Cyclovirobuxine D Ameliorates Experimental Diabetic Cardiomyopathy by Inhibiting Cardiomyocyte Pyroptosis via NLRP3 in vivo and in vitro

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  FIGURE 1. HFD (high-fat and high-glucose diet) combined with STZ (streptozotocin) induces type 2 diabetes model in mice. (A) Insulin resistance index was calculated in mice by detecting FBG (fasting blood glucose) and INS (insulin). (B) After intraperitoneal injection of glucose in fasting condition, the blood glucose of mice at different time points was measured. (C) Histogram of AUC (area under the curve) of blood glucose at different time points in mice. (D) FBG of mice during modeling and treatment. (E,F) Effects of CVB-D on CHO (cholesterol) and TG (triglycerides) contents in serum of DCM (diabetic cardiomyopathy) mice. The data are presented as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01 versus the control group; Δ p > 0.05 versus the model group.

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  FIGURE 2. CVB-D improves cardiac dysfunction in DCM mice. (A) Effects of CVB-D on cardiac hypertrophy in DCM mice. (B) Cardiac function of mice was measured by cardiac ultrasound. (C–G) The effect of CVB-D on EF (left ventricular ejection fraction), FS (fractional shortening), IVSd (interventricular septal thickness at diastole), LVPWd (left ventricular posterior wall dimensions), LVIDd (left ventricular internal diameter at end-diastole) of DCM mice hearts. (H) The content of LDH (lactate dehydrogenase) in serum of mice was detected. The data are presented as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group.

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  FIGURE 3. CVB-D ameliorates the cardiac physiology changes in DCM mice. (A) HE (Hematoxylin-Eosin) staining of mice heart tissues. (B) Masson staining was performed in mice heart tissues. (C,D) Influence of CVB-D on TUNEL positive cell number. The data are presented as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group.

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  FIGURE 4. CVB-D inhibites the expression of pyroptosis-related proteins in the heart tissue of DCM mice. (A,B) Determination of NLRP3 and pyroptosis pathway-related proteins expression in heart tissues of DCM mice by western blotting, and all proteins expression were normalized to GAPDH. (C) The expressions of NLRP3, P-Caspase-1 (Pro-Caspase-1), C-Caspase-1 (Cleaved-Caspase-1), GSDMD&GSDMD-N, IL-18 and IL-1β were observed by immunohistochemistry. (D,E) Effects of CVB-D on the contents of IL-18 and IL-1β in mice serum. The data are presented as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group.

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  FIGURE 5. CVB-D improves the PNRCMs (primary neonatal rat cardiomyocytes) injury induced by HG (high glucose). (A) Immunofluorescence was used to identify the purity of PNRCMs. (B–D) The effect of CVB-D on the decreased viability of PNRCMs induced by HG was detected by MTT assay. (E,F) Western blotting was used to determine the effect of HG on NLRP3 expression in PNRCMs. (G,H) Calcein-AM/PI staining was used to observe cardiomyocyte pyroptosis caused by HG. (I) Effects of CVB-D on pathological changes of PNRCMs induced by HG were observed by Giemsa staining. (J–L) Influence of CVB-D on the contents of IL-18, IL-1β and LDH in the supernatant of PNRCMs culture medium were detected by ELISA and LDH kits. The data are presented as the mean ± SEM (n = 3), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group.

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  FIGURE 6. CVB-D inhibits PNRCMs pyroptosis induced by HG. (A–F) The effect of CVB-D on the expression of pyroptosis pathway proteins was observed by immunofluorescence. (G) The fluorescence values of each group were counted by ImageJ software. (H,I) Western blotting was used to determine the expression level of pyroptosis related proteins after CVB-D treatment. (J,K) Pyroptosis and living cells were differentiated by Calcein-AM/PI staining after CVB-D treatment. The data are presented as the mean ± SEM (n = 3), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group.

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  FIGURE 7. CVB-D ameliorates HG-induced PNRCMs injury by inhibiting cardiomyocyte pyroptosis via NLRP3. (A) Cytotoxicity of MCC950 was detected by MTT assay. (B) After pretreatment with MCC950, LDH content in cell culture supernatant was detected. (C,D) The effect of MCC950 on cardiomyocyte pyroptosis was judged by the number of PI staining positive cells. (E,F) The expression level of pyroptosis related proteins was detected by western blotting with MCC950 pretreatment. The data are presented as the mean ± SEM (n = 3), *p < 0.05, **p < 0.01 versus the control group; #p < 0.05, ##p < 0.01 versus the model group; Δp > 0.05 versus the HG+MCC950 group. (G) Cytotoxicity of BMS-986299 was detected by MTT assay. (H) LDH content in cell culture supernatant was detected after pretreatment with BMS-986299. (I,J) The effect of BMS-986299 on cardiomyocyte pyroptosis was observed by PI staining. (K,L) Western blotting was used to detected proteins expression in pyroptosis pathway with BMS-986299 pretreatment. The data are presented as the mean ± SEM (n = 3), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group; Δ p > 0.05 versus the HG+BMS-986299 group.

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  FIGURE 8. NLRP3-shRNA verifies the regulation mechanism of CVB-D inhibition pyroptosis induced by HG in PNRCMs. (A) LDH release in cell culture supernatant was detected after NLRP3-shRNA lentivirus-infected. (B,C) Western blotting was used to detected proteins expression in pyroptosis pathway after NLRP3-shRNA lentivirus infection with PNRCMs. The data are presented as the mean ± SEM (n = 3), *p < 0.05, **p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the model group; Δ p > 0.05 versus the HG+NLRP3-shRNA group; & p > 0.05 versus the model group.

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  FIGURE 9. The molecular regulation mechanism of CVB-D improving DCM. After HG induced NLRP3 overactivation, it assembles with ASC and pro-caspase-1 to form inflammasome. After Cleaved-caspase-1 is activated, on the one hand, it activates GSDMD-N to cause pore formation on cell membrane. On the other hand, Cleaved-caspase-1 promotes the maturation and secretion of IL-18 and IL-1β, finally leads to cardiomyocyte pyroptosis, accelerating the progress of DCM. CVB-D significantly ameliorates DCM, which may be associated with the inhibition of NLRP3-mediated cardiomyocyte pyroptosis.