| Objective: To explore the effects of yiqiyangyinhuoxue prescription on Nephrin,Nestin and Desmin in kidney tissue of DN,and observe the correlation between the three and reducing proteinuria,improve renal pathological correlation and biochemical indexes,expound the effect of yiqiyangyinhuoxue prescriptions on DN and its effective mechanism,providing experimental evidence and theoretical basis for the clinical application side.Method: 60 healthy male SD rats of clean grade,200±20g,put them in the same environment(room temperature 22°C,relative humidity 50%) of the animal room.After been adaptived feeding for 7 days,they were randomly assigned five groups:Normal control group(N group),Model control group(M group),Valsartan group(Diovan,V group),Valsartan group(Diovan)+Low dose group(V+L group),Valsartan group(Diovan)+ High dose group(V+H group),Model groups were given 45mg/kg disposable intraperitoneal injection of streptozotocin,N group was given the same dose of sodium citrate buffer.Gave the standard,regular disinfection cage,replacement pad,viewed the basic situation.After feeding 3 days,quick tip of the tail blood glucose,three consecutive(0h, 1h,2h),if the blood sugar≥16.7mmol/L,urine strongly positive(+++~++++),urinary protein positive,urine output>150% of the before modeling,s,the model was successful;After the success of modeling,N group and M group rats were given normal saline 1ml?100g-1?d-1,V group were given medicine according to 1.67mg?200g-1?d-1,V+L group and V+H group,ording to the adult dose of 6.25 times,12.5 times,for 6 weeks.Experiment the day before the end of the 24 h urine collection,metabolic cage to be put to death in therat,abdominal aorta in blood,then take the renal pathological detection for.Observed thegeneral condition of rats,the glomerular hypertrophy index,emical index changes,removed the kidney,said the left kidney weight,calculated hypertrophy index;HE and PAS staining,then observed the pathological changes of kidney tissue,Immunohistochemistry(IHC) expression of Nestin and Desmin detection in kidney tissue,Enzymelinked immunosorbent assay(ELISA) to detect kidney tissue Nephrin expression.Result: 1 The basic situation of the rats The basic situation of N group was good,the skin moist,eyes brimming with radiating vigour,activities freely,responsive,the disposition was gentle,appetite was good, water quantity was normal,feces and urine were normal,weight increased come very naturally.Buiding four groups after STZ injection was characterized by the thinning hair and short tempered,listless,no obvious autonomic activity,crouching clannishness, slow response easy to anger,increased vigilance,appetite hyperthyroidism,water quantity increased obviously,soft,urine volume increased obviously,a significant reduction in weight.After drug intervention,the situation of V group,V+L group,V+H group were siowly better and better,V+H group showed the most prominent. 2 The rats body weight,kidney weight,hypertrophy index(kidney weight/body weight) The weigt:N group and building weight had obvious differences between groups, and kidney weight difference was not outstanding.Comparison of hypertrophy inndex: M group,V group,V+L group,V+H group hypertrophy index were all higher than the N group(P<0.01),and the difference was very significant;Treatment group hypertrophy index was less than the N group,the difference was very big(P<0.01);V group compare with V+L group,V+H group,differences were also apparent(P<0.01);or there were differences between the V+L group,V+H group(P<0.01). 3 24 h urine protein excretion of contrast between the groups of rats N group rats compared with V+H group,24 h urine protein quantitative difference was not big,no statistical significance(P>0.05);N group were compared with V group, M group,V+H group,there was a big gap,there was statistically significant(P<0.01);V group and the V+L group,the difference was not statistically significant(P>0.05);V group was significantly higher than V+H group,with statistical significance(P< 01). 4 The influence of biochemical indicators of experimental rats The comparison of groups of rats with ALT,BUN,Scr had no statistical significance.ALB comparison:The N group and modeling groups had significant difference(P<0.01);The difference of V group and M group was significant(P<0.05),with V+L group and V+H group,the difference was still signiificant(P<0.01);among V+L group and V+H group,there was no significant difference(P>0.05).TC comparison:V group, V+L group,M group compared to N group,the difference was significant(P<0.01),while the difference was even greater with the V+H group(P<0.05);V group with M group,the difference was very small,not statistically significant(P>0.05),however,the V+L group and V+H group the difference was significant(P<0.01).TG comparisn:the modeling groups compared with the N group,they were very different(P<0.05);V group and M group was little difference,there was no significant(P>0.05),but the V group with V+L group,V+H group were significantly different(P<0.01).Glucose comparison:N group compared with the modeling of each group,Statistically significant(P< 0.01);compared N group with the treatment groups,there were very significant difference(P<0.05);There was significant difference between V group and V+L group,V+H group(P<0.01);compared with V+L group and V+H group,there was no great difference(P>0.05). 5 Observe the kidney appearance and histopathological changes of the rats Macroscopic observation:double kidney rats in the N group like bean size,color was dark red,the surface smooth and delicate,no coating adhesion,easy tear;M group rats kidney volume increased obviously,the surface slightly and cortex were pale,medulla color deepened,and a clear demarcation between the cortex,coated with adhesive. Medulla color deepened,with clear boundaries cortex,capsular adhesions.HE staining and PAS staining,light microscopy Watch:N group structure intact rat glomerular mesangial matrix normal capillary endothelial cells,tubular,orderly distribution of epithelial cells;The M group rats significantly increased glomerular volume,mesangial region widen,matrix increased,capillary basement membrane thickening,diffuse,renal tubule interstitial appear infiltration of inflammatory cells,renal tubular epitheliaial cells with vacuolar degeneration,glycogen storage in glomerular matrix and capillary endothelium in under. 6 Expression of Nephrin,Nestin and Desmin in renal tissue of rats in each group The expression of Nephrin in podocytes with specificity, Nestin and Desmin in podocytes in cytoplasm of glomerular basement membrane outer expression,brown yel- low positive area;Compared with the N group, the expression quantity modeling of Nephrin of all groups decreased significantly,the expressions of Nestin and Desmin significantly increased,with significant difference;Expression of Nephrin up to the N group,in M group Nestin and Desmin expression amount most,significant difference with the treatment groups;V group and V+L group,V+H were significant difference,t- here was no difference in V+L group and V+H group.Conclusion: 1 Yiqiyangyinhuoxue prescription can improve the basic conditions of DN rats,reduce proteinuria,correct hypoalbuminemia,improve glucolipid metabolic disorder; 2 Yiqiyangyinhuoxue prescription can reduce the DN rat kidney hypertrophy index,inhibit the glomerular mesangial cells and the proliferation of mesangial matrix proliferation,lighten the GBM thickening and the injury of the podocyte,improve the renal pathology; 3 Yiqiyangyinhuoxue prescription may adjust the Nephrin,Nestin and Desmin expresssion in DN rat kidney tissues,reduce in the diaphragm,correct skeletal protein disorder,and play a role of the treatment. |
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