Relieving Effect And Molecular Mechanism Of Betulinic Acid On T-2 Toxin-induced Renal Oxidative Damage By Regulating The Nrf2/NF-κB Signaling Pathway

Oxidative stress,as a potential mechanism for T-2 toxins to exert cytotoxicity,has brought significant economic losses in large-scale breeding,which has led to the continuous development of various antioxidant products.As a plant-derived antioxidant,whether betulinic acid(BA)has a regulatory effect on T-2 toxin-induced kidney damage has not been reported so far.Objective:In this study,T-2 toxin was injected intraperitoneally to establish a model of kidney damage in mice in order to evaluate the effect of BA and further reveal the molecular mechanism of it,which may provide evidence and new ways for the scientific application of BA in the healthy breeding of livestock and poultry.Methods:Sixty healthy male KM mice were randomly divided into 6 groups,namely the control group,the T-2 toxin group,the groups treated with BA(0.25 mg/kg,0.5 mg/kg,1 mg/kg,respectively),and the group treated with 100 mg/kg V-E.After BA was suspended in 1% starch solution and administered orally daily for 14 d,4 mg/kg T-2 toxin was intraperitoneally injected to establish a kidney damage model.After 15 h,mice were sacrificed to collect blood and kidneys.Detection of serum biochemical indexes in mice,including creatinine,Urea,neutrophil gelatinase-associated lipocalin(NGAL)and kidney injury molecule 1(Kim-1);detection of kidney antioxidant indicators in mice,including superoxide dismutase(SOD),glutathione(GSH),glutathione peroxidase(GSH-Px),catalase(CAT)and malondialdehyde(MDA);the pathological changes and ultrastructural changes of the kidney were observed by H & E staining and transmission electron microscopy;the production of reactive oxygen species(ROS)was detected by dihydroethidium(DHE);detection of the m RNA expressions of inflammatory cytokines in kidney by fluorescent quantitative PCR,including interleukin-1β(IL-1β),tumor necrosis factor α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)and transforming growth factor beta 1(TGF-β1);the protein expressions of nuclear factor E2 related factor 2(Nrf-2)signaling pathway were detected by western blot,including Keap1(Epoxy chloropropane Kelch sample related protein-1,Keap1),Nrf2 and heme oxygenase 1(Hemeoxygenase-1,HO-1);protein expression and phosphorylation of IκBα and nuclear factor-kappa B(NF-κB)in the NF-κB signaling pathway were also determined by western blot.Results:1)(0.25 mg/kg,0.5 mg/kg,1 mg/kg)BA pretreatment inhibited the increase of creatinine level caused by T-2 toxin in serum,but had no significant effect on Urea and Kim-1.0.5 mg/kg BA pretreatment significantly reduced NGAL level.BA pretreatment reduced the degree of kidney swelling,restored the kidney to a light reddish-brown color.0.5 mg/kg BA alleviated excessive glomerular hemorrhage and inflammatory cell infiltration caused by T-2 toxin,and at the same time,weakened T-2 toxin-induced mitochondrial vacuolation,restored mitochondrial number,and normalized basement membrane and podocytes in kidney,thereby effectively alleviating kidney damage.2)BA pretreatment promoted the dissociation of Keap1-Nrf2,up-regulated the expression of Nrf2 and HO-1 proteins,enhanced the activities of SOD and CAT and the content of GSH,while down-regulated the content of MDA and inhibited the production of ROS by activating Nrf2/HO-1 signaling pathway,thereby improving T-2 toxin-induced renal oxidative damage.3)BA pretreatment increased the ratio of p-IκBα/IκBα while reduced the ratio of p-NF-κB/NF-κB,and then significantly reduced the m RNA expressions of IL-1β,TNF-α and IL-10,and increased IL-6 m RNA expressions by inhibiting the NF-κB signaling pathway,thereby alleviating the inflammatory response of T-2toxin-induced kidney damage.Conclusion:In T-2 toxin-induced kidney damage in mice,BA exerted potential anti-oxidative and anti-inflammatory effects by improving the kidney’s ability to eliminate ROS and reduce lipid peroxidation,and regulating the expression of inflammatory cytokines.The protective effect of BA on T-2 toxin-induced kidney damage in mice was achieved by activating the Nrf2 signaling pathway and inhibiting the NF-κB signaling pathway.