Daming capsule combined with SGLT2i confers protection against diabetes with myocardial ischemia/reperfusion injury induced ferroptosis via AMPK

Figure 1 Diabetes mellitus with ischemia-reperfusion exacerbated cardiac dysfunction. A: Timeline of animal model construction; B and C: Representative images of echocardiogram and early to late diastolic transmitral flow velocity, ejection fraction, fractional shortening, left ventricular internal dimension in diastole and left ventricular internal dimension in systole were analyzed; D: Representative images of hematoxylin and eosin staining, and representative images of masson staining and statistical graph. Above results are presented as the mean ± SD. The comparison between more groups was conducted by one-way analysis of variance. ^aP < 0.05 vs the wild type group; ^bP < 0.05 vs the diabetes mellitus group. WT: Wild type; I/R: Ischemia-reperfusion; DM: Diabetes mellitus; HFD: High fat diet; STZ: Streptozotocin; FBG: Fasting plasma glucose; LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening; LVID;s: Left ventricular internal diameter at end-systole; LVID;d: Left ventricular internal diameter at end-diastole; E/A: Early to late diastolic transmitral flow velocity.

Figure 2 Diabetes mellitus with ischemia-reperfusion mice exhibited severe glucose and lipid metabolism disorders. A: Statistical analysis of fasting blood glucose; B: The content of total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein in serum; C: The activity of lactate dehydrogenase in serum; D: Representative images of BODIPY 493/503 staining of myocardial tissue and statistical graph. Above results are presented as the mean ± SD. The comparison between more groups was conducted by one-way analysis of variance. ^aP < 0.05 vs the wild type group; ^bP < 0.05 vs the diabetes mellitus group. WT: Wild type; I/R: Ischemia-reperfusion; DM: Diabetes mellitus; LDH-c: Lactate dehydrogenase cholesterol; LDL-c: Low-density lipoprotein cholesterol; TC: Total cholesterol; TG: Triglyceride.

Figure 3 Daming capsule combined with sodium-dependent glucose transporters 2 inhibitor improved cardiac function and metabolism caused by diabetes mellitus with ischemia-reperfusion. A: Representative images of echocardiogram and ejection fraction, fractional shortening, left ventricular internal diameter at end-diastole and left ventricular internal diameter at end-systole were analyzed; B: Statistical analysis of fasting blood glucose; C: The activity of lactate dehydrogenase in serum; D: The content of total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein in serum; E: Representative images of hematoxylin and eosin staining, and representative images of masson staining and statistical graph. Above results are presented as the mean ± SD. The comparison between more groups was conducted by one-way analysis of variance. ^aP < 0.05 vs the NULL group (diabetes mellitus + ischemia-reperfusion model). DMC: Daming capsule; SGLT2i: Sodium-dependent glucose transporters 2 inhibitor; I/R: Ischemia-reperfusion; DM: Diabetes mellitus; LDL-c: Low-density lipoprotein cholesterol; LDH: Lactate dehydrogenase; LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening; TC: Total cholesterol; TG: Triglyceride; LVID;s: Left ventricular internal diameter at end-systole; LVID;d: Left ventricular internal diameter at end-diastole; HE: Hematoxylin and eosin.

Figure 4 Sodium-dependent glucose transporters 2 inhibitor combined with daming capsule suppressed ferroptosis in diabetic mice with ischemia-reperfusion. A: Western blot was performed to detect the protein expression of glutathione peroxidase 4 and ferritin heavy chain 1 in mice; B: Iron deposition in cardiac tissue was detected by Prussian blue staining; C: Representative images of BODIPY 493/503 staining of myocardial tissue and statistical graph; D: Transmission electron micrographic image of mitochondrial ultrastructure in mice myocardial tissue. Above results are presented as the mean ± SD. The comparison between more groups was conducted by one-way analysis of variance. ^aP < 0.05 vs the wild type group; ^bP < 0.05 vs the diabetes mellitus + ischemia-reperfusion group or NULL group (diabetes mellitus + ischemia-reperfusion model). WT: Wild type; DMC: Daming capsule; SGLT2i: Sodium-dependent glucose transporters 2 inhibitor; I/R: Ischemia-reperfusion; DM: Diabetes mellitus; GPX4: Glutathione peroxidase 4; FTH1: Ferritin heavy chain 1.

Figure 5 Ferroptosis induces myocardial damage in vitro model of diabetes mellitus with ischemia-reperfusion. A: Timeline of cell model construction; B: CCK-8 detects cell activity to determine the protective effect of drugs in vitro; C: Representative images of BODIPY 493/503 staining and statistical graph in model groups; D and E: Immunofluorescence was conducted to detect the expression of glutathione peroxidase 4 and 4-hydroxynonenal in model groups; F: Ion-specific fluorescent probe was used to detect the content of iron in model groups; G: Lipid peroxidation sensor BODIPY™ 581/591 C11 was employed to detect lipid ROS levels in model groups; H: The changes of mitochondrial membrane potential in the model groups were analyzed by JC-1 fluorescent probe. Above results are presented as the mean ± SD. The comparison between more groups was conducted by one-way analysis of variance. ^aP < 0.05 vs the control group or the low glucose group; ^bP < 0.05 vs the high glucose + palmitic acid group. LG: Low glucose; HG: High glucose; PA: Palmitic acid; DAPI: 4,6-diamidino-2-phenylindole; CTRL: Control; H/R: Hypoxia/reoxygenation; 4-HNE: 4-hydroxynonenal; GPX4: Glutathione peroxidase 4.

Figure 6 Sodium-dependent glucose transporters 2 inhibitor combined with daming capsule inhibited ferroptosis in vitro and alleviated diabetes mellitus with ischemia-reperfusion. A: Flow chart of medicated medicine extracted; B: CCK-8 detects cell activity to determine the protective effect of drugs in vitro; C: Representative images of BODIPY 493/503 staining and statistical graph in treatment groups; D and E: Immunofluorescence was conducted to detect the expression of glutathione peroxidase 4 and 4-hydroxynonenal in treatment groups; F: Ion-specific fluorescent probe was used to detect the content of iron in treatment groups; G: Lipid peroxidation sensor BODIPY™ 581/591 C11 was employed to detect lipid reactive oxygen species levels in treatment groups; H: The changes of mitochondrial membrane potential in the treatment groups were analyzed by JC-1 fluorescent probe. Above results are presented as the mean ± SD. The comparison between more groups was conducted by one-way analysis of variance. ^aP < 0.05 vs the NULL group (diabetes mellitus + ischemia-reperfusion model); ^bP < 0.05 vs the high glucose + palmitic acid + hypoxia/reoxygenation group. HG: High glucose; PA: Palmitic acid; H/R: Hypoxia/reoxygenation; DMC: Daming capsule; SGLT2i: Sodium-dependent glucose transporters 2 inhibitor; GPX4: Glutathione peroxidase 4.

Figure 7 The combination therapy of Sodium-dependent glucose transporters 2 inhibitor combined with daming capsule relieved oxidative stress through adenosine monophosphate-activated protein kinase pathway to mitigate diabetes mellitus with ischemia-reperfusion effectively. A: Western blot was performed to detect the protein expression of phospho-adenosine monophosphate-activated protein kinase (p-AMPK) and adenosine monophosphate-activated protein kinase (AMPK) in mice; B: Western blot was performed to detect the protein expression of p-AMPK and AMPK in H9C2 cells. Above results are presented as the mean ± SD. The comparison between two groups was conducted by t-test. ^aP < 0.05 vs the wild type group or the control group; ^bP < 0.05 vs the diabetes mellitus + ischemia-reperfusion group or the high glucose + palmitic acid + hypoxia/reoxygenation group. WT: Wild type; DMC: Daming capsule; SGLT2i: Sodium-dependent glucose transporters 2 inhibitor; I/R: Ischemia-reperfusion; DM: Diabetes mellitus; HG: High glucose; PA: Palmitic acid; H/R: Hypoxia/reoxygenation; CTRL: Control.

Figure 8 Diagram of sodium-dependent glucose transporters 2 inhibitor and daming capsule protect diabetic myocardial ischemia-reperfusion through adenosine monophosphate-activated protein kinase pathway. Daming capsule, in synergy with sodium-dependent glucose transporters 2 inhibitor, reduced lipid droplet accumulation through the adenosine monophosphate-activated protein kinase signaling pathway, mitigated oxidative stress and thereby effectively inhibited diabetes mellitus with ischemia-reperfusion induced ferroptosis. DMC: Daming capsule; SGLT2i: Sodium-dependent glucose transporters 2 inhibitor; AMPK: Adenosine monophosphate-activated protein kinase; p-AMPK: Phospho-adenosine monophosphate-activated protein kinase; 4-HNE: 4-hydroxynonenal; GPX4: Glutathione peroxidase 4; FTH1: Ferritin heavy chain 1; GSH: Glutathione; GS-SH: Glutathione persulfide; DM: Diabetes mellitus; I/R: Ischemia-reperfusion.