| Part one Study on the changes of blood and urine indexes in DN rats and the intervention effect of different compatibility of huayu-tongluo Chinese herbs.Objective: DN rat model was established in this part,the indexes of glucose and lipid metabolism,excretion of urine protein,platelet release and fibrinolytic system were measured to observe the intervention effect of different compatibility of huayu-tongluo Chinese herbs.The aim was to provide basis for improving clinical efficacy and drug screeningMethods: Seventy-five SD rats were adaptive fed for a week,10 rats were randomly selected as normal group(C group),which were fed with normal diet,and the rest rats were fed with high fat diet combined with small dose of STZ to replicate the DN model.The model rats were randomly divided into model group(M group,n=15),Huayu Tongluo group(Z group,n=15,treatment with Danshen,Chuanxiong,Dilong,Shuizhi,Quanxie),Huayu group(H group,n=14,treatment with Danshen,Chuanxiong),Tongluo group(T group,n=14,treatment with Dilong,Shuizhi,Quanxie).After the success of the model,the rats were given the corresponding medicine.Chinese drug dosage: Danshen 1.41 g/kg,Chuanxiong 1.12 g/kg,Dilong 0.94 g/kg,Shuizhi 0.56 g/kg,Quanxie 0.56 g/kg.Rats in group C and group M were treated with purified water of the same volume,once a day,and then continuously for a period of 20 weeks.Fasting blood glucose(FBG)was measured every 4 weeks(3rd day,4th week,8th week,12 th week,16 th week,20 th week).At the end of the 16 th week,hemoglobin A1c(HbA1c)were detected by rats’ tail vein blood and 24 hour urine protein quantitation(urine total protein,UTP)were measured.The rats were anesthetized and blood samples were taken to measure blood lipids,thromboglobulin β(β-TG),platelet Factor 4(PF4),tissue type Plasminogen Activator(t-PA)and plasminogen Activator Inhibitor 1(PAI-1).Results:1 General stateDuring the experiment,there was no death in the C group,3 rats died in M group and T group,2 rats died in Z group,and 4 rats died in H group.One rat was relieved in M group,and each of 2 rats blood glucose remission in Z,H,T group.2 Fasting blood glucose(FBG)at different time pointsCompared with C group at the same time point,M,Z,H,T rats were significantly elevated in FBG(P<0.01);compared with M group at the same time point,there were no significant difference among Z,H and T group in FBG.3 Hemoglobin A1cCompared with C group,M,Z,H,T group of HbA1 c were significantly higher(P<0.01);compared with M group,the levels of Hb A1 c in Z,H and T groups there were no statistical difference(P>0.05).4 24 hour urine total protein(24h UTP)Compared with C group,the level of 24 h UTP in M group was significantly higher(P<0.01);compared with M group,the levels of 24 h UTP in rats of Z,H and T were significantly lower(P<0.01);compared with Z group,the levels of 24 h UTP in rats of H and T were significantly higher(P<0.05).5 blood lipidCompared with C group,TC,TG,HDL-C,LDL-C,VLDL-C in M group were significantly increased(P<0.01);compared with M group,the levels of TC and HDL-C in Z,H and T groups were no significantly difference(P>0.05),The levels of TG,LDL-C and VLDL-C were significantly lower(P<0.05 or P<0.01);compared with Z group,the levels of TC,TG,HDL-C and VLDL-C in H and T group were not significantly different(P>0.05),the level of LDL-C in H group was significantly higher than that in Z group(P<0.05).6 β-TG,PF4,t-PA,PAI-1 contents in rats’ plasmaCompared with C group,the content of β-TG in rats’ plasma was significantly increased in M group(P<0.05);compared with M group,the levels of β-TG in plasma of Z and T groups were significantly decreased(P<0.05 or P<0.01);compared with Z group,the level of β-TG in plasma was significantly increased in the H group(P<0.01),and there was no significant difference in T group(P>0.05).There was no significant difference of PF4 in plasma among the 5 groups(P>0.05).Compared with C group,the t-PA content of M group was significantly reduced,and the content of PAI-1 was significantly increased(P<0.05);compared with M group,the content of t-PA in each intervention group increased significantly(P<0.05),the content of PAI-1 in Z group and T group was significantly decreased(P<0.05),and there was no significant difference between H group and M group(P>0.05);compared with Z group,there was no significant difference in t-PA content between H group and T group(P>0.05),the content of PAI-1 in H group was significantly increased(P<0.05).Part two Effects of different compatibility of huayu-tongluo Chinese herbs on renal injury in DN rats.Objective: To verify whether the different compatibility of Chinese medicine can improve the protective effect of DN on the renal injury by improving the lipid metabolism,platelet release and the related indicators of fibrinolytic system in DN ratsMethods: Ninety SD rats were adaptive fed for a week,10 rats were randomly selected as normal group(C group)and the modeling method ibid.The model rats were randomly divided into model group(M group,n=15),Irbesartan group(I group,n=14,treatment with Irbesartan),Huayu Tongluo group(Z group,n=15),Huayu group(H group,n=14),Tongluo group(T group,n=14).Irbesartan group dosage was 14.12 mg/kg and the Chinese herbs intervention method as above.At the end of the third day after STZ injection and the end of 4th,8th,12 th,16th,20 th week the rats weight,water intake,food intake and urine volume were measured.Intervention for twentieth weeks,blood collection for liver function,renal function,the remaining part of the renal cortex for light microscopy and transmission electron microscopy.Results:1 General stateDuring the experiment,3 rats died in M,I,T group,2 rats died in Z group,4 rats died in H group,one rat was relieved in M group,and each of 2 rats blood glucose remission in Z,H,T group.Three days after STZ injection,there was no significant difference in weight among the groups(P>0.05);after 4 weeks of intervention,the weight of rats in M group was significantly lower than that in C group at the same time point(P<0.01);compared with M group at the same time point,the weight of rats in Z group was significantly higher than that in M group after 16 weeks(P<0.01),rats in H group and T group body weight of rats were significantly increased at the end of 20 weeks(P<0.05).Compared with C group at the same time point,the food intake of rats in M group increased significantly(P<0.01);compared with M group at the same time point,there was no significant difference in the food intake of each intervention group(P>0.05);there was no significant difference among the intervention groups(P>0.05).Compared with C group at the same time point,the water intake and urine volume of M group were significantly increased(P<0.01);compared with M group at the same time point,the water intake and urine volume of Z group significantly reduced after 16 weeks(P<0.01),and the water intake and urine volume of H,T group of rats were significantly lower than M group at the end of 20 weeks(P<0.05);compared with I group at the same time point,the water intake of Z group was significantly lower at the end of 16 weeks(P<0.05),rats of Z group and H group drinking water and urine volume were significantly reduced(P<0.05).2 kidney hypertrophy index(KI)Compared with C group,KI in M group was significantly increased(P<0.01);compared with M group,KI in Z group and I group was significantly decreased(P<0.05 or P<0.01);3 Liver function and renal functionThere was no significant difference in total protein(TP)and albumin(ALB)among the two groups(P>0.05).Compared with C group,serum creatinine(Scr),urea nitrogen(BUN)and uric acid(UA)were significantly higher in M group(P<0.01);compared with M group,there was no statistical difference in Scr among the intervention groups(P>0.05),BUN in Z group was significantly lower than that in M group(P<0.05),UA in I,Z,T group were significantly lower than that in M group(P<0.01).compared with I group,BUN in Z group was significantly decreased(P<0.01),BUN in H and T groups was higher than that in Z group(P<0.05),UA in H group was higher than that in Z group(P<0.05).4 24 hour urine total protein(24h UTP)There was no statistical difference in 24 h UTP 3 days after STZ injection(P>0.05).The 24 h UTP of rats in M group was higher than that in C group at the same time point(P<0.01);compared with M group at the same time point,the urinary protein excretion in each intervention group was decreased(P<0.01 or P<0.05);compared with I group at the same time point,the 24 h UTP of Z group was significantly reduced after 16 weeks(P<0.01);the urine protein of Z group was significantly lower than that in H group at the end of 16 and 20 weeks(P<0.05),24 h UTP in Z group was lower than that in T group(P<0.05).5 Creatinine clearance(Ccr)Compared with C group,the Ccr level of M group was significantly higher(P<0.01);compared with M group,the Ccr of each intervention group was significantly decreased(P<0.01 or P<0.05).6 Optical microscope observationC group rats had normal kidney tissue structure;in the M group rats,the glomerular hypertrophy,narrow renal capsul,some segments of the balloon occlusion,increased the number of mesangial matrix,basement membrane thickening,renal tubular epithelial cell swelling,renal interstitial fibrosis;compared with M group,each treatment group was significantly improved.Glomerular volume(GV)was calculated and compared,GV significantly increased in M rats(P<0.01);compared with M group,the GV of each intervention group was significantly decreased(P<0.01 or P<0.05);compared with I group,GV in Z group and T group was significantly reduced(P<0.01 or P<0.05).7 Electron microscope observation The ultrastructure of rats in C group was normal.In the M group,the glomerular basement membrane was thickened,the fusion of the podocyte foot process was serious,and the mesangial matrix proliferation.Compared with M group,the pathological changes of each intervention group were reduced,the basement membrane thickening was reduced and partial fusion of foot process.Part three Effects of different compatibility of huayu-tongluo Chinese herbs on the expression of integrin alpha 3 beta 1 and podocyte density in kidney tissue of rats with diabetic nephropathy.Objective: In this part,we focused on the expression of integrin alpha 3 beta 1 in renal tissue and the relationship between its expression and podocyte density of rats.And whether the target of different compatibility of huayutongluo Chinese herbs reducing urinary protein is related to it.Methods: Inducing model and intervention method was same as above.The ITGA3,ITGB1,WT1 mRNA and protein expression were detected by Real-time PCR and immunohistochemistry in twentieth weeks after anesthesia.WT-1 was used to mark the nucleus of podocytes,count the number of podocytes,and calculate the density of podocytes.Results:1 Detection of ITGA3,ITGB1,WT1 mRNA expression in rats’ kidney by Real-Time PCRCompared with C group,the expression of itga3,itgb1 and WT1 mRNA in M group were significantly decreased(P<0.01);compared with M group,itga3,itgb1,WT1 mRNA in I group and Z group significantly increased(P<0.01 or P<0.05),the expression of itga3 and WT1 mRNA in T group were increased(P<0.05);compared with I group,the expression of itga3 mRNA decreased in H group(P<0.05),and the itgb1 mRNA increased in Z group(P<0.05);compared with Z group,the expression of itga3,itgb1 and WT1 mRNA in H group were significantly decreased(P<0.05),itgb1 mRNA in T group was also decreased(P<0.05).2 Detection of ITGA3,ITGB1,WT1 protein expression in rats’ kidney by IHCItga3 and itgb1 were mainly located in the glomerular basement membrane and mesangial matrix,and they were highly expressed in the C group,mainly in the basement membrane and podocyte,a small amount of distribution in the cytoplasm of podocytes.Compared with C group,the expression of itga3 and itgb1 was significantly decreased in M group,Compared with M group,I group,Z group and T group their expression increased,but H group was no significant difference.WT1 is a specific protein of podocytes,which is expressed in glomeruli and located in the nucleus of podocytes.Compared with C group,the expression of WT1 decreased in M group;Compared with M group,the expression of WT1 in I group,Z group and T group increased,but there was no significant difference in H group.3 Detection of podocyte density in glomeruli of ratsCompared with C group,the density of podocytes in M group was significantly decreased(P<0.01);compared with M group,the density of podocytes in I group,Z group and T group was increased(P<0.01 or P<0.05);compared with I group,the density of H group decreased(P<0.05);compared with Z group,the H group had reduced podocyte density(P<0.05),and there was no significant difference in T group(P>0.05).Part four The expression of wnt/beta-catenin pathway in diabetic nephropathy rats and the intervention effect of different compatibility of huayu-tongluo Chinese herbs.Objective: In this part,we prepared DN rat model to explore whether the kidney of the rat has abnormal expression of Wnt/β-catenin signaling pathway? Moreover,it is unclear whether the huayu-tongluo Chinese herbs could protect podocytes and reduce renal fibrosis is associated with Wnt/β-catenin pathway?Methods: Inducing model and intervention method was same as above.At the end of the twentieth week intervention,the rats were anesthetized and part renal cortex were taken using Real-time PCR method to detect ILK,Wnt4,GSK3β,β-catenin,E-cadherin mRNA expression,Wnt4,p-GSK3(S9),β-catenin,E-cadherin protein expression were detected by immunohistochemical,ILK,Wnt4,p-GSK3β(S9),GSK3β,β-catenin,E-cadherin protein expression were detected by western-blot.Results:1 Detection of ILK,Wnt4,GSK3β,β-catenin,E-cadherin mRNA expression in rats’ kidney by Real-Time PCRCompared with C group,the expression of ILK,Wnt4,β-catenin mRNA in M group were significantly increased,and the expression of E-cadherin mRNA was significantly decreased(P<0.01 or P<0.05);compared with M group,ILK,Wnt4,β-catenin mRNA expression in each intervention group decreased significantly(P<0.01 or P<0.05),and the expression of E-cadherin mRNA in I group,Z group and T group was increased(P<0.01 or P<0.05);compared with I group,the expression of β-catenin mRNA in H group was increased(P<0.05),and the expression of E-cadherin mRNA in Z group was increased(P<0.05);compared with Z group,the expression of β-catenin mRNA in H group was significantly increased,and the expression of Ecadherin mRNA was decreased(P<0.05).There was no significant difference in the expression of GSK3β mRNA in renal tissue of rats(P>0.05).2 Detection of Wnt4,p-GSK3β,β-catenin,E-cadherin protein expression in rats’ kidney by IHCIn C group,Wnt4 protein was only weakly expressed in some renal tubular epithelial cells,p-GSK3β protein was expressed in glomeruli and tubules,β-catenin protein was expressed in some glomeruli,renal tubular epithelial cells and distal tubules.Compared with C group,the expression of Wnt4,p-GSK3β,β-catenin in glomerular and renal tubular epithelial cells in M group was significantly increased;compared with M group,the expression of each intervention group was decreased.The expression of E-cadherin protein in C group was mainly expressed in renal tubular epithelial cells and mesangial cells.Compared with C group,the expression of E-cadherin protein in M group was significantly decreased;compared with M group,the expression of E-cadherin protein was increased in different degrees.3 Detection of ILK,Wnt4,p-GSK3β,GSK3β,β-catenin,E-cadherin protein expression in rats’ kidney by Western BlotCompared with C group,the expression of ILK,Wnt4,p-GSK3β,β-catenin protein was significantly increased in M group,and the expression of E-cadherin protein was down regulated(P<0.01 or P<0.05);compared with M group,the expression of ILK,Wnt4,β-catenin protein in each intervention group decreased significantly(P<0.01 or P<0.05),the level of p-GSK3β protein in I group,Z group and H group decreased significantly(P<0.01 or P<0.05),and the expression of E-cadherin protein in I group,Z group and T group was increased(P<0.01 or P<0.05);compared with Z group,the expression of E-cadherin protein in H group was significantly reduced(P<0.05).There was no significant difference in the expression of GSK3β protein in renal tissue of rats(P>0.05).Conclusions:1 DN rats have obvious disorder of glucose and lipid metabolism,the enhancement of function of platelet release,the low activity of fibrinolytic system,significant proteinuria and renal pathological damage,which proved that the DN rat model was successfully reproduced.2 Huayu-tongluo Chinese herbs,huayu Chinese herbs and tongluo Chinese herbs can improve lipid metabolism disorder in DN rats,inhibit abnormal platelet activation,improve platelet release function and fibrinolytic dysfunction,reduce urinary protein excretion,reduce glomerular hyperfiltration in high perfusion,reduce kidney damage.It has exact renal protective effect.3 Huayu-tongluo Chinese herbs and tongluo Chinese herbs can up regulation the expression levels of integrin alpha 3 and beta 1,reduce podocyte detachment,reduce podocyte injury,maintain normal glomerular filtration function.This may be one of the mechanisms by which the two herbs reduce proteinuria of DN rats.The application of huayu Chinese herbs alone has no obvious influence on the shedding of podocyte.4 Huayu-tongluo Chinese herbs can inhibit ILK activation,inhibit the high expression of wnt/β-catenin pathway in kidney of DN rats,which may be one of the main ways the herbs to protect podocytes,reduce urinary protein excretion.5 The effect of combined application of huayu-tongluo Chinese herbs is superior to huayu Chinese herbs and tongluo Chinese herbs.The two herbs can be applied simultaneously,and there may be synergistic relationship.Tongluo Chinese herbs is better than huayu Chinese herbs on some efficacy,such as protect renal function,improve the abnormal platelet activation and fibrinolytic function etc. |
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