| Objective: Diabetic nephropathy （DN） is one of the most commonchronic complications of diabetes mellitus （DM）, and also one of main causesleading to death. Diabetic nephropathy has become the first primary diseasedeveloped chronic renal failure. In our country, with the rapid increase of theincidence of diabetes, end-stage renal failure is increasing caused by DN.Expected in the next20years DN will become the most important cause ofend-stage renal failure, but so far there is no effective method to prevent theoccurrence and development of DN, which has become an urgent problem.Furthermore, DN has great impact on life quality and prognosis of the patientsand increases the suffering of patients. So, it has important significance toexplore the pathogenesis of DN and effective methods for prevention of DN.The main pathological manifestation of DN is glomerular capillarybasement membrane thickening, mesangial matrix proliferation and nodularglomerulosclerosis. The main clinical manifestation of DN is proteinuria,which is microalbuminuria at early manifestations and proteinuria at clinicalperformance. Pathogenesis of diabetic nephropathy is very complex, includingprotein non-enzymatic glycation, oxidative stress and podocyte injury and soon. DN is the result of multiple factors and multiple paths synergy. Therefore,treatment for diabetic nephropathy must also be multiple factor, multipletargets. At present, the modern medical prevention and control measures ofDN are mainly in the following aspects: strict control of blood glucose, bloodpressure, lipid disorders correction and application of ACEI and ARB. There isno specific drug for controlling DN. And western medicine has more sideeffects, which can not satisfy the patients in the control of clinical symptoms.However, Chinese medicine may develop effect on many aspects of DN andshow great potential and broad application prospect in the clinical prevention and treatment of DN with its unique overall adjustment, syndromedifferentiation system and three for individualized treatment plan system.Our research group arrival at a conclusion that the pathogenesis of DN ismainly blood stasis in the kidney collateral combined with years of clinicaltreatment experience of DN and TCM etiology and pathogenesis theory,especially the collateral disease theory. And the treatment should be adoptedhuoxue-huayu-tongluo therapy. We compose huoxue-huayu-tongluo Chineserecipe using salvia, chuanxiong, earthworm, leech, Scorpio and so on, whichadopting compatibility of traditional Chinese medicine. Clinical observationand preliminary experiments show that the recipe has a good effect on DN.This study adopts the model of DN rats with a single high dose streptozotocinintraperitoneal injection. First of all, we make an overall study on theprotective effect on the DN model in kidney of rats of huoxue-huayu-tongluoChinese medicine. Secondly, we develop a cell experiment using mice livingsystem condition of podocyte. And we observe the effect ofhuoxue-huayu-tongluo Chinese medicine decoction drug-containing serum ofrats and drug monomer-tanshinone II A on podocyte, in order to furthervalidate the efficacy of the party, to investigate the mechanism of DN, and theeffect of measurement, and provide reference for clinical application.Methods:1Renal protective effects of huoxue-huayu-tongluo Chinese medicine on DNratsHealthy male4～5weeks old50SD rats with body weight120g～150gafter adaptive feeding for1week were grouped randomly. And they were fedfor one week after making sure the rats’ urine protein negative. Ten rats werenormal control group. Other40rats were fed with high fat diet throughout thestudy. The animals were randomly divided into normal group （NG） for ten ratsand DM model group for forty rats. The DM model group was given a singledose of60mg·kg-1streptozotocin intraperitonally and rats in NG were ip vehic.Blood glucose levels were measured for three times continuously (≥16.7mmol·L-1) on the third day after streptozotocin injection to confirm the development of diabetes. We divide the model rats randomly into three groups:model group （MG）, irbesartan group （XG） and huoxue-huayu-tongluo Chinesemedicine group （ZG）. Rats in XG were orally administered with15mg·kg-1·d-1irbesartan, while the ZY group with decoction-free herbal granules of salvia,chuanxiong, earthworm, leech and Scorpio by1.0g·kg-1·d-1, and the left groupswith the same dose of Sodium Chloride everyday for sixteen weeks.At the end of4Weeks,8Weeks,12Weeks and16Weeks, rats wereplaced into metabolic cages for24hours to collected24h urine. Rats′waterintake, diet intake, urine output, and body weight were measured.FBG and HbA1c were measured with sampling from tail vein. Then ratswere anesthesiaed with chloraldurate and blood sample was collected fromaortaventralis and serum was separated. Serum creatinine, urinary creatinine,serum lipid （including total choleaterol, triglyceride, high density lipidcholeaterol and very low density lipid choleaterol） were measured byfull-automatic biochemistry checker. Renal weight was weighed. Kidneyindexes （KI） were calculated. Renal cortex samples were stored for rulemicroscopy or transmission electron microscopy and glomerular basementmenbrane thickness was measured by transmission electron microscopes.2Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin andthe expression of p-cadherin in DN rats2.1Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin inDN ratsAt the end of4Weeks,8Weeks,12Weeks and16Weeks, rats wereplaced into metabolic cages for24hours to collected24h urine. Urine sampleswere centrifuged at speed of3500rpm for10minutes, and supernate ofsamples were stored at-20℃. Urinary albumin concentration was measuredby Beckman cx7full automatic biochemical analyzer.2.2Effects of huoxue-huayu-tongluo Chinese medicine on the expression ofp-cadherin in DN ratsRenal cortex samples were cut fast and stored at-80℃. We extract totalRNA in renal tissue and use reverse transcriptase polymerase chain reaction （RT-PCR） and fluorescence quantitative polymerase chain reaction （Real-TimePCR） to detect expression of p-cadherin mRNA in renal tissue. Also, theexpression of p-cadherin protein was detected by Western blot.3Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein andfactors associated with renal fibrosis in DN rats3.1Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein inDN ratsAccording to the special instrument manual, urinary protein in24hoursin rats in each group was determined by using Beckman cx7automaticbiochemical analyzer.3.2Effects of huoxue-huayu-tongluo Chinese medicine on factors associatedwith renal fibrosis in DN ratsRenal cortex samples were cut fast and expression of BMP-7and TGF-β1protein and mRNA were detected using immunohistochemistry, Western blot,reverse transcriptase polymerase chain reaction and fluorescence quantitativereal-time polymerase chain reaction. We also detected the expression of OPNmRNA by RT-PCR and Real-Time PCR and the expression of Smad7proteinby Western-blot. Correlation of BMP-7and TGF-β1also analyzed.4Effects of huoxue-huayu-tongluo serum containing medicine and drugmonomer-tanshinone II A on the proliferation of podocyteMice living system condition of podocyte were cultured in vitro using thetechnique of cell culture and observing the effect on the proliferation ofpodocyte of huoxue-huayu-tongluo serum containing medicine and theconcentration of the monomer-tanshinone II A, using MTT method.5Statistical analysesAll results were expressed as±s. Repeated measures and multivariateanalysis of variance （ANOVA） process of the general linear model was used toidentify comparison among different groups and different measure timepairwise; One-Way ANOVA was used to compare mean values among groups,and SNK to compare mean values between each two group. And the datawhich was not conforming to the normal distribution was analyzed by rank sum test, then One-Way ANOVA to rank. Correlations between variables usePerson linear correlation analysis. Analysis was carried out using SAS V8.P-value of <0.05was considered to be statistically significant.Results:1Renal protective effects of huoxue-huayu-tongluo Chinese medicine on DNrats1.1Comparison of FBG in each group at different timeCompared with NG, FBG in MG, ZG and XG increased notably （P<0.05）at different time. Compared with MG, there was no significant difference ofFBG in ZG and XG （P>0.05）. Furthermore, within4months of MG, ZG andXG in rat FBG levels have been stable, no obvious change.1.2Comparison of body weight in each group at different timeBody weight of NG rats gradually increased. Compared with NG, bodyweight of MG, ZG and XG rats decreased notably （P<0.05） at different time.Compared with MG, body weight of rats in ZG and XG decline eased. Andbody weight of ZG and XG rats was significantly higher than that of MG from8W, there was significant difference （P<0.05）. Furthermore, there was nosignificant difference of body weight in ZG and XG at different time （P>0.05）.1.3Comparison of water intake in each group at different timeCompared with NG, water-intake in MG, ZG and XG increased notably（P<0.05） at different time. Compared with MG, water-intake of ZG rats wassignificantly lower than that of MG from12W （P<0.05）, however, there wasno significant difference of water-intake between ZG and XG rats （P>0.05）.1.4Comparison of food intake in each group at different timeCompared with NG, diet-intake in MG, ZG and XG increased notably（P<0.05） at different time. Compared with MG, diet-intake in ZG and XGincreased less, but the effect is slow. Until12W the increase of diet-intake inZG and XG began to improve significantly （P<0.05）. However, there was nosignificant difference of water-intake between ZG and XG rats （P>0.05）.1.5Comparison of urine output in each group at different timeCompared with NG, urine-output in MG, ZG and XG increased notably （P<0.05） at different time. Compared with MG, urine-output in ZG increasedless, but the effect is slow. Until16W the increase of urine-output in ZG beganto improve significantly （P<0.05）. And there was significant difference ofurine-output between ZG and XG rats （P<0.05）.1.6Comparison of HbA1c, KI, BUN, SCR, TP and ALB in each groupCompared with NG, HbA1c and KI in MG, ZG and XG increased notably（P<0.05）. Compared with MG, there was no significant difference of FBG inZG and XG （P>0.05）. Compared with NG, BUN in MG, ZG and XG ratsincreased notably （P<0.05）, and compared with MG, BUN in ZG and XG ratsdecreased notably （P<0.05）, and there was no significant difference of BUNbetween ZG and XG （P>0.05）. Compared with NG, SCr in MG, ZG and XGrats decreased notably （P<0.05）, and compared with MG, SCr in ZG and XGrats increased notably （P<0.05）, and SCr in XG rats increased notablycompared with ZG （P<0.05）.1.7Comparison of serum lipid （including TC, TG, HDL, and VLDL） in eachgroupThere was no significant difference of TC and HDL in all groups（P>0.05）. Compared with NG, TG and VLDL in MG, ZG and XG ratsincreased notably （P<0.05）, and compared with MG, TG and VLDL in ZG andXG rats decreased notably （P<0.05）, and there was no significant difference ofTG and VLDL between ZG and XG （P>0.05）.1.8Comparison of changes of pathomorphology observations in kidney ineach groupObservations under light microscope: in the normal group, there were noobvious hypertrophy of renal glomerulus, thickening of GBM and the changeof the MCs quantity. In model group, there were obvious hypertrophy of renalglomerulus, thickening of GBM, expansion of mesangial matrix andvacuolar/granular degeneration of renal tubular cell. In irbesartan group andZY groups, the pathomorphology changes above were improved comparedwith that of the model group. There was no obvious difference between twotreated groups. Observations under transmission electron microscopy: In the normalgroup, the structure of glomerular capillary basement membrane was clear andcomplete, microcirculatary endothelial cell, foot processes was normal. Inmodel group, there were obvious thickening of GBM, the fusion of the footprocesses was extensive, microcirculatary endothelial cell confluence andwindow structure disappeared. In irbesartan group and ZY groups, thepathomorphology changes above were improved compared with that of themodel group. There was no obvious difference between two treated groups.1.9Comparison of GBM thickness in each groupCompared with NG, GBM in MG, ZG and XG thickened notably（P<0.05）. Compared with MG, the thickness of GBM in ZG and XG reducednotably （P<0.05）, however, which in ZG reduced notably compared with XG（P<0.05）.2Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin andthe expression of p-cadherin in DN rats2.1Comparison of urinary albumin in each group at different timeCompared with NG, U-Alb in MG, ZG and XG increased notably（P<0.05） at different time. Compared with MG, U-Alb in ZG and XGincreased less, but the effect is slow. Until12W to16W the increase of U-Albbegan to improve significantly （P<0.05）. However, there was no significantdifference of U-Alb between ZG and XG rats （P>0.05）.2.2Comparison of expression of p-cadherin mRNA in renal tissue in eachgroupCompared with NG, expression of p-cadherin mRNA in MG, ZG and XGdecreased notably （P<0.05）. Compared with MG, expression of p-cadherinmRNA in ZG and XG induced notably （P<0.05） and There was no obviousdifference between two treated groups （P>0.05）.2.3Comparison of expression of p-cadherin protein in renal tissue in eachgroupIHC staining showed: in NG, brown yellow particles took on a lineardistribution along glomerular basement membrane. Compared with NG, the brown yellow particles in MG significantly reduced. However, compared withMG, the brown yellow particles increased significantly in ZG and XG.3Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein andand factors associated with renal fibrosis in DN rats3.1Comparison of24h urine protein in each group at different timeCompared with NG,24h Upro in MG, ZG and XG increased notably（P<0.05） at different time. Compared with MG,24h Upro in ZG and XGincreased less, but the effect is slow. Until12W to16W the increase of24hUpro began to improve significantly （P<0.05）. However, there was nosignificant difference of24h Upro between ZG and XG rats （P>0.05）.3.2Comparison of the expression of BMP-7protein and mRNA in each groupAfter detection with IHC, Western blot and Real-Time PCR, comparedwith NG, expression of BMP-7protein and mRNA in MG, ZG and XGdecreased notably （P<0.05）. Compared with MG, expression of BMP-7protein and mRNA in ZG and XG increased notably （P<0.05）. There was noobvious difference between two treated groups （P>0.05）.3.3Comparison of the expression of TGF-β1protein and mRNA in each groupAfter detection with IHC, Western-blot and Real-Time PCR, comparedwith NG, expression of TGF-β1protein and mRNA in MG, ZG and XGincreased notably （P<0.05）. Compared with MG, expression of TGF-β1protein and mRNA in ZG and XG decreased notably （P<0.05）. There was noobvious difference between two treated groups （P>0.05）.3.4Comparison of the expression of Smad7protein in each groupAfter detection with Western-blot, compared with NG, expression ofSmad7protein in MG, ZG and XG decreased notably （P<0.05）. Comparedwith MG, expression of Smad7protein in ZG and XG increased notably（P<0.05）. There was no obvious difference between two treated groups（P>0.05）.3.5Comparison of the expression of OPN mRNA in each groupAfter detection with Real-Time PCR, compared with NG, expression ofOPN mRNA in MG, ZG and XG increased notably （P<0.05）. Compared with MG, expression of OPN mRNA in ZG and XG decreased notably （P<0.05）.There was no obvious difference between two treated groups （P>0.05）.3.6Analysis of correlation of BMP-7and TGF-β1in MGAnalysis results showed that relationship between the expression ofBMP-7and TGF-β1in renal tissue was as follows: the correlation coefficientbetween the expression of BMP-7and TGF-β1in nucleic acid and proteinwere-0.9269and-0.9055. That means that there was a negative correlationbetween the expression of BMP-7and TGF-β1（P<0.01）.4Effects of huoxue-huayu-tongluo serum containing medicine and drugmonomer-tanshinone II A on the proliferation of podocyte4.1Effects of huoxue-huayu-tongluo serum containing medicine on theproliferation of podocyteThere was no obvious difference between huoxue-huayu-tongluo serumcontaining medicine group and low glucose control group （P>0.05）.Compared with high glucose control group, the proliferation of podocyteobviously promote in huoxue-huayu-tongluo serum containing medicine group（P<0.05）.4.2Effects of drug monomer-tanshinone II A on the proliferation of podocyteThere was no obvious difference between tanshinone II A group and lowglucose control group after incubation for12hours （P>0.05）. Compared withlow glucose control group, tanshinone II A with1×10-7mol/L and1×10-8mol/L concentration developed an obvious role in promoting proliferationafter incubation for24hours （P<0.05） and tanshinone II A with1×10-6mol/L,1×10-7mol/L and1×10-8mol/L concentration developed an obvious role inpromoting proliferation after incubation for36hours （P<0.05） and tanshinoneII A with all concentration developed an obvious role in promotingproliferation after incubation for48hours （P<0.05）.There was no obvious difference between tanshinone II A group and highglucose control group after incubation for12hours （P>0.05）. Compared withhigh glucose control group, tanshinone II A with1×10-7mol/L and1×10-8mol/L concentration developed an obvious role in promoting proliferation after incubation for24hours （P<0.05） and tanshinone II A with1×10-5mol/L,1×10-6mol/L,1×10-7mol/L and1×10-8mol/L concentration developed anobvious role in promoting proliferation after incubation for36hours （P<0.05）and tanshinone II A with all concentration developed an obvious role inpromoting proliferation after incubation for48hours （P<0.05）.Conclusions:1A single high dose of STZ ip can repulate DN rat model succesfully.2Huoxue-huayu-tongluo Chinese medicines could attenuate polydipsia,polyphagia, polyuria, and body weight reduction notably, as well as metabolicdisorder of lipid. It also could decrease U-Alb and24h Upro extenuate thepathological lesion attenuate glomerular hyrerfiltration and glomerularhypertrophy, thickened GBM, so, it has a renal protective role.3Huoxue-huayu-tongluo Chinese medicines could up-regulate theexpression of p-cadherin protein and mRNA significantly, so as to decreasethe proteinuria, make further efforts to protect the kidney function and delaythe occurrence and development of glomerulosclerosis.4Huoxue-huayu-tongluo Chinese medicines could up-regulate theexpression of BMP-7and Smad7, and could down-regulate the expression ofTGF-β1and OPN significantly, so as to decrease the accumulation ofextracellular matrix, make further efforts to delay the occurrence anddevelopment of glomerulosclerosis.5Huoxue-huayu-tongluo serum containing medicine developed anobvious role in promoting proliferation of podocyte. Drug monomer-tanshinone II A also developed an obvious role in promoting proliferation ofpodocyte, which could enhance the ability to repair itself of podocyte, so as todecrease the proteinuria and make further efforts to protect the kidney function.This result was consistent with the overall experiments. Thus it gave furtherevidence from the cell biological mechanism of huoxue-huayu-tongluo herbsin the treatment of DN. And provide a basis for the application of traditionalChinese medicine monomer.6To postulate the essence of DN according to the results of present study, it is blood stasis syndrome.7Huoxue-huayu-tongluo Chinese medicines have a therapeuticequivalence compared with Western medicine. |
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