Regulation And Mechanism Of Salidroside On Mastitis Of Dairy Cows

Mastitis causes decreased milk yield,changes in milk composition,apoptosis of mammary epithelial cells and impaired blood-milk barrier function of dairy cows,which has a negative impact on cows and even leads to the elimination of cows,bringing serious economic losses to the breeding industry.Our previous study found that Salidroside(SAL)can reduce inflammation of liver cells and alleviate liver damage,and has potential utilization value as an alternative to antibiotics in preventing mastitis.Therefore,the blood and milk samples of cows were firstly collected and the biomarkers related to mastitis were detected to evaluate the degree of inflammation and the integrity of the blood-milk barrier,which reflected the regulation effect of SAL on mastitis of cows.Subsequently,the protective effect of SAL on mammary gland was further explored through the mastitis models construction of mice and primary bovine mammary epithelial cells(BMECs),and the specific mechanism was explored through transcriptome analysis,which provided theoretical basis for exploring nutritional regulation means of cow mastitis and revealing its mechanism.Section 1:The application effect of SAL in cow productionHealthy dairy cows with 50～150 days of lactation were selected as control group(CON group),and clinical mastitis cows were randomly divided into mastitis group fed normal diet(CM group)and treatment group supplemented with 3%SAL(SAL group),4 g/d/cow.Each group had 10 cows.The experiment lasted for 40 days.The main experiment results are as follows:At the early stage of mastitis(Day 0),the levels of inflammatory biomarkers LDH(P<0.01),NAG(P<0.01)and blood-milk barrier integrity biomarkers Na~+/K~+(P<0.05)in CM and SAL groups with mastitis were significantly increased,while the changes of other biomarkers were not significant.Without intervention,the degree of mastitis increased with time,and the levels of SAA and IL-1βin serum of mastitis cows also showed a significant increase(P<0.01),while there was no significant difference in the direct indicators between the SAL group and the CON group at Day 40(P>0.05),indicating that the addition of SAL in TMR can alleviate mammary inflammation.Protect the integrity of the blood milk barrier.Section 2:Protective effect and mechanism of salidroside on mastitisIn the mouse experiment,BALB/c mice at 5 to 7 days of delivery were randomly divided into CON group,CM group and SAL group(n=8).The mastitis model of mice was established by injecting LPS into milk catheter.Serum and breast tissue were collected to evaluate the degree of inflammation and blood-milk barrier function.The cell experiment was grouped in the same way as the mouse experiment.Mastitis of BMECs was induced by LPS.The secretion of inflammatory cytokines and cell apoptosis were detected after the experiment.And transcriptome sequencing was performed.The main experiment results are as follows:(1)In the mouse experiment,the mammary gland tissue of CM group showed redness,swelling,congestion and inflammatory cell infiltration,while these symptoms were improved in SAL group.Compared with CON group,the levels of pro-inflammatory cytokines TNF-α,IL-1βand IL-6 in mammary gland tissue of mice in CM group were significantly increased(P<0.01),and the expressions of tight junction proteins ZO-1,Claudin-3 and Claudin-5 in mammary tissue of mice were significantly decreased(P<0.01).The expression of Occludin decreased significantly(P<0.05).The level of proinflammatory cytokines in SAL group was significantly lower than that in CM group(P<0.01),the expression of Occludin and Claudin-3 was significantly higher than that in CM group(P<0.05),and the expression of ZO-1 and Claudin-5 was significantly higher than that in CM group(P<0.01).The results showed that SAL could alleviate breast inflammation and protect the integrity of the blood-milk barrier.(2)In the cell experiment,10μg/mL LPS and 100μmol/L SAL were selected as experimental concentrations according to cell viability.The relative m RNA expression levels of TNF-α,IL-1βand IL-6 in CM group were significantly higher than those in CON group(P<0.01),and the apoptosis rate was significantly higher than that in CON group(P<0.01),while the above indexes in SAL group were significantly lower than those in CM group(P<0.01),indicating that SAL could alleviate mammary gland inflammation and the apoptosis induced by mastitis was improved.(3)Transcriptome sequencing and analysis of CM group and SAL group showed that the protective effect of SAL on LPS-induced mastitis may be related to several signaling pathways,including classical inflammatory signaling pathways MAPK and NF-κB,apoptosis related pathway PI3K/AKT and oxidative stress related pathway FoxO.In addition,signal transduction processes such as EMC receptor communication,tight junction and adhesive junction,which are related to cell-cell communication,movement and adhesion,may play an important role in the regulation of SAL against mastitis.In conclusion,mastitis leads to mammary tissue inflammatory injury and blood-milk barrier function impairment in dairy cows.SAL comprehensively regulates several signaling pathways such as MAPK,NF-κB,PI3K/AKT and FoxO to inhibit the secretion of pro-inflammatory cytokines,relieve mammary tissue inflammatory injury,protect the integrity of blood-milk barrier,and have a protective effect on mastitis.