抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响

doi:10.11843/j.issn.0366-6964.2023.01.032

Abstract

Abstract: This study aimed to investigate the effect of endoplasmic reticulum stress on lipopolysaccharide (LPS) induced autophagy in bovine mammary epithelial cells (BMECs). Firstly, the experiment was divided into control group (CON) and LPS group (LPS, 4 μg·mL^-1) to study the effects of LPS on endoplasmic reticulum (ER) stress and autophagy in BMECs. Then, cells were pretreated with different concentrations of PERK inhibitor GSK2606414 (GSK), and the mRNA expressions of PERK, ATF4, eIF-2α and CHOP were measured to determine the optimal inhibitory concentration of GSK. Finally, the BMECs were divided into 4 groups: control group (CON), LPS group (LPS, 4 μg·mL^-1), GLPS group (GSK+LPS) and GSK group, to study the effects of PERK inhibition on LPS-induced autophagy in BMECs were investigated. The expressions of endoplasmic reticulum stress and autophagy related genes and proteins were analyzed by RT-qPCR and Western blot. The fluorescence intensity of GRP78 and p62 was measured by immunofluorescence. The results showed as follows: 1) Compared with the control group, the mRNA and protein expressions of PERK, IRE1α, ATF6, GRP78 and CHOP in LPS group were significantly (P<0.05) or extremely significantly (P<0.01) increased. LPS significantly also increased the mRNA and protein expressions of LC3, ATG5, ATG14 and Beclin1 (P<0.01), and significantly decreased the mRNA and protein expressions of p62 (P<0.01). In addition, LPS reduced the fluorescence intensity of p62, increased the fluorescence intensity of GRP78 and lysosome formation; 2) Compared with LPS group, GSK pretreatment significantly reduced the protein expressions of PERK, ATF4, eIF-2α and CHOP (P<0.05 or P<0.01), and also significantly reduced the mRNA and protein expressions of LC3, ATG14, ATG5, Beclin1 and increased the mRNA and protein expressions of p62 (P<0.01 or P<0.05). The immunofluorescence results further indicated that GSK pretreatment could increase the fluorescence intensity of p62. Therefore, in this study, LPS induced endoplasmic reticulum stress and autophagy, and inhibition of the PERK/eIF-2α/ATF4 signaling pathway can alleviate LPS-induced autophagy in BMECs.

Key words: bovine mammary epithelial cells, LPS, autophagy, PERK

CLC Number:

S857.26

MENG Meijuan, WANG Yan, HUO Ran, LI Xuerui, CHANG Guangjun, SHEN Xiangzhen. Effect of Inhibition of PERK on LPS Induced Autophagy in Bovine Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 351-360.

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Effect of Inhibition of PERK on LPS Induced Autophagy in Bovine Mammary Epithelial Cells

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