Klotho+33.3 mmol/L葡萄糖组培养基),分别处理48 h。SA-β-半乳糖苷酶染色法检测各组HUVECs细胞衰老情况;RT-PCR方法检测各组HUVECs中人端粒酶逆转录酶mRNA的表达情况;流式细胞术检测各组HUVECs的细胞凋亡率;Western-blot法检测各组HUVECs中凋亡相关蛋白p53、Bax、Bcl-2表达情况。结果与PBS对照组相比,高浓度葡萄糖组中HUVECs细胞中SA-β-半乳糖苷酶染色阳性率显著上升(P<0.05),端粒酶逆转录酶mRNA表达显著下降(P<0.05)。而当不同浓度Klotho蛋白和高糖同时作用HUVECs时,细胞中SA-β-半乳糖苷酶染色阳性率逐渐下降(P<0.05),端粒酶逆转录酶mRNA表达则逐渐上升(P<0.05);高浓度葡萄糖组中HUVECs细胞凋亡率显著上升(P<0.05),抑制凋亡Bcl-2蛋白表达显著下降(P<0.05),而促进凋亡Bax、p53蛋白表达显著上升(P<0.05)。不同浓度Klotho蛋白和高糖同时作用HUVECs时,细胞凋亡率逐渐降低(P<0.05),抑制凋亡Bcl-2蛋白表达显著上升(P<0.05),而促进凋亡Bax、p53蛋白表达显著下降(P<0.05)。结论抗衰老Klotho蛋白能够抑制高糖诱导的HUVECs细胞衰老和凋亡,并通过诱导人端粒酶逆转录酶mRNA、Bcl-2蛋白表达,抑制Bax、p53蛋白表达发挥作用。

Objective To investigate the inhibitory effects of anti -aging Klotho protein on apoptosis and senes- cence of human umbilical vein endothelial cell（HUVECs） induced by high glucose and its mechanism of action. Methods HUVECs cultured in vitro were divided into PBS control group, low glucose group （5.5 mmol/L） , high glucose group （33.3 mmol/L）, 0. 1 mol/L Kltho ＋33.3 mmol/L glucose group, 1 mol/L Kltho ＋33. 3 mmol/L glucose group, 10 mol/L Kltho ＋ 33.3 mmol/L glucose group, and were treated accordingly for 48 h. Senescence of HUVECs in each group was de- termined by SA-β -galactosidase staining. The human telomerase reverse transcriptase（hTERT） mRNA expression was detected by RT - PCR. The apoptosis rate was measured by flow cytometry. Expression of apoptosis related protein, Bcl - 2, Bax, P53 were detected by Western - blot. Results Compared with the PBS control group, SA - β - galactosidase positive rate of the high glucose group increased significantly （ P 〈 0. 05 ）, and the hTERT mRNA expression was signifi- cantly lower（ P 〈 0.05 ）. However, when HUVECs were treated with different concentrations of Klotho protein plus high glucose , the SA - β - galactosidase staining positive rate decreased with rising Klotho protein level（ P 〈 0.05 ）, while ex- pression of hTERT mRNA increased （ P 〈 0. 05 ）. The apoptosis rate of the high glucose group was significantly higher （ P 〈 0. 05 ）, with expression of the apoptosis - inhibiting protein, Bcl - 2, significantly diminished （ P 〈 0.05 ）, and that of the apoptosis - promoting protein, Bax and p53, signicanfly increased （ P 〈 0. 05 ）. Conclusion Anti - aging Klotho protein can inhibit high glucose - induced senescence and apoptosis of HUVECs, possibly through promoting hTERT mR- NA and Bcl -2 protein expression and inhibiting Bax and P53 protein expression.