| PART1 1,25(OH)2D3 inhibits the High glucose-induced Hypertrophy of Neonatal and H9C2 CaidiomyocytesObjective: to confirm the hypertrophy inducing role of high glucose in rats cardiomyocytes; to explore the optimum concentration of 1,25(OH)2D3 at which could anti- high glucose induced cardiomyocytes hypertrophy; to find wheher the hypertrophy inducing role of high glucose is associated with PARP1.Methords: the neonatal and H9C2 cardiomyocytes were clutured and divided into 5 groups, and were combined or mono-treated with different glucose and 1,25(OH)2D3(10-9、10-8、10-7mol/L) concentrations. The area of cardiomyocytes were calculated by Image Pro Plus, PARP1 m RNA expression was determined by RT-PCR, and its protein expression was tested by Western bolt. Then the cells were sent into other 4 groups: LG, LG+INO1001(PARP1inhibitor), HG, HG+INO1001, and the area of cardiomyocytes was calculated, and both the m RNA and protein expression were analyzed.Results: 25mmol/L high glucose could induce hypertrophy on cardiomyocytes, as well as the m RNA expression of cardiac hypertrophy indicators, such as β-MHC, ANF, BNP were increased. 1,25(OH)2D3 could inhibit the high glucose induced hypertrophy on cardiomyocytes in a concentration dependent way. High glucose could increase the PARP1 expression and its hypertrophy inducing role was associated with PARP1 activation.Conclusion: high glucose could conduct cardiomyocytes hypertrophy, and 1,25(OH)2D3 treatment could anti-high glucose induced cardiomyocytes hypertrophy by PARP1 inhibition.PART2 The effects and mechainsms of VDR silencing by lentivirus transfection on H9C2 cells hypertrophyObjective: to construct the stable transfective VDR silencing cardiomyocytes; to explore the effects of VDR silencing on H9C2 cells; to confirm whether the anti-hypertrophy role of 1,25(OH)2D3 is through the VD/VDR classic pathway, and associated with PARP1 activation.Methods: transfected H9C2 cells with lenti-sh VDR, and found the multiplicity of infection and selected cells with puromycin, finally, to built a stable-transfected cardiomyocytes. The m RNA and protein expression were detected by RT-PCR and Western bolt. The H9C2 cells were treated with low glucose, high glucose, and INO-1001 respectively or combined, and the area of cells, m RNA expression of cardiac hypertrophy indicators, and the protein expression of VDR and PARP1 were examed.Resultes: lenti-sh VDR had no significant toxiciy on H9C2 cells, and the MOI of 50 could efficiently infected the H9C2 cells and silenced the VDR expression over 70%. Compared with normal control group, the area of cardiac cells, m RNA expression of cardiac hypertrophy indicators, and proteins expression of PARP1 were increased by VDR silencing with or without high glucose treatment.Conclusion: lenti-sh VDR could safely and efficiently infected the H9C2 cells and silenced the VDR expresion over 70%。VDR silencing conducted hypertrophy of H9C2 cells and might associated with PARP1.PART3 The anti-diabetic cardiomyopathy role of 1,25(OH)2D3 on rats and its mechanismsObjective:to explore the effects of VDR silencing on diabetic rats heart function; to test the anti-diabetic cardiomyopathy role of 1,25(OH)2D3 of rats and its mechanisms.Methods: sixty SD-rats were randomly assigned into following 5 gourps: control group, diabetic group, diabetic+1,25(OH)2D3 treatment group, diabetic+1,25(OH)2D3 +sh VDR treatment group, diabetic+sh VDR treatment group, each contained 12 rats. The diabetic rat model was constructed by single interperitoneal injection with STZ. The cardiac function was evaluated by echocardiography, the cardiac fibrosis was tested by HE and Sirus red stain, m RNA and protein expression of β-MHC, ANF, BNP, VDR, PARP1, SIRT1, TSC2, m TOR, and p70s6 k were detected by RT-PCR or Western bolt.Results: the diabetic cardiomyopathy model has been established by the obove methods. Compared with control group, diabetic group displayed a bigger LVIVS, LVPW, LVEDd/s(P<0.05), and these indicators were improved in diabetic+vd group. However, when treated with lenti-sh VDR, these indicators were increased with or without VD3 treatments. Consistant with these, the m RNA expression of β-MHC, ANF, BNP were changed in the same trends. Meanwhile, the CVF and PCA/LA were increased in PART3 diabetic group and lenti-sh VDR groups(P<0.01), and 1,25(OH)2D3 treatment could decrease these factors in diabetic rats, but had no effect on VDR silencing rats. Besides, the PARP1 expressions, phosphorylation level of m TOR and p70s6 k were increased in diabetic rats and VDR silenced rats, while the SIRT1 expression and phosphorylation level of TSC2 were decreased.Conclusion: once interperitoneal injection of STZ could successfully establish the diabetic cardiomyopathy rat model; lenti-sh VDR could safely and effeciently tranfected the diabetic rats and could silence the VDR stably. 1,25(OH)2D3 could improve the myocardial hypertrophy, cardiac fibrosis, and the cardiac function. The 1,25(OH)2D3 treatment might be a new pathway in the prevention and therapy of diabetic cardiomyopathy. |
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