The Effect And Mechanism Of Artemisinin On Milk Metabolites In Dairy Cows

In order to study the effect of Artemisinin(ART)on milk metabolites of dairy cows and its regulation mechanism,in this experiment,Artemisinin of suitable concentration was fed in vivo to detect the changes of dairy production performance,milk metabolites and metabolic pathway.At the same time,different concentrations of artemisinin were added to cow mammary epithelial(bMECs),The effects of bMECs activity,intracellular lipid droplet synthesis,PI3K-AKT-mTOR and AMPK-mTOR signaling pathway on the expression of mRNA and protein related to milk fat synthesis genes were studied to study their effects on the regulation of milk fat synthesis.The results showed that:(1)Through the in vivo feeding test:12 healthy Holstein cows with similar parity,body weight,lactation volume and lactation days were used,were randomly divided into 2 groups and each 6 head/group was fed for a feeding trial.Control group(CON)was fed with a totally mixed diet(TMR).test group(ART)TMR was added with 120 g/d of Artemisinin.In the early feeding 10 days trial period of 35 days.Milk samples were collected at 0,14 and 28 days after the start of the official period(mixed at 4:3:3 in the morning,middle and evening),collected three times.Milk samples were analyzed by milk composition and metabolomics,respectively.The results show that,Compared with the CON,the average milk yield of dairy cows fed 120g/d Artemisia increased by 3.27%which significantly increased the milk fat correction yield and milk fat ratio(P<0.05),and the number of SCC in dairy cows decreased to a certain extent(0.05<P<0.1),which decreased 18.95%.The results of milk metabolomics showed that the milk metabolites of Artemisinin group and control group were different by PCA and OPLS-DA analysis.A total of 33 significantly different metabolites were detected in this assay(VIP>1,P<0.05).The main metabolites were glycerophospholipids and glycerol lipids,followed by flavonoids metabolites.The results of KEGG pathway enrichment analysis of different metabolites showed that compared with the control group,the addition of Artemisinin mainly affected lipid metabolism in milk of dairy cows,which may be significantly down-regulated by inhibiting the increase of lysophospholipid,phosphatidylcholine,α-linolenic acid metabolism,linoleic acid metabolism,glycerol phospholipid metabolism(P<0.05).thus affecting the metabolic pathway of lipids in dairy milk.(2)Activity of bMECs and the synthesis of lipid droplets:The effect of different concentrations of Artemisinin(10,20,40,60,80,100μM)on bMECs activity after co-culture with bMECs for 1,3,6,9,12,24 h by MTT,red O staining and TG kit.Compared with the CON,Artemisinin was co-cultured with bmecs for 12h as the best action time,of which 20～80μM of Artemisinin were suitable for addition range.(3)Signaling pathway genes:detection of key genes related to milk fat synthesis in two signaling pathways of PI3K-AKT-mTOR and AMPK-mTOR by qRT-PCR and Western-blot,To study the molecular mechanism of Artemisinin on bMECs milk synthesis.The results showed that 40μM Artemisinin and bMECs co-cultured for 12 h significantly up-regulated the transcription levels of the key genes AMPK、mTOR、PPARγ、SREBP1-c、ACC、SCD1、FASN、PI3K、AKT1、CyclinDl(P<0.05),whereas at the translation level,the addition of 40μM Artemisinin significantly up-regulated the expression of AMPK、mTOR、p-mTOR、PPARγ、SREBP1-c(P<0.05).Conclusion:By adding 120 g/d of Artemisinin,the lipid metabolism pathway of dairy cow mammary gland was affected,the synthesis of fatty acid was promoted,and the milk fat rate was increased.40μM Artemisinin can up-regulate the expression of milk protein,milk fat and cell proliferation related genes and improve the lactation ability of bMECs.Artemisinin promotes milk lipid synthesis by activating AMPK-mTOR signaling pathway and increasing protein level expression such mTOR、PPARγ、SREBP1-c.