| Diabetes Mellitus (DM) and its complications are diseases which severely jeopardize health and quality of life. Diabetic nephropathy (DN) is one of the most severe complications, which is also an important cause of death. The earlier manifestations of DN are hypertension, hypertransfusion and hyperfiltration of renal glomerulus, and further the GBM thickening of glomerular capillary loop and the increasing of ECM, the final is glomerular sclerosis. The GMCs and podocytes have metergasis with the effects of high glucose, advanced glycosylation end-products (AGEs), D-glucitol and so on in the development of DN. The change of MMP-2, 9/TIMP-1, oxidative stress and antioxidation system is hot spot at present.Valsartan (Val) is an antagonist of angiotensin II, LMWH posseses the effects of anticoagulation and antithrombgenesis, and both are used in the treatment of DN. This research aimed to the effects of Val and LMWH on the expression of MMP-2,9/TIMP-l, oxidative stress and the proliferation induced by high glucose of GMC and podocytes to support the theoric basement for clinical application. 1. Effect of Valsartan and LMWH on podocyte proliferation induced by high glucoseMPC5 cells(podocyte) were cultured in vitro. The effects of drugs on proliferation were determined by MTT. The third generation cells at the concentration of 5×103/well were added into 96-well plate, then devided into: Normal group(treated with normal glucose); HG group (treated with high glucose); Val group (10-6mol·L-1); LMWH group (25IU·L-1); Val+LMWH group (Val: 10-6mol·L-1 + LMWH: 25IU·L-1). And then the cells were cultured for 24, 48, 72 hours respectively. The proliferation was measured by MTT. The results showed that the proliferation was strengthened by Val+LMWH at 72h compared with HG group (P<0.05).2. Effect of Valsartan and LMWH on the secretion of MMP-2/TIMP-1 of podocytes induced by high glucose detected by ELISAThe result showed that the secretion of MMP-2 was increased and TIMP-1 was decreased by Val or LMWH compared with HG group, and it was more effective of Val+LMWH group compared with Val or LMWH (P<0.05).3. Effect of Valsartan and LMWH on the expression of MMP-2, 9 of podocytes induced by high glucose detected by gelatin zymopraphyThe result showed that the zone pellucidas of treatment groups were brighter than HG group (P<0.01, P<0.05), and it was more significant when Val+LMWH compared with Val or LMWH (P<0.01).4. Effect of Valsartan and LMWH on the antioxidation system of podocytes induced by high glucoseThe result showed that the level of MDA was significantly attenuated when treatment groups compared with that of HG group (P<0.01, P<0.05). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.05). The level of SOD was significantly increased when treatment groups compared with that of HG group (P<0.05). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.01). The level of GSH-PX was significantly increased when treatment groups compared with that of HG group (P<0.05). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.05). 5. Effect of Valsartan and LMWH on the oxidative stress of podocytes induced by high glucose detected by fluorescence microscopeThe result detected by fluorescence microscope showed that the green fluorescence was strongest in the HG group; in the treatment groups, the green fluorescence was weeker compared with HG group. And it was most obvious in Val+LMWH groups. The result detected by fluorescence microplate reader showed that the absorbance was weak when the treatment groups compared with HG group (P<0.01). The significantly differences were found when Val+LMWH groups compared with Val or LMWH (P<0.05).6. Effect of Valsartan and LMWH on the proliferation of GMCs induced by high glucose (in vitro)The proliferation of GMCs induced by high glucose was determined by MTT method. The GMCs were added into 96-well plate at the concentration of 5×103/well, and then divided into: Normal group(treated with normal glucose); HG group (treated with high glucose); Val groups, treated with high glucose and four doses of Valsartan(10-4, 10-5, 10-6, 10mol·L-1); LMWH groups, treated with high glucose and four dose of LMWH(250, 25, 5, 2.5IU·L-1); Val+LMWH groups, treated with high glucose and Valsatan, LMWH(Val: 10-6 mol·L-1+LMWH: 25IU·L-1). And then the GMCs were cultured for 24, 48, 72 hours respectively. MTT assay was used to test the amounts of the GMCs. The results showed: the high glucose induces the proliferation of GMCs (P<0.01); the results showed that the proliferation of GMCs in these three treatment groups (Val, LMWH, Val+LMWH) were significantly attenuated compared with HG group (P<0.01, P<0.05) and it was a concentration-dependented, and furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.05).7. Effect of Valsartan and LMWH on the level of MMP-2/TIMP-1 of GMCs induced by high glucoseThe result showed that the level of MMP-2 was increased and the level of TIMP-1 was attenuated when the treatment group compared with HG group (P<0.01). The effect was not significant when Val+LMWH compared with Val or LMWH (P>0.05).8. Effect of Valsartan and LMWH on the level of MMP-2/TIMP-1mRNA of GMCs induced by high glucose detected by RT-PCRThe result showed that the level of MMP-2mRNA of GMCs was significantly attenuated, and TIMP-1mRNA significantly increased compared with controls (P<0.01). The change of MMP-2/TIMP-1mRNA was attenuated when the treatment groups compared with HG group (P<0.01, P<0.01). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.01).9. Effect of Valsartan and LMWH on the level of MMP-2, 9 of GMCs induced by high glucoseThe result showed that the zone pellucidas of treatment groups were brighter than HG group (P<0.01), and it was more significant when Val+LMWH compared with Val or LMWH (P<0.01, P<0.05).10. Effect of Valsartan and LMWH on the level of MCP-1 of GMCs induced by high glucose detected by immunohistochemistryThe result showed that the level of MCP-1 was attenuated when the treatment group compared with .HG group (P<0.05). The effect was significant when Val+LMWH compared with Val or LMWH (P<0.05).11. Effect of Valsartan and LMWH on the level of MCP-1mRNA of GMCs induced by high glucose detected by RT-PCRThe result showed that the level of MCP-1mRNA was attenuated when the treatment groups compared with HG group (P<0.01). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.01, P<0.05).12. Effect of Valsartan and LMWH on the antioxidation system of GMCs induced by high glucoseThe results showed that the level of MDA was significantly attenuated when treatment groups compared with that of HG group (P<0.01, P<0.05). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.05). The level of SOD was significantly increased when treatment groups compared with that of HG group (P<0.05). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.01). The level of GSH-PX was significantly increased when treatment groups compared with that of HG group (P<0.05). And furthermore, the significantly differences were found when Val+LMWH compared with Val or LMWH (P<0.05). 13. Effect of Valsartan and LMWH on the oxidative stress of GMCs induced by high glucoseThe result detected by fluorescence microscope showed that the green fluorescence was strongest in the HG group; in the treatment groups, the green fluorescence was weeker compared with HG group. And it was most obvious in Val+LMWH groups.The result detected by fluorescence microplate reader showed that the absorbance was weak when the treatment groups compared with HG group (P<0.05). The significantly differences were found when Val+LMWH groups compared with Val or LMWH (P<0.05). |
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