Shuangshen Ningxin capsules ameliorate diabetic cardiomyopathy in mice by inhibiting ferroptosis via the NRF2/HO-1 signaling pathway.

OBJECTIVE: To investigate the effects of Shuangshen Ningxin Capsules (SSNX) on myocardial function in patients with diabetic cardiomyopathy (DCM) and its underlying mechanism. METHODS: A streptozotocin (STZ)-induced DCM model was established in C57BL/6J mice. The mice were administered low or high doses of SSNX (90 mg/kg/d or 180 mg/kg/d). Cardiac function was evaluated by echocardiographic parameters, H&E staining, Masson staining, and TUNEL assays. Transcriptomics analysis and Western blotting analysis were performed to explore potential molecular mechanisms. An in vitro high glucose (35 mmol/L)-induced DCM cell model was also established. Cells were treated with SSNX (40 μg/mL or 80 μg/mL) alone or in combination with the ferroptosis activator erastin (10 μM) or the NRF2 inhibitor ML385 (20 μM). Biochemical assays, EdU staining, and Western blotting were performed to investigate the effects of SSNX on cell proliferation, ferroptosis, and the NRF2/HO-1 pathway in DCM cells. RESULTS: SSNX significantly improved cardiac dysfunction and attenuated cardiomyocyte hypertrophy, myocardial fibrosis, and apoptosis in DCM mice. Transcriptomics analysis revealed that after SSNX intervention, 16 originally upregulated genes were downregulated, while 33 originally downregulated genes were upregulated. These differentially expressed genes (DEGs) were associated with ferroptosis-related pathways. In vitro experiments showed that SSNX inhibited ferroptosis in the myocardium of DCM mice through activating the NRF2/HO-1 signaling pathway. Moreover, SSNX significantly reversed high glucose-induced suppression of the proliferation of cardiomyocytes, inhibition of the NRF2/HO-1 signaling pathway, and induction of ferroptosis. CONCLUSION: SSNX alleviates myocardial injury in DCM mice, and the mechanism underlying the effect of SSNX may involve activation of the NRF2/HO-1 signaling pathway to inhibit ferroptosis.