Esmolol alleviates lipopolysaccharide-induced intestinal injuries by enhancing autophagy through the AMPK/mTOR/ULK1 pathway

Figure 1 Schematic of the study design. A: Rats in the Sham group continuously received normal saline (NS) for 3, 6, 12, or 24 hours after surgery; B: Rats in the lipopolysaccharide (LPS) group continuously received NS for 3, 6, 12, or 24 hours, and LPS was administered 30 minutes after the start of the NS infusion; C: Rats in the LPS + ES group continuously received ES for 12 or 24 hours, and LPS was administered 30 minutes after the start of ES and LPS treatment; D: Rats in the LPS + 3-methyladenine (3-MA) and LPS + rapamycin (RAPA) groups continuously received with NS for 12 or 24 hours as well as an intraperitoneal injection of 3-MA or RAPA, followed by an intraperitoneal injection of LPS 30 minutes after the start of the NS infusion. 3-MA: 3-methyladenine; ES: Esmolol; NS: Normal saline; RAPA: Rapamycin; LPS: Lipopolysaccharide.

Figure 2 Interstitial tissue pathological changes in sepsis model rats at 12 and 24 hours (n = 6). The interstitial tissues from rats in different groups were stained with hematoxylin-eosin (H&E). A: No significant tissue destruction or other symptoms were observed in the Sham group; B: Whereas significant pathological tissue changes were observed in the lipopolysaccharide group; C: After treatment with esmolol, the impairments were reduced; D: Significant pathological tissue changes were observed in rats treated with 3-methyladenine; E: The impairments were reduced in rats treated with rapamycin. H&E, × 100 magnification, scale bar = 50 μm; × 400 magnification, scale bar = 20 μm. 3-MA: 3-methyladenine; ES: Esmolol; NS: Normal saline; RAPA: Rapamycin; LPS: Lipopolysaccharide.

Figure 3 Effects of esmolol on lipopolysaccharide-induced shortening of the interstitial villus (n = 6). A: The length of the interstitial villus in each group at 12 hours; B: The length of the interstitial villus in each group at 24 hours. Each bar presents the mean ± SE of the mean. n = 6 in each group. LPS: Lipopolysaccharide; ES: Esmolol; 3-MA: 3-methyladenine; RAPA: Rapamycin.

Figure 4 Esmolol suppressed the levels of fatty acid-binding protein, diamine oxidase, interleukin-1, and interleukin-6 and increased the levels of interleukin-10 in rats with lipopolysaccharide-induced sepsis. A: Fatty acid-binding protein (I-FABP) concentration at 12 hours; B: I-FABP concentration at 24 hours; C: Diamine oxidase (DAO) concentration at 12 hours; D: DAO concentration at 24 hours; E: Interleukin (IL-1) level at 12 hours; F: IL-6 level at 12 hours; G: IL-10 level at 12 hours. Lipopolysaccharide (LPS) significantly increased the production of I-FABP and DAO, whereas esmolol (ES) and rapamycin markedly decreased their serum levels (A-D). LPS significantly increased the production of IL-1 and IL-6 and decreased that of IL-10 at 12 hours. ES suppressed the levels of IL-1 and IL-6 and increased those of IL-10 in rats with LPS-induced sepsis at 12 hours. n = 6 in each group. I-FABP: Fatty acid-binding protein; DAO: Diamine oxidase; ES: Esmolol; 3-MA: 3-methyladenine; IL: Interleukin; RAPA: Rapamycin.

Figure 5 Effects of lipopolysaccharide on autophagy changed with time in rats with sepsis. A: The expression of Beclin-1 and LC3-II in the intestine was detected by western blotting; B: Quantitative analysis of Beclin-1 protein expression at different time points; C: Quantitative analysis of LC3-II protein expression at different time points. Lipopolysaccharide (10 mg/kg) induced LC3-II at 3 hours (A and C) but inhibited Beclin-1 and LC3-II at 12 hours and 24 hours (A, B, and C). Data in the Sham group were set to 1. n = 6 in each group.

Figure 6 Western blotting was performed for Beclin-1, LC3-II, p-AMPK, p-ULK1, and p-mTOR in intestinal tissue homogenates in the Sham, lipopolysaccharide, lipopolysaccharide + esmolol, lipopolysaccharide + 3-methyladenine, and lipopolysaccharide + rapamycin treatment groups (after 12 and 24 hours of treatment). A-C: The 12-hour protein levels of Beclin-1, LC3-II and β-Actin were determined by western blotting of each group; D-F: The 24-hour protein levels of Beclin-1, LC3-II and β-Actin were determined by western blotting of each group; G-J: The 12-hour protein levels of p-AMPK, p-ULK1, p-mTOR and β-Actin were determined by western blotting of each group; K-N: The 24-hour protein levels of p-AMPK, p-ULK1,p-mTOR and β-Actin were determined by western blotting of each group.β-actin was used as a loading control and for normalization. Representative immunoblots and densitometry analyses are presented. Data in the Sham group were set to 1. n = 6 in each group. 3-MA: 3-methyladenine; ES: Esmolol; RAPA: Rapamycin; LPS: Lipopolysaccharide.

Figure 7 Evaluation of autophagosome counts in the Sham, lipopolysaccharide, lipopolysaccharide + esmolol, lipopolysaccharide + rapamycin, and lipopolysaccharide + 3-methyladenine groups. A: Transmission electron microscopy was used to observe the number of autophagosomes formed in the intestinal tissue after the induction of sepsis. Blue and orange arrows denote autophagosomes. The autophagosome indicated by the orange arrow is magnified in the right panel. Magnification: × 125000; inset: × 500000; B: Autophagosome count based on transmission electron microscopy. Scale bar: 500 nm. n = 3 in each group. 3-MA: 3-methyladenine; ES: Esmolol; RAPA: Rapamycin; LPS: Lipopolysaccharide.

Figure 8 Esmolol significantly improves cell viability and induces autophagy in IEC-6 cells following lipopolysaccharide treatment. A-C: The effect of different concentrations of lipopolysaccharide on the expression of autophagy markers Beclin-1 and LC3-II in IEC-6 cell; D-F: The effect of both different duration of lipopolysaccharide on the expression of autophagy markers Beclin-1 and LC3-II induced by lipopolysaccharide (LPS) in IEC-6 cells; G-I: The effect of different concentrations of ES on the expression of autophagy markers Beclin-1 and LC3-II in LPS-stimulated IEC-6 cells; J: Esmolol (1 × 10^-4 g/mL) significantly increases cell viability in LPS-treated IEC-6 cells. n = 6 in each group. LPS: Lipopolysaccharide; ES: Esmolol.

Figure 9 Western blot analysis was performed for Beclin-1, LC3-II, p-AMPK, p-ULK1, and p-mTOR in IEC-6 cells in the Sham, lipopolysaccharide, lipopolysaccharide + esmolol, lipopolysaccharide + 3-methyladenine, and lipopolysaccharide + rapamycin treatment groups (after 6 hours of treatment). A-D: Protein levels of Beclin-1, LC3-II, p-mTOR and β-Actin were determined by western blotting. The representative blots of Beclin-1, LC3-II, p-AMPK, p-ULK1, and p-mTOR and β-actin after treatment. β-actin was used as loading control and for normalization; E-G: Protein levels of p-AMPK, p-ULK1 and βActin were determined by western blotting. Representative immunoblots and densitometry analyses are presented. Data in the Sham group were set to 1 n = 6 in each group. 3-MA: 3-methyladenine; ES: Esmolol; RAPA: Rapamycin; LPS: Lipopolysaccharide.

Figure 10  Esmolol regulates the expression of genes related to sepsis. A: Heatmap for hierarchical cluster analysis of differentially expressed genes (DEGs) between the groups. Red denotes high expression, and blue denotes low expression; B: Volcano plots of upregulated and downregulated DEGs between the lipopolysaccharide (LPS) and Sham groups; C: Volcano plots of upregulated and downregulated DEGs between the LPS + esmolol and LPS groups; D: Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis; E: Gene Ontology functional enrichment analysis. n = 3 in each group. ES: Esmolol; LPS: Lipopolysaccharide.

Figure 11  Enzyme-linked immunosorbent assay was employed to assess alterations in inflammatory factors within IEC-6 cells after 6 hours of treatment in each group. A: Interleukin (IL)-1 level at 6 hours; B: IL-6 level at 6 hours; C: IL-10 level at 6 hours; D: Tumor necrosis factor-α level at 6 hours; E: IL-17 level at 6 hours. ES: Esmolol; LPS: Lipopolysaccharide; IL: Interleukin; TNF-α: Tumor necrosis factor-α; CC: Compound C.

Figure 12  Esmolol ameliorates lipopolysaccharide-induced intestinal injury via the AMPK/mTOR/ULK1 autophagy pathway. A: The MTT assay was used to detect cell viability; B-D: Western blotting analysis was performed to analyze Beclin-1, LC3-II expression in IEC-6 cells in the Sham, lipopolysaccharide (LPS), LPS + esmolol (ES), LPS + Compound C (CC), and LPS + ES + CC treatment groups (after 6 hours of treatment); E-H: Western blotting analysis was performed to analyze p-AMPK, p-ULK1, and p-mTOR expression in IEC-6 cells after 6 hours of treatment in each group. β-actin was used as a loading control and for normalization. Representative immunoblots and densitometry analyses are presented. Data in the Sham group were set to 1. n = 6 in each group. ES: Esmolol; CC: Compound C; LPS: Lipopolysaccharide.

Figure 13  Possible molecular mechanism by which esmolol influences signaling crosstalk in lipopolysaccharide-induced intestinal injury. Esmolol, as a selective β1-adrenoceptor antagonist, alleviates intestinal injury in rats with sepsis by augmenting autophagy, downregulating interleukin (IL-1), IL-17, TNF-α, and IL-6, and upregulating IL-10 through the AMPK/mTOR/ULK1 pathway. IL: Interleukin; LPS: Lipopolysaccharide.