| Objectives: Diabetic nephropathy(DN) is a common microvascular complication in diabetes mellitus(DM) patients, and it is one of the main cause of end stage renal disease that bring heavy burden to society and families. Modern medicine is still lack of effective means to delay the occurrence and development of DN. Traditional Chinese medicine(TCM) is one of the features of clinical medicine in China, and recent years, it has made a good curative effect in kidney disease treatment. Most of the TCM experts affirm that the basic pathogenesis of diabetic nephropathy is deficiency of qi and Yin, stasis-blood-blocking-collaterals. Through repeated Clinical screening, we identified a stasis-removing-and-collaterals-dredging drug formula to treat DN.Proteinuria is the most important clinical manifestation in early stage DN.Modern medical science confirmed that urine filtration must pass vascular endothelial cells, glomerular basement membrane(GBM) and podocytes, and the three layer structure is known as the glomerular filtration barrier. The glomerular filtration barrier have mechanical barrier and charge barrier function. Each of the barrier function injury can lead to proteinuria. Under the normal state, plasma albumin with negative charge can’t through the glomerular filtration barrier, and when the filtration barrier negative charge reduced, proteinuria appears even if the structure is integrated. This study intends to test laboratory indexes to verify whether there is stasis-bloodblocking-collaterals in DN rats, and to investigate weather the curative effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on DN proteinuria is related to glomerular charge barrier through in vivo and in vitro experiments. In addition, we intend to verify the different effect ofstasis-removing drug and collaterals-dredging drug on DN.Methods:1 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on stasis-blood-blocking-collaterals related laboratory indexes in DN rats75 SD rats were adaptive fed for a week, 10 rats were randomly selected as normal control group(C group), the rest ones were fed with high sugar high fat diet for 4 weeks. Then modelling rats were injected intraperitoneally with1% streptozocin(STZ) 35 mg/kg, and modelling rats whose random blood glucose in tail venous ≥ 16.7mmol/L after 72 hour were considered as successfull DN models. 60 DN rats were randomly divided into model group(M group, n=15), stasis-removing-collaterals-dredging drug group(Q group,n=15), stasis-removing drug group(H group, n=15), collaterals-dredging drug group(T group, n=15). Chinese drug dosage: Danshen 1.35g/kg, Chuanxiong1.08g/kg, Dilong 0.9g/kg, Shuizhi 0.54g/kg, Quanxie 0.54g/kg; C group and M group was given equivalent amount of water by intragastric administration once a day for 16 weeks. At the end of the 16 th week, hemoglobin A1c(Hb A1c) were detected by rats’ tail vein blood. After anesthesia, blood was taken from abdominal aortic for blood glucose, blood lipid, blood regular test,blood coagulation, von Willebrand factor(v WF) and CD62 p detection.2 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on renal injury in DN rats90 SD rats were adaptive fed for a week, 10 rats were randomly selected as normal control group(C group), the rest ones were induced for DN model as above. 74 DN rats were randomly divided into model group(M group,n=15), Irbesartan group(I group, n=14), stasis-removing-collaterals-dredging drug group(Q group, n=15), stasis-removing drug group(H group, n=15),collaterals-dredging drug group(T group, n=15), intervention method as above. Each rat’s food intake, water intake, unrie volume was recorded after72 h STZ injection(0 weeks) and at the end of the 2nd, 4th, 8th, 12 th, 16 th week. Urinary protein concentration were detected by ELISA. At the end of the 16 th week, rat’ blood was taken for liver and kidney function detection and kidney was preserved for optical microscope and transmission electron microscope detection.3 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on glomerular charge barrier in DN ratsInducing model and intervention method was same as above. At the end of the 16 th week, rat’ kidney was preserved for HSPG, HPA, PCX, GLEPP1 expression detection by Real-Time PCR, immunohistochemistry, Western Blot and part of the kidney was preserved in 0.5% polyethylenimine(PEI) for GBM anionic sites(AS) detection.4 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on glomerular charge barrier related proteins in podocytes under high glucose conditionMouse podocytes were cutured in DMEM-F12 medium containing γ-IFN in 33℃ incubator. when cell proliferated to a sufficient number, replace for DMEM-F12 medium excluding γ-IFN at 37℃ incubator for cell differentiation. Screened out the optimal stasis-removing-collaterals-dredging medicated serum concentration according to MTT. Podocytes were divided into normal glucose group(NG), high glucose group(HG), mannitol group(MG) and Chinese drugs group(ZG). After intervention for 24 h, 48 h, 72 h, detect podocytes PCX, GLEPP1 expression by immunocytochemistry and Real-Time PCR, Western Blot.Results:1 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on stasis-blood-blocking-collaterals related laboratory indexes in DN rats1.1 General state3 rats died and 2 rats blood glucose was lower than 16.7mmol/L within72 h after STZ injection. 3 in M group, 3 in Q group, 3 in H group, 2 in T group blood glucose was lower than 16.7mmol/L and 1 in C group, 5 in M group, 3 in Q group, 4 in H group, 3 in T group died within 16 weeks’ intervention.1.2 Hemoglobin A1 c testCompared with C group, Hb A1 c in modelling groups were significantly increased(P < 0.01); there was no statistical difference among modelling groups(P≥0.05).1.3 Fasting blood glucose, blood lipid test Compared with C group, FBG in modelling groups were significantly increased(P < 0.01); there was no statistical difference among modelling groups(P≥0.05).Compared with C group, CHO in modelling groups were significantly increased(P < 0.01); there was no statistical difference among modelling groups(P≥0.05). Compared with C group, TG in M group was increased(P<0.01); compared with M group, Q group, H group, T group were decreased(P<0.05). Compared with C group, HDL-C in M group was increased(P<0.01); compared with M group, T group were decreased(P<0.05). Compared with C group, LDL-C in M group was increased(P<0.01); compared with M group, Q group, T group were decreased(P<0.05). Compared with C group,VLDL-C in M group was increased(P<0.01); compared with M group, Q group, H group, T group were decreased(P<0.05).1.4 Platelet parameter testPLT in each group had no statistical difference(P≥0.05). Compared with C group, MPV in M group was increased(P<0.05); compared with M group,Q group, H group, T group were decreased(P<0.05). PCT in each group had no statistical difference(P≥0.05). Compared with C group, PDW in M group was increased(P<0.05); compared with M group, Q group were decreased(P<0.05).1.5 Blood coagulation testCompared with C group, PT in M group was significantly shortened(P<0.01); compared with M group, Q group, H group, T group were extended(P<0.05). Compared with C group, INR in M group was significantly decreased(P<0.01); there was no statistical difference among modelling groups(P≥0.05). Compared with C group, APTT in M group was significantly shortened(P<0.01); compared with M group, Q group were extended(P<0.05). TT in each group had no statistical difference(P≥0.05). Compared with C group,FIB in M group was increased(P<0.01); there was no statistical difference among modelling groups(P≥0.05). Compared with C group, AT-III activity in M group was significantly decreased(P<0.01); compared with M group, Q group, H group, T group were increased(P<0.05).1.6 Detection of v WF, CD62 p content in rats’ plasma by ELISACompared with C group, v WF in M group was significantly increased(P<0.01); compared with M group, Q group, H group, T group were decreased(P < 0.05); there was no statistical difference among Q group, H group, T group(P≥0.05).Compared with C group, CD62 p in M group was significantly increased(P < 0.01); compared with M group, Q group, H group, T group were decreased(P<0.05); CD62 p in Q group, T group was lower than H group(P<0.05).2 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on renal injury in DN rats2.1 General stateCompared with C group at the same time points, food intake, water amount, urine volume in M group were significantly increased(P < 0.01);there was no statistical difference among modelling groups(P≥0.05).2.2 Detection of 24-hour urine protein excretion(24h UPE) in rats’ urine by ELISA24h UPE in each group had no statistical difference 72 h after modelling(P≥0.05). At the end of the 2nd week, compared with C group, 24 h UPE in M group was increased(P<0.05); compared with M group, I group and Q group was decreased(P<0.05). At the end of the 4th week, compared with C group,24 h UPE in M group was increased(P <0.01); compared with M group, I group and Q group was decreased(P < 0.05). At the end of the 8th week,compared with C group, 24 h UPE in M group was increased(P < 0.01);compared with M group, four treatment groups was decreased(P<0.05). At the end of the the 12 th and 16 th week, compared with C group, 24 h UPE in M group was increased(P < 0.01); compared with M group, four treatment groups was decreased(P<0.05); Q group was lower than I group, Q group, H group and T group(P<0.05).2.3 Liver function, renal function testTP and ALB in each group had no statistical difference(P≥0.05).Compared with C group, SCr in M group was significantly increased(P< 0.01); compared with M group, Q group was decreased(P < 0.05).Compared with C group, BUN in M group was significantly increased(P<0.01); there was no statistical difference among modelling groups(P≥0.05).Compared with C group, UA in M group was significantly increased(P <0.01); compared with M group, Q group and T group was decreased(P <0.05).2.4 Body weight, kidney weight and kidney hypertrophy index(KI)Compared with C group, body weight in M group was significantly decreased(P < 0.01); there was no statistical difference among modelling groups(P≥0.05). Kidney weight in each group had no statistical difference(P≥0.05). Compared with C group, KI in M group was significantly increased(P<0.01); compared with M group, Q group and T group was decreased(P<0.05).2.5 Optical microscope observationC group rats had clear and normal kidney tissue structure; M group rats had enlarged glomerular, narrow renal capsule, thick GBM, and increased mesangial matrix; compared with M group, each treatment group was significantly improved; among the treatment groups, H group pathological performance was heavier than other treatment groups.2.6 Transmission electron microscope observationC group rats had clear and normal kidney tissue structure; M group rats had thick GBM, increased mesangial matrix and podocyte foot process fusion;compared with M group, each treatment group pathological change significantly improved; among the treatment groups, H group pathological performance was heavier than other treatment groups.3 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on glomerular charge barrier in DN rats3.1 Detection of HSPG, HPA, PCX, GLEPP1 m RNA expression in rats’ kidney by Real-Time PCRHSPG m RNA expression in each group had no statistical difference(P≥0.05). Compared with C group, HPA m RNA in M group was significantly increased(P<0.01); compared with M group, I group, Q group and T group were decreased(P<0.05). Compared with C group, PCX m RNA in M group was significantly increased(P<0.05); compared with M group, I group and Q group were decreased(P<0.05). Compared with C group, GLEPP1 m RNA in M group was significantly increased(P < 0.05); there was no statistical difference among treatment groups(P≥0.05).3.2 Detection of HSPG, HPA, PCX, GLEPP1 protein expression in rats’ kidney by IHCHSPG protein was expressed in GBM and mesangial matrix, and HSPG protein expression in each group had no obvious difference. In C group, HPA protein was no obvious expression in glomerular and low expression in tubular epithelial cells; compared with C group, HPA protein expression in M group was significantly increased in tubular epithelial cells, and expressed in glomerular endothelial cells; compared with M group, HPA protein expression was declined in treatment groups. PCX and GLEPP1 are the specific proteins in podocyte. Compared with C group, PCX and GLEPP1 protein expression in M group was reduced; compared with M group, I group and Q group was increased.3.3 Detection of HPA, PCX, GLEPP1 protein expression in rats’ kidney by Western BlotCompared with C group, HPA protein expression in M group was significantly increased(P<0.01); compared with M group, I group, Q group,T group was decreased(P < 0.05). Compared with C group, PCX protein expression in M group was decreased(P<0.05); compared with M group, I group and Q group was increased(P < 0.05). Compared with C group,GLEPP1 protein expression in M group was decreased(P<0.05); compared with M group, I group and Q group was increased(P<0.05).3.4 Detection of PCX content in rats’ urine by ELISACompared with C group, PCX content in M group was significantly increased(P<0.01); compared with M group, PCX content in each treatment group were decreased(P<0.05); compared among each treatment group, PCX content in I group, Q group and T group was less than H group(P<0.05).3.5 Detection of AS in GBM by transmission electron microscopeCompared with C group, AS in M group were significantly reduced(P<0.01); compared with M group, I group, Q group and T group was increased(P<0.05).4 The effect of stasis-removing-and-collaterals-dredging Chinese herbal drug on glomerular charge barrier related proteins in podocytes under high glucose condition4.1 Podocytes identificationAdult podocytes look like branches in the tree, and express specific protein WT-1 in nucleus.4.2 Screen the optimal medicated serum concentration by MTTCompared with NG at the same time point, OD in HG were significantly lower(P<0.01), MG had no statistical difference(P≥0.05). 5%, 10%, 15%medicated serum had similar function to promote cell proliferation, superior to2.5% medicated serum. So we used 5% medicated serum for follow-up study.4.3 Detection of PCX, GLEPP1 protein expression in podocytes by ICCICC results showed that PCX, GLEPP1 protein expressed in podocyte membrane, NG and MG PCX, GLEPP1 protein expression had no obvious difference; compared with NG, PCX, GLEPP1 protein expression in HG were decreased significantly; compared with HG, PCX, GLEPP1 protein expression in ZG were increased.4.4 Detection of PCX, GLEPP1 m RNA expression in podocytes by Real-Time PCRNG and MG PCX mRNA expression had no statistical difference(P≥0.05); compared NG at the same time point, HG PCX m RNA expression decreased significantly(P<0.01); compared with HG at the same time point,ZG PCX m RNA expression increased(P<0.01).NG and MG GLEPP1 mRNA expression had no statistical difference(P≥ 0.05); compared with NG at the same time point, HG GLEPP1 m RNA expression decreased significantly(P<0.01); compared with HG at the same time point, after 24 h, 48 h medicated serum intervention, GLEPP1 m RNA expression had no statistical difference(P≥0.05), after 72 h medicated serum intervention, GLEPP1 m RNA expression increased(P<0.05).4.5 Detection of PCX, GLEPP1 protein expression in podocytes by Western BlotNG and MG PCX protein expression had no statistical difference(P≥0.05); compared NG at the same time point, HG PCX protein expression decreased(P < 0.05); compared with HG at the same time point, after 24 h medicated serum intervention, PCX protein expression had no statistical difference(P ≥ 0.05), after 48 h, 72 h medicated serum intervention, PCX protein expression increased(P<0.05).NG and MG GLEPP1 protein expression had no statistical difference(P≥ 0.05); compared NG at the same time point, HG GLEPP1 protein expression decreased significantly(P<0.01); compared with HG at the same time point, ZG GLEPP1 protein expression increased(P<0.01).Conclusions:1 DM model rats dealt with high sugar high fat diet combined with STZ appear high blood glucose, DM clinical symptoms, and gradually to proteinuria and renal pathological changes of DN. And model rats appear hyperlipidemia, injured vascular endothelial cell, abnormal activated platelet and hypercoagulable state, conform to stasis-blood-blocking-collaterals in TCM.2 Stasis-removing-and-collaterals-dredging drug can reduce DN proteinuria, relieve renal pathological injury and delay renal disease progress.Besides, it can improve stasis-blood-blocking-collaterals in DN rats.3 The mechanism of reducing DN proteinuria may be related to stasisremoving-and-collaterals-dredging drug inhibiting renal HPA expression,upregulating podocyte PCX, GLEPP1 expression, and maintaining glomerular charge barrier integrity.4 In vitro experiments, compared with high glucose cultured podocytes,high glucose plus 5% stasis-removing-collaterals-dredging medicated serum intervention can upregulate podocyte PCX and GLEPP1 expression, and reduce podocyte surface negative charge loss.5 Comprehensive analysis, stasis-removing-collaterals-dredging drug is superior to stasis-removing drug and collaterals-dredging drug. There is synergy between stasis-removing drug and collaterals-dredging drug. |
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