Melatonin ameliorates ox-LDL-induced lipid accumulation and enhances ABCA1 expression in VSMCs via inhibition of the TRPV1-Ca2+-calpain signaling pathway

Abstract

Oxidized low-density lipoprotein (ox-LDL) has been shown to induce significant lipid accumulation and disrupt cholesterol homeostasis in vascular smooth muscle cells (VSMCs), which may contribute to the development of atherosclerosis (AS). In this study, we investigated the effects of ox-LDL on lipid accumulation, and the expression of key proteins involved in cholesterol transport, such as ATP-binding cassette transporter A family member 1 (ABCA1). Our results demonstrated that ox-LDL treatment led to a concentration-dependent increase in intracellular lipid content, as evidenced by Oil Red O and BODIPY staining. Additionally, ox-LDL elevated intracellular calcium (Ca²⁺) levels and activated the Ca²⁺-Calpain signaling pathway, which in turn suppressed the expression of ABCA1, a critical protein responsible for cholesterol efflux. We further explored the therapeutic potential of melatonin (MLT) in mitigating the harmful effects of ox-LDL. Treatment with MLT significantly improved cell viability and reduced lipid accumulation in VSMC. Moreover, MLT, along with specific inhibitors of the TRPV1-Ca²⁺-Calpain pathway (Capsazepine, EGTA, and Calpeptin), successfully lowered intracellular Ca²⁺ levels and calpain activity, while restoring ABCA1 expression at protein levels but not mRNA. These findings suggest that MLT exerts a protective effect by inhibiting the TRPV1-Ca²⁺-Calpain signaling pathway, thereby promoting cholesterol efflux and reducing lipid accumulation in VSMCs. To validate these findings in vivo, ApoE^−/− mice were fed a high-fat diet (HFD) for 12 weeks to induce atherosclerotic lesions, with MLT administered via intraperitoneal injection during the final 6 weeks. Histological analyses of the aortic root revealed that MLT treatment significantly reduced atherosclerotic plaque areas compared to untreated HFD-fed mice. Furthermore, Western blot analyses demonstrated that MLT increased ABCA1 expression and decreased TRPV1 expression in aortic tissues, aligning with our in vitro observations. In conclusion, our study highlights the role of the TRPV1-Ca²⁺-Calpain signaling pathway in ox-LDL-induced lipid dysregulation and identifies MLT as a potential therapeutic agent for reversing these effects, offering new insights into the molecular mechanisms underlying lipid accumulation in VSMCs.