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

摘要/Abstract

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<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。

关键词: 奶牛乳腺上皮细胞, LPS, 自噬, PERK

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

中图分类号:

S857.26

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.

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.

参考文献 37

[1]	HERTL J A, GRÖHN Y T, LEACH J D G, et al. Effects of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on the probability of conception in New York State Holstein dairy cows[J]. J Dairy Sci, 2010, 93(4):1551-1560.
[2]	FOGSGAARD K K, BENNEDSGAARD T W, HERSKIN M S. Behavioral changes in freestall-housed dairy cows with naturally occurring clinical mastitis[J]. J Dairy Sci, 2015, 98(3):1730-1738.
[3]	BONNEFONT C M D, RAINARD P, CUNHA P, et al. Genetic susceptibility to S. aureus mastitis in sheep:differential expression of mammary epithelial cells in response to live bacteria or supernatant[J]. Physiol Genomics, 2012, 44(7):403-416.
[4]	KORHONEN H, MARNILA P, GILL H S. Milk immunoglobulins and complement factors[J]. Br J Nutr, 2000, 84 Suppl 1:S75-S80.
[5]	ZHANG B B, LI M, YANG W, et al. Orai calcium release-activated calcium modulator 1(ORAI1) plays a role in endoplasmic reticulum stress in bovine mammary epithelial cells challenged with physiological levels of ketone bodies[J]. J Dairy Sci, 2020, 103(5):4691-4701.
[6]	WANG, MONIRUZZAMAN M, SHUFFLE E, et al. Immune regulation of the unfolded protein response at the mucosal barrier in viral infection[J]. Clin Transl Immunol, 2018, 7(4):e1014.
[7]	CNOP M, FOUFELLE F, VELLOSO L A. Endoplasmic reticulum stress, obesity and diabetes[J]. Trends Mol Med, 2012, 18(1):59-68.
[8]	HALE A N, LEDBETTER D J, GAWRILUK T R, et al. Autophagy:regulation and role in development[J]. Autophagy, 2013, 9(7):951-972.
[9]	SAKAKI K, WU J, KAUFMAN R J. Protein kinase Cθ is required for autophagy in response to stress in the endoplasmic reticulum[J]. J Biol Chem, 2008, 283(22):15370-15380.
[10]	YU X L, LONG Y C. Autophagy modulates amino acid signaling network in myotubes:differential effects on mTORC1 pathway and the integrated stress response[J]. FASEB J, 2015, 29(2):394-407.
[11]	MEDINA D L, DI PAOLA S, PELUSO I, et al. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB[J]. Nat Cell Biol, 2015, 17(3):288-299.
[12]	VICENCIO J M, LAVANDERO S, SZABADKAI G. Ca2+, autophagy and protein degradation:thrown off balance in neurodegenerative disease[J]. Cell Calcium, 2010, 47(2):112-121.
[13]	LIM J H, KIM H W, KIM M Y, et al. Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy[J]. Cell Death Dis, 2018, 9(3):270.
[14]	WANG S H, SHIH Y L, KO W C, et al. Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway[J]. Cell Mol Life Sci, 2008, 65(22):3640-3652.
[15]	ZHAO K, LIU H Y, ZHOU M M, et al. Establishment and characterization of a lactating bovine mammary epithelial cell model for the study of milk synthesis[J]. Cell Biol Int, 2010, 34(7):717-721.
[16]	MA N N, CHANG G J, HUANG J, et al. cis-9, trans-11-conjugated linoleic acid exerts an anti-inflammatory effect in bovine mammary epithelial cells after Escherichia coli stimulation through NF-κB signaling pathway[J]. J Agric Food Chem, 2019, 67(1):193-200.
[17]	MA N N, WEI G Z, ZHANG H Z, et al. Cis-9, Trans-11 CLA alleviates lipopolysaccharide-induced depression of fatty acid synthesis by inhibiting oxidative stress and autophagy in bovine mammary epithelial cells[J]. Antioxidants (Basel), 2021, 11(1):55.
[18]	GAO Q Y, WANG Y, MA N N, et al. Sodium valproate attenuates the iE-DAP induced inflammatory response by inhibiting the NOD1-NF-κB pathway and histone modifications in bovine mammary epithelial cells[J]. Int Immunopharmacol, 2020, 83:106392.
[19]	CHENG X Y, AABDIN Z U, WANG Y, et al. Glutamine pretreatment protects bovine mammary epithelial cells from inflammation and oxidative stress induced by γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP)[J]. J Dairy Sci, 2021, 104(2):2123-2139.
[20]	SIWECKA N, ROZPDEK W, PYTEL D, et al. Dual role of endoplasmic reticulum stress-mediated unfolded protein response signaling pathway in carcinogenesis[J]. Int J Mol Sci, 2019, 20(18):4354.
[21]	LOUESSARD M, BARDOU I, LEMARCHAND E, et al. Activation of cell surface GRP78 decreases endoplasmic reticulum stress and neuronal death[J]. Cell Death Differ, 2017, 24(9):1518-1529.
[22]	SCHRÖDER M, KAUFMAN R J. ER stress and the unfolded protein response[J]. Mutat Res, 2005, 569(1-2):29-63.
[23]	GORMAN A M, HEALY S J M, JÄGER R, et al. Stress management at the ER:regulators of ER stress-induced apoptosis[J]. Pharmacol Ther, 2012, 134(3):306-316.
[24]	LEE A S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress[J]. Methods, 2005, 35(4):373-381.
[25]	季英磊, 闫骏, 王艳莎, 等. 内质网应激参与介导脂多糖诱导的大鼠肝细胞凋亡[J]. 法医学杂志, 2014, 30(1):13-18.JI Y L, YAN J, WANG Y S, et al. Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte[J]. Journal of Forensic Medicine, 2014, 30(1):13-18. (in Chinese)
[26]	LAMARK T, KIRKIN V, KIKIC I, et al. NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets[J]. Cell Cycle, 2009, 8(13):1986-1990.
[27]	BJØRKØY G, LAMARK T, BRECH A, et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death[J]. J Cell Biol, 2005, 171(4):603-614.
[28]	ASHKENAZI A, BENTO C F, RICKETTS T, et al. Polyglutamine tracts regulate beclin 1-dependent autophagy[J]. Nature, 2017, 545(7652):108-111.
[29]	LIU K, JIAN Y L, SUM X J, et al. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion[J]. J Cell Biol, 2016, 212(2):181-198.
[30]	PATTINGRE S, TASSA A, QU X P, et al. Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy[J]. Cell, 2005, 122(6):927-939.
[31]	徐倩, 许银丰, 杨杰杰, 等. 内质网应激与细胞自噬的关系[J]. 中国细胞生物学学报, 2020, 42(8):1489-1500.XU Q, XU Y F, YANG J J, et al. Relationship between endoplasmic reticulum stress and autophagy[J]. Chinese Journal of Cell Biology, 2020, 42(8):1489-1500.
[32]	HØYER-HANSEN M, JÄÄTTELÄ M. Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium[J]. Cell Death Differ, 2007, 14(9):1576-1582.
[33]	DING W X, NI H M, GAO W T, et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability[J]. Am J Pathol, 2007, 171(2):513-524.
[34]	WEIDBERG H, SHVETS E, ELAZAR Z. Biogenesis and cargo selectivity of autophagosomes[J]. Annu Rev Biochem, 2011, 80:125-156.
[35]	HUANG G H, SHI L Z, CHI H B. Regulation of JNK and p38 MAPK in the immune system:signal integration, propagation and termination[J]. Cytokine, 2009, 48(3):161-169.
[36]	KOUROKU Y, FUJITA E, TANIDA I, et al. ER stress (PERK/eIF2α phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation[J]. Cell Death Differ, 2007, 14(2):230-239.
[37]	KIM K W, MORETTI L, MITCHELL L R, et al. Endoplasmic reticulum stress mediates radiation-induced autophagy by perk-eIF2α in caspase-3/7-deficient cells[J]. Oncogene, 2010, 29(22):3241-3251.