The Effect Of Mechanism Of Choline Against Apoptosis In Heat Stress-Induced Bovine Mammary Epithelial Cells

Heat stress is one of the important factors that affect the performance of dairy cows.Holstein cow is afraid of high temperature,the sudden increase of the external ambient temperature causes the cow to develop heat stress,reducing the cow’s feed intake,thus resulting in a cow’s milk production and milk quality decline,for the dairy cattle industry has a huge economic loss.Therefore,how to effectively alleviate heat stress in dairy cattle is now the focus of attention.Choline,as a nutrient regulating osmotic pressure,has important functions to maintain normal cell morphology.A large number of studies have shown that choline has an antioxidant effect which can significantly improve the immune function of the body.However,the molecular mechanism of choline action on heat stress-induced mammary gland in dairy cattle is not clear at present.In addition,mammary epithelial cells are important functional cells in the synthesis of milk from the breast.The state of their cells is closely related to milk quality and milk production,indicating the physiological health state of dairy cows.Therefore,in this study,bovine mammary epithelial cell line（MAC-T）was used as a cell model for in vitro study of cow heat stress to explore the effect of choline on heat stress-induced apoptosis and oxidative damage in mammary epithelial cells,to elaborate the mechanism of choline inhibiting heat stress-induced apoptosis in mammary epithelial cells of cows,and to provide a theoretical basis for the comprehensive prevention and control of subsequent cow heat stress.Experiment 1 Effects of choline on proliferation and apoptosis of bovine mammary epithelial cells induced by heat stressHeat stress can lead to breast dysfunction.Many studies have shown that heat stress can reduce the proliferative activity of dairy cow mammary epithelial cells.The cell proliferation,apoptosis and gene expression of HSP70 and HSP90 were detected by CCK-8,flow cytometry,caspase-3 cell activity assay kit,q RT-PCR and Western blot.Results:the bovine mammary epithelial cells in the heat stress group appeared round and crumpled significantly compared with the normal control group,and cell viability significantly decreased,however,when the concentration of choline pretreatment was 100μmol·L^-1,cell proliferation significantly increased,therefore,the 100μmol·L^-1choline was chosen as the optimal choline pretreatment concentration according to the results of cell proliferation.In order to further explore the effect of choline on heat stress-induced apoptosis in dairy mammary epithelial cells,it was found that heat stress stimulation significantly increased the rate of apoptosis,and up-regulated the m RNA expression level of apoptosis marker Caspase-3,while choline pretreatment effectively alleviated heat stress-induced apoptosis and inhibited the m RNA expression of Caspase-3.The results of Caspase-3 cell activity assay kit were consistent with the above results.Increased levels of HSP70 family proteins in cells can inhibit the activation of stress-induced apoptotic pathways,thereby reducing cell apoptosis.The results showed that the expression of HSP70 and HSP90 in the heat stress group increased significantly compared with the control group,while choline pretreatment significantly reversed the above gene expression.To sum up,this study has confirmed that choline can slow down the apoptosis of dairy cow mammary epithelial cells induced by heat stress,but its mechanism is explained in the following chapters.Experiment 2 Mechanism of choline on heat stress-induced apoptosis of mammary epithelial cells in dairy cattleHeat stress is one of the important factors that leads to rapid accumulation of ROS,and excessive ROS is also a major factor that induces cells to undergo oxidative and endoplasmic reticulum stress,which leads to apoptosis.To investigate the mechanism of choline alleviating heat stress-induced apoptosis in bovine mammary epithelial cells,in this test,based on the grouping of test one,we investigated the effect of choline on heat stress-induced oxidative versus endoplasmic reticulum stress by immunofluorescence staining,Western blot and q RT-PCR.The results showed that the levels of ROS and MDA were significantly increased,and the activities of SOD and GSH-PX were significantly decreased in the heat stress group compared with the control group,meanwhile,the expression of GRP78 m RNA and protein were significantly increased,prompting perk to dissociate from GRP78 and obviously increase perk phosphorylation levels.However,choline pretreatment significantly reversed the above results.The above results indicated that choline pretreatment had antioxidant effect on heat stress-induced cow mammary epithelial cells and could inhibit the occurrence of heat stress-induced cow mammary epithelial cells endoplasmic reticulum stress.To further explore the mechanism by which choline protects against heat stress-induced apoptosis in the bovine mammary epithelium cows,the results,which were examined by immunofluorescence staining,Western blot and q RT-PCR,showed that compared with the control group,heat stress group significantly increased the ratio of Bax/Bcl-2,while choline pretreatment significantly downregulated the expression level of Bax and increased the expression of Bcl-2.Moreover,heat stress significantly up-regulated the expression of e IF2α,ATF-4 and CHOP,the downstream genes of PERK,which activate the apoptotic pathway,while choline pretreatment significantly reversed the above phenomenon.It was further verified that choline could slow down the damage of apoptosis induced by heat stress by inhibiting the e IF2α/ATF-4/CHOP signaling pathway.Secondly,compared with the control group,cells treated with heat stress showed increased Nrf-2incorporation into the nucleus and upregulated the expression of Nrf-2 and HO-1 in the nucleus,while the expression of Keap1,an inhibitor protein,was significantly reversed by choline pretreatment.In conclusion,choline can alleviate apoptosis induced by heat stress in dairy cow mammary epithelial cells by regulating apoptosis signal pathway e IF2α/ATF-4/CHOP and antiapoptotic pathway Nrf-2/HO-1 expression.