| 抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响 |
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| 引用本文： | 孟梅娟,汪艳,霍然,李雪睿,常广军,沈向真.抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响[J].畜牧兽医学报,2023,54(1):351-360. |
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| 作者姓名： | 孟梅娟 汪艳 霍然 李雪睿 常广军 沈向真 |
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| 作者单位： | 南京农业大学动物医学院, 南京 210095 |
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| 基金项目： | 国家自然科学基金项目(31872528；32172933；31702301)；宁夏回族自治区自然科学基金重点项目(2022AAC02072)；宁夏回族自治区重点研发计划项目(21BEF02019)；江苏省高等学校重点学科发展计划(PAPD) |
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| 摘 要： | 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...
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| 关 键 词： | 奶牛乳腺上皮细胞 LPS 自噬 PERK |
| 收稿时间： | 2022-07-07 |
Effect of Inhibition of PERK on LPS Induced Autophagy in Bovine Mammary Epithelial Cells |
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| 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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| Authors: | MENG Meijuan WANG Yan HUO Ran LI Xuerui CHANG Guangjun SHEN Xiangzhen |
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| Institution: | College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China |
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| 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. |
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| Keywords: | bovine mammary epithelial cells LPS autophagy PERK |
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