| Objective: Observe the protective effect of Tibetan medicine Siwei Jianghuangtang powder with different doses on diabetic nephropathy,to screen the optimal dose ratio for preventing and treating diabetic nephropathy,and explore its the effects and mechanisms in preventing and treating diabetic nephropathy.Methods: 1 Study on the Relationship of Dosage ratio of Siwei Jianghuangtang powder on DN Model Rats:(1)The method of quality control of Siwei Jianghuangtang powder : The content of 7 ingredients of Siwei Jianghuangtang powder Measured by HPLC method.(2)To explore the formation time of diabetic nephropathy model: Rats were injected intraperitoneally with 1% STZ(60mg/kg)for modeling,by measuring blood sugar and collecting urine to measure urinary protein content once a week,to determine the DN model formation time.(3)Protective Effect of Siwei Jianghuangtang powder on DN Rats:(1)The glucose meter was used to measure fasting blood glucose in rats and chemical method was used to detect serum urea nitrogen(BUN),uric acid(UA),creatinine(Scr)and total protein(TP)content.(2)Serum transforming growth factor-?1(TGF-?1)and vascular endothelial growth factor(VEGF)levels detected by ELISA kit.(3)Observation of pathological changes of renal tissue and determination of glomerular basement membrane thickness under electron microscope.(4)Multivariate analysis methods such as principal component analysis(PCA)and multiple stepwise regression analysis(MSRA)were used to analyze the dose-ration relationship between the ratio-dose and pharmacodynamic results,screen for the best dose compatibility.2 Effect of Siwei Jianghuangtang powder on db/db mice model:(1)To explore the formation time of diabetic nephropathy model: By measuring blood sugar and collecting urine to measure urinary protein content once a week,to determine the DN model formation time.(2)Protective Effect of Siwei Jianghuangtang powder on db/db mice:(1)The glucose meter was used to measure fasting blood glucose in mice and chemical method was used to detect serum Scr,BUN,UA and urine albumin excretion rate(UAE)and urinary microalbumin(UAlb)levels.(2)Serum transforming growth factor-?1(TGF-?1) and vascular endothelial growth factor(VEGF)levels detected by ELISA kit.(3)HE staining and PASM-specific staining were used to observe the normal pathological changes of renal tissue and glomerular basement membrane thickness.(3)Study on Protective Effect and Mechanism of Siwei Jianghuangtang powder on db/db mice:(1)RT-PCR was used to detect the m RNA expression of HIF-1?,TGF-?1 and VEGF in renal tissue.(2)Immunohistochemical was used to detect the expression of HIF-1?,TGF-?1 and VEGF in renal tissue.(3)Detection of HIF-1?,TGF-?1 and VEGF Protein Expression in renal tissue by Western Blot.Results:(1)Study on the Relationship of Dosage ratio of Siwei Jianghuangtan g powder on DN Rat Model:(1)The mass percentages of gallic acid,magnofl orine,ellagic acid,Jatrorrhizine hydrochloride,palmatine hydrochloride,berberine hydrochloride,curcumin in Siwei Jianghuangtang powder were determined to be 7.6482mg/g,13.8024mg/g,3.7290mg/g,0.9316mg/g,0.6789mg/g,4.6780mg/g and 3.7890mg/g,respectively.(2)Results showed that after 12 weeks of model establishment,the blood sugar and urinary protein content of the model group were significantly higher(P<0.01),compared with the normal group.(3)MS RA results showed that BUN,renal index,FBG,glomerular basement membrane thickness,VEGF,Scr,and UA 7 levels and dose ratios were multivariate linear or multivariate nonlinearities relationships,the regression equation has significant significance(P<0.05).Between TGF-?1 and TP levels and doses have no significant linear or nonlinear relationship,the regression equation has no sign ificant significance(P>0.05).In the global optimization comparison around the range of uniform design,The optimal dosage of the rats model was Curcumae Longae Rhizoma: Berberidis dictyophyllae Cortex: Phyllanthi Fructus: Tribuli Fructus=1:2:1:2.(2)The effect of Tibetan medicine Siwei Jianghuangtang powder on renal tissue of db/db mice:(1)The results showed that compared with the db/m group,the blood glucose and urinary protein levels were significantly increased in the 10-week-old db/db group,there was a significant difference(P<0.01).(2)After administration for 4 weeks,compared with the db/db group,the tibetan medicine Siwei Jianghuangtang powder(3.914g/kg,1.957g/kg,0.978g/kg)decreased the blood glucose in mice,but there was no significant differenc e(P>0.05).After 8 weeks of administration,Siwei Jianghuangtang powder(3.914g/kg,1.957g/kg)significantly reduced blood glucose(P<0.05).Siwei Jianghuang tang powder(3.914g/kg)had a better effect of lowering blood glucose than Siwei Jianghuangtang powder(1.957g/kg).The other pharmacodynamic parameters o f db/db mice were also improved to varying degrees(P<0.01 or P<0.05)by Si wei Jianghuangtang powder(3.914g/kg,1.957g/kg,0.978g/kg).(3)Renal histopat hology test results:the size of the glomeruli in the db/m mice was relatively uniform,the renal capsule is clear,and has no significant accumulation in the c yst and obvious histopathological changes.In the db/db group,the glomerular volume increased,and the blood vessels dilated and congested;glomerular base ment membrane diffuse thickening,mesangial matrix widening;eosinophilic stai ning material(increased glomerular permeability)in the renal capsule,occasion ally red blood cells;the mesangial cells were slightly hyperplastic and the renal tubular epithelial cells were denatured;each administration can effectively reduce renal tissue lesions in db/db mice.(4)Renal histological observations: After PASM staining,the glomerular and glomerular basement membranes of the db/m mice showed fewer black parts.However,there are more black parts in the db/db group mice.Glomerular basement membrane changes were analyzed by image pro-plus software(The greater the average integrated optical density(IOD),the more obvious the thickening of the basement membrane): Compared with the db/m group,the mean IOD of the glomerular basement membrane in the db/db mice increased,with significant difference(P<0.01).It was shown that the glomerular basement membrane of mice in db/db group was significantly thickened.Compared with the db/db group,the average IOD value of the glomerular basement membrane in the other groups was decreased,there was sign ificant difference(P<0.01).It was suggested that the thickness of glomerular ba sement membrane in db/db mice was significantly decreased by each administra tion(P<0.01).(5)Gene expression results show: Compared with the db/m grou p,the m RNA contents of HIF-1?,TGF-?1 and VEGF in the kidney of db/db mice increased,there was a significant difference(P<0.01).Siwei Jianghuangtan g powder(0.978g/kg)significantly reduced the mRNA contents of HIF-1?,TGF-?1 and VEGF in the kidney of db/db mice(P<0.01).Siwei Jianghuangtang po wder(3.914g/kg,1.957g/kg)significantly decreased the m RNA contents of TGF-?1 and VEGF in the kidney of db/db mice(P<0.01).(6)Immunohistochemistry results indicate: The immunohistochemical reactions of HIF-1?,TGF-?1 and V EGF in renal tissue of db/m mice were weakly positive,the immunohistochemical reactions of HIF-1?,TGF-?1 and VEGF were positive in the renal tissue o f mice in the db/db group.Compared with the db/m group,HIF-1?,TGF-?1,VEGF expression in the db/db group of mouse kidney tissue expression is high,there was a significant difference(P<0.01).Compared with db/db group,each administration could significantly reduce the expression of HIF-1?,TGF-?1 a nd VEGF in kidney tissue of db/db mice(P<0.01 or P<0.05).(7)Protein expre ssion results show:Compared with the db/m group,the expression of HIF-1?,T GF-?1,VEGF protein in the renal tissue of the db/db group was significantly increased,there was a significant difference(P<0.01).Compared with db/db gro up,each administration could significantly reduce the expression of HIF-1?,TG F-?1,VEGF protein in renal tissue of db/db mice(P<0.01 or P<0.05).Conclusion:Tibetan medicine Siwei Jianghuangtang powder has a good preventive effect on DN,the mechanism may be through inhibiting the expression of HIF-1?,TGF-?1,VEGF factors,regulating renal endothelial cell function,and improving diabetic kidney disease kidney tissue damage. |
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