PI3K/Akt调控糖尿病冠状动脉平滑肌细胞BK通道的作用机制
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1.无锡市转化医学研究所 南京医科大学附属无锡人民医院;2.南京医科大学附属无锡人民医院;3.无锡市转化医学研究所;4.美国Mayo Clinic细胞电生理研究室

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江苏省自然科学基金(BK20171151)


PI3K/Akt pathway mediates large conductance calcium-activated potassium in diabetic coronary smooth muscle cells
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the Natural Science Foundation of Jiangsu Province( Grants No BK20171151

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    摘要:

    目的 探讨PI3K/Akt对糖尿病冠状动脉平滑肌细胞BK通道的调节作用。方法 采用正常糖浓度(5mM)、高糖浓度(15mM)、活性氧(Reactive Oxygen Species,ROS)抑制剂GKT137831、PI3K抑制剂Wortmannin作用于人冠状动脉平滑肌细胞(Human Coronary Artery Smooth Muscle Cell,HCASMC)。以GKT137831喂饲链脲霉素(Streptozocin,STZ)诱导的糖尿病大鼠,免疫印迹测定HCASMC、大鼠冠状动脉PI3K超家族DNA-Pkcs、Akt、p-Akt(S473)及BK-α亚基、BK-β1亚基的表达, 2',7'-二氯二氢荧光素二乙酸酯测定HCASMC及大鼠冠状动脉ROS表达。结果 高糖浓度下HCASMC及糖尿病大鼠冠状动脉ROS、DNA-Pkcs、Akt、p-Akt(S473)表达量增高,BK-β1表达量降低(p<0.05)。 GKT137831作用后的HCASMC及糖尿病大鼠冠状动脉中,ROS、DNA-Pkcs、Akt、p-Akt(S473)表达量降低,BK-β1表达量明显增高(p<0.05)。Wortmannin作用后的HCASMC p-Akt(S473)表达量降低,BK-β1表达量明显增加(p<0.05)。结论 PI3K/Akt通路参与糖尿病冠状动脉平滑肌细胞BK通道的调控。

    Abstract:

    【Abstract】Objective To investigate the effect of PI3K/Akt pathway on the regulation of large conductance Ca2+-activated K+ channel(BK channel) in diabetic coronary smooth muscle cells. Methods Human coronary artery smooth muscle cells(HCASMC) were incubated with different concentrations of glucose(5mM and 15mM).In all cases, controls were performed with cells treated with the corresponding vehicle alone with a final concentration of dimethylsulfoxide (DMSO) at 0.1% (w/w).For DNA-PKcs inhibitor treatment, cells were preincubated with wortmannin (100nM) for 30 min in serum-free medium before treatment with glucose.For reactive oxygen species (ROS) inhibitor treatment, cells were incubated with GKT137831 (20μM) and high concentration of glucose(15mM) for 48h. Expression of DNA-Pkcs、Akt、p-Akt (S473)、 BK-αsubunit and BK-β1 subunits were determined by Western blot.ROS generation in HCASMC was measured in terms of fluorescence using the cell-permeable fluorogenic probe DCFH-DA. Streptozotocin-induced diabetic rats were established successfully by intraperitoneal injection.GKT137831 were fed to diabetic rats and the expression of ROS、DNA-Pkcs、Akt、p-Akt (s473) and BK-α and BK-β1 subunits in rats coronary artery smooth muscle cells were determined.ROS in diabetic rats was measured with cell-permeable fluorogenic probe DCFH-DA. Results Compared with that of HCASMC incubated in 5mM glucose concentration, the expression of DNA-Pkcs、Akt、p-Akt (S473) increased significantly and BK-β1 subunit decreased significantly ( p<0.05), there was no significant change in BK-α subunit expression (p>0.05) of the HCASMC with 15 mM glucose concentration. Compared with the control group,the expression of p-Akt (s473) decreased significantly(p<0.05) and BK-β1 increased significantly (p<0.05) after pretreated with Wortmannin for 30 minutes. The expression of ROS、DNA-Pkcs、Akt、p-Akt (S473) decreased (p<0.05),BK-β1 expression increased significantly after HCASMC incubated with 15mM glucose and GKT137831 for 48h. Compared with normal rats, expression of DNA-Pkcs、Akt、p-Akt(S473)of the diabetic coronary arteries increased by 50%、30% and 30% respectively, and that of BK-β1 subunit decreased by 45%. The expression of ROS、DNA-Pkcs、Akt、p-Akt(S473) decreased (p<0.05) and that of BK-β1 subunit recovered to normal level after the diabetic rats were fed with GKT137831 for 2 weeks. Conclusion PI3K/Akt channel is involved in the regulation of BK channel expression in diabetic coronary smooth muscle cells, and ROS inhibitor GKT137831 can inhibit the generation of ROS and protect coronary smooth muscle cells from injury.

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  • 收稿日期:2020-07-10
  • 最后修改日期:2020-12-08
  • 录用日期:2022-02-25
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