Screening Of Key Proteins And Study On Its Action Mechanism Of Dairy Cows With Postpartum Anestrus Caused By Subclinical Ketosis

Subclinical ketosis（SCK）is a common nutritional and metabolic disease during the transition period in high-yield dairy cows.Although the estrus synchronization and timed-AI technology has been widely used in scale-up pastures,some SCK cows still lack estrus manifestations after the estrus synchronization and timed-AI treatment.Therefore,the increased prevalence of anestrus induced by SCK in high-yield dairy cows has become an urgent problem to be solved in the field of metabolism and reproduction at home and abroad.This current study focuses on this practical clinical problem.In vivo experiments were conducted by tracking and detecting plasma metabolites,hormones,liver function indexes,and the largest follicle diameter and proteomics analysis of liver tissues in SCK-anestrus（SCK-A）cows.Subsequently,the effects of a key differentially expressed protein（DEP）retinol binding protein 4（RBP4）on proliferation,apoptosis,and steroid hormone synthesis of bovine follicular granulosa cells（FGCs）cultured under non-esterified fatty acids（NEFA）were explored in vitro.This study aimed to（1）clarify the relationship between metabolic and hormonal disorders,and liver function injury and SCK and postpartum anestrus.（2）To clarify the proteomic change characteristics of SCK-A cows and the relationship between RBP4 and SCK and postpartum anestrus,as well as its role in early warning and monitoring of postpartum anestrus in SCK cows.（3）To reveal the molecular mechanism of RBP4affecting the follicular growth and development in dairy cows.To provide theoretical basis for future research on the mechanism of SCK-induced anestrus in dairy cows.（1）In this experiment,61 healthy control（HC）cows[β-hydroxybutyrate（BHBA）<1.20 m M]and 57 SCK cows（1.20 m M≤BHBA≤2.90 m M,and no clinical symptoms of ketosis）were investigated during the transition period.The plasma metabolites,hormones,liver function indexes,and the largest follicle diameter were tracked and detected in HC-estrus（HC-E）cows（n=15）and SCK-A cows（n=15）.Data were analyzed by independent sample t-test,chi-square test,binary logistic regression analysis,and repeated measures analysis of variance,where applicable.The results showed that the SCK cows in this experiment presented the characteristics of excessive negative energy balance,excessive mobilization of adipose tissue,and lower milk yield.Compared with HC cows,the calving to first estrus interval,estrous cycle,calving interval,calving to conception interval,and number of services per conception in SCK cows were significantly increased（P<0.01）,estrus rate and conception rate was significantly decreased（P<0.01）,and these had a 5.641-fold increased risk of postpartum anestrus（OR=6.641,P=0.023）.On day 21postpartum,compared with HC-E cows,SCK-A cows had greater concentrations of plasma BHBA,NEFA,triglyceride,aspartate aminotransferase,alanine aminotransferase,γ-glutamyl transferase,and total bilirubin and lesser concentrations of plasma insulin,insulin-like growth factor-1,insulin-like growth factor binding protein-3,glucose（Glu）,total cholesterol,albumin,and total protein（P<0.05）.These changes persisted until 55 to 60 days postpartum（P<0.05）,resulting in lower plasma estradiol（E₂）and progesterone（P₄）levels and a lower follicular growth rate for cows in the SCK-A group than the HC-E group（P<0.05）.Histological analysis exhibited that there was remarkable hepatic steatosis in SCK-A cows.（2）In this experiment,the liver tissues of HC-E cows（n=6）and SCK-A cows（n=6）were analyzed by TMT proteomic analysis,combined with bioinformatics analysis and statistical analysis.The results showed that 39 DEPs were downregulated and 41 DEPs were upregulated in liver tissues of cows in SCK-A group compared with those in HC-E group（FC>1.2,P<0.05）.The down-regulated DEPs are mainly concentrated in tryptophan metabolism,metabolism of xenobiotics by cytochrome P450,and glycolysis/gluconeogenesis pathways.Up-regulated DEPs are mainly concentrated in mineral absorption,autoimmune thyroid disease,and typeⅠdiabetes mellitus.Three common DEPs（RBP4,transthyretin,and serum amyloid P component）were identified by Veen analysis and were significantly downregulated in liver tissue,follicular fluid,and ovarian tissue in SCK-A cows（P<0.05）.Moreover,the western blotting and ELISA results of the common DEPs were consistent with the trend of their proteomic results（P<0.01）,which verified the accuracy of the omics data.Similarly,the plasma ELISA results of the common DEPs were consistent with the trend of their proteomic result,and significantly decreased in SCK-A cows during the transition period（P<0.01）,which continued into the breeding period（P<0.05）.Person correlation analysis showed that plasma RBP4 levels at 21 days postpartum were significantly negatively correlated with SCK during the transition period（higher BHBA and NEFA and lower Glu levels）（P<0.05）.The plasma RBP4 levels at 55 days postpartum were significantly positively correlated with postpartum anestrus（lower E₂ and P₄ levels,and follicle growth rate）（P<0.05）.ROC analysis confirmed that the plasma RBP4≤45.2μg/m L at 21 days postpartum and the plasma RBP4≤53.0μg/m L at 55 days postpartum could be used for early warning and monitoring of postpartum anestrus in cows with SCK,respectively.（3）In vitro,FGCs isolated from dairy cows were transfected with RBP4 knockdown adenovirus（Ad-sh RBP4）,combined with immunofluorescence,q RT-PCR,western blotting,CCK8,flow cytometry,and radioimmunoassay,and one-way analysis of variance.The results showed that compared with the negative control（Ad-GFP）group,the cell viability of FGCs,the contents of E₂ and P₄ in supernatant,and the protein expression levels of cyclin（Cyclin D1 and CDK4）,anti-apoptotic protein Bcl-2,steroid hormone synthetases（St AR,CYP11A1,HSD3B1,and CYP19A1）,and key proteins of PI3K/Akt and Erk1/2 signaling pathways（p-PI3K,p-Akt,and p-Erk1/2）in FGCs in the Ad-sh RBP4 group were significantly decreased（P<0.05）,while apoptosis rate of FGCs,protein expression levels of cell cycle suppressor proteins（P27 and P21）and pro-apoptosis proteins（Bax,Cytochrome c,and Cleaved caspase-3）,and the Bax/Bcl-2 ratio were significantly increased（P<0.05）.These results indicated that RBP4 could regulate FGCs proliferation,apoptosis,and steroid hormone synthesis in dairy cows.In vitro,bovine FGCs were treated with 0,0.6,1.2,and 1.8 m M NEFA.The results showed that NEFA reduced RBP4expression in FGCs and subsequently showed the same result as after transfection with Ad-sh RBP4.After transfection with RBP4 overexpressed adenovirus（Ad-RBP4）,1.2 m M NEFA was added.The results showed that overexpression of RBP4 alleviated the effects of NEFA on FGCs proliferation,apoptosis,and steroid hormone synthesis.These results suggested that NEFA inhibited RBP4 expression,subsequently inhibited FGCs proliferation and steroid hormone synthesis,and induced FGCs apoptosis.After transfection with Ad-RBP4,PI3K inhibitor（20μM LY294002）and MEK inhibitor（50μM PD98059）were added,respectively,and 1.2 m M NEFA was added.The results showed that the addition of LY294002 and PD98059 attenuated the remission of negative effects of RBP4 overexpression on NEFA,including inhibition of FGCs proliferation and steroid hormone synthesis,and induction of FGCs apoptosis.In conclusion,SCK during the transition period is an important risk factor for postpartum anestrus in dairy cows.The metabolic and hormonal disorders and liver injury caused by it will continue into the breeding period,inhibiting the follicular growth and development and thus leading to the occurrence of postpartum anestrus in dairy cows.Moreover,the continuation of liver injury changes the liver proteome of anestrus cows.Among which,circulating RBP4 levels were decreased,and were significantly negatively correlated with SCK during the transition period,and significantly positively correlated with postpartum anestrus,which could be used for early warning and monitoring of postpartum anestrus of cows with SCK.In addition,RBP4 regulates FGCs proliferation,apoptosis,and steroid hormone synthesis through PI3K-Akt and Erk1/2 signaling pathways,thus regulating follicular growth and development.