Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis
Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis
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AMPK对糖尿病胃轻瘫大鼠胃平滑肌细胞凋亡及能量代谢的影响
DOI：
10.1007/s12013-019-00870-9
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发表时间：
2019-06-01
影响因子：
2.6
通讯作者：
Jin, Zheng
中科院分区：
文献类型：
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作者：
Zhang, Mo-han;Fang, Xue-sen;Jin, Zheng
本研究旨在观察AMPK对糖尿病大鼠胃平滑肌细胞凋亡及能量代谢的影响，探讨AMPK在糖尿病胃轻瘫（DGP）发病机制中的作用。建立糖尿病大鼠模型后，将大鼠分为正常对照组（NC）、糖尿病4周组（DM 4W）、糖尿病6周组（DM 6W）和糖尿病8周组（DM 8W）。检测各组大鼠胃内色素残留率、小肠推进率和小肠推进率，以证实DGP模型建立成功。实验观察了NC组和DM8W组大鼠离体胃平滑肌条的自发性收缩。Western blot检测大鼠胃平滑肌组织中磷酸化AMPK、AMPK、磷酸化LKB1、LKB1、磷酸化TAK1、TAK1和CaMMK β的表达; Elisa检测ADP、AMP、ATP含量和能荷;流式细胞术检测胃平滑肌细胞凋亡。体外培养大鼠胃平滑肌细胞，并用AMPK抑制剂和激动剂处理。在24和48 h观察AMPK对胃平滑肌细胞凋亡和能量代谢的影响。糖尿病大鼠胃平滑肌组织自发收缩减少、AMPK活化、细胞凋亡和能量代谢紊乱，AMPK活化与ADP/ATP、AMP/ATP比值、LKB 1活性和CaMMK β表达增加相关。从体外细胞培养实验中，我们发现AMPK激活的高糖条件下促进细胞凋亡。抑制AMPK对高糖早期细胞凋亡无明显影响，但对高糖晚期细胞凋亡有明显抑制作用。AMPK可以调节高糖条件下的线粒体代谢和糖酵解途径。在高糖早期，AMPK是线粒体代谢途径的主要促进因子，但不增加ATP的产生，AMPK还促进糖酵解途径。在高糖后期，AMPK是线粒体途径的主要抑制剂，但仍对糖酵解途径起促进作用，是糖酵解途径的主要调节剂。DGP发生时胃平滑肌细胞存在凋亡和能量代谢紊乱。在高糖条件下，AMPK被激活，可促进细胞凋亡，改变细胞能量代谢途径，抑制线粒体能量代谢，促进糖酵解。
This study aimed to investigate the effect of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells in diabetic rats and to explore the role of AMPK in the pathogenesis of diabetic gastroparesis (DGP). After establishment of a diabetic rat model, rats were divided into normal control (NC), 4-week (DM4W), 6-week (DM6W), and 8-week (DM8W) diabetic model groups. The gastric residual pigment ratio, intestinal transit rate, and intestinal propulsion rate in each group were detected to confirm the successful establishment of the DGP model. The spontaneous contraction in isolated gastric smooth muscle strips of the NC and DM8W groups was experimentally observed. The expression of phospho-AMPK, AMPK, phospho-LKB1, LKB1, phospho-TAK1, TAK1, and CaMMKβ in rat gastric smooth muscle tissues was detected by western blot analysis; ADP, AMP, ATP contents, and the energy charge were detected using Elisa; and apoptosis of gastric smooth muscle cells was detected by flow cytometry. The rat gastric smooth muscle cells were cultured in vitro, and treated with an AMPK inhibitor and an agonist. At 24 and 48 h, the effects of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells were observed. Reduced spontaneous contractions, AMPK activation, cell apoptosis, and energy metabolism disorders were observed in gastric smooth muscle tissues of a diabetic rat, and AMPK activation was associated with an increased ratio of ADP/ATP, AMP/ATP, LKB1 activity, and CaMMKβ expression. From in vitro cell culture experiments, we found that AMPK activation of high-glucose conditions promoted cell apoptosis. Inhibition of AMPK had no obvious effect on apoptosis at the early stage with high glucose, but the inhibitory effect was significant at the late stage with high glucose. AMPK can regulate both mitochondrial metabolism and glycolysis pathways under high-glucose conditions. During the early stage with high glucose, AMPK was the main promotion factor of the mitochondrial metabolism pathway, but did not increase the ATP production, AMPK also promoted the glycolysis pathway. During the late stage with high glucose, AMPK was a major inhibitor of the mitochondrial pathway, and still played a role in promoting the glycolytic pathway, which acted as the main regulator. Apoptosis and energy metabolism disorders were present in gastric smooth muscle cells during the occurrence of DGP. Under high-glucose condition, AMPK was activated, which can promote apoptosis, change the energetic metabolism pathway of cells, inhibit mitochondrial energy metabolism, and promote glycolysis.