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.

References 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.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 37

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SUN Wenli, WANG Haoqi, ZE Licuo, GAO Yufan, ZHANG Feifan, ZHANG Jian, DUAN Mengqi, SHANG Peng, QIANG Bayangzong. Polymorphism of Pro-Inflammatory Factors (IL-1β, IL-6, TNF-α) in Tibetan Pigs and Its Association Analysis with Immune Traits [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1958-1969.
[2]	YANG Xiaofeng, QIN Xiaowei, LÜ Lihua. Protective Effect of a Derivative of MNQ Against LPS-Induced Inflammatory Injury in Bovine Ovarian Follicular Granulosa Cells in Vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2032-2041.
[3]	LI Feifei, ZHANG Chenmiao, TONG Jinjin, JIANG Linshu. Research Progress on the Mechanism of Mitochondrial Autophagy Regulating the Activity of NLRP3 Inflammatory Corpuscles to Improve Animal Health [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1446-1455.
[4]	KANG Fangyuan, LIU Zhentao, WU Kuixian, NI Han, ZHONG Kai, LI Heping, YANG Guoyu, HAN Liqiang. Regulation of Lipophagy on the Size of Lipid Droplets in Bovine Mammary Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1095-1101.
[5]	QIU Wenyue, SU Yiman, YE Jiali, ZHANG Xinting, PANG Xiaoyue, WANG Rongmei, XIE Zimao, ZHANG Hui, TANG Zhaoxin, SU Rongsheng. Study on Asiatic Acid Alleviates LPS-induced Acute Kidney Injury by Regulating Apoptosis and Autophagy of Broilers [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 809-821.
[6]	LIU Yueyang, LI Mengyuan, NIE Xueyi, MA Yabo, HOU Yuxin, MA Boli, YANG Yi, XU Jinrui. The Regulation of Calcium-binding Protein S100A4 on Autophagy in THP-1 Cells Infected with Bacillus Calmette-Guérin [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 311-322.
[7]	WANG Chongnian, YU Jialin, GONG Zhaoqian, WU Xiaoling, DENG Guangcun. Regulation of BCG-induced Autophagy in Macrophages RAW264.7 by PLIN2 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2134-2146.
[8]	WANG Han, MENG Lijie, LIU Wenjiao, XU Yongjian, GONG Ting. Effect of TAS1R3 Gene Interference on Autophagy Related Factors in Leydig Cells of Xiang Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1525-1534.
[9]	CAI Mingyu, ZHANG Hailong, HAI Zhenzhen, QIAO Yarui, DU Jun, ZHOU Xuezhang. The Inflamed Molecular Mechanism Induced by Recombined 14-3-3 Protein of Candida krusei on Bovine Mammary Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1679-1689.
[10]	FAN Lei, SHEN Yu, YOU Liuchao, TIAN Xinyu, LUO Hao, WANG Xin, ZHANG Tingting, SHEN Liuhong. Research Progress on Abnormal Glucose and Lipid Metabolism in Dairy Cows Induced by Lipopolysaccharide (LPS) [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 484-493.
[11]	ZHANG Chengcheng, SUN Jiahao, WANG Xiuling, ZHANG Xiaorong, WU Yantao. Beclin1 Interacts with the Nonstructural Protein NS5A of CSFV and Promotes Virus Proliferation [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 715-725.
[12]	MA Tianwen, YU Yue, LÜ Liangyu, JIA Lina, RUAN Hongri, WANG Haoran, WANG Xinyu, ZHANG Yuxin, ZHANG Jiantao, GAO Li. Effects of Bilobalide on Autophagy, Proliferation and Apoptosis of IL-1β-induced ATDC5 Chondrocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 837-846.
[13]	ZHAO Xin, WANG Ying, LI Chunting, WANG Wei, SANG Rui, LI Haitao, ZHANG Xuemei. Alleviating Effect and Mechanism of Dandelion Extract on LPS-Induced Mastitis in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2773-2781.
[14]	LIU Lihua, ZHONG Zhenyu, ZHENG Yujie, WANG Xin. lncRNA EPB41L4A-AS Inhibits the Proliferation of Bovine Mammary Epithelial Cells by Regulating the Expression of ErbB3 Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2152-2159.
[15]	MA Boli, NIE Xueyi, LIU Yueyang, MIAO Shenao, CHEN Tong, YANG Yi, XU Jinrui. Regulation of PERK/ATF4/CHOP Pathway on NLRP3 Inflammasome Activation Induced by BCG in THP-1 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2268-2281.