Effect Of GABA On The Proliferation And Apoptosis Induced By LPS In MAC-T Cell Line

Endogenous lipopolysaccharide(LPS)produced by subacute ruminal acidosis(SARA)can activate the hypothalamic-pituitary-adrenocortical axis(HPA),induce immunological stress,promote apoptosis of mammary epithelial cells and reduce milk production.Gamma-aminobutyric acid(GABA)can regulate the HPA axis to reduce the inflammatory response,improve the body’s immunity and production performance.Whether GABA can modulate the immune stress induced by LPS and improve the milk performance of SARA damage is unclear.The proliferation and apoptosis of mammary epithelial cells play an important role in the development of mammary glands and maintenance of milk production performance.This study investigated the effect of GABA on LPS-induced proliferation and apoptosis of breast mammary epithelial cells(MAC-T)at the cellular level.Firstly,we used MTT,CCK-8 and LDH to detect the effect of GABA on LPS induced proliferation of MAC-T cells.Secondly,RT-PCR,Western blot and flow cytometry were used to detect and analyze the changes of inflammatory response and apoptosis signal,and evaluate the effect of GABA on LPS induced inflammatory response and apoptosis of MAC-T cells.Finally,MAC-T cells were treated with GABA receptor inhibitor(CGP35348)and PI3 K inhibitor(LY294002),based on the changes of inflammatory molecules and apoptotic signals to evaluate the molecular mechanism of GABA regulating inflammatory response and apoptosis of mammary gland cells,providing new ideas for improving the reduction of milk production caused by SARA in ruminants.The result is as follows:GABA can promote the proliferation of MAC-T cell and increase the cell viability induced by LPS: GABA can promote the proliferation of MAC-T cells,and depend on the concentration of GABA and the duration of action.It could significantly promote the proliferation of MAC-T cells when the cells were treated by 12 ~ 36 h with the concentration is 50 μmol/L of GABA.GABA could significantly increase the viability of MAC-T cells decreased by LPS and reduce the LDH release significantly induced by LPS.It suggests that GABA can promote LPS-mediated proliferation of MAC-T cells and reduce cell mortality.GABA attenuates LPS-induced MAC-T cell apoptosis: Annexin V FITC/PI results show that LPS caused the number of apoptotic cells increased,GABA can significantly reduce LPS-induced MAC-T cell apoptosis,GABA could not reduce the number of apoptotic cells induced by LPS in MAC-T cells after blocking the receptor effect of GABA.RT-PCR and Western blot results showed that LPS increased the expression of pro-apoptotic genes Caspase3,Caspase4 and Bax,and decreased the survival gene’s expression level of Bcl-2.GABA could reverse the effect of LPS on apoptosis gene expression.GABA receptor antagonist significantly attenuates the effect of GABA to the expression of apoptosis gene on LPS-mediated MAC-T cell.It is suggested that GABA can attenuate LPS-induced MAC-T cell apoptosis.GABA inhibits LPS-induced MAC-T cell inflammatory response: RT-PCR results showed that LPS increased the transcription levels of inflammatory signaling molecules(TNF-ɑ,IL-6,IL-8)is obvious,while GABA significantly reduced the expression of inflammatory signaling molecules activated by LPS.Western blot results showed that LPS induced expression of IL-6 protein,GABA can reduce the increased effect that LPS-induced expression of IL-6 protein.It suggests that GABA inhibits LPS-induced MAC-T cell inflammatory response.GABA regulates LPS-induced MAC-T cell apoptosis through PI3K/AKT and TLR4/NF-κB pathways: RT-PCR results indicate that LPS significantly enhances the Transcription levels of key factors(TLR4,NF-κBp65,My D88,TRAF6,and IRF3)in TLR4/NF-κB signaling pathway,but GABAergic,significantly decreased the expression of key factors and inflammatory signaling molecules of LPS-activated TLR4/NF-κB signaling pathway.Western blot results showed that LPS induced the expression increase of IRF3 protein,but GABA reduced the expression increase of IRF3 protein induced by LPS.PI3K/AKT pathway inhibitor LY294002 can increase inflammatory and apoptotic gene m RNA expression.It suggests that GABA regulates LPS-induced MAC-T cell apoptosis through PI3K/AKT and TLR4/NF-κB signaling pathways.