黄连素抗缺氧复氧诱导的人肾小管上皮HK-2细胞凋亡的机制

Mechanism of Berberine Inhibiting Apoptosis in Human Proximal Tubular Epithelial HK-2 Cells Induced by Hypoxia/reoxygenation

目的:探索鞘氨醇激酶1(sphingosine kinase 1,SPHK1)在黄连素抑制再灌注复氧(hypoxia/reoxygenation,H/R)诱导的人肾小管上皮细胞(HK-2细胞)细胞凋亡中的作用。方法:建立HK-2细胞H/R模型,分别复氧0 h、3 h、6 h、12 h。用黄连素预先处理HK-2细胞,随后进行H/R处理。检测SPHK1表达水平、HK-2细胞活性、HK-2细胞凋亡,及p-P38,caspase3,caspase9、Bcl-2、Bax的表达水平。结果:H/R损伤伴随着HK-2细胞中SPHK1水平的上升,呈时间依赖型,12 h时到达最大值(P<0.01)。黄连素能够显著下调SPHK1的mRNA水平,呈浓度依赖型,在10μmol/L时下调作用达到最大(P<0.01)。黄连素能够明显促进H/R诱导的HK-2细胞增殖、减弱HK-2细胞凋亡,呈浓度依赖型,在浓度为10μmol/L作用最大。黄连素能降低p-P38、caspase-3、caspase-9和SPHK1水平,提高Bcl-2/Bax的比率。SPHK1过表达显著抑制HK-2细胞增殖、促进HK-2细胞凋亡,而黄连素能减轻SPHK1过表达对HK2细胞增殖的抑制及其促凋亡作用。结论:黄连素能明显改善缺氧复氧诱导的HK-2细胞中SPHK1的高表达引起的细胞凋亡,其机制包括P38蛋白激酶的活化作用和线粒体应激途径。

Objective:To investigate whether SPHK1 participated in the anti-apoptosis process of berberine in HK-2 cells after H/R injury.Methods:The H/R model in human proximal tubular epithelial HK-2 cells was established and reoxygenated for 0,3,6 or 12 h,respectively.Cells were treated with berberine before subjected to H/R.The expression level of SPHK1,HK-2 cell activity,HK-2 cell apoptosis,and p-P38,caspase3,caspase9、Bcl-2、Bax expression were detected.Results:SPHK1 expression was increased accompanied with H/R-induced injury in HK-2 cells time-dependently with the maximum level at 12 h(P<0.01).Berberine can significantly decrease SPHK1 mRNA level in a dose-dependent manner with maximum reduction at 10μmol/L(P<0.01).Berberine significantly promoted H/R-induced HK-2 cell proliferation and inhibited HK-2 cell apoptosis in a dose-dependent manner with maximum effect at 10μmol/L(P<0.01).Berberine remarkably down-regulated p-P38,caspase-3,caspase-9 and SPHK1 levels while up-regulated Bcl-2/Bax ratio.The overexpression of SPHK1 significantly inhibit the proliferation of HK-2 cells and promote the apoptosis of HK-2 cells,while berberine reduce its effect of inhibiting HK-2 cells proliferation and promoting HK-2 cells apoptosis.Conclusion:Berberine exhibited an anti-apoptosis effect in H/R-induced HK-2 cells through down-regulating SPHK1 expression by the mechanisms of blocking mitochondrial stress and p38 MAPK pathways.