Effects Of Different Concentration Of High Glucose On The Expression Of Collagen Typeâ… and Visfatin In Neonatal Rat Cardiac Fibroblasts In Vitro

Objective: Diabetes is one of the serious threats to the human health.Various complications of diabetes, especially the cardiovascular complicationsin diabetic patients are closely related to the high mortality. Diabeticcardiomyopathy (DCM),as an early complication of the diabetes, is mainlycharactered as left ventricular hypertrophy, impaired left ventricular diastolicfunction and the gradual emergence of ventricular systolic dysfunction, andthen ultimately, congestive heart failure(CHD). DCM is one of the mostimportant causes of death in diabetic patients. The major pathological changesof DCM is myocardial necrosis, hypertrophy and myocardial interstitialreconstruction and fibrosis. Excessive proliferation of cardiac fibroblast anddeposition of extracellular matrix (ECM) is an important part of myocardialfibrosis. The most of ECM are collagens,of which80%is collagen typeⅠ inthe cardiac tissue.Studies have shown that hyperglycemia in diabeticcardiomyopathy, which induces oxidative damage and the excessivedeposition of ECM,then leads to ventricle dysfuction,is an important factorinducing myocardial necrosis, hypertrophy and fibrosis and plays a importantrole in the development of DCM.Visfatin, as a novel adipocytokine, not only it can mimick theglucose-lowering effects of insulin, but also it is involved in the process ofinflammation, immune regulation and energy metabolism and are closelyrelated to the complex diseases of the obesity, diabetes, coronary heartdisease,metabolic syndrome, and congestive heart failure. Whether Cardiacfibroblasts are capable of secreting visfatin and the effects of visfatin on themechanisms of Diabetes induced myocardial damage are still unknown. Theobjectives of our study are visfatin and collagen typeⅠexpression of thecardiac fibroblasts in the hyperglucose environment in vitro to clarify the potential influence of visfatin in diabetic induced doposition ofECM,interstitial reconstruction and fibrosis to comprehend the mechanism ofDCM.Methods: In the asepsis condition, we extracted4-day-old male orfemale Sprague-Dawley (SD) rat cardiac fibroblasts and Subcultured them,then took the3rd generation of cardiac fibroblasts to our experiment. After48hours with serum-containing medium, the cells were cultured in serum-freemedium for24hours, then we selected the cells in good growth staterandomly. Experimental groups: the control (NG:containing5.5mmol/Lglucose),high glucose group (containing10mmol/L glucose)，high glucosegroup (containing30mmol/L glucose)， high glucose group (containing50mmol/L glucose), the hypertonic group (OSM: containing5.5mmol/Lglucose+19.5mmol/L mannitol), each of them divided into three wells. Afterculturing the cells in different concentrations of glucose for24hours, we usedReal time PCR to detect the levels of visfatin and collagen typeⅠmRNAexpression of cardiac fibroblasts and detected the amount of visfatin andcollagen typeⅠ expression in the medium by ELISA.Results:1. high concentration of glucose induces the expression of thecollagen typeⅠ in cardiac fibroblastsCompared with the control, in the high glucose group (10mmol/L,30mmol/L,50mmol/L)expression of the procollagen type Ⅰ mRNA wassignificantly increased (P<0.01). Compared with the hypertonic group, in thehigh glucose group (10mmol/L,30mmol/L,50mmol/L),the expression of theprocollagen type Ⅰ mRNA was significantly increased (P<0.01).There was nostatistically significant observed on procollagen type Ⅰ gene expression in thehypertonic group and the control(P>0.05). Procollagen type Ⅰ mRNAexpression of high glucose group30mmol/L increased compared with10mmol/L group, with a significant difference observed in statistics(P<0.01).Expression of procollagen type Ⅰ mRNA in50mmol/L group was greater thanthe30mmol/L group, which is not significant in statistics(P>0.05).At the same time, enzyme-linked immunosorbent assay(ELISA) determination assay was used to detect the amount of type Ⅰ collagen productin the medium.The results showed a significant up-regulation of type Ⅰcollagen in cardiac fibroblasts of SD rats in hypergloucose groups(10mmol/L,30mmol/L,50mmol/L), when compared with controls. Compared with thehypertonic group, in the high glucose group,the expression of collagen type Ⅰwas significantly increased(P<0.01).Similar findings were noted in othergroups of hypergloucose,where the cardiac fibroblasts had significantly higherexpression of type Ⅰ collagen in higher levels of glucose solution(P<0.01).Interestingly, type Ⅰ collagen was not seen to be significantly up-regulated in50mmol/L group when compared with corresponding30mmol/Lgroup(P>0.05), whereas there was no significant difference in the expressionof type Ⅰ collagen in the hyperosmosis group and controls (P>0.05).2.high glucose increased the expression of visfatin in cardiac fibroblaststhe amount of the visfatin mRNA in the high glucose group (30mmol/L,50mmol/L) increased compared to controls (P<0.01). In10mmol/L group,theamount of the visfatin mRNA also increased compared to controls with astatistical significance(P<0.05).Compared with the hypertonic group, in thehigh glucose group(30mmol/L,50mmol/L),the expression of the visfatinmRNA was significantly increased(P<0.01).the mRNA of visfain in the10mmol/L group is greater than the hypertonic group,but nosignificance(P>0.05).Visfatin gene expression in the hyperosmosis group andcontrols was not statistically significant (P>0.05). Visfatin mRNA expressionin30mmol/L group was greater than10mmol/L group significantly (P<0.01).The level of visfatin mRNA expression in50mmol/L group was a little lowerthan the30mmol/L group without a significant difference (P>0.05).The application of ELISA on the visfatin content in the medium showed thatcompared with the control, in high glucose groups (10mmol/L,30mmol/L and50mmol/L), the expression of visfatin increased significantly.Compared withthe hypertonic group, in the high glucose group,the expression of visfatin wassignificantly increased (P<0.01).There was no significant difference inexpression of visfatin in the hyperosmosis group and the control (P>0.05). The amount of30mmol/L group is more than10mmol/L group on the visfatinexpression, and a significant difference was observed(P<0.01). The level ofthe visfatin expression in50mmol/L group was lower than30mmol/Lgroup,without a statistical difference (P>0.05).Conclusion: The high glucose induces collagen type Ⅰ synthesis of cardiacfibroblasts, significantly increases the accumulation of type Ⅰ collagenproducts, and as the elevation of glucose concentration, the expression ofcollagen type Ⅰ also increases in cardiac fibroblasts. In cardiac fibroblastssynthesis and expression of visfatin could be induced in high glucosecondition, and the cardiac fibroblasts had significantly higher expression ofvisfatin in higher levels of glucose solution.