Research On The Treatment Of DN Proteinuria With Qiditangshen Recipe And Its Separate Prescriptions Based On The Pathogenesis Of "Jingluobi"

Background and objective:1 Diabetic nephropathy(DN)is one of the most common micro vascular complications in patients with diabetes mellitus(DM),which main clinical manifestation is(micro)albuminuria.Diabetic kidney disease(DKD)is the clinical diagnosis of DN.With the prolongation of the course of the disease,it can lead to renal failure,and it has become the main cause of dialysis due to end stage renal disease(ESRD)worldwide.Our team's previous research found that"essence depletion and collateral impediment" is the core pathomechanism of diabetic nephropathy,which combining essence deficiency and collateral obstruction.The mainly pathogenesis of diabetes is qi and yin deficiency.In the case of deficiency of both qi and yin and deficiency of yin and essence for a long time,DM will reach the kidney position and develop to DKD.Protein is a subtle substance of the human body,which is firmly sealed by the kidney.In DKD kidney essence deficiency,the kidney loses its function,and it is difficult to seal the subtle kidneys,resulting in proteinuria.Current research indicates that DKD microalbuminuria is a critical period leading to differences in patient prognosis,and podocyte damage is associated with the production of diabetic nephropathy proteinuria.This experiment focused on exploring the distribution characteristics of TCM syndromes in patients with DKD kidney essence deficiency syndrome and the correlation between syndromes and nephrin and podocin in urine.2 Aiming at this pathomechanism,a prescription for combination therapy with supplementing essence(Tianjing,TJ)and unblocking the collaterals(TongLuo,TL)is proposed——QiDiTangShen recipe(QDTS).Early studies of QDTS showed that it could improve the insulin sensitivity of kidney tissue of db/db mice by inhibiting the activation of MAPK signaling pathway,restoring IRS-1 Ser/Tyr phosphorylation balance Role of kidney damage caused by DM.However,the effects of supplementing essence method and unblocking the collaterals method on DN have not been elucidated,and the research on the mechanism of this side needs to be supplemented and improved.Therefore,this experiment analyzes the effect of QDTS and its separated prescriptions(TJ and TL)on db/db mice and podocyte damage.In this experiment,the network pharmacology analysis of QDTS was used to analyze the specific mechanism of action,and the role of possible effective pathways(AGEs-RAGE-RhoA/ROCK signaling pathway)analyzed by network pharmacology was verified.The purpose is to explore the possible targets and mechanisms of QDTS and lay the foundation for elucidating the core pathogenesis of DN essential damage.Methods:1 Clinical researchThe patients with DKD microalbuminuria were taken as the research object,and the patients with simple DM were used as the control group.Use the "Chinese Medicine Syndrome Judgment Scale" developed by the research group through the previous study to score the patients' symptoms,including all the symptoms contained in the syndromes related to the deficiency of kidney essence(deficiency of kidney essence,deficiency of kidney qi,deficiency of kidney yin,deficiency of kidney yang),and standard syndrome(resent heat syndrome,damp heat syndrome,blood stasis syndrome,phlegm and turbidity syndrome).The content of nephrin and podocin in podocytes in morning urine of patients with DKD microalbuminuria kidney essence deficiency syndrome was detected by ELISA.The laboratory indexes,TCM syndrome scores related to kidney essence deficiency and urine podocyte-specific protein content were compared between the two groups.The purpose of this experiment was to analyze the correlation of TCM syndromes with laboratory indicators and podocyte-specific proteins in patients with DKD microalbuminuria with kidney essence deficiency syndrome.2 Internet pharmacology researchUse online pharmacology to integrate multiple online databases to predict the target information of seven herbs of QDTS.Cytoscape software was used to construct the herbs-component interaction network and component-target interaction network of QDTS.Use the online database to search the target information about diabetic nephropathy,and merge with the drug target network to get the core PPI network.The Metascape database was used to analyze KEGG enrichment of the core PPI network.3 Experimental research8-week-old male db/db mice were used as the DN animal model,and the same-week-old male C57BL/6 mice were used as the normal control group.After all animals were adaptively fed for 1 week,they were placed in a metabolic cage,and urine was retained for 8 hours.The urine microalbumin content was detected by ELISA and the urine microalbumin excretion rate(UAER)was calculated.The db/db mice with a higher UAER than the normal group were randomly divided into 5 groups based on body weight,blood glucose and UAER,8 in each group,namely:model group(db/db group),valsartan group(db/db+valsartan Group),QDTS group(db/db+TJ&TL Group),TJ group(db/db+TJ group)and TL group(db/db+TL group).The mice were continuously given an equal volume of the corresponding drug solution for 12 weeks,and then the material was taken.Evaluate kidney damage by UAER,serum renal function(BUN,Cr,UA,Ccr)and pathological examination.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)to evaluate liver function.Serum total cholesterol(TC)and triglyceride(TG)to assess blood lipid function.Observe the ultrastructure of podocytes and calculate the fusion rate of podocyte foot processes using electron microscopy;use immunohistochemistry to detect the expression of nephrin and desmin in the kidney tissue of mice in each group.The experiment aims to evaluate podocyte damage.The purpose of the experiment is to evaluate the effect of QDTS and its separated prescriptions(TJ and TL)on podocyte injury.In order to verify some of the mechanism results obtained by network pharmacology analysis.Western blot,immunohistochemistry and other methods were used to detect the protein expression levels of AGEs,RAGE,RhoA and ROCK1 in the kidney tissues of mice in each group.The purpose was to evaluate the effect of QDTS on AGEs-RAGE signaling pathway.Results:1 Clinical researchCompared with patients in the DM group,urine microalbumin(UALB)and urine microalbumin/creatinine ratio(UACR)in the DKD microalbuminuria group were significantly higher(P<0.01).In this experiment,the DKD microalbuminuria group patients with kidney Qi deficiency syndrome,kidney yin deficiency syndrome,heat syndrome and blood stasis syndrome had higher syndrome scores than the DM group(P<0.05).The deficiency syndrome of kidney essence accounts for a relatively high proportion in patients with DM and DKD microalbuminuria group.In addition,syndrome of kidney essence deficiency,damp heat syndrome,blood stasis syndrome were positively correlated with UACR(P<0.05).Kidney essence deficiency syndrome,damp heat syndrome,phlegm and turbidity syndrome were positively correlated with podocyte-specific protein podocin(P<0.05).2 Internet pharmacology researchHerbs of QDTS were obtained through network pharmacology,and 63 component targets were obtained after drug screening.Using STITCH and Uniprot to get 74 targets associated.The target of QDTS and the target of diabetic nephropathy were intersected to obtain a core PPI interaction network containing 23 intersected targets.Using the Metascape database,KEGG enrichment analysis of the core PPI network,sorted by P value,the top 20 signal pathways related to diabetic nephropathy are:oxidative stress,apoptosis,lipopolysaccharide reaction,AGEs-RAGE signaling pathway,Fatty acid metabolism,vascular endothelial cell migration,etc.3 Experimental researchCompared with the db/db mice in the model group,the UAER levels of the QDTS group(TJ&TL),TJ group and TL group mice were significantly reduced,and the renal pathological damage was significantly reduced.In terms of ultrastructure,compared with mice in the db/db group,the GBM thickening and foot fusion rate of the QDTS group(TJ&TL group),TJ group and TL group mice were significantly reduced(P<0.01).QDTS and its separated prescriptions(TJ and TL)can reduce the expression of desmin protein in kidney tissue of db/db mice(P<0.05),while just QDTS can increase the expression level of nephrin protein in the kidney tissue of db/db mice(P<0.05).In order to verify the partial mechanism results of network pharmacology,the protein expression levels of AGEs,RAGE,RhoA and ROCK1 in the kidney tissues of each group of mice were detected.The results showed that compared with the db/db group,the expressions of AGEs and RAGE proteins in the kidney tissues of the db/db+QDTS group significantly decreased(P<0.05),and the expressions of RhoA and ROCK1 proteins also decreased significantly(P<0.05).Conclusion:1 Deficiency of kidney essence is a common feature of the distribution of diabetes and diabetic nephropathy syndrome.Symptoms related to kidney deficiency and deficiency appear after many years of DM(especially middle-aged and elderly patients),and deficiency of kidney essence runs through the course of DKD.The deficiency syndrome of kidney essence and the "kidney syndrome" that cause "collaterals" do not exist independently.Due to the deficiency and stagnation,the kidneys are blocked,which forms an important pathogenesis of diabetic nephropathy.2 The combination therapy with supplementing essence and unblocking the collaterals can improve the podocyte damage of diabetic nephropathy.QDTS and its separated prescriptions(TJ and TL)can reduce the fusion rate of foot process and reduce the expression of podocyte injury marker protein(desmin),while only QDTS can protect the SD structure of podocytes.It shows that the effect of supplementing essence and unblocking the collaterals is more significant.3 QDTS may likely improve diabetic nephropathy through AGEs-RAGE-RhoA/ROCK signaling pathway.Network pharmacological studies have shown that the possible mechanism of QDTS in the treatment of DN is the AGEs-RAGE signaling pathway.Experimental studies have shown that QDTS can reduce the podocyte damage of db/db mice and improve the kidney damage caused by T2DM through the AGEs-RAGE-RhoA/ROCK signaling pathway.It further shows that the mechanism of action of Chinese herbal compound can be preliminarily analyzed through network pharmacology analysis.