The Mechanism Of IFN-γ-induced Arginine Depletion Mediated By CARNS1 In Bovine Mammary Epithelial Cells

IFN-γ is a pleiotropic cytokine that plays an important role in regulating the body’s immune and nutritional metabolism.The research team found earlier that daily feeding of cow corn stalk feed can increase the level of IFN-γ in the body.The increased IFN-γ further induces the depletion of arginine in bovine mammary epithelial cells(BMECs)and becomes Follow-up cows’ lactation ability,cell malignant proliferation and other causes of a series of diseases.However,the mechanism of IFN-γ-induced BMECs arginine depletion is unknown.Therefore,based on the in vitro culture of MAC-T cell lines,this study studied the mechanism of IFN-γ induced arginine depletion from two aspects,namely,arginine transport and metabolism,by referring to the transcriptome data of IFN-γ on BMECs and the control group.This study enriched the molecular mechanisms of nutrition and immunity,and provided a certain theoretical basis for improving the lactation ability and disease resistance of cows.First,in order to determine the effect of IFN-γ on extracellular arginine transport into the cell,this study used fluorescence quantitative PCR and ELISA to detect the expression changes of molecules involved in arginine transport after IFN-γ action.The results showed that IFN-γ can inhibit the transport of extracellular arginine into the cell by reducing the expression of CAT-1.In addition,the research team found earlier that IFN-γ can inhibit ASS1 activity involved in arginine synthesis.On the basis of this study,ELISA method was used to enrich the detection of the effect of IFN-γ on the activity of enzymes related to arginine metabolism,and through fluorescence quantitative PCR and Western blot,it was further found that IFN-γ inhibited ASS1 expression in MAC-T cells.In order to further study the mechanism of IFN-γ-induced BMECs arginine depletion,this study referred to the transcriptome data made by the research group in the previous period,and screened for differentially expressed genes(DEGs)involved in arginine metabolism after IFN-γ action.Quantitative PCR confirmed DEGs.In order to determine the correlation between the screened genes and the intracellular arginine content,our study by transfecting the interfering plasmids of the corresponding genes into the cells and determining their inhibitory effects,the intracellular arginine was detected by high performance liquid chromatography mass spectrometry Acid content.The results showed that CARNS1,CARS2,and LAP3 are involved in IFN-γ-induced intracellular arginine depletion.In addition,quantitative PCR and immunofluorescence showed that blocking IFNGR2 by antibodies or inhibiting the p38 signaling pathway with p38 MAPK inhibitors can significantly reduce the effects of IFN-γ on CARNS1 and ASS1.Finally,through RNAi technology,quantitative PCR,Western blot,and ELISA and other experimental techniques,it was found that knockdown of CARNS1 can significantly inhibit the activity and expression of ASS1.In summary,this study found that the increase in IFN-γ caused by daily feeding of corn straw feed to dairy cows can inhibit the endogenous synthesis of arginine through the IFNGR2-p38-CARNS1-ASS1 pathway and induce arginine depletion in BMECs.This study revealed the mechanism of IFN-γ-induced arginine depletion of BMECs,which provided a theoretical basis for the treatment of various diseases caused by arginine depletion.