| Part one Clinical study on therapeutic effects of Warming yangDispelling turbidity-Dredging collaterals formula on patients with early-middle stage chronic renal failureObjective: To comprehensively evaluate the clinical efficacy of Warming yang-Dispelling turbidity-Dredging collaterals formula in the treatment of early-middle stage chronic renal failure(CRF).Methods: Eighty patients with early-middle stage CRF were randomly divided into control group and treatment group,administrated with Niaoduqing particles and Warming yang-Dispelling turbidity-Dredging collaterals formula respectively for 12 weeks on the basis of general treatment.The efficacy indicators(symptom degrees,Scr,BUN,GFR,24 h UTP),as well as security indexes were recorded and tested before and after the treatment.Results:1 Baseline information of the two groupsThere was no significant difference in sex,age,disease course,symptom degrees,renal function,and 24 h UTP between the two groups of patients before treatment(P>0.05).2 Analysis of curative effect after treatmentIn the control group,10 cases were significantly effective,14 cases were effective,7 cases were stable,and 6 cases were ineffective after treatment,with a total effective rate of 83.78%.While,in the treatment group,19 cases were significantly effective,12 cases were effective,4 cases were stable,and 3cases were ineffective after treatment,with a total effective rate of 92.11%.Compared with control group,efficiency rate in treatment group were significantly higher(P<0.05).3 Comparison of symptom scores before and after treatmentCompared before treatment,symptom scores of the two groups both significantly decreased(P<0.05).Compared with control group,symptom scores in treatment group were significantly lower after treatment(P<0.05).4 Comparison of Scr,BUN and GFR before and after treatmentCompared before treatment,Scr and BUN of the two groups both significantly decreased(P<0.05),GFR increased meanwhile.Compared with control group,Scr and BUN in treatment group were significantly lower after treatment(P<0.05),while GFR higher(P<0.05).5 Comparison of 24 h UTP before and after treatmentCompared before treatment,24 h UTP of the treatment group significantly decreased(P<0.05),while that of control group had no significant change(P>0.05).Compared with control group,24 h UTP in treatment group were significantly lower after treatment(P<0.05).6 Safety indicatorsThere were no significant changes in body temperature,pulse,respiration,blood pressure,blood routine,stool routine,ALT,AST,electrolyte and electrocardiogram before and after treatment in the two groups,and no adverse events occurred during treatment.Part two Effects of Warming yang-Dispelling turbidity-Dredging collaterals formula on biochemical indexes and renal pathological changes in rats with CRFObjective: To observe the effects of the Formula on serum biochemical indexes and pathological changes in CRF rats,and to verify the renal protective effects of the Formula.Methods: Eight of the 40 SD rats were randomly selected into Sham group,which accepted open surgery without renal parenchymal injury.While,the remaining 32 rats underwent 5/6 nephrectomy.After successful modeling,they were divided into model group(5/6NX),Formula group(WXT)and losartan group(LST),administrated with corresponding drugs.After theintervention of 12 weeks,the urine were collected to test 24 h UTP,and the blood samples for Scr,BUN,UA,TP,ALB,blood lipid,and electrolyte.Meanwhile,the renal tissues were removed for HE and Masson staining to observe the pathological changes and analyze fibrosis degree.Results:1 general condition of the ratsDuring the experiment,2 rats died in the 5/6NX group,2 in the LST group,and no rats died in the WXT group or Sham group.Rats in Sham group were in good mental state,with normal diet,quick reaction,soft body hair and bright color.While,rats in 5/6NX group showed poor mental performance,decreased activity and food intake,and rough and dull body hair.The rats in the WXT group showed gradual improvement in spirit,activity,food intake and body hair luster.However,the LST group showed no significant improvement.The weight of rats in 5/6NX group was significantly reduced compared with the Sham group(P<0.05).Compared with the 5/6NX group,the weight of the WXT group was significantly increased(P<0.05),and the weight of the LST group had no significantly change(P>0.05).Besides,the weight of WXT group was significantly higher than LST group(P>0.05).2 Comparison of renal function related indicatorsScr and BUN were significantly increased in 5/6NX group compared with the Sham group(P<0.05).Compared with 5/6NX group,the Scr and BUN levels in the WXT group significantly decreased(P<0.05),while only BUN decreased in the LST group(P>0.05).Notably,the Scr and BUN of WXT group were significantly lower than those in LST group(P<0.05).3 Comparison of 24 h UTP in rats of each group24h UTP significantly increased in 5/6NX group compared with the Sham group(P<0.05).Compared with 5/6NX group,24 h UTP in WXT and LST group both significantly decreased(P<0.05).While,24 h UTP of WXT group was much lower than that in LST group(P<0.05).4 Comparison of TP and ALB in rats of each groupTP and ALB of 5/6NX group were significantly decreased compared with the Sham group(P<0.05).Compared with the 5/6NX group,TP and ALB significantly increased in the WXT group(P<0.05),but the LST group had no significant change(P>0.05).Interestingly,TP and ALB of WXT group were significantly higher than LST group(P<0.05).5 Comparison of blood lipid in rats of each groupTC,TG,LDL of 5/6NX group were significantly increased compared with those in Sham group(P<0.05).Compared with 5/6NX group,TC,TG and LDL of the WXT group all decreased significantly(P<0.05),while the LST group showed no significant change(P>0.05).Naturally,TC,TG and LDL of WXT group were significantly lower than LST group(P<0.05).There was no significant difference in HDL among the groups(P>0.05).6 Comparison of electrolytes in rats of each groupSerum P and Ca were respectively increased and decreased in 5/6NX group(P<0.05).Compared with the 5/6NX group,serum P and Ca in the WXT group were significantly decreased and increased(P<0.05),while the LST group showed no significant change(P>0.05).Compared with LST group,serum P in WXT group were significantly decreased(P<0.05)and and serum Ca significantly increased(P<0.05).There was no significant difference in the contents of Na,Cl and K in serum among the groups(P>0.05).7 Renal pathological and morphological changesThe renal structure of Sham group was normal.5/6NX group showed mild to moderate proliferation of mesangial cells and matrix,segmentary sclerosis and partial glomerular ischemic shrinkage,tubular vacuolar granule degeneration,focal atrophy,focal interstitial fibrosis with lymphocyte infiltration,arteriole wall intensification and lumen stenosis.Compared with5/6NX group,the kidney injury of the WXT group and the LST group were reduced to some extent,particularly the WXT group.8 Comparison of renal fibrosis degreesThe degree of renal fibrosis was significantly increased in 5/6NX group(P<0.05).Compared with the 5/6NX group,the fibrosis degree of the WXTgroup and the LST group was significantly reduced(P<0.05).The degree of fibrosis in WXT group was obviously lower than LST group(P<0.05).Part three Effects of Warming yang-Dispelling turbidity-Dredging collaterals formula on renal fibrosis related factors in rats with CRFObjective: To observe the effects of the Formula on fibrosis related factors TGF-?1,Col ?,Col ? and FN,and to further explore the intervention effects of the Formula on renal fibrosis.Methods: Rat modeling,grouping and intervention methods were as the same as before.After the intervention of 12 weeks,the blood were collected to test TGF-?1 contents,and the kidneys were removed to detect Col ? protein expression by IF,Col ? and FN protein expression by IHC,and Col ? and FN mRNA expression by qPCR.Results:1 Comparison of TGF-?1 contents in serumSerum TGF-?1 level in 5/6NX group was significantly increased(P<0.05).Compared with 5/6NX group,the TGF-?1 content in the WXT group and the LST group was significantly decreased(P<0.05)and there was no significant difference between the two groups(P>0.05).2 Comparison of Col ?,Col ? and FN protein expression in kidneysCol ?,Col ? and FN protein expression in 5/6NX group significantly increased.Compared with those of 5/6 NX group,Col ?,Col ? and FN protein expression in WXT group and LST group were reduced.Compared with LST group,Col ?,Col ? and FN protein expression in 5/6NX group decreased obviously.3 Comparison of Col ? and FN mRNA expression in kidneysCol ? and FN mRNA expression increased significantly in 5/6NX group(P<0.05).Compared with 5/6 NX group,Col ? and FN mRNA expression were significantly lower in WXT group(P<0.05),while no significant change in LST group(P>0.05)..Part four Study on the mechanisms of Warming yang-Dispelling turbidity-Dredging collaterals formula based on renal tubular EMT and Wnt/?-catenin pathwayObjective: To observe the effects of the Formula on renal tubular epithelial EMT and Wnt/?-catenin pathway,and to explore the cellular and molecular mechanisms of the Formula in improving renal function and fibrosis.Methods: Rat modeling,grouping and intervention methods were as the same as before.After the intervention of 12 weeks,the protein expression of EMT related proteins(?-SMA,Vimentin,E-cadherin)were detected by IHC,meanwhile,the the protein expression of EMT related proteins(?-SMA,Vimentin,E-cadherin)and Wnt/?-catenin pathway related proteins(Wnt4,?-catenin,TCF-4)were tested by western-blot.Besides,the mRNA expression of EMT(?-SMA,E-cadherin)related proteins and Wnt/?-catenin pathway related proteins(Wnt4,?-catenin,TCF-4)were tested by qPCR.Results:1 Protein expression of EMT related proteins ?-SMA,Vimentin and E-cadherin tested by IHCProtein expression of ?-SMA and Vimentin in 5/6NX group significantly increased,meanwhile the protein expression of E-cadherin significantly decreased.Compared with the 5/6NX group,the protein expression of ?-SMA and Vimentin were decreased in the WXT group and the LST group,and the protein expression of E-cadherin was increased.2 Protein expression of EMT related proteins ?-SMA,Vimentin and E-cadherin tested by western-blotProtein expression of ?-SMA and Vimentin in 5/6NX group significantly increased(P<0.05),at the same time,E-cadherin significantly decreased(P<0.05).Compared with 5/6NX group,protein expression of ?-SMA and Vimentin significantly decreased both in the WXT group and LST group(P<0.05),meanwhile the protein expression of E-cadherin significantly increased(P<0.05).Besides,the protein expression of ?-SMA,Vimentinand E-cadherin between WXT group and LST group had no significant difference(P<0.05).3 Expression of EMT related proteins ?-SMA,Vimentin and E-cadherin mRNA tested by qPCRExpression of ?-SMA mRNA in 5/6NX group increased(P<0.05),in the meantime,the expression of E-cadherin mRNA decreased(P<0.05).Compared with 5/6NX group,the expression of ?-SMA mRNA significantly decreased(P<0.05),and E-cadherin mRNA increased(P<0.05)in both the WXT group and LST group.And,there was no significant difference between the two groups(P>0.05).4 Protein expression of Wnt/?-catenin pathway related proteins Wnt4,?-catenin and TCF-4 tested by western-blotProtein expressions of Wnt4,?-catenin and TCF-4 in 5/6NX group significantly increased(P<0.05).Compared with the 5/6NX group,the protein expressions of Wnt4,?-catenin and TCF-4 in the WXT group and LST group were both decreased(P<0.05).Notably,expressions of Wnt4,?-catenin andTCF-4 in WXT group were lower in WXT group(P<0.05).5 Expression of Wnt/?-catenin pathway related proteins Wnt4,?-catenin and TCF-4 mRNA by qPCRThe mRNA expression of Wnt4 and ?-catenin increased in 5/6NX group(P<0.05),while there was no significant difference among 5/6NX,WXT and LST group(P>0.05).There was no significant difference in mRNA expression of Wnt4 among the four groups(P>0.05).Conclusions:1 The Warming yang-Dispelling turbidity-Dredging collaterals formula can decrease symptom scores,improve renal function,decrease urinary protein,and significantly rise curative effect of disease in patients with early-middle stage chronic renal failure,which confirmed that the Warming yang-Dispelling turbidity-Dredging collaterals formula has good therapeutic effect in treating early-middle stage CRF.2 The Warming yang-Dispelling turbidity-Dredging collaterals formula can improve the renal function of CRF rats,reduce urine protein,improve renal pathological injury and renal fibrosis,and has the effect of regulating malnutrition,blood lipid and electrolyte disorders,and the effect is significantly better than losartan,confirming that the Formula has the exact protective effects on kidney,and has obvious advantages in the overall regulation.3 The Warming yang-Dispelling turbidity-Dredging collaterals formula has the effect of reducing expression of fibrosis related proteins TGF-?1,Col ?,Col ? and FN,so as to improve renal fibrosis.4 The Warming yang-Dispelling turbidity-Dredging collaterals formula can inhibit EMT at the level of protein and gene transcription,which may be one of the cellular mechanisms to improve renal fibrosis and protect renal function.5 The Warming yang-Dispelling turbidity-Dredging collaterals formula can inhibit the abnormal activation of Wnt/?-catenin pathway,which may be one of the molecular mechanisms to improve renal fibrosis and protect renal function. |
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