Huang Gan Formula Inhibits The Effect Of Advanced Oxidation Protein Products(AOPPs) On The Regulation Of Ages Receptors Expression Via The JAK2/STAT3 Pathway

Background and objective:Chronic kidney disease(CKD),with high prevalence and low cure rate has been a significant health issue.Oxidative stress is common in CKD processes due to metabolic disorders,Inflammation,and impaired renal function,which can change healthy proteins into advanced oxidation protein products(AOPPs)aggravating the progression of oxidative stress in vivo.Advanced glycation end product receptor(AGER)has different functions in the toxicity and disposal(detoxification)in advanced glycation end products(AGEs).The AGE binding receptor class encompasses a number of members which could be categorized into two forms:1)one linked to the enhanced oxidant stress,growth,as well as the inflammatory effect that are finely represented by AGE(RAGE)receptors and 2)the other involving detoxification of AGE including scavenger receptors class A type ?,as well as class B type ?,and AGE receptors 1,2,and 3.Among these scavenger receptors,AGE receptor 1(AGE-R1)promotes the uptake and removal of AGEs and blocks cellular AGE-mediated ROS generation and inflammation.Because AGE-R1 and RAGE compete for AGEs,the binding of AGEs to RAGE may increase when AGE-R1 levels decrease,resulting in the induction of increased ROS.Huang Gan formula(HGF),a compound in the second generation of Niaoduqing particles products,is a novel traditional Chinese herbal medicine formulas on the basis of basic theories of traditional Chinese medicine.The characterization of chemical compositions of the HGF compound was achieved by using HPLC-Q-TOF-MS spectrometry.In total,8 major compounds were found and measured through the comparison of reference standards.In our previous study,we found that HGF could significantly decrease oxidative stress,improve renal function,and delay renal fibrosis development in CKD rat models that were caused by adenine or 5/6 nephrectomy.However,the precise HGF mechanisms toward divergent regulation of RAGEs expression have not yet been thoroughly clarified.This research was aimed to investigate the influences of HGF on eliminating the effects of oxidative stress induced by AOPP and the underlying mechanisms related to whether AOPPs affect AGEs receptors expression via JAK2/STAT3 cellular pathway.Methods and results:In this experiment,we employing western blotting,quantitative polymerase chain reaction,and biochemical assays:We use UV spectrophotometry of Witko-Sarsat to detect the AOPPs relative concentration of chloramine-T(?M);Flow cytometry and DCFH-DA fluorescent probe detection of AOPPs on the ROS levels in HMCs and the intervention of HGF;Griess reagent method to measure the effect of AOPPs on NO level in HMCs as well as the intervention effect of HGF;ELISA kits to measure the effect of AOPPs on MDA?SOD and GSH-Px in HMCs;Real-Time Quantitative PCR to detect the expression of AOPPs induce RAGE and AGE-R1 in HMCs as well as the intervention effect of HGF;Western blot to detect the expression of AOPPs induce RAGE?AGE-R1?JAK2?P-JAK2?STAT3 and P-STAT3 in HMs as well as the intervention effect of HGF.To further examine the possibility that AOPP-dependent proliferative responses were mediated through the JAK2/STAT3 cascade,serum-starved HMC were divided into two groups:1)cells were stimulated with AOPPs(200?g/ml),or HGF(800?g/ml)for 24 h.Control cells received no treatment.2)cells were pretreated with AG-490(lOuM)for 12 h before the addition of AOPPs(200?g/ml),or HGF(800?g/ml)for an additional 24h,and then RAGE?AGE-RI?JAK2?P-JAK2?STAT3 and P-STAT3 were detected by western blot.We found that HGF significantly reduced AOPP-induced oxidative stress injury.In addition,the protective effect of compound Huanggan may be related to the up-regulation of advanced glycation end-product receptor 1(AGE-R1)and the down-regulation of RAGE by the receptor.Treatment with HGF and Janus kinase 2(JAK2)inhibitor AG490 significantly attenuated AOPP-induced P-JAK2/P-STAT3 protein levels.ConclusionsHuang Gan formula can effectively protect the Human mesangial cells against oxidative stress induced by AOPPs,significantly increase SOD,GSH-PX,NO content,reduce the contents of MDA and ROS,and increase the ability of anti-oxidative stress.Huang Gan formula can inhibit the expression of RAGE and up-regulate the expression of AGE-R1 from mRNA and protein levels,protect AOPPs-mediated chronic kidney disease injury.HGF can inhibit the expression of P-JAK2 and P-STAT3 protein induced by AOPPs,and then regulate the balance of AGE receptor.The protective mechanism of HGF may be through blocking the activation of AOPPs and JAK2/STAT3 pathway,inhibited expression of RAGE,up-regulate expression of AGE-R1,thus effectively delay the progress of chronic kidney disease.