Protective Effect And Mechanism Of Betulinic Acid On Intestinal Mucosal Oxidative Damage Induced By T-2 Toxin In Mice

Betulinic acid(BA)is a pentacyclic lupane-type triterpene which widely distributed in the plant kingdom.BA exerts a wide variety of biological and pharmacological activities including immunomodulation,antioxidant,anti-inflammatory and etc.Previous study has showed that dietary BA supplement increased intestinal antioxidant capacity,restored the morphology of intestinal mucosa,and reduced the diarrhea in weaned piglets.It was suggested that BA may have a protective effect on intestinal mucosa barrier function by inhibiting inflammation and enhancing anti-oxidant activity.However,no reports at present have investigated the effects of BA on the intestinal mucosa barrier function and its possible mechanism.Therefore,the objective of the current study was to explore the protective effect and molecular mechanisms of BA on T-2 toxin-induced intestinal oxidative damage by regulating the mRNA expression of intestinal junction proteins and protein expression of NF-κB signaling pathway in mice.Objectives:To elucidate the protective effect and mechanism of BA on oxidative damage of intestinal mucosa induced by T-2 toxin in mice,thus it may provide theoretical basis for a novel nutritional intervention for gut health through improving anti-oxidative capability on the intestines by BA.Methods:60 healthy male mice were randomly divided into 6 groups,namely,the control group,T-2 toxin group,low,medium and high doses of BA(0.25 mg / kg,0.5 mg / kg,1 mg / kg)+ T-2 groups,V-E + T-2 group.BA and V-E were suspended in 1% soluble starch,and the control group and T-2 group were given the same amount of 1% soluble starch once a day and continuously administered for 14 days.Animals were received T-2 toxin which dissolved in mixed solution of alcohol and PBS(alcohol: PBS = 1:12.5)intraperitoneally at the dose of 4 mg/kg b.w.to induce oxidative damage,while the control group given the mixed solution of alcohol and PBS injections of the equal volume.After fasting for 15 h(free drinking water),mice were executed to collect blood and the small intestines.The levels of malondialdehyde(MDA),glutathione(GSH),catalase(CAT),and glutathione peroxidase(GSH-Px)in the duodenum,jejunum and ileum were determined by commercial assay kits.The levels of intestinal fatty acid binding protein(IFABP),secretory immunoglobulins A(SIgA),Complement C3(C3),Complement C4(C4)in jejunum,and the levels of immunoglobulin M(IgM),immunoglobulin G(IgG),and diamine oxidase(DAO)in serum were detected by ELASA.The morphological changes of intestinal mucosa were observed by H&E stain and transmission electron microscopy.RT-PCR was used to detect the mRNA expression of tight junction proteins such as Occludin,zonula occludens-1(ZO-1),and the mRNA expression of cytokines such as interleukin-1 beta(IL-1β),IL-6,IL-10 and tumor necrosis factor-α(TNF-α)in intestinal mucosa.Western blot was used to detect the protein expressions and phosphorylation of nuclear transcription factor-kappa B(NF-κB)p65 and protein kinase alpha(IKB-alpha)in the NF-κB signaling pathway.Results:(1)BA pretreatment alleviated the decrease of CAT and GSH-Px activities in intestine caused by T-2 toxin,increased the content of GSH,and decreased the content of MDA,thereby improving the antioxidant capacity of intestine;(2)BA pretreatment enhanced SIgA level in intestine and IgG and IgM levels in serum,restored the intestinal C3 and C4 levels,and thus increasing intestinal immune barrier;(3)Pretreatment with BA reduced the level of IFABP in intestine,and decreased the level of DAO in serum,thereby improving intestinal chemical barrier;(4)BA pretreatment increased the mRNA expressions of ZO-1 and Occludin in intestinal mucosa,improved the morphological of intestinal villi caused by T-2 toxin,and thus boosting the physical barrier of intestine;(5)Pretreatment with BA at the dose of 1mg/kg decreased the protein expression and phosphorylation of P-NF-κB /NF-κB,and P-IKB-a/IKB-a protein in intestine,while all doses of BA pretreatment suppressed the mRNA expression of pro-inflammatory cytokines such as IL-1β,IL-6 and TNF-α,and increased mRNA expressions of anti-inflammatory cytokine such as IL-10 in intestine induced by T-2 toxin.It was suggested that BA inhibited the activation of NF-κB signaling pathway.Conclusions:BA has a protective effect on intestinal oxidative damage induced by T-2 toxin in mice by enhancing intestinal antioxidant capacity,inhibiting the secretion of inflammatory cytokines and repairing intestinal mucosal barrier functions,which was related to inhibiting the activation of NF-κB signaling pathway.