猪肺炎支原体乳酸脱氢酶在诱导猪支气管上皮细胞凋亡中的作用

doi:10.11843/j.issn.0366-6964.2024.05.037

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 2195-2205.doi: 10.11843/j.issn.0366-6964.2024.05.037

猪肺炎支原体乳酸脱氢酶在诱导猪支气管上皮细胞凋亡中的作用

王吉英^1,2,3,4, 尹蕊如^2,3,5, 谢星^2,3,5, 王海燕^3,6, 刘胡栋⁷, 胡辉⁴, 熊祺琰^2,3,5, 冯志新^2,3,5, 邵国青^2,3,5, 于岩飞^2,3,5*

1. 湖南农业大学动物医学院, 长沙 410125;
2. 江苏省农业科学院兽医研究所, 南京 210014;
3. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225327;
4. 怀化职业技术学院动物科技学院, 怀化 418099;
5. 南京农业大学动物医学院, 南京 210095;
6. 江苏省农业科学院动物免疫工程研究所, 南京 210014;
7. 湖南医药学院医学人文与信息管理学院, 怀化 418000

收稿日期:2023-08-10 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 于岩飞，主要从事动物呼吸道病原尤其是支原体的致病机制、免疫逃逸机制及防控技术研究，E-mail：yuyanfeihaha@163.com
作者简介:王吉英(1988-)，女，湖南邵阳人，博士生，主要从事动物病原致病机制研究，E-mail：wjyhhzy@163.com
基金资助:
国家重点研发计划(2022YFD1800903)；湖南省教育厅科学研究项目(22B1055)；湖南省自然科学基金项目(2022JJ50321)；国家自然科学基金项目(32172860；32273011)；江苏省科协青年科技人才托举工程(JSTJ-2023-XH031)

Effects of LDH in Mesomycoplasma (Mycoplasma) hyopneumoniae on Apoptosis of Porcine Bronchial Epithelial Cells

WANG Jiying^1,2,3,4, YIN Ruiru^2,3,5, XIE Xing^2,3,5, WANG Haiyan^3,6, LIU Hudong⁷, HU Hui⁴, XIONG Qiyan^2,3,5, FENG Zhixin^2,3,5, SHAO Guoqing^2,3,5, YU Yanfei^2,3,5*

1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410125, China;
2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
3. Guotai(Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225327, China;
4. Department of Animal Science and Technology, Huaihua Polytechnic College, Huaihua 418099, China;
5. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
6. Institute of Veterinary Immunology and Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
7. School of Medical Humanity and Information Management, Hunan University of Medicine, Huaihua 418000, China

Received:2023-08-10 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 猪肺炎支原体感染会引起宿主上皮细胞的损伤及凋亡。上皮屏障的破坏往往导致细菌和病毒的继发感染，给养猪业造成严重的经济损失，但具体的致病机制及毒力因子尚未完全阐明。前期研究发现猪肺炎支原体乳酸脱氢酶(lactate dehydrogenase, LDH)不仅参与猪肺炎支原体的代谢还与猪肺炎支原体的毒力相关。因此，本研究通过原核表达及亲和层析获得猪肺炎支原体乳酸脱氢酶重组蛋白(recombinant LDH, rLDH)，之后分别用不同浓度的rLDH与猪支气管上皮细胞(porcine bronchial epithelial cell, PBEC)孵育，经显微镜观察rLDH对细胞形态的影响，通过CCK-8法检测rLDH对细胞活力的影响。选取对细胞形态及活力影响不显著的50和 100 μg·mL^-1 rLDH分别与PBEC孵育，通过流式细胞仪检测细胞凋亡率，反转录荧光定量PCR(reverse transcription-quantitative polymerase chain reaction，RT-qPCR)法和免疫印记检测凋亡相关基因 caspase 3、caspase 8、caspase 9、Bax、BCL-2的转录水平和蛋白水平。结果显示：猪肺炎支原体rLDH呈剂量依赖性地诱导PBEC凋亡，其作用与猪肺炎支原体菌株一致。用rLDH及Mhp 168孵育后，PBEC的促凋亡因子Bax、caspase 3和caspase 9 的mRNA水平显著上调，凋亡抑制因子BCL-2 的mRNA水平显著下调，caspase 8的mRNA水平未发生显著变化。促凋亡因子Bax、caspase 3的蛋白表达水平显著上调，凋亡抑制因子BCL-2的蛋白表达水平显著下调。结果表明，猪肺炎支原体可能通过LDH诱导PBEC内源性细胞凋亡，凋亡相关因子caspase 3、caspase 9、Bax和BCL-2在其中发挥重要作用。

关键词: 猪肺炎支原体(Mhp), 乳酸脱氢酶(LDH), 猪支气管上皮细胞(PBEC), 凋亡

Abstract: Mesomycoplasma (Mycoplasma) hyopneumoniae (Mhp) infection can cause damage and apoptosis of host epithelial cells, and the breakdown of the epithelial barrier often leads to the secondary infection of other bacteria and viruses, causing serious economic losses to the swine industry. However, the pathogenicity and virulence determinants of Mhp are not yet completely elucidated. Previous studies have found that Mhp lactate dehydrogenase (LDH) not only involved in the metabolism of Mhp, but also related to its virulence. Therefore, in this study, recombinant LDH (rLDH) was obtained through prokaryotic expression and affinity chromatography, and different concentrations of rLDH were used to interact with porcine bronchial epithelial cell (PBEC). The effect of rLDH on cellular morphology was observed by microscope, and the effect of rLDH on cellular viability was detected by CCK-8 method. The rLDH concentrations (50 and 100 μg·mL^-1) were selected for further analysis by that there was no significant effect on cellular morphology and viability when it incubated with PBEC, and the apoptosis rate of cells was detected by flow cytometry. The transcriptional and expression levels of apoptosis-related genes caspase3, caspase 8, caspase 9, Bax and BCL-2 were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. The results showed that rLDH induced apoptosis of PBEC in a dose-dependent manner, the function of that was similar with Mhp. The mRNA levels of caspase 3, caspase 9, and Bax of PBEC after incubation with rLDH and Mhp 168 were significantly up-regulated and the mRNA level of BCL-2 was significantly down-regulated, while the mRNA of caspase 8 was not changed significantly. The protein expression levels of caspase 3 and Bax were significantly up-regulated and the protein expression level of BCL-2 was significantly down-regulated. The results showed that Mhp could induce the endogenous apoptosis of PBEC through LDH, in which the apoptosis-related factors caspase 3, caspase 9, Bax and BCL-2 playing important roles.

Key words: Mesomycoplasma hyopneumoniae, lactate dehydrogenase, PBEC, apoptosis

中图分类号:

S858.285.3

王吉英, 尹蕊如, 谢星, 王海燕, 刘胡栋, 胡辉, 熊祺琰, 冯志新, 邵国青, 于岩飞. 猪肺炎支原体乳酸脱氢酶在诱导猪支气管上皮细胞凋亡中的作用[J]. 畜牧兽医学报, 2024, 55(5): 2195-2205.

WANG Jiying, YIN Ruiru, XIE Xing, WANG Haiyan, LIU Hudong, HU Hui, XIONG Qiyan, FENG Zhixin, SHAO Guoqing, YU Yanfei. Effects of LDH in Mesomycoplasma (Mycoplasma) hyopneumoniae on Apoptosis of Porcine Bronchial Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2195-2205.

参考文献

[1] SIMIONATTO S, MARCHIORO S B, MAES D, et al. Mycoplasma hyopneumoniae:from disease to vaccine development[J]. Vet Microbiol, 2013, 165(3-4):234-242.
[2] 宁雅茹, 丁红雷. 猪肺炎支原体与宿主相互作用研究进展[J]. 生物工程学报, 2020, 36(9):1741-1753.
NING Y R, DING H L. Interaction between Mycoplasma hyopneumoniae and host—a review[J]. Chinese Journal of Biotechnology, 2020, 36(9):1741-1753. (in Chinese)
[3] RANA N F, SAUVAGEOT N, LAPLACE J M, et al. Redox balance via lactate dehydrogenase is important for multiple stress resistance and virulence in Enterococcus faecalis[J]. Infect Immun, 2013, 81(8):2662-2668.
[4] OEHMCKE-HECHT S, NASS L E, WICHURA J B, et al. Deletion of the L-lactate dehydrogenase gene ldh in Streptococcus pyogenes leads to a loss of SpeB activity and a hypovirulent phenotype[J]. Front Microbiol, 2017, 8:1841.
[5] GASPAR P, AL-BAYATI F A Y, ANDREW P W, et al. Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood[J]. Infect Immun, 2014, 82(12):5099-5109.
[6] CHEN W C, WEI L L, ZHANG Y, et al. Involvement of the two L-lactate dehydrogenase in development and pathogenicity in Fusarium graminearum[J]. Curr Genet, 2019, 65(2):591-605.
[7] HE L, BASTOS R G, YU L, et al. Lactate dehydrogenase as a potential therapeutic drug target to control Babesia bigemina[J]. Front Cell Infect Microbiol, 2022, 12:870852.
[8] LEAL ZIMMER F M A, PAES J A, ZAHA A, et al. Pathogenicity & virulence of Mycoplasma hyopneumoniae[J]. Virulence, 2020, 11(1):1600-1622.
[9] YU Y F, LIU M J, HUA L Z, et al. Fructose-1, 6-bisphosphate aldolase encoded by a core gene of Mycoplasma hyopneumoniae contributes to host cell adhesion[J]. Vet Res, 2018, 49(1):114.
[10] KANG H G, LEE S, JEONG P S, et al. Lycopene improves in vitro development of porcine embryos by reducing oxidative stress and apoptosis[J]. Antioxidants (Basel), 2021, 10(2):230.
[11] CHEN H L, YANG Y F, WANG Y L, et al. Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro[J]. J Ovarian Res, 2019, 12(1):90.
[12] QIAO N, YANG Y Y, LIAO J Z, et al. Metabolomics and transcriptomics indicated the molecular targets of copper to the pig kidney[J]. Ecotoxicol Environ Saf, 2021, 218:112284.
[13] 李保良, 庞胜美, 吕林芬, 等. 产肠毒素大肠杆菌诱导肠上皮细胞凋亡的研究进展[J]. 微生物学报, 2023, 63(6):2340-2349.
LI B L, PANG S M, LÜ L F, et al. Research progress in enterotoxigenic Escherichia coli-induced apoptosis of intestinal epithelial cells[J]. Acta Microbiologica Sinica, 2023, 63(6):2340-2349. (in Chinese)
[14] VITALE I, PIETROCOLA F, GUILBAUD E, et al. Apoptotic cell death in disease-Current understanding of the NCCD 2023[J]. Cell Death Differ, 2023, 30(5):1097-1154.
[15] 高铎, 孔令聪, 贾博岩, 等. 牛支原体致病机制研究进展[J]. 动物医学进展, 2015, 36(7):90-94.
GAO D, KONG L C, JIA B Y, et al. Progress on pathogenic mechanism of Mycoplasma bovis[J]. Progress in Veterinary Medicine, 2015, 36(7):90-94. (in Chinese)
[16] 刘艳红, 李艳, 刘东瀛, 等. 猪鼻支原体抗原对K562细胞增殖的影响[J]. 肿瘤防治研究, 2003, 30(1):12-14.
LIU Y H, LI Y, LIU D Y, et al. Effects of Mycoplasma hyorhinis antigens on K562 cell proliferation[J]. Cancer Research on Prevention and Treatment, 2003, 30(1):12-14. (in Chinese)
[17] 张焱焱. 猪肺炎支原体ES-2株的特性研究及猪链球2型菌荚膜多糖合成相关基因的研究[D]. 武汉:华中农业大学, 2019.
ZHANG Y Y. The characteristic of M. hyopneumoniae ES-2 strain and study on the genes involved in synthesis of capsular polysaccharide in Streptococcus suis type 2[D]. Wuhan:Huazhong Agricultural University, 2019. (in Chinese)
[18] YU Y, ZHANG L, LI Y. 鸡毒支原体GroEL蛋白诱导宿主细胞凋亡的机制[J]. 中国预防兽医学报, 2019, 41(8):875.
YU Y, ZHANG L, LI Y. The mechanism of host cell apoptosis induced by Mycoplasma gallisepticum GroEL protein[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(8):875. (in Chinese)
[19] 王艳歌, 董辉, 黄兵. 寄生虫乳酸脱氢酶研究进展[J]. 中国动物传染病学报, 2014, 22(1):80-86.
WANG Y G, DONG H, HUANG B. Progress in research on lactate dehydrogenase of parasites[J]. Chinese Journal of Animal Infectious Diseases, 2014, 22(1):80-86. (in Chinese)
[20] ADAMS J M. Ways of dying:multiple pathways to apoptosis[J]. Genes Dev, 2003, 17(20):2481-2495.
[21] YUAN Z, DEWSON G, CZABOTAR P E, et al. VDAC2 and the BCL-2 family of proteins[J]. Biochem Soc Trans, 2021, 49(6):2787-2795.
[22] WOLF P, SCHOENIGER A, EDLICH F. Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane[J]. Biochim Biophys Acta Mol Cell Res, 2022, 1869(10):119317.

猪肺炎支原体乳酸脱氢酶在诱导猪支气管上皮细胞凋亡中的作用

Effects of LDH in Mesomycoplasma (Mycoplasma) hyopneumoniae on Apoptosis of Porcine Bronchial Epithelial Cells

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