Metformin Inhibits the NLRP3 Inflammasome via AMPK/mTOR-dependent Effects in Diabetic Cardiomyopathy

Figure 1. Cardiac function and morphology in diabetic mice treated with or without metformin. (A) M-mode echocardiograms of the left ventricles are shown. EF (B) and FS values (C) are shown. (D) The results of HE and Masson's trichrome staining are shown. Scale bar, 60 μm. (E and F) Western blot analysis of collagen I and collagen III expression in the different groups. *P<0.05 compared to the control group, #P<0.05 compared to the DM group. n=5.

Figure 2. NLRP3, caspase-1 and IL-1β expression levels in vivo. (A) Immunohistochemistry analysis was performed to detect the expression of NLRP3, caspase-1 and IL-1β. Scale bar, 60 μm. The results were quantified and statistically analyzed. Western blotting analyses were conducted to detect the expression levels of NLRP3 (B), caspase-1 (C) and IL-1β (D). *P<0.05 compared to the control group, #P<0.05 compared to the DM group. n=5.

Figure 3. NLRP3, caspase-1, IL-1β and GSDMD-N expression levels in vitro. Primary cardiomyocytes were treated with 5.5 mmol/L (control) and 30 mmol/L glucose (HG). The cardiomyocytes in the HG group were treated with or without 2 mM metformin. Western blotting was used to detect the expression levels of NLRP3 (A), caspase-1 (B) and IL-1β (C). The expression levels of GSDMD-N were detected by an immunofluorescence assay (D) and western blotting (E). Scale bar, 100 μm. *P<0.05 compared with the control group, #P<0.05 compared to the HG group. n=3.

Figure 4. Metformin activates mitochondrial complex I/p-AMPK expression. (A) The activity of mitochondrial complex I was detected in cardiomyocytes. (B) The protein expression levels of p-AMPK and AMPK in cardiomyocytes were detected by western blotting. GAPDH was used as internal control. n=3. (C) The expression levels of p-AMPK and AMPK in mice were detected. n=5. *P<0.05 compared to the control group. #P<0.05 compared to the HG or the DM group.

Figure 5. An AMPK inhibitor blocked the suppressive effect of metformin on the NLRP3 pathway in cardiomyocytes. In HG-induced cardiomyocytes treated with 2 mM metformin or 2 mM metformin and 10 μM compound C, the expression levels of p-AMPK and AMPK (A), NLRP3 (B), caspase-1 (C) and IL-1β (D) were detected by western blotting. The expression levels of GSDMD-N were determined by western blotting (E) and immunofluorescence staining (F). Scale bar, 100 μm. LC3 (G) and mTOR (H) were detected by western blotting. *P<0.05 compared to the control group, #P<0.05 compared to the HG group. &P<0.05 compared to the HG+Met group. n=3.

Figure 6. An AMPK inhibitor blocked the protective effect of metformin on cardiac function. (A) M-mode echocardiograms of the left ventricles are shown. (B) EF and FS values are shown. (C) HE and Masson's trichrome staining are shown. Scale bar, 60 μm. (D and E) Western blot analysis of collagen I and collagen III expression in the different groups. *P<0.05 compared to the control group, #P<0.05 compared to the DM group, &P<0.05 compared to the DM+Met group. n=5.

Figure 7. Metformin inhibits the NLRP3 pathway via AMPK/mTOR-dependent effects in diabetic mice. The expression levels of p-AMPK and AMPK (A), LC3 (B), mTOR (C), NLRP3 (D), caspase-1 (E) and IL-1β (F) were detected by western blotting. (G & H) Immunohistochemistry analysis was performed to detect NLRP3, caspase-1 and IL-1β expression. Scale bar, 60 μm. *P<0.05 compared to the control group, #P<0.05 compared to the DM group, &P<0.05 compared to the DM+Met group. n=5.

Figure 8. Graphical abstract of how metformin inhibits the NLRP3 pathway via AMPK/mTOR-dependent effects in DCM.