Clinical And Experimental Studyof Astragalus Membranaceus On Improving The Early Renal Tubular Injury In Type 2 Diabetic Mellitus By Regulating The Nrf2/ARE/L-FABP Signaling Pathway

Background:Diabetic nephropathy(DN)has become the major cause of end-stage renal disease increasing the mortality risk of diabetes.At present,urinary microalbumin,which is generally recognized as a biological index to predict the occurrence and prognosis of DN,is considered to be short of early warning and prognosis defects.In the treatment of DN,there is no particularly effective treatment for the cause,especially enter large amount of urinary albumin stage,the treatment effect is very poor,so it is expected that simple and noninvasive indexes can predict DN earlier,so as to intervene treatment earlier.Liver-type fatty acid-binding protein(L-FABP),secreted from proximal tubules during oxidative stress or ischemia events.L-FABP has an important role in kidney injury and repair.L-FABP is considered to be a biomarker for predicting the prognosis of DN.Research has demonstrated that the oxidative damage and apoptosis of renal tubular cells is present during DN.Chinese medicinal plant Astragalus membranaceus has been widely used for the treatment of kidney diseases,however,the role and mechanism by which Astragalus membranaceus may ameliorate high glucose-induced apoptosis and oxidative stress of DN remains largely unknown.In the present study,the effect of AS-IV on high glucose-induced HK-2 cell apoptosis and oxidative stress was investigated.Findings may provide sufficient evidence that Astragalus membranaceus has potential target as a therapeutic for the treatment of DN.Objective:To verify the effect of Chinese medicinal plant Astragalus membranaceus on early renal damage landmark in Type 2 Diabetes mellitus,and try to clarify the molecular mechanism.Search for the therapeutic target in DN.Methods:Clinical study:62 T2DM patients with UACR≤300mg/g were randomly divided into control group(30 cases)and treatment group(32 cases).The control group received basic treatment.Treatment group was treated with Astragalus membranaceus on the basement of control group.The period of observation is three weeks.The UL-FABP,U β 2-MG,UACR,GSP and TCM clinical symptom score were observed.Experimental research parts:Cell viability,apoptosis and protein expression were detected by Trypan blue staining,Cell Counting Kit-8 assay,terminal deoxynucleotidyl transferase 2’-deoxyuridine-5’-triphosphate nick-end labelling,flow cytometry and western blot analysis.In addition,enzymatic activities,including superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and lipid peroxide(LPO),were measured with the corresponding detection kits.DCFH-DA assay and flow cytometry were performed to detect the production of reactive oxygen species(ROS).Western blot analysis and reverse transcription-quantitative polymerase chain reaction were conducted to evaluate protein and mRNA expressions of the nuclear factor erythroid 2 like 2(Nrf2)/antioxidant response element(ARE)signaling pathway.Result:Clinical research:L-FABP in T2DM expressed higher than nomorl person.It’s associated with TG、TC、LDL、U β 2-MG and Cystatin C,especially with U β 2-MG.After treatment,the decrsase of urine indicators in treatment group was better than that in control group.There were significant differences in TCM clinical symptom score after treatment in treatment group than control group.Experimental research:The results demonstrated that AS-IV significantly enhanced HK-2 cell viability induced by high glucose in a dose-dependent manner.In addition,AS-IV notably inhibited HK-2 cell apoptosis stimulated by high glucose,which may be associated with inhibition of Bax,Cleaved-caspase-3 and Cleaved-caspase-9,expression and the promotion of Bcl-2.AS-IVsignificantly increased the activities of antioxidant enzymes SOD,GSH-Px and CAT,and decreased the high-glucose-induced ROS production in HK-2 cells,in a dose-dependent manner.Finally,it was determined that AS-IVregulated the Nrf2/ARE signaling pathway and inhibited the expression of liver-type fatty acid binding protein.Conclusion:1.L-FABP in T2DM expressed higher than nomorl person.It’s associated with TG、TC、LDL、U β 2-MG and Cystatin C,especially with U β 2-MG.2.Astragalus membranaceus can effectively improve early renal damage in T2DM,decrease the level of UL-FABP and UACR.3.Astragalus membranaceus can decrease TCM clinical symptom score in early renal damage of T2DM.4.The main component of Astragalus membranaceus------AstragalosideⅣ(AS-Ⅳ)can improve the high glucose-induced HK-2 cell apoptosis and oxidative stress.5.AS-IV decreases high glucose-induced HK-2 cell apoptosis and oxidative stress by regulating the Nrf2/ARE signaling pathway.Furthermore,L-FABP may inhibit glucose-induced HK-2 cell apoptosis and oxidative independently of the AS-IV mediated Nrf2/ARE signaling pathway.AS-IV may have a potential role in the treatment of DN.