The Repair Effect Of Betulinic Acid On F-2 Toxin-induced Uterine Injury In Mice

F-2 toxin,also known as Zearalenone,is a kind of mycotoxin widely existed in feed raw materials.Its estrogen-like effect can cause damage to various organs of livestock and poultry,especially reproductive organs,and then interfere with reproductive performance.Betulinic acid is a polyphenolic substance that can be extracted from many plants such as birch.Its special pharmacological activity and structural multiplicity make it have many biological activities such as anti-tumor,anti-oxidative stress,immunomodulatory,anti-inflammatory and anti-HIV.Therefore,it has been widely paid attention by people.Objective:In this study,female mice of the Kunming family were used as the research object,and the mouse model of uterine injury was induced by intragastric administration of F-2 toxin.Objective to explore the repair effect of betulinic acid on the uterus injury induced by F-2toxin in mice and its possible regulatory mechanism,so as to provide theoretical and experimental basis for the development of betulinic acid as the antidote of F-2 toxin.Methods:A total of 90 healthy female 7W KM mice were randomly divided into 6 groups with 15mice in each group:control group,20 mg/kg·bw F-2 toxin group,F-2 toxin+low and high dose betulinic acid groups（0.25 and 0.5 mg/kg·bw）,F-2 toxin+100 mg/kg·bw V_Egroup and0.5 mg/kg·bw betulinic acid control group.At the 1st week of the formal experiment,the control group and the Betulinic acid control group were given 5%ethanol solution by gavage,and the other groups were given F-2 toxin（dissolved in 5%ethanol）once a day for consecutive 7 days to induce the reproductive system injury model.At the second week,control group and F-2 toxin group were gavaged with 1%soluble starch paste,and the remaining groups were gavaged with corresponding doses of Betulinic Acid and V_E（uniformly suspended in 1%soluble starch paste）,once a day,for consecutive 7 days.At the third week,control group,F-2 toxin group,V_Egroup and betulinic acid control group were gavaged with 1%soluble starch paste,while betulinic acid low-dose and high-dose groups were gavaged with betulinic acid（uniformly suspended in 1%soluble starch paste）at the corresponding dose,once a day,for consecutive 7 days.The mice were then killed and blood and uterine tissue were collected.Serum levels of sex hormones Estradiol（E₂）and Progesterone（P₄）were determined.The levels of Reactive oxygen species（ROS）,Superoxide dismutase（SOD）,Glutathione（GSH）,Catalase（CAT）and Malondialdehyde（MDA）in the uterus of mice were detected.The morphological changes of uterine tissue were observed by H&E staining.The ultrastructure of endometrial epithelial cells was observed by electron transmission microscope.Uterine cell apoptosis rate was detected by TUNEL assay.Immunohistochemistry was used to detect the distribution and expression of estrogen receptor ERα,ERβ,Proliferating cell nuclear antigen（PCNA）in mouse uterus.The m RNA levels of ERα,ERβand PCNA in mouse uterus were detected by q RT-PCR.The expression of estrogen receptor ERαprotein in mouse uterus was detected by Western blot.The expression of PCNA,a marker protein of proliferation;Mitochondrial apoptosis signaling pathway Bax,Bcl-2,Caspase-3,Caspase-9 protein expression;Expression and phosphorylation levels of extracellular regulated protein kinases（ERK1/2）,P38mitogen-activated protein kinase（P38 MAPK）and c-Jun N-terminal kinase（JNK）in the MAPK signaling pathway.Results:1)Betulinic acid treatment group can effectively alleviate the uterine appearance enlargement caused by F-2 toxin,and significantly reduce the uterine organ coefficient（P<0.05）;2)The uterine H&E staining showed that the betulinic acid treatment group significantly improved the endometrial and glandular epithelial hyperplasia caused by the F-2 toxin group,and significantly decreased the thickness of the lamina prima and the muscular layer of the uterus（P<0.01）;3)Ultrastructural observation showed that betulinic acid could improve the morphology of mitochondria and increase the number of microvilli and cilia in uterine cells;4)0.25 mg/kg and 0.5 mg/kg betulinic acid treatment could significantly recover the decrease of serum E₂content caused by F-2 toxin and increase of serum P₄content（P<0.05）;5)Compared with the control group,the MDA content in F-2toxin group was significantly increased（P<0.01）,the GSH content and ROS level were significantly increased（P<0.05）,and the contents of GSH,MDA and ROS levels were significantly decreased after treatment with all doses of betulinic acid（P<0.01）;6)TUNEL assay showed that betulinic acid treatment significantly inhibited F-2 toxin-induced apoptosis of endometrial epithelial cells（P<0.01）;7)Immunohistochemical results showed that the betulinic acid treatment group was able to significantly reduce the F-2 toxin caused by the increase in uterine ERαand PCNA immunopositive reaction（P<0.01）;8)q RT-PCR showed that betulinic acid treatment group could significantly reduce the increase of relative expression levels of ERαand PCNA m RNA caused by F-2 toxin（P<0.01）;9)Western blot analysis showed that betulinic acid treatment group could significantly down-regulate the expressions of uterine ERα,PCNA,Caspase-3 and Caspase-9 caused by F-2 toxin and the increase of Bax/Bcl-2（P<0.05）,and could significantly down-regulate the phosphorylation levels of ERK1/2 and P38 protein in uterine MAPK signaling pathway caused by F-2 toxin（P<0.05）.Conclusion:Betulinic acid has a repairing effect on uterine injury caused by F-2 toxin.It can achieve the repairing effect by restoring the level of related sex hormones,inhibiting the abnormal hyperplasia and hypertrophy of endometrial epithelium,improving the antioxidant capacity of uterus,clearing the level of ROS in uterus and reducing the apoptosis rate of endometrial epithelial cells.This repair effect may be related to the inhibition of the activation of estrogen receptor pathway,proliferation and apoptosis pathway and MAPK signaling pathway by betulinic acid.