| Background:Proliferation and hypertrophy of mesangial cells,extracellular matrix(ECM) secretion too much are main pathological features of diabetic nephropathy(DN), transforming growth factor beta (TGF-β) play an important role in the matrix accumation. Thus,inhibit TGF-βexpression and ECM secretion,could help to retard the development and progression of renal disease.People are trying to find the best way to treat DN for many years.Studies on models of DN showed that instead of animal protein,soy protein could ameliorates proteinuria, recent studies also showed the beneficial effect of soy protein might be related to its isoflavone content. Genistein,the major isoflavone in soy products, has attracted considerable attention in recent years. It has many bioavailability effects,for example: modify the activation of estrogen receptor,have antioxidant effect, inhibit protein tyrosine kinases and so on. Studies showed that genistein affect cell growth related to its concentrations and estrogen receptor.While other studies showed that genistein could suppress mesangial cell collagen synthesis, reduce ECM accumulation,and retard the progression of renal disease.Non-renal research showed that genistein could inhibit the activation of TGF-β. Nowdays,studies of genistein on DN are very limited,but it seems to havebenefit effect on DN.Objective:The purpose of our studies are try to gain a better insight into the effect of genistein on renal cells.The contents of our studies are to evaluate the effect of proliferation and apoptosis by different concentrations of genistein solution in low glucose and high glucose cultured rat mesangial cells;to explore the possible signal transduction pathways; to examine the synthesis of Ⅳ col,Fn and the expression of TGF-βin cultured mesangial cells stimulated by genistein.Through these studies,we will have some information about the experimental value of genistein in renal disease field.Methods: This study is mainly in vitro study.Genistein was dissolved in dimethyl sulfoxide and diluted in DMEM culture medium at different concentrations(0,1,5,10,30,50μmol·L-1). Control group(low glucose 100mg/dl without genistein) and experiment group(high glucose 450mg/dl with genistein 0,1,5,10,30,50μmol·L-1) were established,each group was incubated for 12,24,36,48hrs. The growth inhibiting effect of genistein on cells was measured by MTT methods,and cell growth curve was observed.Agarose gel electrophoresis of DNA extracted from each group,routine HE staining,the terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling(TUNEL) technique and flow cytometry were performed to observe apoptosis of cells.Flow cytometry was also used to determine the apoptosis index(AI) of cells. Synthesis of fibronectin ,type Ⅳ collagen and TGF-βin cultured mesangial cells were determined by ELISA method,semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was used to examine the expression of TGF-βmRNA in a dose- and time-dependent manner or not.Using lipofectin method,green fiuorescence protein (GFP) gene was transient transfected into rat mesangial cells to explore the best transfection condition, at the same condition, NF-κB -Luc was transfected into rat mesangial cells to observe transient gene expression,by fluorescent assay methods, theactivation change of NF-κB in low glucose, high glucose conditions and different concentrations of genistein were observed. We detected the expression of estrogen receptor (ER)α,βin cultured rat mesangial cells by immunohistochemical technique.Results: MTT methods showed that hyperglycemia stimulated MC proliferation in 48 hrs,low concentrations of genistein (1μmol·L-1)have no obvious effects on MCs, 5μmol·L-1 led to weak inhibition to the cells and high concentrations of genistein (10μmol·L-1,30μmol·L-1,50μmol·L-1) strongly inhibited cells growth in a dose- and time-dependent manner. Agarose gel electrophoresis of DNA extracted from cells of control group and experiment gr...
|
PDF Full Text Request