Wnt5a介导Wnt/Ca2+通路对BCG诱导牛原代肺泡上皮细胞自噬的调控作用

doi:10.11843/j.issn.0366-6964.2022.11.032

摘要/Abstract

摘要： 旨在探讨体外分离培养牛肺泡上皮细胞（bovine alveolar epithelial cells，BAECs）的方法及Wnt5a对牛结核分枝杆菌卡介苗（bacille Calmette-Guérin，BCG）感染BAECs细胞自噬的调控机制。试验选用酶联合消化法和机械刮刷法分离细胞，差速贴壁法纯化BAECs，免疫荧光染色检测上皮细胞标志物角蛋白14（cytokeratin 14，CK14）和角蛋白5（cytokeratin 5，CK5）的表达；BCG感染BAECs，并用Box-5抑制Wnt5a的表达，Western blot和免疫荧光染色检测自噬相关蛋白及非经典Wnt信号通路相关蛋白的表达。结果表明，采用酶联合消化法和机械刮刷法能够成功分离纯度较高的BAECs，细胞经CK14和CK5鉴定为阳性；BCG感染BAECs促进Wnt5a表达，增加细胞自噬，Box-5预处理下调BCG诱导的细胞自噬相关蛋白LC3II、P62、Atg7及Atg5的表达，且抑制非经典Wnt/Ca²⁺信号通路相关蛋白Wnt5a、CaMKII及NFAT的表达。综上，试验成功建立BAECs分离培养方法，BCG感染增加BAECs内Wnt5a表达和细胞自噬，抑制Wnt5a下调BCG诱导的BAECs细胞自噬，且Wnt5a是通过非经典Wnt/Ca²⁺信号通路调控BCG诱导的BAECs细胞自噬。

关键词: Wnt5a, BCG, 牛肺泡上皮细胞, 细胞自噬

Abstract: The aim of this study was to investigate the method of in vitro isolation and culture of bovine alveolar epithelial cells (BAECs) and the regulatory mechanism of Wnt5a on the autophagy of BAECs infected with Mycobacterium tuberculosis bacille Calmette-Guérin (BCG). BAECs were isolated by enzyme combined digestion method and mechanical scraping method, and purified by differential adhesion method. The expression of epithelial cell markers CK14 and CK5 were detected by immunofluorescence staining. BAECs were infected with BCG and the expression of Wnt5a was inhibited by Box-5. The expression of autophagy-related proteins and nonclassical Wnt signaling pathway related proteins were detected by Western blot and immunofluorescence staining. The results showed that BAECs were successfully isolated with high purity by enzyme combined digestion and mechanical scraping. Isolated cells were identified as positive by CK14 and CK5. BCG infection with BAECs promoted the expression of Wnt5a and increased autophagy. Box-5 pretreatment down-regulated the expression of BCG-induced autophagy-related proteins LC3II, P62, Atg7 and Atg5, and inhibited the expression of nonclassical Wnt/Ca²⁺ signaling pathway related proteins Wnt5a, CaMKII and NFAT. In conclusion, the isolation and culture method of BAECs was successfully established. BCG infection promoted the Wnt5a expression and autophagy of BAECs, and the inhibition of Wnt5a expression down-regulated the BCG-induced autophagy in BAECs. Moreover, Wnt5a regulates the autophagy induced by BCG in BAECs through the nonclassical Wnt/Ca²⁺ signaling pathway.

Key words: Wnt5a, BCG, bovine alveolar epithelial cells, autophagy

中图分类号:

S852.4

陈琪, 杜长征, 郑雪迪, 马伯利, 徐金瑞, 杨易. Wnt5a介导Wnt/Ca²⁺通路对BCG诱导牛原代肺泡上皮细胞自噬的调控作用[J]. 畜牧兽医学报, 2022, 53(11): 4071-4080.

CHEN Qi, DU Changzheng, ZHENG Xuedi, MA Boli, XU Jinrui, YANG Yi. Regulation on BCG-induced Autophagy by Wnt5a-mediated Wnt/Ca²⁺ Pathway in BAECs[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4071-4080.

参考文献 39

[1]	World Health Organization.Global tuberculosis report 2021[R].Geneva:WHO, 2021.
[2]	ALIPOOR S D, ADCOCK I M, TABARSI P, et al.MiRNAs in tuberculosis:their decisive role in the fate of TB[J].Eur J Pharmacol, 2020, 886:173529.
[3]	LARA-ESPINOSA J V, SANTANA-MARTÍNEZ R A, MALDONADO P D, et al.Experimental pulmonary tuberculosis in the absence of detectable brain infection induces neuroinflammation and behavioural abnormalities in male BALB/c mice[J].Int J Mol Sci, 2020, 21(24):9483.
[4]	YAMAMOTO K, AHYI A N N, PEPPER-CUNNINGHAM Z A, et al.Roles of lung epithelium in neutrophil recruitment during pneumococcal pneumonia[J].Am J Respir Cell Mol Biol, 2014, 50(2):253-262.
[5]	TOMIOKA H, SATO K, SANO C, et al.Intramacrophage passage of Mycobacterium tuberculosis and M.avium complex alters the drug susceptibilities of the organisms as determined by intracellular susceptibility testing using macrophages and type II alveolar epithelial cells[J].Antimicrob Agents Chemother, 2002, 46(2):519-521.
[6]	RAVANAN P, SRIKUMAR I F, TALWAR P.Autophagy:the spotlight for cellular stress responses[J].Life Sci, 2017, 188:53-67.
[7]	陈琪, 李勇, 徐金瑞, 等.Wnt5a在肺相关疾病发生发展过程中作用的研究进展[J].吉林大学学报:医学版, 2021, 47(1):237-242.CHEN Q, LI Y, XU J R, et al.Research progress in roles of Wnt5a in occurrence and development of lung-related diseases[J].Journal of Jilin University:Medicine Edition, 2021, 47(1):237-242.(in Chinese)
[8]	ZHANG H, QI Y H, GENG D C, et al.Expression profile and clinical significance of Wnt signaling in human gliomas[J].Oncol Lett, 2018, 15(1):610-617.
[9]	ZINS K, SCHÄFER R, PAULUS P, et al.Frizzled2 signaling regulates growth of high-risk neuroblastomas by interfering with β-catenin-dependent and β-catenin-independent signaling pathways[J].Oncotarget, 2016, 7(29):46187-46202.
[10]	JI T R, ZHANG C W, MA L L, et al.TRPC6-mediated Ca2+ signaling is required for hypoxia-induced autophagy in human podocytes[J].Cell Physiol Biochem, 2018, 48(4):1782-1792.
[11]	SHARMA S, MEENA L S.Potential of Ca2+ in Mycobacterium tuberculosis H37Rv pathogenesis and survival[J].Appl Biochem Biotechnol, 2017, 181(2):762-771.
[12]	LIU F, CHEN J X, WANG P, et al.MicroRNA-27a controls the intracellular survival of Mycobacterium tuberculosis by regulating calcium-associated autophagy[J].Nat Commun, 2018, 9(1):4295.
[13]	陈琪, 郑雪迪, 聂雪伊, 等.Wnt5a调节BCG感染肺泡上皮细胞自噬的作用研究[J].中国病原生物学杂志, 2021, 16(8):878-883.CHEN Q, ZHENG X D, NIE X Y, et al.Effects of Wnt5a on autophagy induced by BCG in alveolar epithelial cells[J].Journal of Pathogen Biology, 2021, 16(8):878-883.(in Chinese)
[14]	XU W L, JANOCHA A J, LEAHY R A, et al.A novel method for pulmonary research:Assessment of bioenergetic function at the air-liquid interface[J].Redox Biol, 2014, 2:513-519.
[15]	廖伟, 钱桂生, 雷撼, 等.人气道上皮细胞的体外原代培养研究[J].解放军医学杂志, 2007, 32(6):565-567.LIAO W, QIAN G S, LEI H, et al.Studies on primary culturing of human bronchial epithelial cells[J].Medical Journal of Chinese People's Liberation Army, 2007, 32(6):565-567.(in Chinese)
[16]	GREEN M D, HUANG S X L, SNOECK H W.Stem cells of the respiratory system:From identification to differentiation into functional epithelium[J].Bioessays, 2013, 35(3):261-270.
[17]	HASHMI A A, HUSSAIN Z F, IRFAN M, et al.Cytokeratin 5/6 expression in bladder cancer:Association with clinicopathologic parameters and prognosis[J].BMC Res Notes, 2018, 11(1):207.
[18]	CAO W Y, LI J H, YANG K P, et al.An overview of autophagy:Mechanism, regulation and research progress[J].Bull Cancer, 2021, 108(3):304-322.
[19]	HARRIS R C, SUMNER T, KNIGHT G M, et al.Age-targeted tuberculosis vaccination in China and implications for vaccine development:A modelling study[J].Lancet Glob Health, 2019, 7(2):e209-e218.
[20]	GUILLOT L, NATHAN N, TABARY O, et al.Alveolar epithelial cells:Master regulators of lung homeostasis[J].Int J Biochem Cell Biol, 2013, 45(11):2568-2573.
[21]	ARDAIN A, MARAKALALA M J, LESLIE A.Tissue-resident innate immunity in the lung[J].Immunology, 2020, 159(3):245-256.
[22]	INVERNIZZI R, LLOYD C M, MOLYNEAUX P L.Respiratory microbiome and epithelial interactions shape immunity in the lungs[J].Immunology, 2020, 160(2):171-182.
[23]	ROWE R K, BRODY S L, PEKOSZ A.Differentiated cultures of primary hamster tracheal airway epithelial cells[J].In Vitro Cell Dev Biol Anim, 2004, 40(10):303-311.
[24]	白东梅, 李玉霞, 王薇, 等.一种原代肺上皮细胞体外培养方法[J].生物技术通讯, 2014, 25(5):676-681.BAI D M, LI Y X, WANG W, et al.One method of primary alveolar epithelial cells in vitro[J].Letters in Biotechnology, 2014, 25(5):676-681.(in Chinese)
[25]	NAKAI T, ICHIHARA S, KADA A, et al.The unique luminal staining pattern of cytokeratin 5/6 in adenoid cystic carcinoma of the breast may aid in differentiating it from its mimickers[J].Virchows Arch, 2016, 469(2):213-222.
[26]	RICKMEYER T, JAGER K, SCHONIGER S, et al.Cytokeratin expression profiles of canine epithelial tissues[J].Histol Histopathol, 2019, 34(6):683-696.
[27]	ROCK J R, ONAITIS M W, RAWLINS E L, et al.Basal cells as stem cells of the mouse trachea and human airway epithelium[J].Proc Natl Acad Sci USA, 2009, 106(31):12771-12775.
[28]	ZUO W, ZHANG T, WU D Z, et al.p63+Krt5+ distal airway stem cells are essential for lung regeneration[J].Nature, 2015, 517(7536):616-620.
[29]	ONORATI A V, DYCZYNSKI M, OJHA R, et al.Targeting autophagy in cancer[J].Cancer, 2018, 124(16):3307-3318.
[30]	YUAN K, SHAMSKHOU E A, ORCHOLSKI M E, et al.Loss of endothelium-derived Wnt5a is associated with reduced pericyte recruitment and small vessel loss in pulmonary arterial hypertension[J].Circulation, 2019, 139(14):1710-1724.
[31]	JATI S, KUNDU S, CHAKRABORTY A, et al.Wnt5A signaling promotes defense against bacterial pathogens by activating a host autophagy circuit[J].Front Immunol, 2018, 9:679.
[32]	WONG K W.The role of ESX-1 in Mycobacterium tuberculosis pathogenesis[J].Microbiol Spectr, 2017, 5(3), doi:10.1128/microbiolspec.TBTB2-0001-2015.
[33]	JAYACHANDRAN R, SUNDARAMURTHY V, COMBALUZIER B, et al.Survival of mycobacteria in macrophages is mediated by coronin 1-dependent activation of calcineurin[J].Cell, 2007, 130(1):37-50.
[34]	CAVALLOTTI C, FRATI A, SAGNELLI P, et al.Re-evaluation and quantification of the different sources of nerve fibres supplying the rat eye[J].J Anat, 2005, 206(3):217-224.
[35]	BLANC E, GOLDSCHNEIDER D, DOUC-RASY S, et al.Wnt-5a gene expression in malignant human neuroblasts[J].Cancer Lett, 2005, 228(1-2):117-123.
[36]	FARRERA-HERNÁNDEZ A, MARÍN-LLERA J C, CHIMAL-MONROY J.WNT5A-Ca2+-CaN-NFAT signalling plays a permissive role during cartilage differentiation in embryonic chick digit development[J].Dev Biol, 2021, 469:86-95.
[37]	CHEN Y F, ZHOU X P.Research progress of mTOR inhibitors[J].Eur J Med Chem, 2020, 208:112820.
[38]	SUN B, OU H, REN F, et al.Propofol inhibited autophagy through Ca2+/CaMKKβ/AMPK/mTOR pathway in OGD/R-induced neuron injury[J].Mol Med, 2018, 24(1):58.
[39]	SHI W W, XU D C, GU J H, et al.Saikosaponin-d inhibits proliferation by up-regulating autophagy via the CaMKKβ-AMPK-mTOR pathway in ADPKD cells[J].Mol Cell Biochem, 2018, 449(1-2):219-226.

Wnt5a介导Wnt/Ca²⁺通路对BCG诱导牛原代肺泡上皮细胞自噬的调控作用

Regulation on BCG-induced Autophagy by Wnt5a-mediated Wnt/Ca²⁺ Pathway in BAECs

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