Qihuang Zhuyu formula modulates mitochondrial function to inhibit platelet activation via endocannabinoid signaling mediated by the SIRT1/PPARα pathway

Ethnopharmacological relevance: Developing effective drugs is urgent for preventing platelet activation and atherosclerotic diseases. Qihuang Zhuyu formula (QHZYF) is a curative medical Plants formula in clinic while its molecular mechanism remains to be further elucidated.

Aim of the study: To investigate how QHZYF, via the Sirtuin 1 (SIRT1)/Peroxisome Proliferator-activated Receptor alpha (PPARα) pathway, mediates the endocannabinoid system, regulates mitochondrial function, and inhibits platelet activation, thereby offering novel strategies for the prevention and treatment of cardiovascular diseases.

Methods: The chemical constituents of QHZYF were characterized by ultra-performance liquid chromatography tandem with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS). For the in vivo experiments, an atherosclerosis rat model was developed. Platelet activation and endocannabinoid levels were assessed by ELISA, while the expression of PPARα was evaluated by immunofluorescence. Additionally, the expression of endocannabinoid-degrading Enzymes was analyzed by Western blotting. In the in vitro studies, the protein-protein interaction between SIRT1 and PPARα was initially investigated using co-immunoprecipitation (Co-IP). Thereafter, platelets were co-cultured with human umbilical vein endothelial cells (HUVECs) stimulated by oxidized low-density lipoprotein (ox-LDL). The mitochondrial function of platelets was examined by flow cytometry. Platelet activation and endocannabinoid levels were quantified using ELISA, and the expression of PPARα, endocannabinoid-degrading Enzymes and proteins reflecting mitochondrial function were determined by Western blotting. Subsequently, QHZYF was incorporated into the aforementioned system, and the entire experimental protocol was replicated to explore how QHZYF influences platelet activation through the SIRT1/PPARα pathway-mediated endocannabinoid system.

Results: In the in vivo experiments, it was observed that QHZYF significantly augmented the levels of PPARα, concurrently with a decrease in the expression of endocannabinoid-degrading Enzymes. This was paralleled by a rise in endogenous cannabinoid levels and a reduction in platelet activation, a process that was found to be regulated by SIRT1. The co-immunoprecipitation (Co-IP) analysis substantiated the cooperative interaction between SIRT1 and PPARα, underscoring their role in mediating the endocannabinoid system, regulating mitochondrial function, and consequently influencing platelet activation. In the in vitro studies, a co-culture system involving platelets and HUVECs stimulated with ox-LDL was employed. It was noted that the SIRT1/PPARα pathway mediates the endocannabinoid system in regulating mitochondrial function, which in turn affects platelet activation. The incorporation of QHZYF-containing serum into this system produced outcomes consistent with those observed in the in vivo experiments. These findings suggest that QHZYF operates by modulating the endocannabinoid system via the SIRT1/PPARα pathway, thereby controlling mitochondrial function and inhibiting platelet activation.

Conclusion: SIRT1 may mediate endocannabinoid signaling through its interaction with PPARα, regulating mitochondrial function and subsequently influencing platelet activation. QHZYF effectively modulates the endocannabinoid system via the SIRT1/PPARα pathway, thereby inhibiting platelet activation.

Endocannabinoid system; Mitochondrial function; Platelet activation; Qihuang Zhuyu; SIRT1/PPARα.