Effects Of Ghrelin On Homocysteine-induced Dysfunction And Inflammatory Response In Rats Cardiac Microvascular Endothelial Cells

BackgroundGhrelin is a newly discovered hormone, produced principally in the stomach. Ghrelin can inhibit inflammatory response and apoptosis of endothelial cells effectively, promote angiogenesis, reduce ischemia reperfusion injury, relax vessels and alleviate heart failure.It is widely believed that hyperhomocysteinemia (HCY) is one of cardiovascular risk factors and may contribute to the pathogenesis of atherosclerosis by altering endothelial functions. One of the mechanisms of HCY-induced atherosclerosis is inflammatory response.However, research of effects of HCY on CMECs is still relatively insufficient at present, our study examined the effects of Ghrelin on homocysteine-induced CMECs, and explored its mechanism preliminarily.Methods:PartⅠ:CMECs culture. Rat left ventricular was harvested under the aseptic condition, and 0.02% collagenase typeⅡand 0.25% trypsin were used to digest heart tissues. After being centrifuged, sedimentation of the solution was re-suspended with complete culture medium (low glucose DMEM,15% FCS,1% streptomycin,100U/L penicillin).PartⅡ:The effects of HCY on rat CMECs. CMECs were isolated from rats left ventricle and administrated with HCY in concentration of 0,0.1,0.25,0.5,1 mmol/L respectively for 24 hours, MTT was used to detect the cell viability. NO secretion was measured by Griess reaction. The morphological assessment of apoptosis was studied by Hoechst 33258 staining. ELISA was used to detect the contents of the cell inflammatory factors.PartⅢ:The effects of Ghrelin on homocysteine-induced CMECs. CMECs were isolated from rats left ventricle and cultured and pretreated with Ghrelin at concentrations of 0,10,100, 1000ng/ml respectively for one hour. Then added HCY(0.25 mmol/L) to CMECs culture system. MTT was used to detect the cell viability. NO secretion was assessed by Griess reaction. The morphological assessment of apoptosis was studied by Hoechst 33258 staining. ELISA was used to detect the contents of the cell inflammatory factors. Western blot was used to examine the protein expression of the PCNA and NF-κB.Results1. CMEC were isolated and cultured in vitro successfully. 2. After treatment with HCY for 24h, the survival rate and the activity of NO of CMECs reduced significantly(P<0.05 vs. control), cell apoptosis ratio rose significantly(P<0.05 vs. control), and the secretion of inflammatory factors increased significantly(P<0.05 vs. control). The effect of HCY was dose-dependent.3. Compared with single HCY-treated group, in the group pretreated with Ghrelin, the activities of CMECs were significantly higher, the activity of NO significantly increased, the expression of cytokines obviously reduced, Western blot showed the expression of NF-κB elevated after treatment with HCY, whereas reversed significantly after pretreatment with Ghrelin.Conclusions1. HCY can induce dysfunction and inflammatory response in rats CMECs dose-dependently.2. Ghrelin inhibits the homocysteine-induced CMECs impairment and inflammatory response, likely mediated by inhibition of NF-κB activation.