Protective Effect Of Betulinic Acid On Testicular Injury Induced By T-2 Toxin In Mice And Its Mechanism

Objective: To study the protective effect of Betulinic acid(BA)on testicular injury induced by T-2 toxin in mice and its molecular mechanism,and provide an important theoretical basis for the use of BA as a mycotoxin antidote.Methods: Sixty healthy male Kunming mice were randomly divided into six groups: control group,T-2 toxin group,low,medium and high dose(0.25,0.50,1.00 mg/kg·bw)BA group and 100mg/kg vitamin E group.The control group and T-2 toxin group were fed with 1% soluble starch solution.The other low,medium and high dose BA groups were fed with BA suspended in soluble starch at different doses.The positive control group was fed with Trolox of 100 mg/kg.bw once a day for 14 days.After 14 days,except 75% ethanol and PBS mixture(75% ethanol: PBS = 1:12.5),the other five groups were intraperitoneally injected with T-2 toxin(4 mg/kg).· The oxidative damage model was induced by bw,1 ml 75% ethanol and 12.5 ml PBS dilution.Fifteen hours later,anesthetized mice were sacrificed after eyeball blood collection.Testicles and epididymis were taken to detect the following related indicators:(1)The protective effect of BA on oxidative damage of testis in mice induced by T-2 toxin: the activity of SOD,CAT and T-AOC in testis tissue and the content of GSH and MDA were detected.(2)The protective effect of BA on T-2 toxin-induced apoptosis in mouse testicular tissue: Western Blot detection of apoptosis-related proteins STAT3,P-STAT3,JAK2 and P-JAK2 and their downstream Bax,Bcl-2,caspase3 proteins The expression of BA on the apoptosis of mouse testis tissue induced by T-2 toxin: TUNEL method was used to detect apoptosis of testicular cells;QPCR method was used to detect the expression of MALAT-1 gene.(3)Protective effect of BA on testosterone(T)secretion abnormality induced by T-2 toxin: The testosterone level of mice was detected by enzyme-linked immunosorbent assay(ELISA).(4)The protective effect of BA on T-2 toxin-induced testicular morphological damage in mice: Paraffin sections were prepared and HE staining was performed to observe the morphology and structure of testicular cells.(5)The protective effect of BA on sperm motility and morphological damage induced by T-2 toxin: taking epididymis,detecting sperm motility,mortality and deformity rate.Results:(1)Compared with the control group,T-2 toxin decreased the activity of testicular SOD,CAT and T-AOC and the content of GSH,and increased the content of MDA;compared with the T-2 toxin group,different doses(0.25,0.5 and 1mg/kg·bw)BA and T-2 toxin can increase the activity of testicular SOD,CAT and T-AOC and GSH content,while reducing the content of MDA.(2)Compared with the control group,P-JAK2/JAK2 ratio and P-STAT3/STAT3 ratio in T-2 toxin group increased significantly(P<0.05);Bax/Bcl-2,Cleaved caspase 3/Procaspase 3 ratio increased significantly(P<0.01,P<0.01);apoptotic cells increased significantly in T-2 toxin group;MALAT-1 gene expression increased significantly(P<0.01).Compared with T-2 toxin group,the ratio of P-JAK2/JAK2,P-STAT3/STAT3,Bax/Bcl-2,Cleaved caspase 3/Procaspase 3 in low,medium and high dose BA group decreased significantly(P < 0.05),the number of apoptotic cells decreased in a dose-dependent manner,and the expression of MALAT-1 gene decreased significantly(P < 0.01).(3)Compared with the control group,the secretion of testosterone in T-2 toxin group was decreased,while the secretion of testosterone in low,medium and high doses of BA increased in a dose-dependent manner compared with that in T-2 toxin group.(4)Compared with the control group,the structure of testis in T-2 toxin group was disordered and testicular cell necrosis disappeared.Compared with T-2 toxin group,the structure of testicular cell gradually returned to normal with the increase of dose in low,medium and high dose BA group,especially in high dose group.(5)Compared with the control group,T-2 toxin can reduce the sperm survival rate,increase the sperm mortality rate and abnormality rate,and can effectively prevent testicular damage caused by T-2 toxin by adding low,medium and high dose BA pretreatment.Conclusion: BA can prevent testicular damage caused by T-2 toxin by repairing oxidative stress,regulating the expression of proteins related to mitochondrial apoptosis signaling pathway and the secretion of testosterone.