The Effect And The Potential Mechanism Of Betulinic Acid On The Functional Recovery Of Testicular Injury In ZEA-challenged Mice

Zearalenone(ZEA),also known as F-2 toxin,is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum,which is mainly derived from the grain and feed contaminated by Fusarium during field,farm or storage.ZEA is a mycoestrogen that has been receiving increasing attention for its estrogen-like effects on the reproductive system.Betulinic acid(BA)is a natural lupane-type pentacyclic triterpene antioxidant.Previous studies found that BA effectively alleviated the testicular damage caused by T-2 toxin through anti-oxidative stress.It is worth exporing whether BA has a protective effect on testicular oxidative damage induced by ZEA,no literatures have been reported so far.Objective:The aim of the study was to explore the protective effect and the underlying mechanisms of BA on the testicular damage of mice treated with ZEA,which may contribute to an viable alternative to alleviate ZEA-induced oxidative stress and male reproductive toxicology,thus improving animal health.Methods:Sixty male KM mices were randomly divided into 6 groups: control group,BA(0.5mg/kg)group,ZEA(20mg/kg)group,ZEA and BA(0.25 mg/kg or 0.5 mg/kg)cotreatment groups,and ZEA and vitamin E(100 mg/kg)co-treatment group.ZEA was dissolved in 5% ethanol solution and given by gavage for 1W to induce testicular injury model,and then different doses of BA or vitamin E were suspended in 1% soluble starch and adminstrated by gavage for 2W.(1)The effect of BA on the functional recovery of testicular injury in mice Firstly,the histological changes of testis were observed,including testis gross observation and organ coefficient analysis;The pathological changes of testis were observed by H&E staining;and the percentage of cell apoptosis in testis was detected by TUNEL.Secondly,the redox system of testis was analysized such as the levels of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),glutathione(GSH)and glutathione peroxidase(GHS-Px).Thirdly,the indexes of the male reproductive system involve the expression of estrogen receptor alpha(ERα),the testosterone(T)level and sperm motility were measured,the ultrastructure of blood testis barrier(BTB)was observed by transmission electron microscopy,and the m RNA expressions of the main extracellular plasmid specialization protein N-cadherin 2(CDH2),claudin 11(CLDN11)and vimentin(Vim),which are key structural proteins of tight junction,were detected by Real-time quantitative PCR(q PCR).Finally,the m RNA expressions of inflammatory cytokines such as interleukin-1 beta(IL-1β),IL-6,IL-10 and tumor necrosis factor-α(TNF-α)were mresured by q PCR.(2)BA alleviated ZEA-induced testicular injury in mice by regulating MAPK and Nrf2 signaling pathways The expression and phosphorylation of p38,extracellular regulated protein kinases 1/2(ERK1/2),and c-Jun N-terminal kinase(JNK)proteins in the signaling pathway of mitogen-activated protein kinase(MAPK),and the protein expressions of epoxy chloropropane kelch sample related protein-1(Keap1),nuclear factor erythroid-2 related factor 2(Nrf2)and heme oxygenase-1(HO-1)in the Nrf2 signaling pathway in testis were detected by Western blot.The content and distribution of Nrf2 and HO-1 in testis were determined by immunohistochemistry.Results:(1)BA mitigated the pathological changes caused by ZEA,such as atrophy of spermatogenic tubules,abscission of lumen cells,blockage of lumen,dilatation and congestion of the testicular vascular tissues,vacuolization of intercellular stroma,and reduction of the cell apoptosis in testis,suggesting that BA ameliorated ZEA-triggered testicular damage by restoring the histological changes and reducing cell apoptosis.(2)Pretreatment with BA significantly increased the activities of SOD,CAT,and the content of GSH in testis,and significantly reduced the levels of ROS and MDA in testis of ZEAexposed mice.These results suggested that BA could alleviate the oxidative stress in testis by improving the antioxidant capacity.(3)After treatment with BA,the m RNA and protein expression of ER-α in testis were up-regulated,the T level in serum was increased,the sperm motility was restored,and the relative m RNA expressions of CDH2,CLDN11 and Vim were up-regulated which contribute to reconstruct the BTB,indicating that BA alleviated ZEA-triggered testicular damage by restoring hormone level and BTB.(4)BA pretreatment significantly increased the expression of anti-inflammatory cytokine IL-10 m RNA and lowered the expression of pro-inflammatory cytokines such as IL-1β,IL-6 and TNF-α,showing that BA moderated ZEA-evoked testicular damage by anti-inflammation effect.(5)BA down-regulated the phosphorylation of p38 and ERK,and un-regulated the protein expressions of Nrf2 and HO-1,indicating that BA ameliorated ZEA-triggered testicular damage by inhibiting MAPK signaling pathway and activating Nrf2 signaling pathway.Conclusion:BA has a therapeutic protective effect on the testicular injury caused by ZEA by improving the antioxidant capacity,inhibiting the inflammatory response,up-regulating the expression of ERα,increasing the T level in serum and rebuilding the BTB.This protective effect is mainly regulated through inhibition of MAPK signaling pathway and activation of Nrf2 signaling pathway.