关键词: 司美格鲁肽, 缺氧复氧, 心肌细胞, 炎症, 自噬

Abstract: Objective:To investigate the effects of semaglutide on inflammation and autophagy in human AC16 cardiomyocytes under hypoxia/reoxygenation conditions. Methods:AC16 cells in logarithmic growth phase were randomly assigned to 4 groups:control group (CON), H/R group (H/R), H/R+ semaglutide group (H/R+SEM), and H/R+ semaglutide +3MA group (H/R+SEM+3-MA). Except for the control group, the cells in the other 3 groups were treated with 8 h hypoxia and 12 h reoxygenation. Cell viability was detected by CCK-8 assay. IL-1βand TNF-αwere detected by ELISA kits. Western blot was used to detect the the expression of pathway-related protein AMPK, p-AMPK, mTOR, p-mTOR, ULK1, p-ULK1 and autophagy-related proteins Beclin-1,LC3 in each group. The fluorescence patterns of AC16 cells infected with mRFP-GFP-LC3 adenovirus were observed through laser confocal microscopy, and the intracellular autophagosomes were quantitatively analyzed. The structure of autophagosome and autolysosome in AC16 cells was observed by transmission electron microscopy.Results: Compared with the CON group, the H/R group had significantly increased expressions of IL-1β and TNF-α (P < 0.0001). Compared with the H/R group, the H/R + semaglutide group had significantly decreased expressions of IL-1β and TNF-α (P < 0.001). Compared with the H/R + semaglutide group, the expression of inflammatory factors in the H/R + semaglutide +3-MA group was significantly increased (P < 0.05). Western blot results showed that compared with the CON group, the expression of p-AMPK/AMPK was significantly increased (p < 0.05), the expression of p-mTOR/mTOR was significantly decreased (P < 0.05), and the expression of pULK1/UKL1 was significantly increased (P < 0.001) in the H/R group. Compared with the H/R group, the expression of p-AMPK/AMPK was significantly increased (p < 0.05), the expression of p-mTOR /mTOR was significantly decreased (P < 0.01), and the expression of pULK1/UKL1 was obviously increased (P < 0.001) in the H/R+SEM group. Compared with the H/R+SEM group, the expression of p-AMPK/AMPK was significantly decreased (p < 0.01), the expression of p-mtor /mTOR was significantly increased (P < 0.05), and the expression of pULK1/UKL1 was significantly decreased (p < 0.05) in the H/R+SEM+3MA group. Compared with the CON group , the expressions of Beclin1 and LC3 were significantly increased in group H/R (P < 0.05). Compared with the H/R group, the expressions of Beclin1 and LC3 were significantly increased in group H/R+SEM (P < 0.01; P < 0.001). Compared with the H/R+SEM group, the expressions of Beclin1 and LC3 in H/R+SEM+3-MA group were significantly decreased (P < 0.05; P < 0.01).Laser confocal microscopy was used to observe the fluorescence pattern of mRFP-GFP-LC3 adenovirus transfected human AC16 cells, and the quantitative analysis of intracellular autophagosomes:compared with the CON group, the autophagosomes had significantly increased n the H/R group (3.67±2.31vs 9.67±1.53,*P < 0.05). Compared with the H/R group, the autophagosomes of cardiomyocytes in H/R+SEM group were significantly increased (9.67±1.53 vs 18.67±3.21,**P < 0.01). Compared with the H/R+SEM group, the number of autophagosomes in cardiomyocytes in H/R+SEM+3-MA group was significantly decreased (18.67±3.21vs 12.33±1.53,*P < 0.05). Conclusions: 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