| Objective:To observe the effect of Qingshen Granule on E-cadherin in peripheral blood of patients with chronic renal failure(CRF)damp-heat syndrome.Methods:According to the random number table,70 CRF patients with damp-heat syndrome,that meet the inclusion criteria,were divided into two gtoups(the treatment group and the control group),35 cases in each and 30 cases in the normal group(serum E-cadherin test).In the course of the study,one person was eliminated in the treatment group and the control group,respectively(One person plus Bailing Capsule,another person stopped taking medicine during the operation).68 cases were completed(34 cases in treatment group and control group,respectively),finally.Two groups of patients were given Chinese medicine retention enema and basic treatment of Western medicine,further more,Qingshen granule was added to the treatment group,3 times/day,lOg/times,treatment for 12 weeks.Detection of two groups before and after treatment in patients with Senerum creatinine(Scr)level,the expression of E-cadherin,and to estimate the glomerular filtration rate(eGFR),and to evaluate the two groups of patients with clinical disease efficacy and TCM syndrome efficacy.Results:1.The total effective rate of clinical disease efficacy was 79.41%and 64.71%in the treatment group and the control group respectively,and there was a significant difference between the two groups(P<0.05).2.The total effective of TCM syndrome in the treatment group and the control group was 76.47%and 58.82%respectively,and there was a significant difference between the two groups(P<0.05).3.Before treatment,there was no significant difference in TCM syndrome score between two groups(P>0.05).TCM syndrome score of two groups decreased significantly after treatment(P<0.01,P<0.05);TCM syndrome scores in the treatment group were significantly better than that in the control group at the same time(P<0.01).4.Before treatment,there was no significant difference in Scr and eGFR levels between the two group(P>0.05).After treatment,the level of Scr decreased significantly and the eGFR increased significantly in the treatment group(P<0.01),while the control group showed no significant difference(P>0.05);the level of Scr decreased and eGFR increased in the treatment group,which was significantly better than that in the control group at the same time(P<0.05).5.Before treatment,there was no significant difference in serum E-cadherin levels between the treatment group and control group before treatment(P>0.05),but it was significantly higher than that in the normal group(P<0.01).After treatment,the level of serum E-cadherin in two groups were significantly lower than before treatment(P<0.01),and in the treatment group was lower than that in the control group(P<0.05).6.Safety observation indexes:before and after treatment,safety index such as blood routine,urine routine,feces routine,liver function(ALT,AST)and electrocardiogram showed no obvious changes in treatment group.Conclusion:1.The level of serum E-cadherin was increasingly expressed in CRF patients with damp-heat syndrome.2.Qingshen granule can improve the clinical symptoms of CRF patients with damp-heat syndrome and reduce the level of Scr.3.The level of serum E-cadherin in patients with CRF damp-heat syndrome was reduced by qingshen granules.4.The patients had no adverse reaction after taking the QingShen granules,and the safety indexes of the patients were not obviously changes after treatment compared with before treatment,indicating that the drug was safe.Objective:By detecting of serum containing Qingshen granules in low-does,medium-dose and high-dose groups of Human renal tubular epithelial cells(HK-2)induced by transfroming growth factor-?1(TGF-?1)and the expression of Janus kinase 2(JAK2),signal transducers and activators of transcription 3(STAT3),P-JAK2,p-STAT3,E-cadherin,alphasmooth muscle actin(a-SMA),interleukin-1(IL-1),tumor necrosis factor-?(TNF-?)in each dose;to investigate whether the Qing Shen Granule drug-containing serum in each dose group can inhibit the activation of JAK/STAT signaling pathway,thereby reducing HK-2 cell transdifferentiation(epithelial-mesenchymal transition,EMT),and inhibiting the development of renal interstitial fibrosis(RIF).Methods:6 healthy Japanese albino rabbits weighing 2.5 ± 0.2 kg were all purchased from the Animal Center of Anhui Medical University,randomly divided into drug group and blank group,each group of 3.The Qingshen granule aqueous solution was 1.Og/ml and the animals of drug group were given the Qingshen granule aqueous solution by 5ml/kg gavage,1 time/d,and lasted for 10 days.One hour after the last gavage,no anesthesia,we collected blood by heart puncture with a syringe,made serum containing drug and stored at-20 ? for further detections.The blank group were given the equal amount of normal saline and collected blood as above.HK-2 cells were purchased from China Center for Type Culture Collection(Wu Han),and we used the second or third generation cells for the experiment.The same generation of cells were randomly divided into 6 groups,including normal group,TGF-?1 group,JAK inhibitor group,Qingshen Granule high-dose group,middlie-dose group and low-dose group.The change of cell s EMT was observed;MTT assay were used to evaluate cell growth;Western blot was detected in cells of p-JAK2,p-STAT3 the protein expression;RT-qPCR was detected in cells of JAK2,STAT3,E-cadherin and a-SM the mRNA expression;mmunofluorescence was detected in cells of IL-1,TNF-a expression.Results:1.MTT results showed that after 24 hours:compared with normal group,cell viability of TGF-?1 group,JAK inhibitor group,Qingshen Granule for each dose group decreased,and the difference was significant(P<0.01);compared with TGF-?1 group,cell viability of JAK inhibitor group,Qingshen Granule for each dose group increased significantly(P<0.01);compared with JAK inhibitor group,cell viability of Qingshen granule medium-dose,high-dose group decreased significantly(P<0.01);but no obvious difference was found between Qingshen granule low-dose group and JAK inhibitor group(P>0.05);no obvisous difference was found between Qingshen granule medium-dose and hig-dose group(P>0.05).On the basis of MTT results,we chose 24h for the rest part of the study.2.Comparison of expression of p-JAK2,p-STAT3 protein in each group(Western blot method):the levels of p-JAK2 and p-STAT3 protein in other groups compared with normal group increased significantly(P<0.01);compared with TGF-?1 group,the levels of p-JAK2 and p-STAT3 protein in JAK inhibitor group,each dose group of Qingshen granule decreased significantly(P<0.01);compared with JAK inhibitor group,the levels of p-JAK2 and p-STAT3 protein in Qingshen granule medium-dose and high-dose group increased(P<0.01,P<0.05);but no obvious difference was found between low-dose group and JAK inhibitor group(P>0.05);no obvious difference was found between Qingshen medium-dose and high-dose group(P>0.05).3.Comparison of expression of JAK2,STAT3,a-SMA,E-cadherin mRNA in cells from all groups(RT-qPCR method)(1)Comparison of expression of JAK2,STAT3 and a-SMA mRNA:compared with normal group,the levels of JAK2,STAT3 and a-SMA mRNA in other groups increased significantly(P<0.01);compared with TGF-?1 group,the levels of JAK2,STAT3 and a-SMA mRNA in JAK inhibitor group,each dose group of Qingshen granule decreased significantly(P<0.01);compared with JAK inhibitor group,the levels of JAK2,STAT3 and a-SMA mRNA in Qingshen medium-dose and high-dose group increased(P<0.01,P<0.05);but no obvious difference between Qingshen granule low-dose group and JAK inhibitor group(P>0.05);no obvious difference was found between Qingshen medium-dose and high-dose group(P>0.05).(2)Comparison of expression of E-cadherin mRNA:compared with normal group,the level of E-cadherin mRNAin TGF-?1 group,JAK inhibitor group,each dose group of Qingshen granule decreased significantly(P<0.01);compared with TGF-?1 group,the level of E-cadherin mRNA in JAK inhibitor group,each dose group of Qingshen granule increased significantly(P<0.01);compared with JAK inhibitor group,the level of E-cadherin mRNA in Qingshen medium-dose and high-dose group was decreased(P<0.05);but no obvious difference was found between Qingshen granule low-dose group and JAK inhibitor group(P>0.05);no obvious difference was found between Qingshen medium-dose and high-dose group(P>0.05).4.Comparison of expression of IL-1,TNF-? in cells from all groups(mmunofluoresc-ence):IL-1 and TNF-a have little red fluorescence in normal gruop,but they showed bright red fluorescence in TGF-?1 group.After the interference of JAK inhibitor and different concentrations of Qing Shen Granule,the expression of red fluorescence was weaker than that in TGF-p1group.Conclusion:1.TGF-pl could induce EMT in HK-2 cells;2.Serum containing Qingshen granules could inhibit HK-2 cells EMT induced by TGF-?1;3 JAK/STAT signaling pathway could participate in the progress of EMT and RIF;4.Serum containing Qingshen granules could inhibit JAK/STAT signaling pathway,and reduce the expression of JAK2?STAT3?p-JAK2?p-STAT3?a-SMA?IL-1?TNF-a in HK-2 cells induced by TGF-?1,increase the content of E-cadherin,and to delay the progress of EMT and RIF;5.The low-dose group was better than the medium-dose group and the high-dose group in inhibiting JAK/STAT signaling pathway which was induced by TGF-pl,delaying the progress of EMT and RIF. |
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