| Background: Hypertrophy phenotype change,extracellular matrix(ECM) secretion too much and degradation reduction are main pathological features of diabetic nephropathy(DN).Diet therapy plays an important role in diabetes mellitus(DM) for so long.For many years,people have also been researching into the optical diet therapy for DN.Studies on rat models of DN showed that instead of animal protein,soy protein could ameliorate proteinuria.Recent studies also showed the beneficial effect of soy protein might be related to its isoflavone content.Genistein,the major content of isoflavone in soy products,has much physioactivity,such as inhibiting protein tyrosine kinase,ribosome S6 kinase ,binding with estrogen receptor and playing estrogenoid or antiestrogen role,having antioxidant effect and so on. Studies showed that the expression of SMemb,the mark of mesangial cell's hypertrophy phenotype change, was related very much to protein tyrosine kinase.Another studies showed that genistein could suppress the synthesis of fibronectin,reduce ECM accumulation.Non-renal research showed that genistein could modulate the kinase activity of MMP/TIMP degradation system.Nowdays,there are few reports about genistein on DN,but it seems to have some benefit.Objective: The purpose of our studies are gaining a better insight into the effect of genistein on mesangial cell's phenotype change and ECM expression plus degradation under DN circumstances.We add genistein with different concentrations in high glucose cultured rat mesangial cells;feed DM rats with the single concentration genistein for different days;to explore the expression of SMemb,the mark of mesangial cell's hypertrophy phenotype change;to examine the synthesis of Fn as well as the change of the balance between MMP-2 and TIMP-l;to comprehend its effect on renal pathological changes of DM rats and some related factors;trying toexplore the possible mechanisms.Through these studies,we will have some information about the experimental value of genistein in DN field.Methods: This study was divided into two parts:in vitro and in vivo./n vitro study:The rnesangial cells were divided into control group(low glucose DMEM with 6% BSA without genistein),high glucose group(high glucose DMEM with 6% BSA without genistein),high glucose+different concentration genistein (5, 10, 50, 100, 200 umol/l),each group was incubated for 12,24,48hrs.The expression of SMemb mRNA, MMP-2 mRNA,TIMP-l mRNA was assayed by semi-quantitative RT-PCR.The quantity of fibronectin in cultured supernatants were determined by ELISA.Flow cytometry was peformed to determine the activity of PTK.In vivo study:Spraque-Dawley rats were divided into three groupsxontrol group,DM group,DM+GEN group.Sacriface the rats at the end of 4,8 weeks to get the serum,urine and kidneys.Renal function, serum lipid,blood sugar,urine albumin excretory rate and insulin level were examined.Rat kidneys were dyed with HE. The expression of SMemb mRNA, MMP-2 mRNA, TIMP-1 mRNA in renal tissues was determined by semi-quantitative RT-PCR. Fibronectin in homogenated renal cortex were examined by ELISA. The expression intensity of MMP-2/TIMP-1 in the renal cortex tissue of rats was assayed by immunohistochemical staining technique.Results:1 .In vitro study:The result of RT-PCR showed that rnesangial cells cultured in high glucose could express SMemb mRNA, genistein could down regulate the expression of SMemb mRNA in a dose-dependent and time-dependent manner. Content of Fn detected by ELISA in the cultured supernatants displayed that high glucose might promote MC to secrete more Fn than low glucose group. Genistein could inhibit the effect of high glucose on Fn in dose-dependent manner. The result of RT-PCR also showed that genistein could increase MMP-2 mRNA expression in a dose-dependent manner and decrease TIMP-1 mRNA expression in dose-dependent and time-dependent manner. As a whole effect, genistein could increase the content of MMP-2 mRNA/TIMP-1 mRNA. The activity of protein tyrosine kinase by flow cytometry was inhib... |
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