Research On The Mechanism Of Heat-clearing And Syndrome-eliminating Method In Treating Diabetic Nephropathy Based On SLP

The incidence of diabetic kidney disease(DKD)is increasing year by year and has become a major cause of end-stage renal disease in China and worldwide.However,DKD has an insidious onset and progresses rapidly after entering the clinical proteinuric phase,which is difficult to reverse with conventional treatment.Therefore,early detection,disease evaluation,prognostic assessment,and discovery of therapeutic approaches that can delay the progression of DKD are urgent clinical problems.Previous studies have found that the expression level of secretory leukocyte protease inhibitor(SLPI)increases with increasing renal injury in lesions such as acute kidney injury,which has potential diagnostic value for the disease.However,in diabetic kidney disease,the change pattern of SLPI has not been reported in studies.With many years of clinical experience,Prof.X proposed that the core pathogenesis of DKD is "internal heat causing conglomeration",and treated DKD with the method of Qing-Re-Xiao-Zheng,which has achieved good results.In the preliminary small sample study,the team found that the level of serum SLPI protein increased with the progression of DKD,and the efficacy of the method of Qing-Re-Xiao-Zheng was also initially verified in the preliminary study,but its mechanism of action has yet to be studied in depth.Therefore,is SLPI protein related to kidney damage in diabetic kidney disease?Does the treatment of DKD by Qing-Re-Xiao-Zheng method have a regulatory effect on SLPI protein?To answer these questions,this study firstly investigated the correlation between SLPI protein and DKD disease progression and conventional disease evaluation indexes through a cross-sectional clinical study with a larger sample size,and then investigated its effect on DKD endpoint events and survival time by means of survival analysis through a two-way cohort study.Based on the theory of "internal heat causing conglomeration",a randomized controlled trial was conducted to investigate the efficacy of the method of Qing-Re-Xiao-Zheng in the treatment of DKD and its effect on the regulation of serum SLPI protein.Finally,we investigated the mechanism of the effect of Qing-Re-Xiao-Zheng method on delaying the progression of DKD through animal experiments,taking SLPI protein,inflammation and fibrosis as the entry point.Objective:1 Exploring the role of SLPI protein in disease evaluation and prognosis assessment during DKD disease progression.2 Investigating the regulation of serum SLPI protein in the process of delaying the progression of DKD by Qing-Re-Xiao-Zheng therapy.3 Investigating the mechanism of action of Qing-Re-Xiao-Zheng therapy to regulate SLPI protein levels and delay the progression of DKD.Methods:1 A total of 266 patients with clinically diagnosed DKD were studied and classified into early,middle and late stages according to the staging criteria,and clinical data,physical and chemical examination results and serum SLPI protein levels were collected.We observed the changes of serum SLPI protein level from early to late stage and the differences between groups,analyzed the correlation between serum SLPI protein level and 24-hour urine protein,eGFR and other conventional disease evaluation indexes,drew ROC curve and analyzed the predictive diagnostic effect.In this way,we investigated the correlation between SLPI protein and disease progression of DKD and its predictive diagnostic value for DKD.2 Patients with clinically diagnosed DKD were studied,grouped according to SLPI protein levels,and continued to be followed up to the end of the study cycle based on the follow-up of the previous study.Clinical data,eGFR and other renal function evaluation indexes were collected and recorded,and survival analysis was used to investigate the role of serum SLPI protein levels in assessing the risk of DKD progression and clinical endpoint events.3 Patients with clinically diagnosed DKD were randomly divided into control and experimental groups,and given conventional western medical treatment and the combination of Qing-Re-Xiao-Zheng therapy with conventional western medical treatment,respectively,and followed up for 12 weeks.The clinical data,physical and chemical examination results and serum SLPI protein level were recorded.The therapeutic effect of the Qing-Re-Xiao-Zheng therapy on DKD and the modulating effect on SLPI protein were observed through intra-group and inter-group comparisons.4 The DKD model rats constructed by unilateral nephrectomy combined with streptozotocin duplex method were used for the study,and the Qing-Re-Xiao-Zheng therapy was used as Chinese herbal medicine intervention,and valsartan was used as the positive control,and sham-operated control group,model group,Chinese medicine group and positive control group were established respectively.The expression levels of SLPI,TAK1,P65,P-P65,IκBα and TNF-α were detected by Western blot,and the tissue localization of SLPI and P-P65 protein expression was observed by immunohistochemistry.TGF-β1,Col Ⅳ protein expression levels were detected by WB,and the tissue localization of Col Ⅳ,Col Ⅲ protein expression was observed by immunohistochemical assay.In this way,we observed the mechanism of SLPI protein regulation and prevention of DKD from anti-inflammatory and anti-fibrotic perspectives,respectively.Results:1 The role of SLPI protein in disease evaluation of DKDSerum SLPI levels increased sequentially among the five groups of normal subjects,diabetic group,early,middle and late stages of DKD,and were statistically different among the five groups(H=84.26,P<0.01).Comparison between groups revealed that serum SLPI levels were statistically different between the beginning of the middle stage and the normal subjects group(P=0.01);while in the late stage group,serum SLPI levels were significantly higher compared to the remaining four groups(P<0.01).Serum SLPI levels were significantly and positively correlated with BUN,Scr,UA,24hour urine protein,K+,systolic blood pressure,and duration of proteinuria(P<0.01)with correlation coefficients of 0.532,0.604,0.233,0.372,0,30,0.161,and 0.233,respectively.Significant negative correlations were found with eGFR,HGB,Ca,and CO2CP(P<0.01)with correlation coefficients of-0.564,-0.158,-0.213,and-0.226,respectively.In addition,serum SLPI also had a negative correlation with Alb(P<0.05)with a correlation coefficient of0.126 and a positive correlation with CHO(P<0.05)with a correlation coefficient of 0.135.The sensitivity of serum SLPI protein in the predictive diagnosis of DKD was 73.31%,the specificity was 69.23%,the AUC was 0.76(0.69,0.83),P<0.01,the Jorden index was 0.43,and the cut-off value was 39.11 ng/mL.2 The prognostic role of SLPI protein in the assessment of DKDThe Kaplan-Meier survival curves showed that the low-serum SLPI expression group had significantly better survival time and cumulative survival rate than the high-serum SLPI expression group,and the difference in cumulative survival rate between the two groups was more pronounced as the survival time increased.The survival curves showed that the cumulative survival rate of the group with low expression of serum SLPI was significantly higher than that of the group with high expression of serum SLPI(P<0.01).One-way Cox regression analysis revealed that elevated serum SLPI levels(HR=2.95,P<0.01),elevated BUN levels(HR=1.10,P<0.01),decreased eGFR(HR=3.79,P<0.01),and elevated 24-hour urine protein levels(HR=2.94,P<0.01)may be risk factors for progression of DKD.Elevated HGB(HR=0.74,P<0.01),Alb(HR=0.93,P<0.01),and Ca(HR=0.03,P<0.01)may be protective factors for the progression of DKD.Multifactorial Cox regression analysis identified decreased eGFR(HR=2.34,P<0.01)and elevated 24-hour urine protein(HR=2.12,P<0.05)as independent risk factors for progression of DKD.3 Treatment of DKD with the method of Qing-Re-Xiao-Zheng can reduce serum SLPI protein levelIntra-group comparison revealed that serum SLPI levels were significantly lower in the test group after treatment compared with baseline(P<0.01);no statistical difference was found in the control group before and after treatment.Comparison between groups revealed that the 24-hour urine protein reduction(P<0.05)and serum SLPI level reduction(P<0.05)were better in the test group than in the control group.4 Qing-Re-Xiao-Zheng therapy delays DKD progression through regulating SLPI protein,anti-inflammation and anti-fibrosis4.1 Compared with the control group,the rats in the model group had significantly reduced body weight(P<0.01),significantly increased renal weight-to-weight ratio(P<0.01),significantly increased 24-hour urine protein level,GLU and BUN(P<0.01),and significantly decreased Scr level(P<0.01).After the pharmacological intervention of the Qing-Re-XiaoZheng therapy,the body weight of rats was significantly improved at the end of the experiment compared with the model group(P<0.05),and the renal weight-to-weight ratio was significantly decreased,but no statistical differences were observed.In addition,the 24-hour urine protein and GLU(P=0.01)of the rats in the Qing-Re-Xiao-Zheng therapy group were significantly lower than those in the model group at second month(P<0.05)and fourth month(P<0.01).The renal pathology showed that the Qing-Re-Xiao-Zheng therapy could restore the renal pathological damage in DKD model rats.4.2 WB experiments revealed that TGF-β1(P<0.01)and Col Ⅳ(P<0.01)protein expression were significantly increased in the model rats.The Qing-Re-Xiao-Zheng therapy down-regulated TGF-β1(P<0.01)and Col Ⅳ(P<0.05)protein expression in the model rats.Immunohistochemical experiments showed that the expression of Col Ⅳ protein were increased in the kidney tissues of the model rats.The Qing-Re-Xiao-Zheng therapy could downregulate the expression of Col Ⅳ in the model rats.4.3 WB experiments revealed that TAK1(P<0.05),IκBα(P<0.05),SLPI(P<0.01)protein expression was significantly increased,and P65,P-P65,TNF-α protein expression was increased in the model group rats,but no statistical difference was observed.IκBα(P<0.01)and SLPI(P<0.01)protein expressions were significantly decreased and TAK1,P65,P-P65,TNF-α protein expressions were decreased in the Qing-Re-Xiao-Zheng therapy group,but no statistical differences were seen.Immunohistochemical experiments showed that the expression of SLPI and P-P65 protein was increased in the kidney tissues of the model rats.The Qing-ReXiao-Zheng therapy could down-regulate the expression of SLPI and P-P65 in the model rats.Conclusion:1 Serum SLPI levels gradually increased with the progression of DKD disease,and there was a significant correlation between serum SLPI levels and conventional disease evaluation indicators of DKD,which has a high predictive diagnostic value in DKD.In addition,elevated serum SLPI expression level is a potential risk factor for DKD disease progression.Therefore,serum SLPI protein has potential value for early diagnosis,disease evaluation and prognosis assessment of DKD.2 The treatment of DKD by Qing-Re-Xiao-Zheng therapy can reduce 24-hour urine protein and serum SLPI protein levels,thus delaying the disease progression of DKD.3 Qing-Re-Xiao-Zheng therapy can delay the progression of DKD by regulating SLPI protein,anti-inflammation and anti-fibrosis.