司美格鲁肽通过调控AMPK/mTOR/ULK1通路介导的自噬减轻缺氧/复氧AC16人心肌细胞炎症损伤

doi:10.12092/j.issn.1009-2501.2025.08.005

摘要/Abstract

摘要： 目的：观察司美格鲁肽对缺氧/复氧条件下人AC16心肌细胞炎症和自噬的影响。方法：取对数生长期的AC16细胞,随机分为4组：对照组（CON）、缺氧/复氧组（H/R）、缺氧/复氧+司美格鲁肽组（H/R+SEM）、缺氧/复氧+司美格鲁肽+3-MA组（H/R+SEM+3-MA）。除对照组外,其余3组细胞均进行缺氧8 h、复氧12 h处理。CCK-8法检测细胞活性。ELISA试剂盒检测IL-1β及TNF-α。Western blot检测各组通路相关蛋白AMPK、p-AMPK、mTOR、p-mTOR、ULK1、p-ULK1及各组细胞自噬相关蛋白Beclin-1和LC3的表达。激光共聚焦显微镜观察各组mRFP-GFP-LC3腺病毒感染AC16细胞荧光图,并对细胞内的自噬体定量分析。透射电子显微镜观察各组AC16细胞自噬体及自噬溶酶体结构。结果：与CON组比较,H/R组IL-1β与TNF-α的表达显著增加（P<0.01）;与H/R组比较,H/R+SEM组IL-1β与TNF-α的表达量明显降低（P<0.01）;与H/R+SEM组比较,H/R+SEM+3-MA组炎症因子的表达量显著增加（P<0.05）。Western blot结果显示：与CON组比较,H/R组心肌细胞p-AMPK/AMPK表达明显升高（P<0.05）,p-mTOR/mTOR表达明显降低（P<0.05）,pULK1/UKL1表达明显升高（P<0.01）;与H/R组比较,H/R+SEM组心肌细胞p-AMPK/AMPK表达明显升高（P<0.05）,p-mTOR/mTOR表达明显降低（P<0.01）,pULK1/UKL1表达明显升高（P<0.01）;与H/R+SEM组比较,H/R+SEM+3-MA组心肌细胞p-AMPK/AMPK表达明显降低（P<0.01）,p-mTOR/mTOR表达明显升高（P<0.05）,pULK1/UKL1表达明显降低（P<0.05）。与CON组比较,H/R组心肌细胞Beclin1和LC3表达明显升高（P<0.05）;与H/R组比较,H/R+SEM组心肌细胞Beclin1和LC3表达明显升高（P<0.01）;与H/R+SEM组比较,H/R+SEM+3-MA组心肌细胞Beclin1和LC3表达明显降低（P<0.05,P<0.01）。激光共聚焦显微镜观察各组mRFP-GFP-LC3腺病毒转染人AC16细胞荧光图,并对细胞内的自噬体定量分析：与CON组比较,H/R组心肌细胞自噬体显著增多（3.67±2.31 vs. 9.67±1.53,P<0.05）。与H/R组比较,H/R+SEM组心肌细胞自噬体明显增多（9.67±1.53 vs. 18.67±3.21,P<0.01）。与H/R+SEM组比较,H/R+SEM+3-MA组心肌细胞自噬体数量明显减少（18.67±3.21 vs. 12.33±1.53,P<0.05）。结论：司美格鲁肽可能通过AMPK/mTOR/ULK1通路适度调节自噬,减少炎症因子释放,减轻缺氧/复氧导致的心肌炎症损伤。

关键词: 司美格鲁肽, 缺氧/复氧, 心肌细胞, 炎症, 自噬

Abstract: AIM: To investigate the effects of semaglutide on inflammation and autophagy in human AC16 cardiomyocytes under hypoxia/reoxygenation conditions. METHODS: AC16 cells were randomly divided into four groups: control (CON), hypoxia/reoxygenation (H/R), hypoxia/reoxygenation+semaglutide (H/R+SEM), and hypoxia/reoxygenation+semaglutide+3-MA (H/R+SEM+3-MA). All groups except CON underwent 8 hours of hypoxia followed by 12 hours of reoxygenation. Cell viability was measured by CCK-8. Levels of IL-1β and TNF-α were assessed by ELISA. Western blot analysis evaluated AMPK, p-AMPK, mTOR, p-mTOR, ULK1, p-ULK1, Beclin-1, and LC3. Autophagosomes were analyzed using laser confocal microscopy and transmission electron microscopy. The structure of autophagosome and autolysosome was observed by transmission electron microscopy. RESULTS: IL-1β and TNF-α levels increased significantly in the H/R group compared to CON (P<0.01) but decreased in the H/R+SEM group (P<0.01). In the H/R+SEM+3-MA group, inflammatory levels increased compared to the H/R+SEM (P<0.05). Compared with the CON group, p-AMPK/AMPK and p-ULK1/ULK1 ratios increased significantly in the H/R group (P<0.05, P<0.01). The ratios further increased in the H/R+SEM group compared to H/R (P<0.05, P<0.01). But the two ratios decreased in the H/R+SEM+3-MA group compared to H/R+SEM (P<0.01, P<0.05). The pmTOR/mTOR had the opposite trend to the two previous ratios. The expression of Beclin1 and LC3 were significantly increased in group H/R compared to CON (P<0.05), and which further increased in the H/R+SEM group compared to H/R (P<0.01). But the two indicators decreased in the H/R+SEM+3-MA group compared to H/R+SEM (P<0.01, P<0.05). Laser confocal microscopy revealed increased autophagosome numbers in H/R group compared to CON (P<0.05), and which further increased in the H/R+SEM group compared to H/R (P<0.01). While the H/R+SEM+3-MA group showed a decrease compared to H/R + SEM (P<0.05). CONCLUSION: Semaglutide may moderate regulate autophagy through AMPK/mTOR/ULK1 pathway, reduce the release of inflammatory factors, and alleviate hypoxia/reoxygenation-induced inflammatory injury.

Key words: semaglutide, hypoxia/reoxygenation, cardiomyocytes, inflammation, autophagy

中图分类号:

R965.2

靳丽丽, 郄涛, 李立琴. 司美格鲁肽通过调控AMPK/mTOR/ULK1通路介导的自噬减轻缺氧/复氧AC16人心肌细胞炎症损伤[J]. 中国临床药理学与治疗学, 2025, 30(8): 1058-1066.

JIN Lili¹, QIE Tao², LI Liqin³. Semaglutide alleviates hypoxia/reoxygenation-induced inflammatory injury of AC16 human cardiomyocytes by regulating autophagy through AMPK/mTOR/ULK1 pathway[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2025, 30(8): 1058-1066.

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