Study On Effect Of Serotonin On Mammary Gland Involution In Dairy Ruminant And Its Mechanism

The mammary gland infection of modern high-yielding dairy cows seriously restricts the sustainable development of dairy farming and affects the economic benefits of the dairy farming industry.Promoting the mammary gland involution of dairy cows during the dry period can reduce the incidence of intramammary infection during the dry period,promote the repair and renewal of the mammary glands,and reduce the incidence of mastitis in the next lactation.Previous studies have found that 5-hydroxytryptamine(5-HT)could promote mammary gland involution during the early dry period of dairy cows.Therefore,the current study was firstly to clarify the promoting effect of 5-HT precursor on ruminant mammary gland involution by feeding rumen protected 5-hydroxytryptophan(5-HTP)to goat.Then,the cow mammary epithelial cell MAC-T was used to further explore the mechanism underlying the regulation of 5-HT on mammary gland involution.The research results can provide a new method for the regulation of 5-HT levels in ruminants,and provide new ideas for the prevention of cow mastitis.Experiment 1: 16 adult healthy goats were selected with similar body condition,weight about 30±5.2kg,age 3-4 years,and randomly divide them into 4 groups,with 4repetitions in each group.The rumen-coated 5-HTP was added to the concentrate at 9:00a.m.every day,and the feeding amounts were 0,4,20,100 mg/kg BW for each group.Goats had ad libitum access to feed and water.The behavioral characteristics of the goats in each group were monitored,and the blood of the goats in each group was collected at0,3,6,12,and 24 h after feeding 5-HTP.Serum 5-HTP,5-HT,5-hydroxyindoleacetic acid(5-HIAA)concentrations were detected.Results 1: Serum treated groups fed rumen protected 5-HTP relative to controls: 5-HTP reached the highest level at 6 h,5-HT reached the highest level at 12 h,and 5-HIAA reached the highest level at 12 h.The highest level was reached at 24 h.Feeding low doses of 5-HTP(4,20 mg/kg BW)could have a significant sedative effect on goats(P < 0.05);the serum alkaline phosphatase activity in the 100 mg/kg BW treatment group increased significantly(P < 0.05),alanine aminotransferase activity was significantly decreased(P< 0.05).The results showed that rumen-coated 5-HTP could effectively increase the level of 5-HT in goats,but high concentrations of 5-HTP might cause liver damage in animals.Experiment 2: 16 healthy end-lactating goats(weight 35±5kg)with similar body condition were selected(6-8 parities)and randomly divide them into 2 groups,with 8repetitions in each group.Each goat was fed alone at the same concentrate level(0.8kg/d),and had ad libitum access to silage.From the second day of drying off,0 mg/kg BW and20 mg/kg BW rumen-coated 5-HTP were added to the concentrate at 9:00 am,respectively,and rumen-coated 5-HTP was fed continuously for 5 days at night.Goat were milking at 4 p.m.every day.Blood was collected in the morning of day 5.The roughage intake of goats was recorded,and the somatic cell count(SCC),lactoferrin level,sodium-potassium ion ratio,serum albumin concentration and lactate dehydrogenase activity in milk were detected.Results 2: Rumen-coated 5-HTP increased SCC,lactoferrin levels,sodiumpotassium ion ratio,serum albumin concentration and lactate dehydrogenase activity in milk(P < 0.05);The forage intake of goats in the 5-HTP fed group decreased from the3 rd day onward(P < 0.05).The results showed that 20 mg/kg BW rumen-coated 5-HTP could promote mammary gland involution in goats during early dry period.Experiment 3: MAC-T was cultured in vitro with various concentrations of 5-HT(0,0.2,2,20,200 μg/m L)for 24 h and intracellular 5-HT concentration were detected;the cells were treated for 24 h and 72 h to detect proliferation and transmembrane electrical resistance values of intercellular tight junctions.Then,0,2,200 μg/m L of 5-HT cultured cells for 24 h were subjected to transcriptome analysis and RT-PCR analysis of relevant gene expression.Finally,cells were treated with MDC,an inhibitor of the TG-2 enzyme,the groups were as follows: control(0 μg/m L),2(2 μg/m L 5-HT),200(200 μg/m L 5-HT),MDC(200 μM MDC),2 + MDC(2 μg/m L 5-HT + 200 μM MDC),200 + MDC(200 μg/m L 5-HT + 200 μM MDC)for 24 h,and cell proliferation activity,transmembrane electrical resistance values of intercellular tight junctions,and the expression of related genes were detected.Results 3: 2 、 20 μg/m L 5-HT could significantly increase intracellular 5-HT concentration in MAC-T(P < 0.05);0.2、2、20 μg/m L 5-HT treatment for 24 h could promote cell proliferation(P < 0.05),200 μg/m L treatment inhibited cell proliferation and decreased cell viability(P < 0.05);transmembrane electrical resistance was significantly decreased in 5-HT treated cells(P < 0.05).Through GO functional annotation and KEGG pathway enrichment analysis of the differentially expressed genes,the mechanisms causing mammary gland involution by 5-HT may be related to necroptosis pathway,NOD-like receptor signaling pathway,focal adhesion and PI3K-Akt signaling pathway.H2AC19,STAT family,PYGM,SQSTM1,interferon-α2,interferon-β2,IFI16 may play an important role in serotonin induced mammary gland involution.RT-PCR showed that0.2,2 μg/m L 5-HT increased TG-2 expression(P < 0.05),200 μg/m L 5-HT inhibited TG-2 expression(P < 0.05).200 μg/m L 5-HT treatment significantly increased the expression of TGF-β1 and Caspase-3(P < 0.05),and significantly decreased the expression of cellular tight junction proteins Claudin-1,Claudin-4,and Claudin-8(P < 0.05).Experiment 4: Finally,cells were treated with MDC,an inhibitor of the TG-2 enzyme,the groups were as follows: control(0 μg/m L),2(2 μg/m L 5-HT),200(200 μg/m L 5-HT),MDC(200 μM MDC),2 + MDC(2 μg/m L 5-HT + 200 μM MDC),200 + MDC(200 μg/m L 5-HT + 200 μM MDC)for 24 h,and cell proliferation activity,transmembrane electrical resistance values of intercellular tight junctions,and the expression of related genes were detected.Results 4: Treatment with MDC for 24 h had no effect on cell viability(P > 0.05)and treatment for 72 h significantly inhibited cell proliferation(P < 0.05).5-HT and MDC treatment significantly inhibited MAC-T cell viability(P < 0.05).After MDC treatment,the transmembrane electrical resistance increased on the first day and then decreased significantly(P < 0.05);Transmembrane electrical resistance decreased significantly in all other treatment groups(P < 0.05).In RT-PCR results,the expression of tight junction related genes decreased in the 2+MDC group(P < 0.05),the expression of Claudin-1 in the 200+MDC group was significantly increased(P < 0.05).Compared with the control group,the expression of TGF-β1 in the 200+MDC group was significantly decreased(P< 0.05),and the MDC,2,200,and 2+MDC treatment groups were significantly increased(P < 0.05).In conclusion,it is feasible to feed ruminant ruminant-protected 5-HTP to increase the level of 5-HT in vivo,and 20 mg/kg BW of rumen-transmitted 5-HTP can significantly promote mammary gland involution in ruminants during early dry period.200 μg/m L 5-HT can inhibit the activity of MAC-T and increase the permeability of tight junctions between cells.Transcriptome results showed that 5-HT could affect the expression of genes related to apoptosis-related pathways,PI3K-Akt-TGF-β1 and cell cycle signaling pathways.The above signaling pathways may play a role in the process of 5-HT promoting apoptosis and mammary gland involution.Serotonization may range from 200 μg/m L 5-HT caused apoptosis process and the regulation of tight junction protein claudin-1 plays an important role.