| Objective: To observe the intervention effect of Qingre HuaShi Quyu method on chronic renal failure(CRF),and observation of Qingshen Granule on leptin and monocyte chemoattractant protein-1(MCP-1),Effects of intercellular adhesion molecule-1(ICAM-1)expression.Methods: According to the random number table,70 CRF patients were divided into treatment group and control group,two groups each 35 cases,and normal group 30 cases(for detecting serum Leptin,MCP-1 and ICAM-1).The treatment group and control group were removed in 1 case(Among them,1 case were treated with Bailing Capsule and 1 case were stopped for the operation.),and 68 cases were completed,namely treatment Group 34 cases,control group 34 cases.Both the treatment group and the control group were given retention enemawith traditional Chinese medicine and the basic treatment of Western medicine.Thetreatment group added Qingshen granule with warm water,3 times a day,10 g eachtime for 12 weeks.To compare the clinical disease efficacy and TCM syndrome efficacy in both two groups after treatment.To observe changes of TCM syndromes Integration Value,serum creatinine(Scr),estimate glomerular filtration rate(eGFR),Leptin,Changes of MCP-1 and ICAM-1 in both two groups before and after treatment.Results: 1.Two groups of patients after treatment of clinical disease efficacy comparison: The two groups of patients with total efficiency compared to the difference has a statistically significant(p<0.05),indicating that the treatment group efficacy is superior to the control group.2.Two groups of patients after the treatment of traditional Chinese medicine syndrome comparison: The two groups of patients with total efficiency compared to the difference is statistically significant(p<0.05),indicating that the treatment group of Traditional Chinese medicine syndrome is better than the control group.3.Two groups of patients before and after treatment of Traditional Chinese medicine Syndrome integration Value comparison: There was no significant difference in TCM syndromes in the first two groups(p>0.05),and the value of TCM syndromes in the treatment group was significantly lower than before treatment(p<0.01).The value of TCM syndromes in the control group was statistically different than before treatment(p<0.05)After treatment.The TCM syndromes in the treatment group were statistically different than those in the corresponding control group(p<0.05).4.In two groups of patients before and after the treatment of Scr,eGFR comparison: The treatment of the first two groups of patients with Scr and eGFR level compared with no statistical significance(p>0.05),after treatment of the treatment group Scr,eGFR level compared to the difference before treatment significantly statistically significant(p<0.01),after treatment control group Scr There was no statistically significant difference between the eGFR levels(p>0.05)and the level of Scr and eGFR in the treatment group(p<0.05)compared with the control group.5.Before and after treatment of patients with serum leptin,MCP-1,ICAM-1 levels: before treatment,compared with the normal group,the treatment group,control group of patients with serum Leptin,MCP-1,ICAM-1 levels were significantly elevated(p<0.01);Before treatment,the difference of Leptin,MCP-1 and ICAM-1 in treatment group and control group was not statistically significant(p>0.05),and after treatment,the average of serum Leptin,MCP-1 and ICAM-1 in treatment group and control group was significantly lower than before treatment(p<0.05,p<0.01),and after treatment,patients in treatment group has lower level of Leptin,MCP-1 and ICAM-1 than that in the same control group,and the difference was statistically significant(p<0.05).6.Safety Observation Project analysis: Before and after treatment,the Safety index such as blood routine,urine routine,feces routine,liver function(ALT,AST),electrocardiogram did not chang obviously in test group,so this drug take safety.Conclusion:1.The levels of serum leptin were significantly increased in CRF patients with damp heat syndrome,the expression of MCP-1 and ICAM-1 was increased.2.Qingshen granule could improve the clinical symptoms of CRF patients with damp heat syndrome,and improve the renal function.3.Qingshen granule could reduce the levels of serum leptin,the expression of MCP-1 and ICAM-1 of CRF patients with damp heat syndrome.4.Patients taking Qingshen granule had no adverse change,safety indexs was measured without obviously abnormal,indicating thatthe drug had a good safety.Objective: To detect the leptin(cells)in human renal tubular epithelial cells(HK-2)of leptin induced by serum low,medium and high concentrations of three doses of drug-containing drugs.The content of leptin receptor(OB-R)and the Transforming Growth factor-?1(TGF-?1),hepatocyte growth factor(HGF),Protein expression of monocyte chemoattractant 1(MCP-1),intercellular adhesion molecule-1(ICAM-1);Objective to explore the relationship between HGF and TGF-?1 and to reduce HK-2 cell epithelial-mesenchymal transition(EMT)by regulating the expression of HGF and TGF-?1 in leptin-induced HK-2 cells in the serum of each dosage Group of Qingshen granule,plays a role in inhibiting renal interstitial fibrosis(RIF).Methods: Selected 6 male Japanese albino rabbits,body weight 2.5±0.2kg/each,all purchases of the Animal Center of Anhui Medical University.The rabbits randomly divided into drug group and blank group with 3 rabbits in each.The concentration of clear renal granular aqueous solution was 1.0g/ml and animals in drug group were given clear renal granular aqueous solution by 5ml/kg gavage,1 times a day,10 consecutive days.Last administration,we collected blood by heart puncture with a syringe,stewing 2 hours,then 3000 rpm centrifuged 10 min,got the supernate,filtrated by 0.22?m micropore filter,inactivated by 56?of water bath treatment for 30 min and stored at-20? for further detections.The blank group were given normal saline and collected blood as above.HK-2 cells were purchased from China Center for Type Culture Collection(Wu Han),we used the second and third generation cells.The same generation cells were randomly divided to 7 groups,including control group,mannitol group,high glucose group,Qingshen granule low dose group,Qingshen granule medium dose group,Qingshen granule high dose group,pyrrolidine dithio formic acid(pyrrolidine dithiocarbamate and PDTC)group.Cellular morphology was observed by inverted phase contrast microscope;MTT assay were used to evaluate cell growth;Western blot was detected in cells of Leptin ?OB-R ?HGF?TGF-?1the protein expression;RT-PCR was detected in cells of Leptin?OB-R?HGF?TGF-?1the m RNA expression;To detect the fluorescence expression of HGF,TGF-?1,MCP-1 and ICAM-1 in HK-2 cells by immunofluorescence assay;ELSIA was detected in cells of Leptin?MCP-1?ICAM-1.Results: 1.The MTT assay showed that the drug function was 24 hours later: Compared with the blank control group,the OD value of the group of leptin and kidney granules was lower than that in the blank control group,all of them were statistically significant(p<0.01);compared with the leptin group,the OD value of the group was higher than that of the leptin group,and all of them were statistically significant(p<0.01)The OD value of the low dose group of Qingshen granule was higher than that in the Clear Kidney granule and high dosage group,and there were statistically significant(p<0.01),and there was no difference between the dosage group and the high dose group in the Kidney granules(p>0.05).The drug action time was 24 hours with the result of MTT assay.2.Comparison of expression of Leptin m RNA,OB-R m RNA,HGF m RNA and TGF-?1 m RNA in each group of cells(RT-PCR method)(1)Comparison of Leptin m RNA expression: Compared with the blank control group,the expression of Leptin protein in leptin Group and Qingshen granule groups was higher than that in blank control group,all had statistically significant(p<0.01);compared with leptin group,the expression of Leptin protein in each Qingshen granule dose group was lower;The expression of leptin protein in low dose group of Qingshen granule was the lost(p<0.01),and there was no difference between medium dose group and high dose Group(p>0.05).(2)Comparison of OB-R m RNA expression: Compared with the blank control group,the OB-R m RNA expression of Leptin Group and Qingshen granule group was higher than that in the blank control group,all of them had statistically significant(p<0.01);compared with the Leptin group,OB-R m RNA expression in each Qingshen granule group was lower,and all were statistically significant(p<0.01).The OB-R m RNA expression in low dose group of Qingshen granule was lower than that in the group of medium dose and high dose,and all of them were statistically significant(p<0.01),there was no difference between the Medium Dose Group and the High Dose Group(p>0.05).(3)Comparison of HGF m RNA expression: Compared with the blank control group,the HGF m RNA expression of both Leptin group and Qingshen granule was higher than that in the blank control group,all had statistically significant(p<0.01);compared with the Leptin group,the HGF m RNA expression in each Qingshen granule group was higher than that in the Leptin group,and all of them were statistically significant(p< 0.01).The HGF m RNA expression in the low dose group of Qingshen granule was higher than that in the medium dose and high dose groups,and there were statistically significant(p<0.01).the HGF m RNA expression in the Medium Dose Group was higher chan the High Dose Group(p<0.05).(4)Comparison of TGF-?1 m RNA expression: Compared with the blank control group,the TGF-?1 m RNA expression of Leptin group and Qingshen granule was higher than that in the blank control group,all had statistically significant(p<0.01);compared with Leptin group,the TGF-?1 m RNA expression of each Qingshen granule group was lower than that of Leptin group,There was statistically significant(p<0.01);TGF-?1 m RNA expression in low dose group of Qingshen granule was lower than that in medium dose group and high dose group of Qingshen granule,and all had statistical significance(p<0.01);There was no difference between medium dose group and high dose Group(p>0.05).3.Comparison of the expression of Leptin,OB-R,HGF and TGF-?1 in different groups(Western Blot method):(1)Comparison of expression of Leptin: Compared with the blank control group,the expression of Leptin in Leptin Group and groups of Qingshen granule was higher,and all had statistically significant(p<0.01);compared with Leptin group,the expression of Leptin in each Qingshen granule group was lower(p<0.01);The expression of Leptin in low dose group of Qingshen granule was the lost(p<0.01);There was no difference between the medium dose group and the high dose group in the Qingshen granule(p>0.05).(2)Comparison of expression of OB-R: The expression of OB-R in the groups of Leptin and Qingshen granule was higher than that in the blank control group,all had statistically significant(p<0.01);Compared with the leptin group,the expression of OB-R in all Qingshen granule groups was lower,and all of them were statistically significant(p<0.01);The expression of OB-R in low dose group of Qingshen granule was the lost,and there were statistically significant(p<0.01);There was no difference between the medium dose and high dose group in the Qingshen granule(p>0.05).(3)HGF expression comparison:Compared with the blank control group,the expression of HGF in both leptin group and Qingshen granule was higher(p<0.01);Compared with the leptin group,HGF expression in all groups of Qingshen granule was higher,and all had statistically significant(p<0.01);The expression of HGF in the low dose group of Qingshen granule was higher than that in the Medium Dose Group and the High Dose Group of Qingshen granule,and all of them were statistically significant(p<0.01).There was no difference between the medium and the high dose groups(p>0.05).(4)Comparison of expression of TGF-?1:Compared with the blank control group,the expression of TGF-?1 in both Leptin Group and Qingshen granule groups was higher(p<0.01);Compared with the leptin group,the expression of TGF-?1 in all Qingshen granule groups was lower,and all of them were statistically significant(p<0.01);The expression of TGF-?1 in Low Dose Group was lower than that in the Medium Dose Group and High Dose Group of Qingshen granule,all of them had statistically significant(p<0.01);There was no difference between medium and the high dose groups(p>0.05).4.Comparison of the expression of HGF,TGF-?1,MCP-1 and ICAM-1 in each group of cells(Immunofluorescence method):(1)Comparison of the expression of HGF:HGF is visible in the fluorescent microscope with green light.Compared with the blank control group,the HGF fluorescence intensity of both leptin group and Qingshen granule was higher than that in the blank control group(p<0.01);Compared with the leptin group,HGF fluorescence intensity in each group of Qingshen granule was higher,and all had statistically significant(p<0.01);the fluorescence intensity of HGF in the Low Dose Group was higher than that in the Medium Dosage Group of Qingshen granule,and there were statistically significant(p<0.01);there was a statistically significant difference between the Medium Dose Group and the High Dose Group in the Qingshen granule(p<0.05).(2)Comparison of expression of TGF-? 1:The TGF-? 1 was expressed in the cytoplasm and was red by fluorescence microscope.Compared with the blank control group,the fluorescence intensity of TGF-?1 in both leptin group and Qingshen granule was higher(p<0.01);compared with the leptin group,the fluorescence intensity of TGF-?1 in each Qingshen granule group was lower,and all of them were statistically significant(p<0.01);the fluorescence intensity of TGF-?1 in Low Dose Group was the lost,and there were statistically significant(p<0.01);there was no difference between the Medium Dose Group and the High Dose Group in the Qingshen granule(p>0.05).(3)Comparison of expression of MCP-1:The MCP-1 was green under fluorescence microscope.Compared with the blank control group,the fluorescence intensity of MCP-1 in both leptin group and Qingshen granule was higher,all had statistically significant(p<0.01);compared with the leptin group,the fluorescence intensity of each Qingshen granule groups was lower,and all of them were statistically significant(p<0.01)The fluorescence intensity of MCP-1 in Low Dose Group was the lost in each Qingshen granule groups,and there were statistically significant(p<0.01);there was no significant difference between Medium Dose Group and the High Dose Group in the Qingshen granule(p>0.05).(4)Comparison of expression of ICAM-1:The ICAM-1 was red when detected by immunofluorescence.Compared with the blank control group,the fluorescence intensity of ICAM-1 in both Leptin Group and Qingshen granule groups was higher,all had statistically significant(p<0.01);compared with the Leptin Group,the fluorescence intensity of ICAM-1 in each Qingshen granule groups was lower,and all of them were statistically significant(p<0.01),The fluorescence intensity of ICAM-1 in Low Dose Group was the lost in each Qingshen granule groups(p<0.01);there was no significant difference between the Medium Dose Group and the High Dose Group in the Qingshen granule(p>0.05).5.Comparison of the expression of Leptin,MCP-1 and ICAM-1 in supernatant of cell culture solution(Elsia method):(1)Comparison of expression of Leptin:Compared with the blank control group,the expression of Leptin in the groups of Leptin Group and Qing Shen granule was higher,all had statistically significant(p<0.01);compared with the Leptin group,Leptin expression in all Qing Shen granule groups was lower,and all of them were statistically significant(p<0.01);the expression of Leptin in Low Dose Group of Qingshen granule was significantly lower than that in Medium Dose Group and the High Dose Group(p<0.01);there was no statistically significant difference between the Medium Dose Group and the High Dose Group in the Qingshen granule(p<0.05).(2)Comparison of expression of MCP-1:Compared with the blank control group,the expression of MCP-1 in the group of Leptin Group and Qingshen granule was higher,all had statistically significant(p<0.01);MCP-1 expression in all Qingshen granule groups was lower than that in Leptin group,and all of them were statistically significant(p<0.01);the expression of MCP-1 in Low Dose Group was the lost in Qing Shen granule groups,and there were statistically significant(p<0.01);there was no difference between the Medium Dose Group and the High Dose Group in the Qingshen granule(p>0.05).(3)Comparison of expression of ICAM-1: The expression of ICAM-1 in the group of Leptin Group and Qingshen granule was higher than that in the blank control group,all had statistically significant(p<0.01);compared with the Leptin group,ICAM-1 expression in all Qingshen granule groups was lower,and all of them were statistically significant(p<0.01);the expression of ICAM-1 in Low Dose Group was the lost in Qing Shen granule groups(p<0.01);there was no difference between the Medium Dose Group and the High Dose Group in the Qingshen granule(p>0.05).Conclusion: 1.Leptin can induce HK-2 to be EMT;2.Serum containing Qingshen granule can inhibits the EMT of HK-2 induced by Leptin;3.HGF and TGF-?1 have an antagonistic effect,and leptin can express imbalance between HGF and TGF-?1 in HK-2 through certain signaling pathways,which in turn promotes the expression of two inflammatory factors in MCP-1 and ICAM-1,The serum of Qingshen granule can inhibit the expression of Leptin,OB-R,MCP-1 and ICAM-1 in leptin-induced HK-2 by modulating the imbalance of HGF and TGF-?1 in HK-2.Thus inhibiting the HK-2 process of leptin-induced EMT,the progress of delaying TIF,and finally to reduce the role of CRF.5.The serum Low Dose Group of Qingshen granule drug-containing drugs inhibited the expression of HGF and TGF-?1 in leptin-induced HK-2 and delayed the development of TIF,which was better than the medium and high dosage groups of Qingshen granule. |
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