Effect Of Extract Powder Of Ambrette On Podocyte Injury Induced By High Glucose

ObjectiveDiabetic nephropathy(DN),also known as diabetic glomerulosclerosis,is one of the most common chronic microvascular complications in diabetes.The main clinical manifestations of diabetic nephropathy are impaired glomerular filtration barrier,progressive proteinuria,diffuse kidney injury and functional failure,which eventually leads to end-stage diabetic nephropathy.The occurrence of end-stage diabetic nephropathy not only seriously threatens the health of the patient but also disrupts the normal life of the patient.Huang Kui,also known as Astragalus membranaceus,has been discovered several decades ago.The main component of Huangkui,Hypericin,can improve the symptoms of proteinuria and renal dysfunction in animal models such as early chronic kidney disease and mice.Nowadays,more and more clinical studies have shown that an effective dose of Huangkui Extract powder can reduce micro-urine protein in patients with early diabetic nephropathy and Ig A nephropathy.Despite this,there are still some problems in the mechanism of Huangkui Extract powder in the treatment of early diabetic nephropathy.Podocytes are terminally differentiated,highly specialized renal vesicle epithelial cells and are an important component of the glomerular filtration barrier,which prevents protein loss.Podocyte injury is the main cause of dysfunction of filtration barrier.Glomerular filtration barrier structure and functional damage are the main manifestations of diabetic nephropathy,which indicates that podocyte injury plays a key role in the pathogenesis of diabetic nephropathy.Therefore,in this study,the podocyte MPC5 was used as the research object,and the diabetic nephropathy environment was simulated with high glucose concentration.The effects of different doses of Huangkui extract powder on podocyte injury under high glucose concentration were observed.Subsequently,through cell experiments,the effects of a certain dose of Huangkui extract powder on the biological functions of podocyte cycle,proliferation and apoptosis under high glucose concentration were observed.Finally,q RT-PCR combined with western blot were used to detect the effect of Huangkui extract powder on the expression of nephrin and podocin in podocytes under high glucose concentration,so as to elucidate its possible protective mechanism in diabetic nephropathy and further develop the active constituents of traditional Chinese medicine.The application provides experimental data and reference to provide a solid theoretical basis for future treatment decisions.Methods(1)Screening of high glucose concentration and detection of drug toxicity of HuangkuiHigh glucose concentration screening: The podocyte MPC5 in logarithmic growth phase was divided into the following four groups: blank control normal sugar(NG),high glucose group 1(50 mmol/L),high sugar group 2(25 mmol/L),high glucose Three groups(12.5 mmol/L)were cultured for 24-48 hours,and the growth state of the cells was observed to select a suitable high sugar treatment concentration.After the high sugar concentration was confirmed,the high glucose treated cells were placed in the following 6different yellow sunflower extract powder concentrations(0 g/L,0.3 G/L,0.6 g/L,0.9 g/L,1.2 g/L,Treatment in 1.5 g/L)for 24 and 48 hours.Finally,cytotoxicity was measured according to MTT to screen for the optimal drug treatment concentration.(2)Effect of Huangkui Extract powder on the biological function of podocytes under high glucose concentrationThe experiments were divided into the following four groups according to the purpose of the experiment: 5.6 m M NG;5.6 m M NG + 0.45 g/L HK;25 m M HG;25 m M HG +0.45 g/L HK.The effects of treatment of Huangkui Extract powder on proliferation,apoptosis and cell cycle of podocytes induced by high glucose were detected by MTT and flow cytometry.(3)Biological effects of Huangkui Extract powder on podocytes at high glucose concentrationThe expression levels of nephrin and podocin in each treatment group were examined by q RT-PCR and western blot.Results(1)The screening of high glucose concentration and drug screening experiments of Huangkui Extract powders showed that the effect after treatment with high glucose concentration of 25 m M/L for 24 hours was the closest to that of diabetic nephropathy.In addition,since the concentration of Huangkui extract powder was 0.45 g/L,the toxicity to MPC5 podocytes was the smallest,so this concentration was selected as a follow-up study.(2)The results of cell proliferation experiments showed that podocytes treated with 25 m M/L HG high glucose concentration showed: morphological changes,partially elliptical,reduced foot processes and fusion,compared with 5.6 m M/L NG.At the same time,high glucose concentration can inhibit podocyte proliferation,promote apoptosis and lead to cell cycle arrest.Apoptosis assay showed that the podocyte viability was significantly increased(P<0.05)and the apoptotic rate was decreased in the 25 m M HG+0.45 g/L HK treatment group compared with 25 m M HG.In addition,in the Huangkui extract powder group,the proportion of cells in the G1 phase of the cell cycle decreased significantly,and the proportion of cells in the S phase and G2 phase increased significantly,and it was dose-dependent(P<0.01).(3)The results of q RT-PCR and western blot showed that high glucose concentration significantly inhibited the expression of nephrin and podocin compared with the control group(p<0.01).Under high glucose concentration treatment,the appropriate concentration of Huangkui Extract powder can promote the expression of podocyte-associated proteins Nephrin and podocin(p<0.05).Conclusions(1)Appropriate dose of Huangkui Extract powder can accelerate the cell cycle of podocytes in high glucose environment,promote cell proliferation,inhibit cell apoptosis,and reduce podocyte injury,thus playing an important protective role for podocytes.(2)Apoptosis can be induced by prolonged high glucose concentration treatment,but a certain concentration of Huangkui Extract powder can inhibit high glucose-induced podocyte apoptosis.The possible mechanism is that,under high glucose concentration,Huangkui Extract powder may inhibit the apoptosis of podocytes by regulating the expression of podocyte-associated proteins Nephrin and podocin,thereby playing an important protective role for podocytes.