Sodium Butyrate through G Protein-Coupled Receptor 41 Mediated Protein Kinase B/Mammalian Target of Rapamycin Signaling Pathway to Promote Proliferation of Bovine Mammary Epithelial Cells
This study was to determine the molecular mechanism of sodium butyrate(SB)stimulated the pro-liferation of bovine mammary epithelial cells(BMECs).A single factor experimental design was used to cul-ture BMECs in DMEM/F12 medium(including 10％fetal calf serum)containing different concentrations(0,15,30,45,60 and 75 µmol/L)of SB,the cell activity was determined by cell count kit(CCK-8),and the optimal SB concentration was measured.Then the BMECs were cultured at control(0 μmol/L)and optimal SB concentration,and the signaling pathway and receptor were treated by protein kinase B(AKT)blocker(AKT-IN-1),mammalian target of rapamycin(mTOR)blocker rapamycin(Rap)or small interfering RNA(siRNA)silencing G protein-coupled receptor 41(GPR41),the proliferation and apoptosis of BMECs and the expression of genes and proteins related to GPR41 and Akt/mTOR signaling pathways were detected.The results showed as follows:1)compared with the control group,60 μmol/L SB significantly increased the cell viability of BMECs(P＜0.05),however,75 µmol/L SB significantly inhibited the cell viability of BMECs(P＜0.05);60 μmol/L SB significantly increased the mRNA relative expression levels of proliferating cell nu-clear antigen(PCNA),cyclin A2(CCNA2)and cyclin D1(CCND1)(P＜0.01),and significantly increased the protein relative expression levels of PCNA and cyclin A1(CCNA1)(P＜0.05).2)Compared with the control group,60 µmol/L SB significantly increased the mRNA and protein relative expression levels of B-cell lymphoma 2(BCL2)and the ratio of BCL2/B-cell lymphoma 2 associated X protein(BAX)(P＜0.05 or P＜0.01),significantly decreased the mRNA and protein relative expression levels of BAX,cysteine aspartic acid specific protease-3(Caspase-3)and cysteine aspartic acid specific protease-9(Caspase-9),and significantly increased the phosphorylated protein kinase(p-Akt)/Akt and phosphorylated mammalian target of rapamycin(p-mTOR)/mTOR ratios(P＜0.05 or P＜0.01).Moreover,the activation of Akt and mTOR signaling path-ways caused by 60 µmol/L of SB were obstructed by AKT-IN-1(P＜0.01),and the suppression of mTOR with Rap completely reversed the 60 μmol/L SB modulated promotion of BMECs proliferation and the altera-tion of proliferous genes and protein expressions(P＜0.05 or P＜0.01),but not affected on the mRNA or pro-teins expression related to apoptosis and SB activated Akt signaling pathway(P＞0.05).3)Compared with the control group,60 μmol/L SB significantly increased the mRNA relative expression level of GPR41(P＜0.01),and significantly increased the protein relative expression level of GPR41(P＜0.05).After silencing GPR41 with siRNA completely reversed the stimulative effect of 60 μmol/L SB on BMECs proliferation,and mRNA or protein expression of proliferating genes and proteins associated with the Akt/mTOR signaling path-ways(P＜0.05 or P＜0.01).It is concluded that the proliferation of BMECs is stimulated by 60 μmol/L SB through the GPR41 mediated Akt/mTOR signaling pathways.[Chinese Journal of Animal Nutrition,2024,36(3):1878-1891]
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