Huodan Qinghua formula inhibits CD36 membrane localisation via the TGR5-DHHC4 pathway to ameliorate lipotoxic damage in diabetic cardiomyopathy

Background: Diabetic cardiomyopathy (DCM) is a diabetes-associated cardiac complication with a steadily increasing incidence and remains a major clinical challenge due to its complex and recurrent pathogenesis. Huodan Qinghua formula (HDQH), a proprietary herbal formulation, has been extensively employed in clinical practice for over a decade in the treatment of diabetic cardiovascular diseases, with demonstrated efficacy in improving cardiac function and prognosis. Nevertheless, the underlying mechanisms of action and pharmacological effects of HDQH in the amelioration of DCM remain to be fully elucidated.

Objective: To investigate whether HDQH alleviates cardiac lipotoxicity by reducing fatty acid uptake (FAU), thereby elucidating its underlying mechanism in the treatment of DCM.

Methods: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to identify HDQH and its bioactive constituents in vivo. Network pharmacology and molecular docking techniques were utilized to predict the key therapeutic targets and signaling pathways through which HDQH exerts its effects on DCM. These predictions were subsequently validated using both in vivo and in vitro experiments. A diabetic mouse model was established in C57BL/6 J mice via a high-fat diet combined with streptozotocin (STZ) administration, followed by daily oral gavage of HDQH for 12 weeks. Cardiac function and morphology were assessed via echocardiography, Masson's trichrome staining, and hematoxylin and eosin (HE) staining. Myocardial lipid accumulation and FAU were examined using Oil Red O staining and fluorescently labeled fatty acids (FAs), respectively. Levels of total bile acids, Insulin, NT-proBNP, and serum lipids were quantified using ELISA and biochemical assays, while free fatty acids (FFAs) and Reactive Oxygen Species (ROS) were also measured. Potential molecular mechanisms were explored using Western blot and real-time quantitative PCR (RT-qPCR). H9C2 cells were exposed to palmitic acid (PA), and the effects of HDQH were further examined through pharmacological activation and inhibition of target protein expression.

Results: Sixteen compounds and metabolites were identified in the plasma following HDQH administration, with major constituents including palmatine, bavachromanol, loganetin, oxyberberine, and neocryptotanshinone. Network pharmacology and molecular docking analyzes suggested that HDQH might exert potential therapeutic effects on DCM by modulating TGR5 receptor-associated signaling pathways. HDQH significantly improved cardiac function in DCM mice while effectively reducing myocardial hypertrophy, fibrosis, and lipid deposition. Concurrently, it elevated total bile acid levels whilst decreasing FFA and ROS levels. WB and RT-qPCR analyzes demonstrated that the inhibition of cardiac lipotoxicity by HDQH was closely associated with the regulation of TGR5, DHHC4, and CD36 expression at the cardiomyocyte membrane. In vitro, HDQH activated TGR5 expression, leading to suppression of the downstream target DHHC4, which in turn reduced excessive FAU by inhibiting CD36 membrane localization. Notably, the beneficial effects of HDQH were attenuated by the TGR5 inhibitor SBI-115.

Conclusion: HDQH exerts its cardioprotective effects against DCM by regulating the TGR5-DHHC4 pathway, inhibiting CD36 membrane localization, and thereby reducing cardiac FAU and lipid accumulation. These findings provide robust mechanistic evidence supporting HDQH as a promising therapeutic candidate for the treatment of diabetic cardiomyopathy.

CD36; Diabetic cardiomyopathy; Fatty acid uptake; Huodan qinghua formula; Lipotoxicity; TGR5-DHHC4 pathway.