Huang-Lian-Jie-Du Decoction Alleviates Diabetic Nephropathy Through Regulating The AGEs/RAGE Pathway

Diabetic nephropathy(DN)is a chronic kidney disease caused by diabetes,which is the primary cause of end-stage renal disease(ESRD).Huang-Lian-Jie-Du Decoction(HLJDD),a well-known formula for clearing heat and detoxicating,is composed of Rhizoma Coptidis,Radix Scutellariae,Cortex Phellodendri and Fructus Gardenia with a weight ratio of 3:2:2:3.It has been found that HLJDD has a therapeutic effect on DN,however,the underlying mechanisms of HLJDD on DN remain unclear,which needs to be systematically studied.With this purpose,pharmacodynamics,network pharmacology,action pathways and metabolomic were estimated to clarify the mechanism of HLJDD on DN.The research contents are as follows :1.Pharmacodynamics study on the protective effects HLJDD on DNDiabetes nephropathy mice model was established by db/db mice.After 12 weeks of HLJDD intervention,the body weight of mice were recorded,and the biochemical indexes,renal histopathology and expression of Nephrin and TGF-β1 in renal tissue were comprehensively analyzed.The results of biochemical indexes showed that HLJDD could effectively improve glucose and lipid metabolism disorder and renal dysfunction in db / db mice.The results of HE,Masson and PAS staining confirmed that HLJDD has a strong protective effect on abnormal renal morphology,glomerular fibrosis and glycogen accumulation in db/db mice;The results of immunohistochemistry indicated that HLJDD could increase the expression of Nephrin and inhibit the expression of TGF-β1to alleviate podocyte injury and kidney fibrosis of db/db mice.The above results demonstrated that HLJDD could protect against DN.2.Network pharmacology study of HLJDD on DNTaking 31 blood components of HLJDD as the research object,the molecular mechanism of HLJDD on DN was predicted by component-disease-target network,PPI network,GO analysis and KEGG analysis.The results showed that the core targets of HLJDD in the treatment of DN were PIK3R1,PIK3 CA,STAT3,AKT1 and MAPK1.GO and KEGG enrichment analysis showed that HLJDD might play a role through stimulative response,cell composition and receptor activity.Its mechanism was mainly related to AGEs/RAGE signaling pathway.3.Mechanism of HLJDD on DN based on AGEs/RAGE pathwayAccording to the predictive results of network pharmacology,AGEs/RAGE pathway and its related targets were verified and analyzed.By detecting the levels of SOD,GSH and MDA in kidney tissue,it was found that HLJDD could alleviate oxidative stress injury in kidney of db /db mice.ELISA kit results indicated that HLJDD could significantly reduce the level of AGEs in plasma and kidney tissue of db/db mice.The results of immunofluorescence and western blot showed that HLIDD could increase the expression of GLO1,inhibit the expression of RAGE,and enhance the level of Akt phosphorylation,activate the expression of Nrf2 and its downstream antioxidant factors HO-1,SOD2.These findings indicated that HLJDD could improve DN by regulating AGEs/RAGE pathway.4.Urine metabolomic of HLJDD on DNBased on UPLC-Q-Orbitrap HRMS/MS technology,the effect of HLJDD on urine metabolites in db/db mice was explored.The multivariate statistical analysis had shown that there were significant differences in urine metabolites among groups.22 potential biomarkers were identified.Among them,17 metabolites showed a trend towards the normal levels after HLJDD intervention.These metabolites principally involved in phenylalanine metabolism,tryptophan metabolism,glucose metabolism and sphingolipid metabolism.In total,HLJDD had pharmacological effects for improving glucose and lipid metabolism disorders and renal dysfunction,inhibiting glomerular fibrosis and alleviating oxidative stress in db/db mice.Besides,HLJDD could promote DN via regulation of AGEs/RAGE signaling pathways,and phenylalanine metabolism,tryptophan metabolism,glucose metabolism,sphingolipid metabolism.