Low-after-high glucose down-regulated Cx43 in H9c2 cells by autophagy activation via cross-regulation by the PI3K/Akt/mTOR and MEK/ERK1/2 signal pathways
Low-after-high glucose down-regulated Cx43 in H9c2 cells by autophagy activation via cross-regulation by the PI3K/Akt/mTOR and MEK/ERK1/2 signal pathways
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DOI：
10.1007/s12020-017-1251-3
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发表时间：
2017-05-01
期刊：
影响因子：
3.7
通讯作者：
Zhang, Qingyong
中科院分区：
文献类型：
--
作者：
Bi, Yaguang;Wang, Guangyu;Zhang, Qingyong
目的糖尿病低血糖是心血管事件的强预测因子。据报道，高糖改变了连接蛋白43的表达，并促进心肌细胞的自噬。方法H9 c2细胞在33.3mM葡萄糖孵育24 h后，加入2.5mM葡萄糖孵育2、4、6和1/2 h，加入或不加入自噬抑制剂氯喹（Chloroquine，Chloroquine）、MEK 1/2抑制剂U 0126（U 0126）和PI 3 K抑制剂LY 294002（LY 294002）。将在5.5、33.3或2.5 mM葡萄糖（含或不含抑制剂）和甘露醇存在下孵育的细胞用作对照。结果高糖后低浓度暴露可使细胞毒性和早期凋亡增加，细胞增殖降低，氯喹和U 0126可逆转上述作用，LY 294002可加重上述作用。Connexin 43表达以时间依赖性方式下调，并伴有LC 3-II、Beclin-1、p62、p-Akt、p-mTOR和p-ERK 1/2的表达上调。氯喹抑制自噬并逆转连接蛋白43的下调。U 0126抑制ERK激活，降低自噬蛋白表达，但增加连接蛋白43的表达。LY 294002抑制p-Akt，激活自噬，并降低连接蛋白43的表达。结论高糖后低浓度培养H9 c2细胞，通过PI 3 K/Akt/mTOR和MEK/ERK 1/2信号通路促进细胞自噬，从而下调Cx43的表达。
Purpose Hypoglycemia in diabetes is a strong predictor of cardiovascular events. High-glucose have been reported to alter connexin43 expression and to promote autophagy in cardiomyocytes. We investigated whether low-after-high glucose would influence connexin43 expression and autophagy in H9c2 cells.Methods H9c2 cells were incubated in 33.3 mM glucose for 24 h followed by 2.5 mM glucose for 2, 4, 6, or 12 h with or without chloroquine (autophagy inhibitor), U0126 (MEK1/2 inhibitor) or LY294002 (PI3K inhibitor). Cells incubated in 5.5, 33.3, or 2.5 mM glucose with or without inhibitors and in the presence of mannitol were used as controls. Protein expression was assayed by western blot, apoptosis was assayed by flow cytometry, cell proliferation was determined by MTT assays, and cytotoxicity was assayed by lactate dehydrogenase measurement.Results Cytotoxicity and early apoptosis were increased and cell proliferation was decreased after exposure to low-after-high glucose, and these results were reversed by chloroquine and U0126 but were aggravated by LY294002. Connexin43 expression was downregulated in a time-dependent manner and was accompanied by upregulated expression of LC3-II, Beclin-1, p62, p-Akt, p-mTOR, and p-ERK1/2. Chloroquine suppressed autophagy and reversed the downregulation of connexin43. U0126 inhibited ERK activation and decreased autophagy proteins expression but increased connexin43 expression. LY294002 suppressed p-Akt, activated autophagy, and decreased connexin43 expression. Interestingly, MEK1/2 inhibition also increased p-Akt expression, but inhibition of PI3K led to p-ERK downregulation.Conclusion Culturing H9c2 cells under low-after-high glucose downregulated connexin43 by promoting autophagy through a mechanism involving the PI3K/Akt/mTOR and MEK/ERK1/2 signaling pathways.