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.

中文翻译：

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AMPK对糖尿病性胃轻瘫大鼠胃平滑肌细胞凋亡和能量代谢的影响。

本研究旨在探讨AMPK对糖尿病大鼠胃平滑肌细胞凋亡和能量代谢的影响，并探讨AMPK在糖尿病性胃轻瘫（DGP）发病机理中的作用。建立糖尿病大鼠模型后，将大鼠分为正常对照组（NC），4周（DM4W），6周（DM6W）和8周（DM8W）糖尿病模型组。检测每组中的胃残余色素比率，肠运输速率和肠推进速率，以证实成功建立了DGP模型。实验观察到了NC和DM8W组的孤立胃平滑肌条中的自发收缩。蛋白质印迹法检测大鼠胃平滑肌组织中磷酸化AMPK，磷酸化AMPK，磷酸化LKB1，LKB1，磷酸化TAK1，TAK1和CaMMKβ的表达。ADP，AMP，使用Elisa检测ATP含量和能量。流式细胞仪检测胃平滑肌细胞凋亡。在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小时，观察到AMPK对胃平滑肌细胞凋亡和能量代谢的影响。在糖尿病大鼠的胃平滑肌组织中观察到自发性收缩，AMPK激活，细胞凋亡和能量代谢紊乱减少，并且AMPK激活与ADP / ATP，AMP / ATP，LKB1活性和CaMMKβ表达比率增加相关。从体外细胞培养实验中，我们发现高糖条件下的AMPK活化促进细胞凋亡。高糖在早期对AMPK的抑制作用对细胞凋亡无明显影响，而在高糖下对AMPK的抑制作用在晚期显着。在高葡萄糖条件下，AMPK可以调节线粒体代谢和糖酵解途径。在高血糖的早期，AMPK是线粒体代谢途径的主要促进因子，但并未增加ATP的产生，AMPK也促进了糖酵解途径。在高血糖的晚期，AMPK是线粒体途径的主要抑制剂，并且仍在促进糖酵解途径中起着主要的调节作用。DGP发生期间胃平滑肌细胞中存在凋亡和能量代谢异常。在高葡萄糖条件下，AMPK被激活，