Fetal-preserving Effect Of Atractylenolide ? On Threatened Abortion Feline And Preliminary Mechanism Research

Threatened abortion is one of the main causes of pregnancy failure in domestic cats.In this research field,hormone and antibiotic therapy is mainly used.However,in recent years,embryonic malformations,infertility and other side effects caused by the abuse of hormones and antibiotics have become increasingly prominent.Therefore,safe and effective fetal protection products have important clinical application value to reduce the abortion rate of domestic cats and ensure the breeding of fine breeds.In this study,AT-? was used as a starting point,which opened up a new research direction for the prevention and treatment of threatened abortion in domestic cats.The specific research contents are as follows:Study on general toxicity and target animal safety of AT-?.(1)Study on general toxicity and target animal safety of AT-?Acute toxicity test:sixty mice were randomly divided into 5 groups with 12 mice in each group.Groups 1?4 were given AT-? of 6000,4800,3840 and 3072 mg/kg respectively,while the control group was given the same amount of purified water.Within10 days,the poisoning and death were observed,and the median lethal dose(LD₅₀)was calculated.Another twenty Kunming mice were randomly divided into two groups:the experimental group was given 0.2 g/m L AT-? solution 3 times within 18 hours,0.8 m L each time,and the control group was given the same amount of purified water for 7 days,and the maximum tolerance dose(MTD)and tolerance multiple were calculated.Sub-chronic toxicity test:twenty-four female SD rats were randomly divided into low dose group,middle dose group,high dose group and control group(n=6).They were given AT-? of 1200,2400 and 4800 mg/kg/d respectively,while the control group was given the same amount of purified water for 30 days.The clinical signs of rats in each group were recorded every day.On the 31st day,weighing,blood collection,hematological examination,autopsy observation and pathological sections were made.Target animal safety test:twenty female domestic cats aged 2?5 years old were randomly divided into 4 groups(n=5).The low,middle and high dose groups were given2.6,7.8 and 13 mg/kg/d AT-? capsules respectively,and the blank control group was given empty capsule for 7 days.The appetite,mental state and defecation of each group were observed every day.Venous blood samples were collected on the 8th day to detect hematological indexes.Results showed that there was no death in mice in acute toxicity test,and LD₅₀>6000mg Magneto MTD was 24 g/kg,which indicated that the AT-? had no obvious toxicity.In the subchronic toxicity test,there was no significant difference in growth and development,blood routine index and organ coefficient between the middle and high dose groups and the control group(P>0.05).The content of serum total cholesterol decreased significantly(P<0.05),and there was no significant difference in other indexes(P>0.05).The pathological results showed that there was no significant abnormality in the main tissues and organs of rats in the high dose group.In the target animal safety test,the mental state,coat glossiness and feces of domestic cats in each group were not abnormal,and there was no significant difference in hematological indexes between the control group and the control group(P>0.05).(2)Study on the fetal protective effect of AT-? on threatened abortion mouse modelSixty mice 3 days pregnant were randomly divided into:normal pregnancy group(CON,n=10),threatened abortion model group(MOD,n=10),progesterone group(PRO,n=10),low,medium and high dose AT-? groups(AT-L,AT-M,AT-H,n=10).The drug was given by intragastric administration on the 4th day of pregnancy for 5 days.On the 9th day of pregnancy,all groups except CON group were given mifepristone 11.62 mg/kg to establish the mouse model of threatened abortion.On the 14th day,the mice were killed,the number of embryo implantation and fetal development were observed,the contents of PGF_2?,P₄and E₂in serum were detected by ELISA method,the number and relative area of corpus luteum in ovary were observed by HE staining,the expression of St AR m RNA in ovarian tissue was detected by RT-q PCR method,and the content and distribution of St AR in ovarian tissue were detected by immunohistochemical method.The results showed that in MOD group,the uterus had no embryonic attachment,the content of PGF_2?in serum increased significantly,the contents of P₄and E₂decreased significantly,the expression of St AR decreased significantly in luteal dysplasia of ovary(P<0.05).AT-H could increase the embryo implantation rate,decrease the content of PGF_2?and increase the content of P₄,and promote the development of ovarian corpus luteum.There was no significant difference in the expression of St AR between CON group and CON group.It is suggested that AT-H can promote the development of corpus luteum and increase the level of progesterone in blood,so as to alleviate the adverse effects of threatened abortion and reduce the rate of abortion.(3)Study on the fetal protective effect of AT-? on threatened abortion feline modelAglepristone(AGL)of 1,3 and 6 mg/kg was used in the early pregnancy of domestic cats.According to the clinical signs,gestational sac tension,fetal heart activity,vaginal smear and serum progesterone level,the optimal dose for constructing the threatened abortion model of domestic cats was screened.Twenty cats in early pregnancy were randomly divided into normal pregnancy group(CON,n=5),threatened abortion model group(MOD,n=5),progesterone group(PRO,n=5)and atractylenolide group(AT-?,n=5).On the 19 th day of pregnancy,AGL was administered orally at a dose of 3 mg/kg for 6 consecutive days and on the 25 th day of pregnancy.The clinical signs of cats in each group were observed.The gestational sac tension and fetal heart activity were monitored by ultrasound every 5 days until the end of pregnancy,and the final number of live fetuses was recorded.On the 28th day of pregnancy,the serum of cats was collected and the contents of PGF_2?,P₄,E₂and LH were detected by ELISA.Metabolomics analysis of MOD group and AT-? group was performed by LC-MS non-target metabolomics detection technology.The results showed that the fetal gestational sac tension in the 3 mg/kg AGL group was poor,and the fetal heart activity decreased.There were more red blood cells and inflammatory cells in the vaginal smear.The serum progesterone was lower than the average value of progesterone required to maintain pregnancy,which met the criteria for threatened abortion and could be used to construct the threatened abortion model in cats.Compared with the MOD group,the state of abdominal pain in the AT-? group was improved,the appetite was increased,the P₄and LH contents in serum were significantly increased,and PGF_2?was increased.(4)Effect of AT-? on the secretion of estrogen and progesterone in ovarian granulosa cells(FOGCs)of domestic cats and transcriptome studyPrimary feline ovarian granulosa cells were cultured and identified by morphological observation and FSH receptor specific immunofluorescence staining.CCK-8 method was used to determine the growth law and growth curve of FOGCs,and the aging and dose-effect curves of FOGCs under AT-? action.The effect of AT-? on the secretion of P₄and E₂in FOGCs was detected by ELISA.RNA-seq sequencing technology was used for transcriptome analysis of FOGCs control group and FOGCs group treated with AT-?.The results showed that 137 differentially expressed genes(DEGs)were obtained,of which 49DEGs were significantly up-regulated and 88 DEGs were significantly down-regulated.GO(Gene Ontology)enrichment analysis showed that 52 GO terms were enriched throughout the differentiation process.The results of KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis showed that the differentially expressed genes were successfully annotated to 155 signaling pathways,with 23 significantly different signaling pathways(P<0.05).GO and KEGG enrichment analysis showed that the significant signaling pathways related to AT-? promoting FOGCs proliferation and hormone secretion were cholesterol metabolism and ovarian steroid hormone synthesis signaling pathway.From this study,we concluded that:(1)AT-? had no obvious toxicity,and it was safe and reliable for domestic cats to use AT-? at the recommended dose.(2)AT-? can prevent and treat threatened abortion in mice and cats by regulating lipid metabolism to improve luteal function and increase progesterone levels in blood.(3)The mechanism of AT-? was explored based on RNA-seq sequencing technology,and the main signaling pathways related to AT-? promoting the secretion of estrogen and progesterone in FOGCs were cholesterol metabolism pathway and ovarian steroid hormone production pathway.