Mechanisms By Which Heat Stress Acts Through Endoplasmic Reticulum Stress To Mediate The PERK Signalling Pathway To Regulate Apoptosis In Bovine Mammary Epithelial Cells

Heat stress is an important issue that has been limiting the development of China’s dairy industry.Heat stress causes a variety of hazards to cows and seriously affects their performance.Endoplasmic reticulum stress is a protective response to environmental stimulation and has been shown to cause heat shock and apoptosis in the mammary epithelium of cows,but further research is needed to determine if the mechanism of apoptosis is related to endoplasmic reticulum stress in the bovine mammary epithelium under heat stress conditions.The present experiment was performed to investigate the mechanism by which endoplasmic reticulum stress regulates apoptosis in bovine mammary epithelial cells under heat stress conditions by establishing a heat stress model.The mixed growth of dairy fibroblasts and dairy mammary epithelial cells were separated and cultured,and the purified bovine mammary epithelial cells were then identified by immunofluorescence.The endoplasmic reticulum stress inhibitor 4-PBA was then used to treat mammary epithelial cells in heat-stressed cows.q RT-PCR and flow cytometry were used to detect the alleviating effect of endoplasmic reticulum stress inhibitor 4-PBA on apoptosis of the heat-stressed bovine mammary epithelial cells.The endoplasmic reticulum stress inducer Brefeldin A was used to establish an endoplasmic reticulum stress model for bovine mammary epithelial cells,and to investigate the effects on the expression of apoptosis-related genes,cell viability and apoptosis rate of bovine mammary epithelial cells after inhibition of the PERK signaling pathway in the unfolded protein response(UPR)under endoplasmic reticulum stress conditions.The results show that purified bovine mammary epithelial cells were successfully obtained in this experiment.The heat treatment of bovine mammary epithelial cells at 41 ℃ for 2 and 3 h induced endoplasmic reticulum stress,and the expression of endoplasmic reticulum stress-related marker genes GRP78,PERK and XBP1 m RNA was significantly higher(P < 0.05)compared to normal culture conditions at 37 ℃,and the calcium ion content in the cells was also significantly higher(P < 0.05).The expression of Caspase 3 and Caspase 8 mRNA associated with apoptosis was highly significantly increased(P < 0.01),Bcl2 m RNA expression was highly significantly decreased(P < 0.01)and the anti-apoptotic capacity of the cells was reduced.4 mmol 4-PBA reduced the expression of endoplasmic reticulum stress marker genes in bovine mammary epithelial cells during heat stress,and the expression of Bax,Caspase 3 and Caspase 8 m RNA were significantly lower(P < 0.05)and apoptosis rate was significantly lower(P < 0.05)compared to the41 °C heat treatment control group.The expression of endoplasmic reticulum stress-related marker genes GRP78,PERK and e IF2α m RNA was significantly higher(P < 0.05)and cell proliferation was inhibited compared to the control group.After inhibition of the PERK signalling pathway in the UPR under endoplasmic reticulum stress conditions in bovine mammary epithelial cells,the expression of PERK,ATF4 and CHOP m RNA in the cells was significantly lower(P < 0.05)compared to the Brefeldin A-treated group,as well as the expression of Bax and Caspase 3 m RNA(P < 0.05),and the rate of apoptotic cell death was significantly(P < 0.05).In summary,purified bovine mammary epithelial cells were successfully isolated in this experiment.Inhibition of the PERK signalling pathway in dairy mammary epithelial cells at 41 ℃induced endoplasmic reticulum stress and increased apoptosis.4 mmol 4-PBA could alleviate endoplasmic reticulum stress-induced apoptosis in bovine mammary epithelial cells by inhibiting endoplasmic reticulum stress.This provides a new theoretical basis for the study of the mechanism of apoptosis in bovine mammary epithelial cells induced by heat stress.