| Background:The morbidity of DM is increasing year by year in the last decades.DM and its chronic complications have been recognized as one of the great threats to human health.The prevention and cure of diabetic cardiovascular complication has been the focus of clinical practice.The concept has been shifted from glucose and lipid metabolic disorders to immune and inflammatory mechanisms.Some studies have shown the inappropriate activation of innate immunity characterized by TLR4and inflammation might play an important role in DM and cardiovascular disease. For the time being, it is commonly believed that the insufficiency of vitamin D is independently associated with cardiovascular morbidity and mortality. Beyond the classic effects involved in bone mineral metabolism, the impacts of1,25(OH)2D3as immunity modulator to cells of various types has been broadly realized.There is still no report on whether1,25(OH)2D3may have a potential protective effect on endothelial cells,vascular smooth muscle cells(VSMCs) and myocardial cells by downregulating TLR4-mediated inflammatory pathway.Objective:To investigate whether1,25(OH)2D3at various doses may show its protective effect on aorta and heart by downregulating TLR4-mediated inflammatory pathway. Secondly, to investigate whether it is by the same way that1,25(OH)2D3may have a direct effect on VSMCs in vitro.Materials and Methods:The DM rat models were established by intravenous injection of STZ.Then they were randomly divided into seven groups, including normal rats (group C), no-intervention group(group D), DM with low-dose1,25(OH)2D3(group L,0.025μg/kg/d), middle-dose1,25(OH)2D3(group M,0.15μg/kg/d), high-dose1,25(OH)2D3(group H,0.3μg/kg/d), insulin group(group Y, protamine zinc insulin,16U/kg/d) and ARB group(group A, Losartan potassium,10.4mg/kg/d) respectively. Sixteen weeks later, all rats were sacrificed.The blood and urine samples were obtained to test blood glucose, lipid, hepatic and renal function, bone metabolic parameters, CRP and urine microalbumin. By HE and Masson Stain, the pathological changes were investigated in the aorta and heart. The depositions of TLR4, Myd88, NF-κB p65, MCP-1and TRAIL were also investigated by both immunohistochemistry and immunofluorescence methods. At the same time, the expression of mRNA and protein in those tissues were studied by Real-time qPCR and Western blotting as well. In vitro, A7R5VSMCs were cultured in the medium of low glucose (5.5mmol/L) and high glucose (25mmol/L) to observe how the expression of TLR4was affected by glucose at various concentration and different time in culture. When the highest amount of TLR4was determined, the glucose concentration and culture time was identified. Under the fixed condition, the VSMCs was intervened with various doses of1,25(OH)2D3(1×10-9mol/L,1×10-8mol/L,1×10-7mol/L) and Losartan potassium (1×10-5mol/L).Another group of alternative glucose concentration without intervention was also included. Then the VSMCs were collected respectively. As far as TLR4and Myd88were concerned, the expression of mRNA was studied by Real-time qPCR method, while the expression of protein was studied by cell immunofluorescence and Western blotting methods. The results were analysed by one-way analysis of variance.Results:1.In comparison to group D, hypercalciuria was found in group M without dose dependence.2.In group D, rough endothelium, enlargement and derangement of smooth muscle in aortas and degeneration and necrosis of myocardium in hearts were observed. Both inflammatory cell infiltration and fibrosis were involved. Moreover, the expression of TLR4, Myd88, NF-κB p65, MCP-1and TRAIL was increased. Those manifestaions were significantly improved by high-dose1,25(OH)2D3, insulin and ARB. The effects of middle-dose and low-dose1,25(OH)2D3were insignificant.3. In vitro, the expression of TLR4in VSMC was induced by high glucose. In addition, the expression of TLR4was time and dose dependent in a definitive range. Under high-glucose condition, the expression of TLR4and Myd88was inhibited by1,25(OH)2D3(1×10-7mol/L). Such effect was not found in1,25(OH)2D3(1×10-9mol/L,1×10-8mol/L) and ARB treated groups.Conclusion:1. High-dosel,25(OH)2D3exerted its protective effect on aortas and hearts by downregulating TLR4-mediated inflammatory pathway, that could reduce the risk of diabetic cardiovascular complication. The effects of middle-dose and low-dose1,25(OH)2D3were shown to be insignificant. It indicated the protective effect was dependent on appropriate dose of1,25(OH)2D3. When it was fully applied in clinical practice as pluripotent hormone beyond established effects on bone homeostasis, bone metabolic parameters should be monitored simultaneously. When it was unsuitable to be prescribed, the agonists of VDR may be considered.2. When VSMCs were cultured under high-glucose condition, the expression of TLR4and Myd88was directly inhibited by1,25(OH)2D3(1×10-7mol/L). Accompanied by the decrease in downstream inflammatory factors, they may maintain their normal structure and function.3.Losartan potassium showed as same effect as high-dose1,25(OH)2D3in vivo,while it was defective to downregulate the expression of TLR4and MyD88in vitro. It suggested the similar effects of ARB might be through the way by blocking ATII. |
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