Puerarin-V Improve Mitochondrial Respiration and Cardiac Function in a Rat Model of Diabetic Cardiomyopathy via Inhibiting Pyroptosis Pathway through P2X7 Receptors

Abstract

There is a new form of puerarin, puerarin-V, that has recently been developed, and it is unclear whether puerarin-V has a cardioprotective effect on diabetic cardiomyopathy (DCM). Here, we determined whether puerarin-V had any beneficial influence on the pathophysiology of DCM and explored its possible mechanisms. By injecting 30 mg/kg of STZ intraperitoneally, diabetes was induced in rats. After a week of stability, the rats were injected subcutaneously with ISO (5 mg/kg). We randomly assigned the rats to eight groups: (1) control; (2) model; (3) metformin; (4-6) puerarin-V at different doses; (7) puerarin (API); (8) puerarin injection. DCM rats were found to have severe cardiac insufficiency (arrythmia, decreased LVdP/dt, and increased E/A ratio). In addition, cardiac injury biomarkers (cTn-T, NT-proBNP, AST, LDH, and CK-MB), inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α), and oxidative damage markers (MDA, SOD and GSH) were markedly increased. Treatment with puerarin-V positively adjusts these parameters mentioned above by improving cardiac function and mitochondrial respiration, suppressing myocardial inflammation, and maintaining the structural integrity of the cardiac muscle. Moreover, treatment with puerarin-V inhibits the P2X7 receptor-mediated pyroptosis pathway that was upregulated in diabetic hearts. Given these results, the current study lends credence to the idea that puerarin-V can reduce myocardial damage in DCM rats. Furthermore, it was found that the effect of puerarin-V in diabetic cardiomyopathy is better than the API, the puerarin injection, and metformin. Collectively, our research provides a new therapeutic option for the treatment of DCM in clinic.

Keywords: P2X7 receptors; diabetic cardiomyopathy; mitochondrial; puerarin-V; pyroptosis.

Figure 1

Puerarin-V improved the general parameters of DCM rats. (A) The body weight change, (B) the body weight before injection of STZ, (C) the body weight after 6 weeks of drug treatment, (D) the oral glucose tolerance test (OGTT), (E) the area under the OGTT curve, (F) the liver index, (G) the lung index, (H) the spleen index, (I) the kidney index. The data are represented by mean ± SEM (n = 6–8). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 2

Puerarin-V modulated lipid metabolism disturbances. The serum levels of (A) TG, (B) LDL, (C) NEFA, (D) GSP, (E) CHO, and (F) HbA1c were measured. The data are represented by mean ± SEM (n = 6–8). ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 3

Puerarin-V attenuated the electrocardiographic changes induced by diabetes. (A) Representative images of typical electrocardiogram of different groups. (B) ST-segment of different groups. Puerarin-Ⅴ improved the levels of standard deviation of NN intervals (SDNN) (C), root mean square difference between adjacent RR intervals (RMSSD) (D), normalized value of low frequency (LF/(TP-VLF)) (E), normalized value of low frequency (HF/(TP-VLF)) (F), and low frequency (LF)/low frequency (HF) (G) in rats subjected to diabetic cardiomyopathy. The data are represented by mean ± SEM (n = 6–8). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 vs. model group.

Figure 4

Puerarin-V enhanced the function of hemodynamics and left ventricular. (A) The situation of P–V loop of different groups, (B) LVSW, (C) Ea, (D) LVEDP, and (E) dp/dt_max. (F) The content of NO. (G) The activity of NOS. (H) The activity of Na⁺-K⁺-ATPase. (I) The activity of Mg²⁺-ATPase. (J) The activity of Ca²⁺-ATPase. (K) The activity of Ca²⁺Mg²⁺-ATPase in diabetic cardiomyopathy rats. The data are represented by mean ± SEM (n = 6–8). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 vs. model group.

Figure 5

Puerarin-V ameliorated cardiac function in the DCM rats. (A) Representative echocardiographic M-mode records of left ventricular wall and Doppler images of aortic flow. Effects of Puerarin-V on LVIDs (B), LVIDd (C), LVEF (D), LVFS (E), SV (F), CO (G), HR (H), AV peak V (I), and E/A ratio (J). (K) The heart weight/body weight ratio. (L) The left ventricular weight/body weight ratio. (M) The right ventricular weight/body weight ratio. The data are represented by mean ± SEM (n = 6–8). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 6

The DCM rats were protected from myocardial inflammation and necrosis by puerarin-V. (A) Histopathological changes in myocardium of mice subjected to diabetic cardiomyopathy rats (HE stain). Small images are 100×, large images are 200×. Scale bar: 100 μm. (B) Effects of puerarin-Ⅴ on IST. (C) Effects of puerarin-Ⅴ on LVWT. (D–G) The quantitative analyses of IL-6, TNF-α, IL-1β, and IL-18. The data are represented by mean ± SEM (n = 6–8). ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 7

Puerarin-V inhibited the myocardial fibrosis in the DCM rats. (A) Fibrosis changes in myocardium of DCM rats (Masson staining). Left images are 2×, right images are 20×. (B) Effect of puerarin-Ⅴ on left ventricular collagen content. The data are represented by mean ± SEM (n = 6–8). ^## p < 0.01 vs. control group. * p < 0.05 vs. model group.

Figure 8

Puerarin-V preserved the myocardial integrity in the DCM rats. (A) Alteration of ultrastructure of cardiac muscle cells. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) detection of skeleton protein titin (B) and nebulin (C) mRNA expression in myocardial tissue. The data are represented by mean ± SEM (n = 6–8). ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 9

Puerarin-V improved necrosis and antioxidative status of DCM rats. (A–E) The serum levels of cTn-T, NT-proBNP, AST, LDH, and CK-MB. (F–H) The content of MDA, SOD, and GSH activity in the myocardium. The data are represented by mean ± SEM (n = 6–8). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 10

Puerarin-V increased mitochondrial respiration in hearts of DCM rats through complex I/II-related molecular mechanisms. (A) The situation of cabin. (B) The protocol. Summary of mitochondrial respiration data in different groups, including routine measurements (C), CI leak (D), CI oxidative phosphorylation (E), CI plus CII oxidative phosphorylation (F), outer mitochondrial membrane integrity (G), CII electron transfer system (H), CI plus CII electron transfer system (I), residual non-mitochondrial oxygen consumption (J). The data are represented by mean ± SEM (n = 6–8). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 11

Puerarin-V hindered NLRP3-Caspase-1-GSDMD mediated pyroptosis signaling pathway activation. (A–H) Western blot analyses were performed for NLRP3, P2X7, N-GSDMD, Cleaved-caspase-1, ASC, IL-1β, and IL-18 expression, and normalization was performed using β-actin expression. The data are represented by mean ± SEM (n = 6). ^# p < 0.05 and ^## p < 0.01 vs. control group. * p < 0.05 and ** p < 0.01 vs. model group.

Figure 12

A mechanism diagram. Puerarin-V improves mitochondrial respiration and cardiac function in streptozotocin–isoproterenol induced myocardial injury via inhibiting pyroptosis pathway through P2X7 receptors.

Figure 13

Schematic representation of animal experimental design.