| Background and ObjectiveProteinuria is an independent factor that causes renal fibrosis and is an important factor in the progression of diabetic nephropathy(DN).The previous research of the research group showed that:" Kidney essence loss and collaterals obstruction "(which is deficiency of kidney essenceand blood stasis and turbidity originating from interior)is the core pathogenesis of diabetic nephropathy proteinuria,for this pathogenesis,we propose the treatment method of filling essence and dredge collaterals,and In previous animal experiments,it was found that "QI ditangshen particles" under the guidance of filling essence and dredge collaterals can reduce urinary protein in DN rats(P<0.05),but the mechanism is not yet clear.Transforming growth factor ?(TGF-?)is an important factor in the development of fibrosis.In the course of DN,it can induce renal fibrosis through multiple pathways,of which TGF-?1-Smad2/3 is the most classical pathway.Therefore,the purpose of this study was to investigate the effects of "QI ditangshen particles" on the expression of TGF-?1,Smad2,and Smad3 in the TGF-?1-Smad2/3 pathway through experimental studies;and to investigate the correlation of TGF-?1,Smad2,Smad3 with kidney asthenia syndrome,blood stasis syndrome,and cloudiness syndrome through clinical studies.MethodsExperimental research T1D model was produced using a single intraperitoneal injection of STZ 55 mg/kg.Rats with successful modeling were randomly divided into 6 groups:control group,model group,high dose group,medium dose group,and low dose group of "QI ditangshen particles",and valsartan(trade name,Dai Wen)group.The rats in each group were housed in separate cages and fed water freely.The same volume of medicine was fed according to body weight.The model group and the normal control group were given equal doses of carboxymethyl cellulose(CMC),respectively;intragastric administration of high dose,medium dose,and low doses of "QI ditangshen particles" was 5.24g/kg body weight,2.62g/kg body weight,and 1.31 g/kg body weight.The concentration of body weight was formulated into three different concentrations of drug solution;the Dai Wen group was administered at 8 mg/kg body weight and administered for 12 weeks.Blood glucose and body weight were measured at 0,2,4,6,8,10,and 12 weeks;24 h urine was taken before and every 4 weeks after drug administration to detect 24 h urine microalbumin;At the end of the 12th week,blood creatinine(Scr),triglyceride(TG),cholesterol(CHO),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured.HE staining was used to observe the pathological changes of the kidney of each rat by light microscopy.Masson staining was used to observe the pathological changes of renal tubules and interstitium.The expression of TGF-?1,Smad2,Smad3 and a-SMA protein in rat renal tissue was detected by Western-Blot.Clinical research Relying on the project of "Establishment and Validation of Early Prediction Diagnosis Decision Tree for Diabetic Nephropathy"(ID:JD2X2015305)enrolled 57 patients who met the diagnostic criteria for diabetes,collected general data and physicochemical results of patients enrolled in the study:according to the TCM syndrome differentiation table,the patients were diagnosed by TCM syndromes.Then the patients were divided into simple diabetes(DM)group and diabetic kidney disease(DKD)group according to the urinary microalbumin/urine creatinine(ACR)value.The expression levels of serum TGF-?1,Smad2,Smad3 were detected by Elisa method.Compared the differences in the general data and serum TGF?1,Smad2,and Smad3 expression levels between the DM and DKD groups;At last,find correlation between the expression levels of fibrogenic factors TGF-?1,Smad2,and Smad3 and the kidney deficiency,turbid phlegm,and blood stasis syndromes in the DKD group.ResultExperimental research1 General physical and chemical inspection:1.1 Rat kidney index(kidney weight/body weight):Compared with the control group,the kidney index of the model group was significantly increased(P<0.01).Compared with the model group,no significant difference was found in the renal index of each administration group(P>0.05).1.2 Blood glucose:Compared with the control group,The blood glucose level in the model group was significantly higher(P<0.01).Compared with the model group,the blood glucose level in the medium dose group of "QI ditangshen particles" was lower at the 12th week(P<0.05).1.3 Blood lipids and liver function:Compared with the control group,the levels of TG,CHO,ALT and AST in the model group had no difference(P>0.05);Compared with the model group,the TG levels in the high-dose group of "QI ditangshen particles"decreased(P<0.05).no difference in CHO,ALT,AST levels(P>0.05)1.4 Renal function:Compared with the control group,the levels of Scr in the model group had no difference(P>0.05).2 Efficacy indicators24h urinary microalbumin:Compared with the control group,the 24h urine microalbumin level in the model group was significantly higher(P<0.01);Compared with the model group,at the 12th week of administration,the 24h urine microalbumin level in high dose group and medium dose group and low dose group of "QI ditangshen particles" were lower(P<0.01).Compared with the positive drug Dai Wen group,the 24-hour urinary microalbumin levels were lower in the high-dose and middle-dose groups of "QI ditangshen particles"(P<0.05).3 Rat tissue morphology changes3.1 HE staining under light microscope,compared with the control group,the model group of individual kidney glomerular mesangial matrix increased or capillary basement membrane thickening,pathological changes of the renal tubules,see the tubular epithelial cell cytoplasm empty,was blank Samples,vacuolar degeneration of tubular epithelial cells,renal tubular epithelial cells with hypertrophy,or detachment and intraluminal protein droplets,shallow cortical tubular dilatation;compared with the model group,indigestive kidney granules,The incidence of cytoplasmic empty and space-like lesions in renal tubular epithelial cells in the low-dose group was reduced,and the incidence of kidney disease in the middle-dose group was significantly reduced,and the degree of lesions was also reduced;Although there was no decrease in the incidence of kidney disease in the animals,the extent of the lesions was significantly reduced.3.2 There were no obvious abnormalities in collagen fibers stained by Masson staining of kidney tissue sections.Compared with the control group,no glomerular and interstitial fibrosis was observed in the model group.4 Western-Blot method to detect the expression level of TGF-B1,Smad2,Smad3 and a-SMA in renal tissueCompared with the normal group,the expression levels of TGF-B1,Smad2,and Smad3 in the model group increased(P<0.05).Compared with the model group,the level of TGF-B1 in the high-dose group of "QI ditangshen particles' significantly decreased(P<0.01),the level of TGF-BI in the middle dose group of "QI ditangshen particles" was decreased(P<0.05);the level of TGF-B1 in the low dose group had no significant difference(P>0.05);Smad2,Smad3,and a-SMA expression levels in each dose group No significant difference(P>0.05)Clinical research1 There was no statistical difference in the age and physicochemical examination between DM and DKD patients(P>0.05)2 Compared with DM group,the serum TGF-?1 and Smad2 expression levels in DKD patients were increased(P<0.05).3 The expression levels of serum TGF-?1 and Smad2 in patients with DKD were related to phlegm,blood stasis,and dampness(P<0.05);the expression level of TGF-?1 was related to the deficiency of kidney yang(P<0.05);About the expression level of Smad2,there has no correlation in the deficiency of kidney yang(P>0.05).There was no correlation between the expression level of TGF-?1 and Smad2 with the deficiency of kidney yin and deficiency of kidney qi(P>0.05).ConclusionExperimental research1 "QI ditangshen particles can reduce blood glucose,triglyceride,and urinary protein levels in diabetic nephropathy rats2 "QI ditangshen particles" through the TGF-?1-Smad2/3 pathway down-regulate the expression of TGF-?1 to prevent and treat the fibrosis of diabetic nephropathy;no effect on the expression of Smad2,Smad3.Clinical researchThe fibrogenic factors TGF-?1,Smad2 in DKD patients were related to the turbid syndrome,blood stasis syndrome and deficiency of kidney yang syndrome. |
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