Role Of High Glucose And Advanced Glycation End Products On Cultured Skin Fibroblast And Its Possible Mechanism

High glucose is a symbol of diabetes by chronic metabolic diseases and its incidence and the trend of younger age have been rising in recent years.Studies have shown that diabetes lead to the complications of cardiovascular,Eyes, Neuromuscular,Renal,Cutaneous and others and affect human health seriously.Diabetes complications are the main reason for high incidence and the morality which have attracted intensive attention.Clinical studies shown that the diabetic patients'skin can be damaged easily,the thickness of skin reduced obviously and lead to impaired wound healing.The common characters of diabetes mellitus are xerosis and skin itch which affecting the quality of life of patients seriously.But the mechanism is currently remains unclear.Our previous research indicated that the thickness of shin layer was reduced with tenuous collagen distributing disorderly as well as decreased secretion of collagens.Why does diabetes lead to reduction of skin collagen content? What is the specific mechanism? Currently,the investigation on skin lesions of diabetes mellitus was most in clinical reasearch,but less in cellular and molecular level.In this study,we investigated the effects of different concentrations of glucose and AGEs on cell damage and collagen synthesis in cultured rat skin fibroblasts cells which can provide experimental data and theoretical basis for future clinical prevention and treatment of cutaneous complications of diabetes and drug research.1 Effects of high glucose on the collagen synthesis in rat skin fibroblastsTo investigate the effects of high concentrations of glucose on cell injury and collagen synthesis in cultured rat skin fibroblasts cells and to find out the pathogenesis of lessening of skin thickness in the diabetes.After the cell culture,the experimental were divided into 4 groups:control group(25mmol/ L),high glucose 1 group(30mmol/L),high glucose 2 group(35mmol/L)and high glucose 3 group(40mmol / L).After 24h and 48h,we detected the survival rate.Meanwhile,we detected some indicators about oxidative damage(the extent of SOD,GSH and MDA),and the hydroxyproline content in the cultural medium was determined by the commercial kit.At the same time,the expressions of typeⅠ,Ⅲprocollagen,matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2) were measured.The results showed that high concentrations of glucose(30,35,40 mmol /L) inhibit the proliferation of the cultured skin fibroblast cells significantly,and we also found that the enzyme activity of GSH and SOD decreased obviously while MDA content increased significantly,compared with control group(25mmol / L)(P＜0.05 and P＜0.001)The content of collagen synthesis are significantly decreased in a concentration-depended manner(P＜0.001),compared with the controlgroup.RT-PCR results showed that the mRNA levels of procollagenⅠ,Ⅲand TIMP-2 were decreased markedly,while MMP-2 was increased as compared with those of low glucose concentration(P＜0.05).We can conclude that high glucose may induce oxidative stress in skin fibroblasts. Both the downregulation of procollagen and TIMP-2 expression and the upregulation of MMP-2 expression contributed to the decrease of collagen content after exposure to high concentrations of glucose.2:Effects of AGEs on the collagen synthesis in rat skin fibroblastsTo investigate the effects of high concentrations of glucose on cell injury and collagen synthesis in cultured rat skin fibroblasts cells and to find out its relationship with diabetes mellitus.First we set up nonenzymatic glycation system in vitro and prepared advanced glycation end products.50g/L bovine serum albumin(BSA)was incubated with 100mmol/L D-glucose at 37℃for 3 months.The skin fibroblast cells in cell culture were affected by AGEs.The effects on damageing of cells were investigated by MTT,while we detected the contents of SOD,GSH and MDA.The hydroxyproline content in the cultural medium was determined by the commercial kit.The expression of typeⅠ,Ⅲprocollagen mRNA,matrix metalloproteinase-2 (MMP-2)mRNA and tissue inhibitor of metalloproteinase-2(TIMP-2)mRNA was analysed by RT-PCR.The results indicated that AGEs inhibited the proliferation of the cultured skin fibroblast cells in a dose-dependent manner(P＜0.001).The MDA contents of AGEs 1 group,AGEs 2 group and AGEs 3 group were significantly higher than the control group(P＜0.05 and P＜0.001),but the enzyme activity of SOD and GSH content was decreased significantly(P＜0.05 and P＜0.001).Compared with the control group, the amount of collagen secreted by rat skin fibroblast cells cultured in AGEs were decreased seriously in a time-dependent manner.(P＜0.05 and P＜0.001).RT-PCR results showed thatⅠ,Ⅲprocollagen protein mRNA expression levels increased significantly compared with control group(P＜0.05).Compared with control group,the mRNA expression of MMP-2 mRNA increased markedly(P＜0.05)while TIMP-2 mRNA expression decreased in skin fibroblast cellsof(P＜0.05).We can conclude that AGEs can do harm to fibroblast in a dose-dependent manner. The high contents of AGEs have the inhibiting effects on skin fibroblasts,which might be one of the most important reasons leading to diabetes mellitus as well as the secretion of collagens.All in all,we can conclude as follows:1.High concentrations of glucose and AGEs did have damage on skin fibroblasts in a dose-dependent manner,and the damage of AGEs to fibroblast cells is more serious than that of high glucose damage which may be the results from several factors such as oxidative damage and advanced glycation end products pathways lead to the diabetes complications,and the damage of AGEs to fibroblast cells is more serious than that of high glucose damage.2.Both the downregulation ofⅠandⅢprocollagen and TIMP-2 expression and the upregulation of MMP-2 expression contributed to the decrease of collagen content after exposure to high concentrations of glucose and AGEs,which might be one of the most important reasons leading to diabetes mellitus as well as the secretion of collagens.