| ObjectiveTo observe the effects of different doses of Plumula Nelumbinis extract on blood glucose, urine glucose, urine protein, albumin and renal pathological of diabetes rats induced by streptozotocin(STZ).We also observate NADPH oxidase activity of primary cell and glomerular cultured in HG,which pre-treated by Liensinine, isoliensinine. Explore the possible mechanism and the protective effects of Plumula Nelumbinis extract on kidney.Method120 male SD rats,weighting 230-260 g were be selected.After feeding for 1 weeks, we randomly selected 12 rats as control group received citrate buffer intraperitoneal.Type 1 diabetic model was induced by injection of streptozotocin(STZ) at a dose of 39mg/kg on the remaining 108 rats. 72 h later,venous blood were collected in model rats, fasting blood glucose 16.7mmol or greater were selected for the type 1 diabetes mellitus. The successful model rats were randomly divided into five groups: control group, diabetic model, 4 different doses of the Plumula Nelumbinis extract intervention group(20,50,200mg/kg), the Captopril group.Glucose in urine, urinary albumin, urine protein, urine creatinine were determined 0,4,8 weeks at the basal respectively. After the animals were sacrificed,then kidney was removed and calculated the renal index, the left renal tissue was fixed in 10% formalin, stained with HE to observe the pathological changes of renal tissue,1mm3 kidney tissue was fixed on electron fluid,electron microscope observation of the ultrastructural changes of kidney.The right kidney was preserved in liquid nitrogen to detecte the activity of NADPH oxidase and expression of p22 phox,p67phox by Western Blot. Male SD rats were separated, we add different medicine to culture glomerular according to different groups.After cultured for 1, 12 h, Cytochrome C were be used to assay NADPH oxidase activity. We culture primary glomerular for 20 d, and digested cells, according to different groups add medicine, then cultured for 12 h, NADPHoxidase activity were detected by CM-H2 DCFDA probe.Result1.Comparsion of biochemical indexs among the six groupRats after administration of STZ, the weight, sgain rate decreased, there was significant difference compared with the control group. Plumula Nelumbinis extract group and captopril had no significant effect on the body weight of DN rats. After intravenous injection of STZ39mg/kg for 72 hours, the blood glucose levels of rats increased significantly(P<0.01), and has been maintained to the end of the test.Rats After injection of STZ, the urine, urinary albumin, urine protein significantly increased(P<0.01). Plumula Nelumbinis extract had no significant effect on urine glucose levels of DN rat. Plumula Nelumbinis extract can significantly reduce urine microalbumin(P < 0.01). The role of Plumula Nelumbinis extract at 50mg/kg is more siginificant. In addition, Plumula Nelumbinis extract can significantly reduce the urine protein in DN rats(P < 0.01), the role of 4 week is more significant than 8 week.2.The influence of Plumula Nelumbinis extract on renal pathology of DM ratsWhen observe under microscope, the control group rats had normal renal structure,11 cases of rats renal specimens were found renal tubular epithelial vacuolar degeneration; but no obvious thickening of the basement membrane,and no inflammatory cell infiltration and fibrous tissue hypertrophy in the interstitial.The captopril group 10 cases of rats renal specimens were found renal tubular epithelial vacuolar degeneration and no inflammatory cell infiltration and fibrous tissue hypertrophy in the interstitial. 10 cases of rats renal specimens were found renal tubular epithelial vacuolar degeneration in Plumula Nelumbinis extract 20mg/kg group.7 cases of rats renal specimens were found renal tubular epithelial vacuolar degeneration in Plumula Nelumbinis extract 20mg/kg group 8 cases of rats renal specimens were found renal tubular epithelial vacuolar degeneration in Plumula Nelumbinis extract 20mg/kg group. Electron microscope observation:control group glomerular basement membrane structure basically normal uniform thickness,podocyte structure was normal,podocyte withoutapoptosis and shedding. Model group glomerular basement membrane(GBM) thickening was wide,and foot processes become wide.Podocyte loss was also observed. Compared with control group,each Plumula Nelumbinis extract group can significantly reduce the thickness of the glomerular basement membrane(P < 0.01), significantly reduce the foot process width(P < 0.01), and increase the number of podocytes,which represent a dose dependent properties.3. Effect of Plumula Nelumbinis extract on renal NADPH oxidase activity of the ratsDM group renal NADPH oxidase activity significantly increased, the intervention group Plumula Nelumbinis extract can significantly reduce renal NADPH oxidase activity of rats(P <0.01).4. Results of NADPH oxidase subunit p22 phox, p67 phox expressionThe expression of NADPH oxidase p22 phox,p67phox in DM were significantly higher than that in those intervention group(P< 0.01),each Plumula Nelumbinis extract group and captopril can reduce the expression of NADPH oxidase subunit p22 phox, the Plumula Nelumbinis extract group significantiy reduce NADPH oxidase activity(P<0.01); each Plumula Nelumbinis extract group can significantly reduce the expression of p67phox(P < 0.01) 5.Effects of neferine, isoliensinine on glomerular and primary renal cells NADPH oxidase activity20umol neferine can inhibit the activity of NADPH oxidase of glomeruli under the stimulation of HG comparing the CN group and the difference is significant.5, 10,40 umol isoliensinine can significantly attenuate HG stimulated glomerular NADPH oxidase activity after 1h treatment.But after treated for 12 h,there was no significant difference coparing with which treated by HG. 20 umol neferine, isoliensinine has no effect on NADPH oxidase activity of HG treated mesangial cells,which detected by CM-H2 DCFDA probe.The result shows the fluorescence intensity can be enhanced treated by HG,i.e. the NADPH oxidase activity.NADPH oxidase activity of Primary kidney cell can be inhibited by neferine, isoliensinine treated by HG.The intensity of inhibition is weaker thanthat of Apocynin.Conclusion1.Plumula Nelumbinis extract may through reducing the renal NADPH oxidase activity, decrease renal oxidative stress, and play a protect role in renal.2.Neferine,isoliensinine,the main constituents from Plumula Nelumbinis extract,can decrease renal cells NADPH oxidase activity,which under the condition of high glucose. |
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