Observation Of The Effect Of Quercetin On The Transdifferentiation Of TGF-beta 1 Induced Podocyte To Mesenchymal Transition Based On The "Toxic Damaged Kidney Collateral" Theory

Objective: Objective To observe the intervention effect of quercetin(Qu)on TGF-beta 1(Transforming growth factor-beta 1,TGF-1)induced podocyte to mesenchymal transition(Epithelial Mesenchymal Transition).Methods: Used primitive culture of cells in vitro,identified as foot cell,the cell experiment group as control group,model group(TGF-beta1 5ng/mL),quercetin,low dose group(TGF-beta1 5ng/mL+quercetin 25ug/mL),quercetin in dose group(TGF-beta15ng/mL+quercetin 50ug/mL)and quercetin high dose group(TGF-beta1 5ng/mL + quercetin100ug/mL)and control group of conventional culture,corresponding drug intervention groups48 h.The above groups of cells with RTCA real-time dynamic monitoring of sertoli cell growth situation,use AnnexinV-FITC/PI double marker flow cytometry detection of sertoli cell apoptosis,and laser confocal detection used Wilms tumor suppressor cell marker protein factor 1(WT-1),calcium adhesion protein E-cadherin(E-cadherin)and EMT marker protein alpha smooth muscle Actin(alpha smooth muscle Actin,alpha SMA),the expression of protein(Vimentin)waveform,using statistical software comparing differences between groups.Results: 1.Observation of cell morphology by inverted phase contrast microscope and laser confocal technique were used to detect the protein of foot cells and identify the cultured cells as podocytes.Normal foot cells are dendritic,small in volume and around the cell membrane.Protuberances,adjacent foot cell protrusions are intersecting,mononuclear is common,double nucleus is rare.After TGF-beta1 induces the foot cells,the cytoplasm increases,and the protrusion shrinks and even disappears.quercetin low,medium and high dose group,all cell morphological changes,cell increase in the nu quercetin mber of projections,"branches" of the typical forms can be seen in some cells,cells in the three groups of typical for quercetin dose group.2.Cell index:(1)between 0-90 h,compared with the control group,model group cell index growth is weak,the difference was statistically significant(P<0.05),compared with model group,quercetin low,medium and high dose group of cells exponential growth faster,differences were statistically significant(P<0.05),among them with quercetin's fastest-growing dose group of cells in index,followed by quercetin high dose group,thequercetin low-dose group,(2)between 91-118 h,compared with the control group,model group cell index growth is always weak,the difference was statistically significant(P<0.05),compared with model group,low quercetin,cell index of middle and high dose group was obviously higher,differences were statistically significant(P<0.05),among them with quercetin's fastest-growing dose group of cells in index,followed by quercetin low dose group,quercetin high dose group.3.Through both FACS-FITC with PI staining flow cytometry instrument analysis found that in the control group,apoptosis rate is the lowest,compared with the control group,model group,the apoptosis rate increased significantly,the difference was statistically significant(P<0.05),compared with model group,quercetin low,medium and high dose group of apoptosis rate,the difference was statistically significant(P<0.05),of which the lowest dose group the apoptosis rate in quercetin,followed by quercetin high dose group,quercetin low dose group.4.The change of protein: compared with control group,model group of sertoli cell marker protein WT-1,the expression of E-cadherin volume decreased significantly,podocyte EMT protein expression of Vimentin,alpha SMA volume increases,the difference was statistically significant(P<0.05);Compared with model group,quercetin low,medium and high dose group of sertoli cell marker protein expression,E-cadherin WT-1 the quercetinantity increases,podocyte EMT protein decreased the expression of Vimentin,alpha SMA,the difference was statistically significant(P<0.05),in which quercetin mark dose group of cells in the highest amount of protein expression,followed by quercetin high dose group,quercetin low dose group.Conclusion: 1.Low concentration of collagenase digestion and differential screening method can establish a relatively good operation of the primary culture method of rat foot cells with relatively high repeatability.2.TGF-beta1 can not only induce the morphological changes of the foot cells,but also accelerate the development of the foot cell EMT.3.Quercetin can inhibit the occurrence of EMT in the foot cells,and the dose group in quercetin can inhibit TGF-beta1.EMT cell,its mechanism may be related to inhibition of TGF-beta/ Smad signaling pathways of related molecules.