抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响

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

旨在研究PERK在脂多糖(LPS)诱导的奶牛乳腺上皮细胞自噬中的作用。首先,试验设对照组(CON)和LPS组(LPS,4μg·mL^(-1)),研究LPS对奶牛乳腺上皮细胞内质网应激和自噬的影响;随后用不同浓度的PERK抑制剂GSK2606414(GSK)预处理细胞,通过测定PERK、ATF4、eIF-2α和CHOP的mRNA表达,筛选出GSK的最佳抑制浓度;最后将奶牛乳腺上皮细胞分为对照组(CON)、LPS组(LPS)、GSK+LPS组(GLPS)和GSK组4组,研究抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响。采用RT-qPCR和Western blot分析内质网应激、自噬相关基因和蛋白的表达,通过免疫荧光检测GRP78和p62的荧光强度。结果表明:1)与对照组相比,LPS组的PERK、IRE1α、ATF6、GRP78和CHOP的mRNA和蛋白表达均显著(P<0.05)或极显著(P<0.01)升高。LPS还可以显著升高LC3、ATG5、ATG14和Beclin1的mRNA和蛋白表达(P<0.01),并且显著降低p62的mRNA和蛋白表达(P<0.01)。此外,LPS可降低p62的荧光强度、升高GRP78的荧光亮度以及增加溶酶体的形成;2)与LPS组相比,GSK预处理可显著降低PERK、ATF4、eIF-2α、CHOP蛋白表达(P<0.05或P<0.01),并且还可以显著降低LC3、ATG14、ATG5、Beclin1以及升高p62的mRNA和蛋白表达(P<0.01或P<0.05)。免疫荧光试验结果进一步表明,GSK预处理可增加p62的荧光强度。因此,在本研究背景下,LPS可诱导奶牛乳腺上皮细胞发生内质网应激和自噬,并且抑制PERK/eIF-2α/ATF4信号通路可缓解LPS诱导的奶牛乳腺上皮细胞自噬。

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.