唾液链球菌K12对肺炎支原体感染小鼠的预防作用

doi:10.12122/j.issn.1673-4254.2024.12.05

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (12): 2300-2307.doi: 10.12122/j.issn.1673-4254.2024.12.05

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唾液链球菌K12对肺炎支原体感染小鼠的预防作用

苏晓玲¹(), 廖道庸¹(), 李超¹, 陈丽¹, 王婧芸¹, 甘甜¹, 罗浩荡¹, 伍宁³, 何军¹^,²()

^1.南华大学附属南华医院，检验科，湖南衡阳 421001
^2.南华大学附属南华医院，输血科，湖南衡阳 421001
^3.衡阳市第一人民医院医学检验科，湖南衡阳 421001

收稿日期:2024-05-06 出版日期:2024-12-20 发布日期:2024-12-26
通讯作者: 何军 E-mail:xiaoling2020s@163.com;l1326253952@163.com;junhe@usc.edu.cn
作者简介:苏晓玲，在读博士研究生，E-mail: xiaoling2020s@163.com
廖道庸，在读硕士研究生，E-mail: l1326253952@163.com
第一联系人：苏晓玲、廖道庸共同第一作者
基金资助:
国家自然科学基金青年科学基金(82302567);湖南省自然科学基金(2023JJ60055);2023年湖南省卫生健康高层次人才支持计划资助(20240304128);衡阳市科技计划项目(202250045307)

Protective effect of Streptococcus salivarius K12 against Mycoplasma pneumoniae infection in mice

Xiaoling SU¹(), Daoyong LIAO¹(), Chao LI¹, Li CHEN¹, Jingyun WANG¹, Tian GAN¹, Haodang LUO¹, Ning WU³, Jun HE¹^,²()

^1.Department of Laboratory Medicine, Hengyang First People's Hospital, Hengyang 421001, China
^2.Department of Blood Transfusion, Affiliated Nanhua Hospital, University of South China, Hengyang 421001, China, Hengyang First People's Hospital, Hengyang 421001, China
^3.Clinical Laboratory, Hengyang First People's Hospital, Hengyang 421001, China

Received:2024-05-06 Online:2024-12-20 Published:2024-12-26
Contact: Jun HE E-mail:xiaoling2020s@163.com;l1326253952@163.com;junhe@usc.edu.cn
Supported by:
the Young Scientists Fund of the National Natural Science Foundation of China(82302567)

摘要/Abstract

摘要：

目的探讨唾液链球菌K12对肺炎支原体（Mp）感染小鼠是否具有预防作用。方法 20只雄性BALB/c小鼠随机分为：正常对照组、单纯益生菌组（K12+PBS组）、单纯Mp感染组（PBS+Mp组）、益生菌预防组（K12+Mp组），5只/组。Mp感染3 d后进行支气管肺泡灌洗液（BALF）计数；ELISA检测血清中分泌型免疫球蛋白A（sIgA）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的表达；RT-qPCR检测肺组织中Mp的P1和社区获得性呼吸窘迫综合征（CARDS）毒素，肺组织炎症因子TNF-α、IL-6、趋化因子1（CXCL1）、肺部重塑功能基质金属酶9（MMP9）、粘蛋白5ac（MUC5ac）、胶原蛋白3a1（Col3a1）、Toll样受体2（TLR2）及TLR4的mRNA表达；Western blotting检测肺组织中TLR2和TLR4蛋白水平变化；HE染色和AB/PAS染色观察肺组织病理变化和肺部气道重塑变化。结果 K12+Mp组的P1、CARDS mRNA表达和BALF中白细胞数量较PBS+Mp组相比降低（P<0.05）。K12+Mp组血清中的炎症因子差异无统计学意义；但是肺组织中TNF-α（P<0.001）、IL-6（P<0.01）和CXCL1（P<0.05）的mRNA表达都呈下降趋势，且TLR2（P<0.01）和TLR4（P<0.001）的表达也降低。K12+Mp 组肺组织中MMP9（P<0.001）、MUC5ac （P<0.001）和COL3A1（P<0.0001）的mRNA表达呈现下降的趋势，AB/PAS 染色显示粘液分泌降低。结论 K12益生菌在一定浓度和时间范围内预防给药能有效减轻Mp感染小鼠肺部炎症反应，同时能够有效改善肺组织气道重塑功能和肺部损伤，对Mp感染小鼠有一定的预防作用。

关键词: 唾液链球菌K12, 肺炎支原体, 口腔益生菌, 肺炎支原体感染模型, BALB/c小鼠

Abstract:

Objective To investigate the protective effect of the probiotic bacterium Streptococcus salivarius K12 (K12) against Mycoplasma pneumoniae (Mp) infection in mice. Methods Forty male BALB/c mice were randomized into normal control group, K12 treatment group, Mp infection group, and K12 pretreatment prior to Mp infection group. The probiotic K12 was administered daily by gavage for 14 days before Mp infection induced by intranasal instillation of Mp. Three days after Mp infection, the mice were euthanized for analysis of bronchoalveolar lavage fluid (BALF) cell counts and serum levels of secretory immunoglobulin A (sIgA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). RT-qPCR was performed to detect the P1 and community-acquired respiratory distress syndrome ( CARDS ) toxin of Mp in the lung tissues and the mRNA expressions of TNF-α, IL-6, chemokine 1 (CXCL1), matrix metalloproteinase 9 (MMP9), mucin 5ac (MUC5ac), collagen 3a1 (Col3a1), Toll-like receptor 2 (TLR2) and TLR4; the protein expressions of TLR2 and TLR4 in the lung tissue were detected using Western blotting. Pathological changes in the lung tissue and airway remodeling were examined with HE staining and AB/PAS staining. Results Compared with the Mp-infected mice with PBS treatment, the infected mice with K12 treatment showed significantly lowered mRNA levels of P1 and CARDS in the lung tissue and reduced white blood cell counts in the BALF (P<0.05). In spite of the absence of significant differences in serum levels of inflammatory factors between the two groups, the mRNA expressions of TNF‑α, IL-6, CXCL1, MMP9, MUC5ac and COL3A1 and the mRNA and protein levels of TLR2 and TLR4 in the lung tissues were significantly lower in K12-treated mice, in which AB/PAS staining showed obviously decreased mucus secretion. Conclusion K12 pretreatment can effectively reduce pulmonary inflammatory responses, improve airway remodeling and alleviate lung injury in Mp-infected mice.

Key words: Streptococcus salivarius K12, Mycoplasma pneumoniae, oral probiotics, Mycoplasma pneumoniae infection model, BALB/c mice

苏晓玲, 廖道庸, 李超, 陈丽, 王婧芸, 甘甜, 罗浩荡, 伍宁, 何军. 唾液链球菌K12对肺炎支原体感染小鼠的预防作用[J]. 南方医科大学学报, 2024, 44(12): 2300-2307.

Xiaoling SU, Daoyong LIAO, Chao LI, Li CHEN, Jingyun WANG, Tian GAN, Haodang LUO, Ning WU, Jun HE. Protective effect of Streptococcus salivarius K12 against Mycoplasma pneumoniae infection in mice[J]. Journal of Southern Medical University, 2024, 44(12): 2300-2307.

图/表 6

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Primer name	Primer sequence 5'-3'
Mp 16S rRNA	F:TAACGGCCTACCAAGGCAATGA
	R:AGTCAAACTCTAGCCATTACCTGC
Mp 16S rRNA probe	ACGGCCCATACTCCTACGGGAGGCAGCAGT
P1^[14]	F:CGCCGCAAAGATGAATGAC
	R:TGTCCTTCCCCATCTAACAGTTC
CARDS	F:TTCCACTTCAGAAACACCCACAGC
	R:TCAATCAGGGCACGCAAACG
Cyclophilin^[14]	F:AGCACTGGAGAGAAAGGATTTGG
	R:TCTTCTTGCTGGTCTTGCCATT
MUC5ac	F:ACGACACTTTTCAGTACCAATGAC
	R:GCTTCCTTACAGATGCAGTCCT
MMP-9	F:CGGATTTGGCCGTATTGGGC
	R:TGATGGCATGCACTGTGGTC
TNF-α	F:CCACCACGCTCTTCTGTCTAC
	R:TGGGCTACAGGCTTGTCACT
IL-6	F:TTCACAAGTCGGAGGCTTA
	R:CAAGTGCATCATCGTTGTTC
CXCL1	F:TGGCTGGGATTCACCTCAAG
	R:CAAGCCTCGCGACCATTCTT
TLR-2^[15]	F:GCCACCATTTCCACGGACT
	R:GGCTTCCTCTTGGCCTGG
TLR-4^[15]	F:TTTATTCAGAGCCGTTGGTG
	R:CAGAGGATTGTCCTCCCATT
Col3a1^[14]	F:GCCCACAGCCTTCTACAC
	R:CCAGGGTCACCATTTCTC
GAPDH	F:AGGTCGGTGTGAACGGATTTG
	R:TGTAGACCATGTAGTTGAGGTCA

图1 Mp感染小鼠模型的构建及K12益生菌最佳条件的摸索

Fig.1 Construction of M. pneumoniae-infected mouse model and optimization of conditions for K12 probiotic treatment. A: Cultivation of M. pneumoniae on solid plate. B: General condition of the mice 3 days after infection with M. pneumoniae. C: Changes in body weight of the mice at different time points after infection. D: M. pneumoniae DNA detection in mice. E-H: Detection of TNF-α and IL-6 mRNA expressions in mouse lung tissues. *P<0.05, **P<0.01, ****P<0.0001. #P<0.05, ###P<0.001, ####P<0.0001 vs 0. (n=3-5).

图2 K12益生菌预防给药后小鼠肺组织的Mp载量

Fig.2 M. pneumoniae load in the lung tissue of the mice after prophylactic administration of K12 probiotics. A,B: qRT-PCR for detecting M. pneumoniae P1 protein and CARDS mRNA in the lung tissue. *P<0.05, ****P<0.0001 (n=5).

表2 K12益生菌预防给药后小鼠BALF的白细胞分类

Tab.2 Leukocyte classification in BALF of M. pneumoniae mice treated with K12 probiotic

Group	WBC (10⁶/L)	MN^#(10⁶/L)	PMN^#(10⁶/L)	MN (%)	PMN (%)
Control	157.60±92.75	129.60±91.60	28.00±21.52	79.50±19.33	20.50±19.33
K12+PBS	171.00±55.67	157.20±55.83	13.80±0.44	91.08±3.30	8.92±3.30
PBS+Mp	2032.20±399.44****	685.40±185.17****	1346.80±291.22****	33.88±6.19	66.12±6.19
K12+Mp	905.60±665.89^##	372.60±217.14^#	533.00±468.64^##	53.90±26.18	46.10±26.18

图3 K12益生菌预防给药后Mp小鼠血清和肺组织中炎症变化

Fig.3 Changes in levels of inflammatory factors in serum and lung tissue of M. pneumoniae mice with K12 pretreatment. A-C: ELISA for detecting serum levels of sIgA, TNF‑α and IL-6. D-F: qRT-PCR for detecting CXCL1, TNF‑α and IL-6 mRNA expressions in the lung tissue. G-H: HE staining of lung tissues and the pathological scores. I-L: qRT-PCR and Western blotting for TLR2 and TLR4 expression. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ##P<0.01, ###P<0.001, ####P<0.0001 vs Control. (n=5).

图4 肺组织气道重塑因子的转录水平及小鼠肺组织的AB/PAS染色

Fig.4 Transcriptional levels of airway remodeling factors in lung tissues and AB/PAS staining of mouse lung tissue. A-C: mRNA levels of MMP9, MUC5ac and COL3A1 in the lung tissues detected by qRT-PCR. D: AB/PAS staining. E: AB/PAS staining score. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ###P<0.001 vs Control. (n=5).

参考文献 28

1	Waites KB, Xiao L, Liu Y, et al. Mycoplasma pneumoniae from the respiratory tract and beyond[J]. Clin Microbiol Rev, 2017, 30(3): 747-809.
2	Bajantri B, Venkatram S, Diaz-Fuentes G. Mycoplasma pneumoniae: a potentially severe infection[J]. J Clin Med Res, 2018, 10(7): 535-44.
3	Zhang ZK, Wan RJ, Yuan Q, et al. Cell damage and neutrophils promote the infection of Mycoplasma pneumoniae and inflammatory response[J]. Microb Pathog, 2022, 169: 105647.
4	Tamiya S, Yoshikawa E, Ogura M, et al. Vaccination using inactivated Mycoplasma pneumoniae induces detrimental infiltration of neutrophils after subsequent infection in mice[J]. Vaccine, 2020, 38(32): 4979-87.
5	Meyer Sauteur PM, Beeton ML, European Society of Clinical Microbiology and Infectious Diseases Study Group for Mycoplasma and Chlamydia Infections study group. Mycoplasma pneumoniae: delayed re-emergence after COVID-19 pandemic restrictions[J]. Lancet Microbe, 2024, 5(2): e100-1.
6	Chen Y, Zhang Y, Tang QN, et al. Efficacy of doxycycline therapy for macrolide-resistant Mycoplasma pneumoniae pneumonia in children at different periods[J]. Ital J Pediatr, 2024, 50(1): 38.
7	di Pierro F, Colombo M, Zanvit A, et al. Use of Streptococcus salivarius K12 in the prevention of streptococcal and viral pharyngotonsillitis in children[J]. Drug Healthc Patient Saf, 2014, 6: 15-20.
8	Mokhtar M, Rismayuddin NAR, Mat Yassim AS, et al. Streptococcus salivarius K12 inhibits Candida albicans aggregation, biofilm formation and dimorphism[J]. Biofouling, 2021, 37(7): 767-76.
9	Laws GL, Hale JDF, Kemp RA. Human systemic immune response to ingestion of the oral probiotic Streptococcus salivarius BLIS K12[J]. Probiotics Antimicrob Proteins, 2021, 13(6): 1521-9.
10	di Pierro F, Iqtadar S, Mumtaz SU, et al. Clinical effects of Streptococcus salivarius K12 in hospitalized COVID-19 patients: results of a preliminary study[J]. Microorganisms, 2022, 10(10): 1926.
11	MacDonald KW, Chanyi RM, Macklaim JM, et al. Streptococcus salivarius inhibits immune activation by periodontal disease pathogens[J]. BMC Oral Health, 2021, 21(1): 245.
12	徐叔云. 药理实验方法学[M]. 3版. 北京: 人民卫生出版社, 2002.
13	梁珂莹. 应用TaqMan实时荧光定量PCR优化肺炎支原体生长条件的研究[D]. 南华大学,2021.
14	Iannuzo N, Insel M, Marshall C, et al. CC16 deficiency in the context of early-life Mycoplasma pneumoniae infection results in augmented airway responses in adult mice[J]. Infect Immun, 2022, 90(2): e0054821.
15	Daniel S, Phillippi D, Schneider LJ, et al. Exposure to diesel exhaust particles results in altered lung microbial profiles, associated with increased reactive oxygen species/reactive nitrogen species and inflammation, in C57Bl/6 wildtype mice on a high-fat diet[J]. Part Fibre Toxicol, 2021, 18(1): 3.
16	Wang T, Sun HM, Lu ZT, et al. The CARDS toxin of Mycoplasma pneumoniae induces a positive feedback loop of type 1 immune response[J]. Front Immunol, 2022, 13: 1054788.
17	Li YT, Shao FY, Zheng SW, et al. Alteration of Streptococcus salivarius in buccal mucosa of oral lichen planus and controlled clinical trial in OLP treatment[J]. Probiotics Antimicrob Proteins, 2020, 12(4): 1340-8.
18	Tamiya S, Yoshikawa E, Ogura M, et al. Neutrophil-mediated lung injury both via TLR2-dependent production of IL-1α and IL-12 p40, and TLR2-independent CARDS toxin after Mycoplasma pneumoniae infection in mice[J]. Microbiol Spectr, 2021, 9(3): e0158821.
19	Li G, Fan LP, Wang YQ, et al. High co-expression of TNF‑α and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia[J]. Mol Med, 2019, 25(1): 38.
20	Mei XZ, Wang J, Zhang C, et al. Apigenin suppresses mycoplasma-induced alveolar macrophages necroptosis via enhancing the methylation of TNF‑α promoter by PPARγ‑Uhrf1 axis[J]. Phytomedicine, 2023, 108: 154504.
21	Chen M, Deng H, Zhao Y, et al. Toll-like receptor 2 modulates pulmonary inflammation and TNF‑α release mediated by Mycoplasma pneumoniae [J]. Front Cell Infect Microbiol, 2022, 12: 824027.
22	Luo H, He J, Qin L, et al. Mycoplasma pneumoniae lipids license TLR-4 for activation of NLRP3 inflammasome and autophagy to evoke a proinflammatory response[J]. Clin Exp Immunol, 2021, 203(1): 66-79.
23	Johnson MDL, Younis US, Menghani SV, et al. CC16 binding to α4β1 integrin protects against Mycoplasma pneumoniae infection[J]. Am J Respir Crit Care Med, 2021, 203(11): 1410-8.
24	Ma Y, Gu YQ, Zhang XX, et al. High expression of MUC5AC, MUC5B, and layilin plays an essential role in prediction in the development of plastic bronchitis caused by MPP[J]. Front Microbiol, 2022, 13: 911228.
25	Tunçer S, Karaçam S. Cell-free supernatant of Streptococcus salivarius M18 impairs the pathogenic properties of Pseudomonas aeruginosa and Klebsiella pneumonia [J]. Arch Microbiol, 2020, 202(10): 2825-40.
26	Vertillo Aluisio G, Spitale A, Bonifacio L, et al. Streptococcus salivarius 24SMBc genome analysis reveals new biosynthetic gene clusters involved in antimicrobial effects on Streptococcus pneumoniae and Streptococcus pyogenes [J]. Microorganisms, 2022, 10(10): 2042.
27	Garcia-Castillo V, Tomokiyo M, Raya Tonetti F, et al. Alveolar macrophages are key players in the modulation of the respiratory antiviral immunity induced by orally administered Lacticasei-bacillus rhamnosus CRL1505[J]. Front Immunol, 2020, 11: 568636.
28	Zhang NY, Zeng WW, Du TF, et al. Lacticaseibacillus casei CNRZ1874 supplementation promotes M1 alveolar macrophage activation and attenuates Mycoplasma pneumoniae pneumonia[J]. J Appl Microbiol, 2023, 134(3): lxad022.

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唾液链球菌K12对肺炎支原体感染小鼠的预防作用

Protective effect of Streptococcus salivarius K12 against Mycoplasma pneumoniae infection in mice

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