Tumor necrosis factor-α promotes abnormal glucose metabolism after acute pancreatitis by inducing islet β-cell apoptosis via Bax/Bcl-2/caspase-3 signaling pathway

Figure 1 Impaired viability of islet β-cells in acute pancreatitis models. A-F: Cell Counting Kit-8 assay was used to assess the proliferation of islet β-cells (MIN-6) under control conditions, lipopolysaccharide stimulation (5 μg/mL or 10 μg/mL), and co-culture with media from lipopolysaccharide-treated 266-6 cells (A). Apoptosis in islet β-cells was evaluated in both acute pancreatitis cell and mouse models using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (B and E). Quantitative analysis of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive β-cells (C and F). Insulin secretion by islet β-cells in the acute pancreatitis model was quantified with an enzyme-linked immunosorbent assay kit (D). Representative results from three independent replicates are shown (n = 8). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. LPS: Lipopolysaccharide; AP: Acute pancreatitis; MAP: Mild acute pancreatitis; SAP: Severe acute pancreatitis; HE: Hematoxylin and eosin.

Figure 2 Tumor necrosis factor-α levels were elevated in acute pancreatitis models. A: Tumor necrosis factor-α levels in conditioned media from MIN-6 cells were quantified by enzyme-linked immunosorbent assay under control conditions, lipopolysaccharide stimulation (5 μg/mL or 10 μg/mL), and co-culture with media from lipopolysaccharide-treated 266-6 cells; B: Serum tumor necrosis factor-α levels in acute pancreatitis mouse models were quantified using the same methodology. Representative results from three independent replicates are shown. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. AP: Acute pancreatitis; TNF-α: Tumor necrosis factor-α; LPS: Lipopolysaccharide; MAP: Mild acute pancreatitis; SAP: Severe acute pancreatitis.

Figure 3 Acute pancreatitis induces islet β-cell apoptosis through the tumor necrosis factor-α-dependent Bax/Bcl-2/caspase-3 apoptotic signaling pathway. A: Western blot analysis was performed to detect the expression of caspase-3, Bcl-2, and Bax in MIN-6 cells under control conditions, lipopolysaccharide stimulation, and co-culture with media from lipopolysaccharide-treated 266-6 cells, and in acute pancreatitis mouse tissues; B-D: Quantitative analysis of Bcl-2, Bax and caspase-3 relative protein expression in vitro acute pancreatitis model; E-G: Quantitative analysis of Bcl-2, Bax and caspase-3 relative protein expression in vivo acute pancreatitis model. Representative results from three independent replicate assays are shown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Data are presented as the mean ± SD. AP: Acute pancreatitis; LPS: Lipopolysaccharide; MAP: Mild acute pancreatitis; SAP: Severe acute pancreatitis.

Figure 4 Inhibition of tumor necrosis factor-α expression attenuated the impairment of islet β-cell in acute pancreatitis models. A: Tumor necrosis factor-α levels in MIN-6 conditioned media were measured by enzyme-linked immunosorbent assay under control conditions, lipopolysaccharide stimulation (10 μg/mL), and co-culture with media from lipopolysaccharide-treated 266-6 cells, with or without treatment with a tumor necrosis factor-α inhibitor; B: Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay; C: Quantitative analysis of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive β-cells; D: Insulin secretion by islet β-cells in the acute pancreatitis model was quantified using an enzyme-linked immunosorbent assay kit. Representative results from three independent replicate assays are shown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. AP: Acute pancreatitis; TNF-α: Tumor necrosis factor-α; LPS: Lipopolysaccharide.

Figure 5 Inhibition of tumor necrosis factor-α expression reversed the alterations in the Bax/Bcl-2/caspase-3 apoptotic signaling pathway in acute pancreatitis models. A: Western blot analysis of caspase-3, Bcl-2, and Bax expression in MIN-6 cells under control conditions, lipopolysaccharide stimulation (10 μg/mL), and co-culture with media from 266-6 cells, with or without tumor necrosis factor-α inhibitor treatment; B-D: Quantitative analysis of Bcl-2, Bax and caspase-3 relative protein expression in vitro acute pancreatitis model. Representative results from three independent replicate assays are shown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. LPS: Lipopolysaccharide.