| The aim of this study was to investigate the effects of alcohol extract of Artemisia annuli L.on milk protein synthesis in lipopolysaccharide(LPS)induced injury of bovine mammary epithelial cells and its mechanism.The study consisted of four parts.First,a healthy model of bovine mammary epithelial cells was established,and then an inflammatory injury model of bovine mammary epithelial cells was established by inducing LPS.On this basis,the effects of alcohol extract of Artemisia annulus L.on milk protein synthesis and its mechanism of action were further explored.(1)Establishment of healthy model of bovine mammary epithelial cells.The healthy cell lines of bovine mammary epithelial cells were established by trypsin digestion and differential adherent digestion,and the third generation cells were obtained by passage,purification,freeze storage and resuscitation.The physiological activities of the cells were identified and detected by keratin 18 immunofluorescence and MTT assay.The results showed that the 3rd generation cells had strong activity,typical pebble shape,and high purity,which could be used for subsequent experiments.(2)Establishment of inflammatory injury model of bovine mammary epithelial cells induced by LPS.Breast epithelial cells were induced by LPS to establish a cell inflammation model.Cell viability and apoptosis rate were detected to screen the dosage and treatment time of LPS.The results suggested that the apoptosis rate of bovine mammary epithelial cells induced by LPS should be in the range of 30%-50%,and the apoptosis rates of 1 g/m L LPS group and 10 g/m L LPS group were 44.6% and43.96%,respectively.The survival rate of cells treated with different concentrations of LPS for 9 h and 12 h was significantly decreased compared with the control group(P< 0.05),but the decrease trend was more obvious after 10 g/m L LPS treatment for 12 h compared with 1 g/m L LPS group.Therefore,10 g/m L LPS induction treatment for12 h can be used to establish an ideal model of mammary epithelial cell inflammation.(3)Effect of alcohol extract of Artemisia annua L.on LPS-induced milk protein synthesis in bovine mammary gland cells.The experimental groups were as follows:control group: bovine mammary epithelial cells were conventionally cultured in basic medium for 15 h.LPS group: Bovine mammary epithelial cells were first treated with basic medium for 3 h,and then treated with 10 g/m L LPS for 12 h.Artemisia annua alcohol extract +LPS treatment group: firstly,basic medium containing different concentrations(3 g/m L,6 g/m L,12 g/m L)alcohol extract of Artemisia annua was treated for 3 h,and then 10 g/m L LPS was treated for 12 h.Cell cycle,amino acid level,total protein,β-casein and αS1-casein were detected.The results showed that compared with model group,3 g/m L,6 g/m L and 12 g/m L ethanol extract of Artemisia annulata could improve the activity of bovine mammary epithelial cells induced by LPS,promote cell mitosis,and have a certain pre-protective effect.The contents of aspartic acid(Asp)and glutamic acid(Glu)in 3 g/m L Artemisia annua treatment group were significantly increased(P < 0.05),and the content of alanine ALA was significantly decreased(P < 0.05).The concentrations of total essential amino acids(TEAA),total non-essential amino acids(TNEAA),essential amino acids(EAA),non-essential amino acids(NEAA)and total amino acids in 6 g/m L and 12g/m L ethanol extract groups were significantly increased(P < 0.05).These results indicate that the alcohol extract of A.annua can significantly improve the amino acid synthesis of LPS-induced damaged BMECs.Compared with model group,12 g/m L A.annua treatment group had significant preprotective effect,and the total protein content was significantly increased(P <0.05).The content of β-casein in 3 g/m L and 6 g/m L A.A.BMECs in 3 g/m L and 6g/m L A.A.BMECs was significantly higher than that in model group,and returned to the same level as that in control group(P > 0.05).The effect of 3 g/m L Artemisia annua treatment group was not significant(P < 0.05)αS1-casein content in 3 g/m L,6g/m L and 12 g/m L Artemisia annua groups was significantly higher than that in model group(P < 0.05).In conclusion,the alcohol extract of A.annua may increase the content of αS1 and β-casein in the injured BMECs to restore the total protein concentration to normal level.(4)Study on the mechanism of alcohol extract of Artemisia annua L.on milk protein synthesis of LPS-induced injury model.The experimental groups were the same as above.The expression levels of genes related to milk protein synthesis in breast epithelial cells were detected by real-time fluorescence quantitative PCR,and the contents of proteins related to milk protein synthesis in cells were detected by ELISA kit.The results showed that compared with the model group,the alcohol extract of A.annulis could significantly up-regulate the m RNA expression levels of m TOR,S6K1,JSK2,STAT5,CSN1S1,CSN2,4EBP1 and e IF4 E related genes of BMECs milk protein synthesis(P < 0.05).Compared with model group,the m TOR and S6K1 protein contents in cells were significantly increased by ethanol extracts of different concentrations(P < 0.05),and the protein contents of 4EBP1 and JAK2 were significantly increased by 6 g/m L and 12 g/m L ethanol extracts of A.annul(P <0.05).12 g/m L alcohol extract of Artemisia annua increased the protein content of STAT5(P < 0.05).These results indicated that the alcohol extract of A.annulis could up-regulate the expression of genes related to milk protein synthesis of BMECs,and increase the protein contents of m TOR,S6K1,e IF4 E,e IF4EBP1,JAK2 and STAT5 to a certain extent.In conclusion,the alcohol extract of A.annulis has a certain inhibitory effect on the decreased cell activity of LPS-induced inflammatory injury model,and may up-regulate the milk protein synthesis level of LPS-induced injured breast cells through m TOR/S6K1/4EBP1 signaling pathway and JAK2/STAT5 signaling pathway. |
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