Clinical Observation Of Diabetic Nephropathy With Qikui Granule And Study On The Mechanism Of EMT In Renal Tubular Epithelial Cells

?.Objective1.To observe the effect of Qikui granules on clinical treatment and disease progression of early DN microalbuminuria.2.To explore the effect of Qikui granules on prevention of the transition of renal tubular epithelial cells induced by high glucose.?.Methods and contents1.Clinical study80 Cases of early Diabetic Nephropathy(Mogensen III period,deficiency of Qi and Yin,damp stasis syndrome)in accordance with the established inclusion criteria,exclusion criteria and elimination criteria.According to random digital table method,two groups were divided,treatment group(Qikui granules,n=40)and control group(Irbesartan tablets treatment,n=40).In the treatment group,Qigui granules were taken orally,three times a day with one pack each time.The control group was given Irbesartan tablets once a day with 150mg.The treatment period is one year.and the Urinary Albumin/Creatinine Rate(UACR),renal functions(eGFR,BUN,SCr,Cys-c),number of cases entering Mogensen IV,blood glucose(FBG,HbA1C),blood lipid(TC,TG),blood pressure(SBP,DBP)and the change of TCM syndromes integral were observed.2.Cell experimentDrug-containing serum preparation:SD rats were randomly divided into the normal group and the Qikui groups(with low,medium,and high dose).Three rats in each group,once a day for 7 days,blood was taken from the carotid artery on the 7th day and 2h after the last gavage to prepare drug-containing serum.HK-2 cells were taken as the subject,and control group(6%serum containing normal group + 5.5 mmol/L glucose),hypertonic group(6%serum containing normal group + 5.5 mmol/L glucose + 24.5mmol/L mannitol),model group(6%serum containing normal group + 30mmol/L glucose),low dose group of Qikui granules(6%serum containing low doses + 30mmol/L glucose),medium dose group of Qikui granules(6%serum containing medium doses + 30mmol/L glucose),high dose group of Qikui granules(6%serum containing high doses + 30mmol/L glucose),Rho inhibitor group(6%serum containing normal group + 30mmol/L glucose + Y-27632)were divided to culture for 48 hours.The morphologic changes of the cells in each group were observed by inverted phase contrast microscope.Expression of a-SMA,FSP-1,E-cadherin protein and mRNA in each group were detected by fluorescent immunofluorescence and real-time quantitative PCR.Detection of FN Secretion in Cell Supernatant of Each Group by ELISA.Protein expression of RhoA,ROCK1,PMYTP-Thr853,PMYTP-Thr696 were detected by Western blot.?.Results1.Clinical studies:? There were 38 cases in the treatment group and 33 cases in the control group.? Disease efficacy:The total effective rate of treatment group was better than that of control group(P<0.05);? UACR comparison:UACR of both groups decreased after treatment(P<0.05);The difference between two groups before and after treatment was statistically significant(P<0.05);? Number of cases entering Mogensen IV:After treatment,2 cases entered stage IV in the treatment group and 7 cases in the control group.The difference between the two groups was statistically significant(P<0.05);? There were no significant changes in renal function,blood glucose,blood lipid and blood pressure in both groups before and after treatment(P>0.05);? TCM syndromes efficacy:The total effective rate of treatment group was better than that of control group(P<0.01);? The change of the total integral of TCM syndromes:In the treatment group,it became lower compared with itself before treatment and after treatment in the control group(P<0.05),while no significant change was found before and after treatment in the control group(P>0.05);Comparison of single symptom score of TCM syndromes:The curative effect of the treatment group was superior to that of the control group in improving the symptoms of burnout,weakness of waist and knee,swelling of face and limb,and turbid urine(P<0.05,P<0.01);? No significant adverse reactions were observed in both groups during the treatment period.2.Cell experiment:? Morphological changes:Compared with the control group,there was no obvious change of cell morphology in the hyperosmotic group.In the model group,the cells were fusiform and lost their paving-stone-like growth mode.When the cells were growing,they were radial,disordered,and a few cells fell off.The morphology of each group was improved after drug intervention.? The expression of a-SMA,FSP-1,E-cadherin protein:Compared with the control group,the expression of a-SMA,FSP-1 protein significantly increased(P<0.05)while the expression of E-cadherin protein decreased significantly(P<0.05)in the model group;Compared with the model group,the expression of a-SMA,FSP-1 protein decreased significantly(P<0.05)whereas the expression of E-cadherin protein increased significantly(P<0.05)in the Qikui group with low,medium,high dose;Compared with the Rho inhibitor group,the expression of a-SMA,FSP-1,E-cadherin protein were no significant difference(P>0.05)in the Qikui group with low,medium,high dose.? The expression of a-SMA,FSP-1,E-cadherin mRNA:Compared with the control group,the expression of a-SMA,FSP-1 mRNA significantly increased(P<0.05)while the expression of E-cadherin mRNA decreased significantly(P<0.05)in the model group;Compared with the model group,the expression of a-SMA,FSP-1 mRNA decreased significantly(P<0.05)whereas the expression of E-cadherin mRNA increased significantly(P<0.05)in the Qikui group with low,medium,high dose;Compared with the Rho inhibitor group,the expression of a-SMA,FSP-1,E-cadherin mRNA were no significant difference(P>0.05)in the Qikui group with low,medium,high dose.? The FN secretion in the cell supernatant:Compared with the control group,the FN secretion in the cell supernatant in the model group significantly rise(P<0.05);Compared with the model group,the FN secretion in the cell supernatant of the Qikui group with low,medium,high dose and the Rho inhibitor group was significantly decline(P<0.05);Compared with Rho inhibitor group,there was no significant difference in FN secretion in cell supernatant in the Qikui group with low,medium,high dose(P>0.05).? The protein expression of RhoA,ROCK1,PMYTP-Thr853,and PMYTP-Thr696:Compared with control group,the protein expression of RhoA,ROCK1,PMYTP-Thr853,and PMYTP-Thr696 in model group went up significantly(P<0.05);Compared with the model group,the protein expression of RhoA,ROCK1,PMYTP-Thr853,and PMYTP-Thr696 in the Qikui group with low,medium,high dose and the Rho inhibitor group went down significantly(P<0.05);.Compared with the Rho inhibitor group,there was no significant difference in the protein expression of RhoA,ROCK1,PMYTP-Thr853,and PMYTP-Thr696 in the Qikui group with low,medium,high dose(P>0.05).?.Conclusion1.Qikui granules can significantly reduce the microalbuminuria in patients with diabetic nephropathy,delay the progress of the disease and improve the symptoms of fatigue,weak waist,knee edma,urine turbidity.2.Qikui granules can inhibit EMT of renal tubular epithelia cells,and its mechanism may be related to regulate RhoA/ROCK1 signal pathway.