The Effect Of Prolactin Resistant On Lactoprotein Decrease Induce By LPS In BMEC

Mastitis is recognized as one of the three major diseases of dairy cows in the world.It causes high economic losses in dairy industry every year.This inflammatory disease leads to a decrease in milk production and milk quality.However,current domestic and foreign research mostly focuses on the treatment of mastitis and the screening of anti-inflammatory drugs,neglecting the lactation problem of dairy cows after the appearance of mastitis.Therefore,after the occurrence of mastits,the recovery of lactation ability is crucial for the treatment of mastitis.Studies have shown that suppressor of cytokine signaling 3(SOCS3)can inhibit the activation of JAK2-STAT5 signaling pathway,and prolactin(PRL)mainly promotes milk secretion by activating JAK2-STAT5 signaling pathway.However,the existence of a mechanism for the mutual regulation of PRL and SOCS3 is still unclear.The purpose of this study is to isolate and culture primary bovine mammary epithelial cells(BMECs)and establish a lipopolysaccharides(LPS)-induced inflammation model in BMECs.Based on the inflammation model,the follow-up experiment was carried out to explore the interaction of PRL and SOCS3 in BMECs.Experimental contents:In order to determine the optimal concentration of LPS,PRL and SOCS3 when stimulating BMECs,different concentrations of LPS,PRL and SOCS3 were used to treat BMECs respectively.After that,quantitative real-time PCR(q RT-PCR)was used to detect the relative expression of related cytokines,and microscope was used to observe changes in BMECs morphology.To clarified the effects of PRL,LPS and SOCS3 on casein beta(β-casein,CSN2)synthesis signaling pathway and SOCS3,BMECs were divided into three groups: stimulated with 25 ng/m L PRL,stimulated with 10 μg/m L LPS and transfected with 8.5 μL/m L SOCS3.Then,BMECs were collected at different treatment time,the m RNA relative expression and protein expression of SOCS3,Janus Kinase 2(JAK2),signal transducer and activator of transcription 5(STAT5)and CSN2 were detected by q RT-PCR and western blot,respectively.To clarify the role of PRL in resisting the decrease of milk protein synthesis in LPS-induced BMECs,the experiment was divided into two groups:(1)adding 8.5 μL/m L COCS3 to BMECs,followed by adding 125 ng/m L at different time points;(2)stimulating BMECs with 125 ng/m L PRL for 6 hours,followed by adding 10 μg/m L LPS at different time points.the m RNA relative expression and protein expression of JAK2,STAT5,SOCS3 and CSN2 in BMECs were detected by q RT-PCR and western blot,respectively.Experimental results:(1)The optimal concentration of LPS stimulating BMECs was 10 ug/mL,the optimal concentration of PRL stimulating BMECs was 125 ng/m L,and the optimal concentration of SOCS3 transfecting into BMECs was 8.5 μL/m L.(2)After the BMECs were stimulated by LPS and SOCS3,the expression level of SOCS3 was increased,while JAK2/STAT5 signaling pathway was inhibited,resulting in a decrease of CSN2 expression.After stimulation of BMECs with PRL,the expression of JAK2,STAT5 and CSN2 was significantly increased,and the expression of p-JAK2,p-STAT5 and CSN2 was also significantly increased,suggesting that PRL can promote the expression of CSN2 through JAK2-STAT5 signaling pathway.(3)After transfecting the SOCS3 plasmid into BMECs and adding PRL,The expression of JAK2,STAT5 and CSN2 was significantly increased and the expression of SOCS3 was significantly decreased as the prolonging of stimulation time,which indicated that PRL could antagonize SOCS3 to activate the JAK2/STAT5 signaling pathway and promote CSN2 expression.Adding LPS into BMECs after stimulated with PRL,the expression of JAK2,STAT5 and CSN2 was significantly down-regulated and the expression of SOCS3 was significantly up-regulated with the prolongation of LPS stimulation time.It indicates that LPS can antagonize the function of PRL by by promoting SOCS3 expression,which may be accomplished through JAK2/STAT5 signaling pathway.In conclusion,this experiment clarified the mechanism by which SOCS3 inhibits CSN2 synthesis through JAK2/STAT5 signaling pathway,and clarified the role of PRL in resisting the reduction of milk protein content in LPS-induced BMECs.