Curcumin Attenuates Calcification Of Rat Vascular Smooth Muscle Cells Via Inhibition Of Apoptosis

BackgroundVascular calcification refers to calcium deposition within artery wall, leading to arterial stiffness, and reduced elasticity and compliance. It commonly occurs in patients with atherosclerosis, chronic kidney disease and diabetes and highly threatens human health. Vascular calcification is an independent risk factor for cardiovascular mortality and morbidity.Coronary artery calcification can be used as an independent indicator for stroke occurrence. However, there is no efficient clinical prevention and treatment for vascular calcification due to lack of better understanding the mechanisms underlying vascular calcification. Therefore, it is very important to investigate the development and progression of vascular calcification, and especially elucidate the molecular mechanisms underlying vascular calcification.Accumulating studies have demonstrated that vascular calcification is an actively gene-regulated process resembling bone formation. Vascular smooth muscle cells(VSMCs) have been shown to be the major cells involved in vascular calcification. Exposed to harmful stimuli such as oxidative stress and dysregulation of calcium and phosphate metabolism, VSMCs can undergo phenotypic change from contractile to osteoblast-like type, characterized by secreting bone-related proteins including ALP, cbfa1/Runx2, BMPs and OCN, leading to vascular calcification. Osteogenic differentiation of VSMCs is the key step during the development of vascular calcification.Disorders of calcium and phosphate metabolism can induce calcification and apoptosis of VSMCs. Previous studies have demonstrated that apoptosis of VSMCs has played an important role in the process of vascular calcification. Apoptosis can be detected before the onset of calcification of VSMCs. Apoptosis bodies released from VSMCs can concentrate, crystallize calcium and initiate vascular calcification. VSMC apoptosis regulates cell calcification, and inhibition of apoptosis can reduce calcification of vascular smooth muscle cells. Our previous studies have shown that apoptosis of VSMCs regulates cell calcification induced by oxidized low density lipoprotein.Curcumin, derived from the plant curcuma extract, can attenuate vascular lesions including neointimal hyperplasia after vascular injury and atherosclerosis. Curcumin exerts a variety of pharmacological activities including anti-inflammatory, anti-oxidative, anti-migratory and anti-proliferative effects on VSMCs. In addition, curcumin has been shown to inhibit apoptosis and osteoblast calcification. However, it is unclear whether curcumin affects osteogenic differentiation and calcification of VSMCs.We hypothesize that curcumin could inhibit osteogenic differentiation and calcification of VSMCs via regulation of apoptosis. High calcium and phosphate-induced in vitro model of cell calcification will be used in this study. We investigate the effect of curcumin on VSMC differentiation and calcification and the underlying apoptosis mechanism.Aims 1. To investigate whether curcumin inhibits osteogenic differentiation and calcification of VSMCs. 2. To examine whether curcumin inhibits apoptosis of VSMCs. 3. To elucidate the mechanism by which curcumin inhibits osteogenic differentiation, calcification and apoptosis of VSMCs is through regulation of JNK/Bax signaling pathway.Methods Calcifying medium supplemented with BGP(10 m M) and calcium chloride(3 m M) was used to treat VSMCs for 10 days. Alizarin red was used to detect calcium deposition in VSMCs and ocresolphthalein complexone method was used to examine calcium content. ALP activity was assessed by spectrophotometry. q RT-PCR and Western blot analysis were used to examine gene expression. Apoptosis of VSMCs was analyzed by flow cytometry and Caspase3 activity was determined using Caspase3/CPP32 Colorimetric Assay Kit.Results Curcumin inhibits calcifying medium-induced osteogenic differentiation and calcification of VSMCs, as well as cell apoptosis. Additionally, curcumin inhibits the phosphorylation of JNK and Bax expression. Inhibition of JNK can attenuate osteogenic differentiation and calcification of VSMCs.Conclusion Curcumin attenuates apoptosis and calcification of VSMCs, presumably via inhibition of JNK/Bax signaling pathway.