The Effect And Mechanism Of Astragaloside Ⅳ On Renal Tubulointerstitial Fibrosis Through MAPK Signaling Pathway

Renal fibrosis is a major pathological factor of chronic kidney disease(CKD) and can progressively develop to end stage kidney disease (ESRD),which brings huge economic burden to family and society. Astragaloside IV(AS-IV) is one of the main saponins. Clinical practice and modernphamacological studies suggested that Astragalus membranaceus Bge had asignificant role in delaying progression of CKD[1].The effect of AS-IV on renalfibrosis is still unclear. This study is to investigate the mechanism of AS-IV inchronic renal injury.Objective: To determine the effect of AS-IV on renal fibrosis induced byunilateral ureteral obstruction (UUO) and renal tubular epithelial cells injuryinduced by TGF-β1and investigate its mechanism.Methods: Male C57BL/6mice were randomly divided into control group(Sham)(n=8), model group (UUO)(n=10), treatment group(AS-IV)(n=10).UUO and AS-IV groups were performed UUO procedures to build renal injurymodel. Sham animals were subjected to the same operation as theexperimental group without obstrucing the ureter. Mice in the AS-IV groupwere orally administrated AS-IV20mg/(kg×d) for7days after operation. Micein sham and model group were admistrated equal volume of vehichle same asAS-IV group. Bilateral kidneys were collected in postoperation days7,14. Thekidney weight/body weight rate was recorded. TUNEL method was used todetect renal apoptosis. Western Blotting and real-time PCR were respectivelyused to detect the protein and mRNA levels of fibronectin, collagen IV, α-SMA,and TGF-β1. The expression of p-p38/p38, p-JNK/JNK, and p-ERK/ERKMAPK signaling pathways were observed by Western Blotting.In vitro, normal human renal tubular epithelial cells (HK-2) were cultured.Cells were divided into three groups:(1) normal control group (Ctl) given withvehicle same as following groups;(2) TGF-β1group stimulated with recombinant TGF-β1(10ng/ml);(3) AS-IV-treated group stimulated withrecombinant TGF-β1(10ng/ml) and simultaneously treated with differentconcentrations of AS-IV (50,100,200μg/mL) for24or48h. TGF-β1(10ng/ml)was used to stimulate HK-2cells in model and control group, while AS-IVtreatment group was administrated by different concentrations(50,100,200μg/ml) AS-IV. The expression of fibronectin, collagen IV, andα-SMA were investigated by western blotting and real-time PCR. Transversekidney slices were sectioned at2μm, and stained with hematoxylin-eosin andMasson trichrome to evaluate the severity of renal tubule injury. Viability ofcells was determined by cell counting kit-8(CCK-8) and a transferase-mediateddUTP nick-end labeling (TUNEL) assay was performed to detect apoptoticnuclei in kidney sections. The protein and mRNA levels of fibronectin、collagenIV、α-SMA、TGF-β1was detected by western blotting and real-time PCR.Futhermore, SB203580(10μM) was administrated half an hour before givingTGF-β1and the effect of inhibiting p38MAPK signaling was discussed in thisstudy.Results: The kidney weight/body weight (KW/BW) ratio of obstructedkidneys significantly decreased in UUO mice at postoperatin day14(P<0.05),which could be alleviated by AS-IV administration (P<0.05). Compared with Ctlgroup, morphology of obstructed kidneys in UUO group presents changes ofrenal tubulointerstitial fibrosis. The proteins and mRNA expression offibronectin, collagen IV, α-SMA, and TGF-β1were increased significantly inkidney tissues(all P<0.05), which could be reversed by AS-IV administration(all P<0.05). AS-IV inhibited UUO induced renal tubular apoptosis throughinhibiting MAPKs to down-regulate p-p38/p38、p-JNK/JNK、p-ERK/ERKMAPK (all P<0.05).Cell viability was significantly inhibited and cell apoptosis was rosed byTGF-β1stimulating for24h (P<0.05). The expression of fibronectin, collagen IV,and α-SMA increased (all P<0.05). In MAPKs signaling, the ratios of p-p38/p38,p-JNK/JNK protein expression were also increased (all P<0.05). However, p-ERK and ERK did not change significantly (P>0.05). While AS-IV notablyreversed the direction of increasing apoptosis into activating cell proliferationto augment overall cell survival, whereas200μg/ml AS-IV demonstrated theoptimal effect (P<0.05). This anti-apoptotic effect was associated withinhibition of caspase-3activation (all P<0.05) and overexpression medicatedmainly by downturn of phospho-p38and phospho-JNK MAPK signals (allP<0.05). Administration of a dose of SB203580can inhibit p38phosphorylation, which decreased the expression of collagen IV, α-SMA (allP<0.05).Conclusion: AS-IV could improve renal tubulointerstitial fibrosis inducedby UUO and TGF-β1and reduce renal tubular epithelial cell apoptosis. Themechanism of AS-IV protective effect might be associated with inhibition ofp38and JNK MAPKs phosphorylation.