| Objective: To observe the serum levels of hypoxia-inducible factor-1?(HIF-1?),Wnt1,?-catenin,?-smooth muscle actin(?-SMA)and E-cadherin changes in patients with chronic renal failure(CRF)damp heat syndrome before and after taking Qingshen Granules;To investigate whether Qingshen Granule can mediate activation of Wnt/?-catenin pathway by interfering with HIF-1? in chronic kidney disease patients.To improve their renal function and reduce clinical symptoms.Methods: According to the inclusion criteria and random number table,70 cases of CRF patients with damp heat syndrome were divided into two groups(treatment group and control group)each with 35 cases,and 30 cases with normal group(for the detection of serum HIF-1?,Wnt1,?-catenin?-SMA,E-cadherin).The study completed68 cases(treatment group and control group were 34 cases).In the study period,one case was added Bailing Capsule and one case was stopped during the operation.Therefore,two subjects were excluded.Retention enema of traditional Chinese medicine and basic treatment of western medicine were used in two groups of subjects.Subjects in the treatment group were treated with Qingshen Granules(3 times/day,10g/times for 12 weeks).Observe serum creatinine(Scr),HIF-1?,Wnt1,?-catenin,?-SMA,E-cadherin,TCM syndrome integral values before and after treatment in two groups of patients,estimate glomerular filtration rate(e GFR),and evaluate the efficacy of clinical disease and TCM syndromes in both groups of patients.Results:1.Clinical curative effect: The total effective rates of the treatment group and the control group were 79.41% and 64.71% respectively,and the difference between the two groups was statistically significant(P<0.05).2.Efficacy of TCM syndromes: The total effective rates of the treatment group and the control group were 76.47% and 58.82% respectively,and the difference between the two groups was statistically significant(P<0.05).3.There was no significant difference in the number of TCM syndrome scores between the treatment group and the control group before treatment(P>0.05);after 12 weeks of treatment,the TCM syndrome scores of the two groups decreased to different degrees(P<0.01,P<0.05).The scores of TCM syndromes in the treatment group were significantly better than those in the control group at the same time(P<0.01).4.Before treatment,there was no significant difference in Scr between the two groups(P>0.05);after 12 weeks of treatment,Scr in the treatment group was significantly lower than before treatment(P<0.01),and it was significantly lower than the control group at the same time(P<0.01).There was no significant difference in Scr after 12 weeks in the control group(P>0.05).5.Before treatment,there was no significant difference in e GFR between the two groups(P>0.05);after 12 weeks of treatment,e GFR in the treatment group was significantly lower than before treatment(P<0.01),and it was significantly lower than the control group at the same time(P<0.01).There was no significant difference in e GFR after 12 weeks in the control group(P>0.05).6.The serum levels of HIF-1?,Wnt1,?-catenin,?-SMA,and E-cadherin in the treatment group and the control group were significantly higher than those in the normal group(P<0.01),and there was no significant difference between the treatment group and the control group.(P>0.05).The serum levels of HIF-1?,Wnt1,?-catenin,?-SMA,and E-cadherin in the treatment group decreased significantly after treatment(P<0.01),and was significantly lower than that of the control group at the same time(P<0.01).The serum levels of HIF-1?,Wnt1,?-catenin,?-SMA,and E-cadherin in the control group were not significantly different from those before treatment(P>0.05).7.There was a positive correlation between serum HIF-1? levels and Wnt1 and?-catenin studies in patients with damp-heat syndrome(P<0.01).8.Analysis of safety observations: There were no significant changes in the blood routine,urine routine,fecal examination,liver function(mainly ALT,AST)and electrocardiogram(ECG)and other safety indicators of patients in the treatment group after 12 weeks of treatment.Conclusion: 1.Serum HIF-1?,Wnt1,?-catenin,?-SMA and E-cadherin levels were elevated in CRF patients with damp heat syndrome,and the correlation analysis indicated that HIF-1? was positively correlated with Wnt1 and ?-catenin.Confirmed that HIF-1? mediates the activation of Wnt/?-catenin signaling pathway and promotes the progression of chronic renal failure.2.Qingshen Granule can reduce the clinical symptoms of CRF patients with damp heat syndrome,and reduce the serum creatinine level;3.Qingshen Granules can reduce the levels of HIF-1?,Wnt1,?-catenin,?-SMA and E-cadherin in patients with chronic renal failure damp heat syndrome,indicating that Qingshen Granules can intervene in the activation of Wnt/?-catenin signaling pathway mediated by HIF-1?;4.There was no adverse drug reaction in the treatment group after taking Qingshen Granules,and there was no significant change in the safety index after treatment compared with before treatment,indicating that the drug was safety.Objective: By detecting the Qingshen granules in high,medium and low dose groups of the renal interstitial fibrosis in rats 5/6 nephrectomy kidney tissue HIF-1?,Wnt1,?-catenin,p-?-catenin,axin1,axin2,?-SMA and E-cadherin protein expression;To explore whether the Qingshen granules each groups can inhibit HIF-1? mediated Wnt/?-catenin pathway activity to reduce the process of renal tubular epithelial cells transdifferentiation(EMT),and then play the effect of renal interstitial fibrosis.Methods: Selected 70 male SD rats,2 months,clean grade,weighting 200 ± 20 g,all bought from Shanghai jest experimental animal co.LTD,closed management and adaptive feeding for 1 week.After 1 week use the simple random sampling method,randomly divided into 7 groups,including the normal group,sham operation group,model group,Qingshen granules of high,middle,low dose group,XAV939 ?-catenin specific inhibitors)with 10 rats in each.in addition to 10 rats in the normal group,50 rats in the model group were prepared by standard experimental method to prepare 5/6 nephrectomy model.10 rats in the sham operation group did not remove the kidney after entering the abdominal cavity.Animals in each group began to give medicine on the first day after the model was made,with 2ml every day.Qingshen Granule high-dose group,middle-dose group,low-dose group were given Qingshen Aqueous Solution by 4.0g/kg ·d,8.0g/kg·d and 16.0g/kg·d gavage.XAV939 group dissolved XAV939 in DMSO to storage,concentration of 10 mmol/L and keep it in the-20 ? temperature,experiment according to 0.6 ml / 100 g,2 days intraperitoneal injection it.The normal group,the sham operation group and the model group were given the same amount of warm stomach,one time a day,2ml each time.Each group was given 12 weeks.Detect serum Creatinine(Scr),Blood urea nitrogen(BUN)and 24-hoururine protein(24 h Upro)and urine creatinine(Ucr),and use the formula to compute it's Creatinine Clearance(Ccr).Western blot was used to detect the expression of HIF-1??Wnt1??-catenin?p-?-catenin?axin1?axin2?E-cadherin and ?-SMA protein in kidney tissues.Immunohistochemical and immunofluorescence methods were detected in kidney tissue HIF-1? ? Wnt1 ??-catenin?p-?-catenin?E-cadherin and ?-SMA expression;HE staining?masson staining and PAS staining to observe the renal granular of high,medium and low dose group and XAV939 effects on kidney histopathology.Results:1.Comparison of the rats serum Scr,BUN and 24 h Upro in each group:There was no significant difference in serum Scr,BUN,24 h Upro levels between the sham operation group and the normal group(P > 0.05);serum Scr,BUN,and 24 h Upro levels in the model group were significantly higher than those in the normal group and the sham operation group(P<0.01);The levels of serum Scr,BUN,and 24 h Upro in the Qingshen Granule each dose groups and XAV939 group were significantly lower than those in the model group(P<0.01);Compared with Qingshen Granule each dose groups,the levels of Scr,BUN and 24 h Upro in the high-dose group were lowest,and were significantly lower than XAV939 group(P<0.01).2.Comparison of Ccr in each group: There was no significant difference in the level of Ccr between the sham operation group and the normal group(P > 0.05);the Ccr level in the model group was significantly lower than that in the normal group and the sham operation group(P<0.01);The level of Ccr in the Qingshen Granule each dose groups and XAV939 group were significantly higher than those in the model group(P<0.01);Compared with Qingshen Granule each dose groups,the level of Ccr in the high-dose group were highest,and was significantly higher than XAV939 group(P<0.01).3.The expression of HIF-1??Wnt1??-catenin?p-?-catenin?axin1?axin2?E-cadherin and ?-SMA in each group(Western blot method):?Comparison of HIF-1? protein expression: There was no significant difference in the expression of HIF-1? between the sham group and the normal group(P >0.05).The expression of HIF-1? protein in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the HIF-1? protein in the high,middle,and low-dose groups of Qingshen Granule decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the HIF-1? protein expression was the lowest in the high-dose group.There was no significant difference in the expression of HIF-1? protein between the XAV939 group and the model group(P>0.05).The HIF-1? protein expression in the high-dose Qingshen Granule group was significantly lower than that in the XAV939 group(P<0.01).?Comparison of Wnt1 protein expression: There was no significant difference in the expression of Wnt1 between the sham group and the normal group(P > 0.05).The expression of Wnt1 protein in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the Wnt1 protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the Wnt1 protein expression was the lowest in the high-dose group.There was no significant difference in the expression of Wnt1 protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of ?-catenin protein expression: There was no significant difference in the expression of ?-catenin between the sham group and the normal group(P >0.05).The expression of ?-catenin protein in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the ?-catenin protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the ?-catenin protein expression was the lowest in the high-dose group.There was no significant difference in the expression of ?-catenin protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of p-?-catenin protein expression: There was no significant difference in the expression of p-?-catenin between the sham group and the normal group(P >0.05).The expression of p-?-catenin protein in the model group was significantly lower than that in the normal group and the sham group(P<0.01).Compared with the model group,the p-?-catenin protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group increased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the p-?-catenin protein expression was the highest in the high-dose group.There was no significant difference in the expression of p-?-catenin protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of axin1?axin2 protein expression: There was no significant difference in the expression of axin1?axin2 between the sham group and the normal group(P >0.05).The expression of axin1?axin2 protein in the model group was significantly lower than that in the normal group and the sham group(P<0.01).Compared with the model group,the axin1 ? axin2 protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group increased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the axin1?axin2protein expression was the highest in the high-dose group.There was no significant difference in the expression of axin1?axin2 protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of E-cadherin protein expression: There was no significant difference in the expression of E-cadherin between the sham group and the normal group(P >0.05).The expression of E-cadherin protein in the model group was significantly lower than that in the normal group and the sham group(P<0.01).Compared with the model group,the E-cadherin protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group increased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the E-cadherin protein expression was the highest in the high-dose group.The E-cadherin protein expression in the high-dose Qingshen Granule group was significantly lower than that in the XAV939 group(P<0.01).?Comparison of ?-SMA protein expression: There was no significant difference in the expression of ?-SMA between the sham group and the normal group(P > 0.05).The expression of ?-SMA in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the ?-SMA protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the ?-SMA protein expression was the lowest in the high-dose group.The ?-SMA protein expression in the high-dose Qingshen Granule group was significantly lower than that in the XAV939 group(P<0.01).3.3.The expression of HIF-1??Wnt1??-catenin?p-?-catenin?E-cadherin and ?-SMA in each group(Immunohistochemical method):?Comparison of HIF-1? protein expression: There was no significant difference in the expression of HIF-1? between the sham group and the normal group(P >0.05).The expression of HIF-1? protein in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the HIF-1? protein in the high,middle,and low-dose groups of Qingshen Granule decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the HIF-1? protein expression was the lowest in the high-dose group.There was no significant difference in the expression of HIF-1? protein between the XAV939 group and the model group(P>0.05).The HIF-1? protein expression in the high-dose Qingshen Granule group was significantly lower than that in the XAV939 group(P<0.01).?Comparison of Wnt1 protein expression: There was no significant difference in the expression of Wnt1 between the sham group and the normal group(P > 0.05).The expression of Wnt1 protein in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the Wnt1 protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the Wnt1 protein expression was the lowest in the high-dose group.There was no significant difference in the expression of Wnt1 protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of ?-catenin protein expression: There was no significant difference in the expression of ?-catenin between the sham group and the normal group(P >0.05).The expression of ?-catenin protein in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the ?-catenin protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the ?-catenin protein expression was the lowest in the high-dose group.There was no significant difference in the expression of ?-catenin protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of p-?-catenin protein expression: There was no significant difference in the expression of p-?-catenin between the sham group and the normal group(P >0.05).The expression of p-?-catenin protein in the model group was significantly lower than that in the normal group and the sham group(P<0.01).Compared with the model group,the p-?-catenin protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group increased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the p-?-catenin protein expression was the highest in the high-dose group.There was no significant difference in the expression of p-?-catenin protein between the XAV939 group and the Qingshen Granule high-dose group(P>0.05).?Comparison of E-cadherin protein expression: There was no significant difference in the expression of E-cadherin between the sham group and the normal group(P >0.05).The expression of E-cadherin protein in the model group was significantly lower than that in the normal group and the sham group(P<0.01).Compared with the model group,the E-cadherin protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group increased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the E-cadherin protein expression was the highest in the high-dose group.The E-cadherin protein expression in the high-dose Qingshen Granule group was significantly lower than that in the XAV939 group(P<0.01).?Comparison of ?-SMA protein expression: There was no significant difference in the expression of ?-SMA between the sham group and the normal group(P >0.05).The expression of ?-SMA in the model group was significantly higher than that in the normal group and the sham group(P<0.01).Compared with the model group,the ?-SMA protein in the high,middle,low-dose groups of Qingshen Granule and XAV939 group decreased significantly(P<0.01).Compared with the high,medium,and low-dose groups of Qingshen Granules,the ?-SMA protein expression was the lowest in the high-dose group.The ?-SMA protein expression in the high-dose Qingshen Granule group was significantly lower than that in the XAV939 group(P<0.01).4.The expression of HIF-1??Wnt1??-catenin?p-?-catenin?axin1?axin2?E-cadherin and ?-SMA in each group(immunofluorescence method):Immunofluorescence results of HIF-1?: HIF-1? was rarely expressed in the normal group and the sham operation group,with only a trace of red fluorescence;Bright red fluorescence was seen in the model group and XAV939 group.Qing Shen granules in each dose groups showed that the expression of HIF-1? was lower than that of the model group,and the red fluorescence was weakened.Immunofluorescence results of Wnt1: Wnt1 was rarely expressed in the normal group and the sham operation group,with only a trace of red fluorescence;Bright red fluorescence was seen in the model group.XAV939 group and Qing Shen granules in each dose groups showed that the expression of Wnt1 was lower than that of the model group,and the red fluorescence was weakened.Immunofluorescence results of ?-catenin: ?-catenin was rarely expressed in the normal group and the sham operation group,with only a trace of red fluorescence;Bright red fluorescence was seen in the model group.XAV939 group and Qing Shen granules in each dose groups showed that the expression of Wnt1 was lower than that of the model group,and the red fluorescence was weakened.Immunofluorescence results of p-?-catenin: The bright red fluorescence was seen in the normal group and the sham operation group.The model group was expressed rarely,with only faint red fluorescence.XAV939 group and Qing Shen granules in each dose groups showed that the expression of Wnt1 was lower than that of the model group,and the red fluorescence was stronger.Immunofluorescence results of ?-SMA: ?-SMA was rarely expressed in the normal group and the sham operation group,with only a trace of red fluorescence;Bright red fluorescence was seen in the model group.XAV939 group and Qing Shen granules in each dose groups showed that the expression of ?-SMA was lower than that of the model group,and the red fluorescence was weakened.Immunofluorescence results of E-cadherin: The bright red fluorescence was seen in the normal group and the sham operation group.The model group was expressed rarely,with only faint red fluorescence.XAV939 group and Qing Shen granules in each dose groups showed that the expression of E-cadherin was lower than that of the model group,and the red fluorescence was stronger.5.There was a positive correlation between the expression of HIF-1? protein and the expression of Wnt1 and ?-catenin protein in kidney tissue of model rats(P<0.01).6.Kidney pathological changes in each group: There was no obvious abnormality in the pathological structure of the kidney in normal and sham-operated rats.The renal pathology of the model group showed a significant expansion of the tubular lumen,loss of renal tubular epithelial cells,vacuolar degeneration,renal interstitial infiltration of a large number of inflammatory cells and blue-stained collagen fibers deposition,renal interstitial injury index was significantly increased.Compared with the model group,the pathological changes of Qingshen Granules and XAV939 rats had clearly improved.Conclusion:1.Activation of Wnt/?-catenin signaling pathway by HIF-1? is involved in the formation of renal interstitial fibrosis in a 5/6 nephrectomized rat model.2.Qingshen granule can reduce blood Scr,BUN,24 h UPro and increase Ccr in 5/6nephrectomized rats.3.Qingshen Granules can significantly reduce the degree of pathological changes in the kidney tissue of 5/6 nephrectomized rats,and thus play a role in delaying renal interstitial fibrosis;4.Qingshen Granule can reduce the expression of HIF-1?,Wnt1 and ?-catenin in renal tissue of 5/6 nephrectomized rats by inhibiting HIF-1?-mediated activation of Wnt/?-catenin signaling pathway and increasing p-?-Catenin,axin1,axin2 protein expression.And by down-regulating the expression of ?-SMA and up-regulating the expression of E-cadherin,to inhibit the renal tubular EMT in 5/6 nephrectomized rats and delay the progression of renal interstitial fibrosis. |
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