痛风雏鹅肠道菌群通过TLR4/MyD88/NF-κB通路促进肾损伤的研究

doi:10.11843/j.issn.0366-6964.2021.02.027

摘要/Abstract

摘要： 旨在研究痛风雏鹅的肠道菌群对肾损伤的影响及作用机制。本研究选取具有典型痛风特征的雏鹅15只，相同日龄及生长环境下的健康雏鹅15只，利用16S rDNA测序技术分析盲肠内容物中菌群的差异；用改良苦味酸法测定血清肌酐（Cr），用酶比色法测定血清尿酸（UA），用脲酶-谷氨酸脱氢酶法测定血清尿素氮（BUN）；用RT-qPCR和Western blot法测定肾组织Toll样受体4（TLR4）、髓样分化因子（MyD88）、核转录因子-κB （NF-κB）、白细胞介素1β（IL-1β）、肿瘤坏死因子ɑ（TNF-ɑ）及白细胞介素6（IL-6）的mRNA和蛋白表达量。并将肠道菌群移植给无菌小鼠进行验证。结果显示：1）与健康雏鹅比较，痛风雏鹅肠道菌群16S rDNA测序所得操作分类单元（OTUs）数量、Chao 1指数及Shannon指数均显著降低（P<0.01），变形菌门、埃希菌属、变形菌属及肠球菌属等细菌丰度显著增加，痛风雏鹅肠道菌群在免疫系统、细菌感染、膜传输及核苷酸代谢等代谢途径参与度较高；2）与健康雏鹅比较，痛风雏鹅肾组织中TLR4、MyD88、NF-κB、IL-1β、TNF-ɑ及IL-6的mRNA表达及蛋白表达均显著升高（P<0.05或P<0.01），且这些分子表达量与Cr、BUN及UA呈正相关，与埃希菌属、变形菌属及肠球菌属等菌属丰度呈强正相关关系；3）移植痛风雏鹅肠道菌群小鼠的肾小管发生变性，肾组织TLR4、MyD88、NF-κB的mRNA表达量及血中Cr、BUN、肾损伤因子-1（KIM-1）等水平显著高于移植健康雏鹅菌群的小鼠；给予移植痛风雏鹅菌群小鼠TAK-242后，可回调这些变化。综上表明，痛风雏鹅肠道菌群发生了变化，并可激活LTR4/MyD88/NF-κB通路，诱发炎症反应，促进肾损伤的发生。

关键词: 痛风雏鹅, 肠道菌群, 16S rDNA测序, LTR4/MyD88/NF-κB通路, 肾损伤

Abstract: This study aimed to investigate the effect of gut microbiota on renal injury and its mechanism in gout gosling. In this study, 15 goslings with typical gout characteristics and 15 healthy goslings of the same age and growth environment were selected. 16S rDNA sequencing technology was used to analyze the difference of bacterial diversity in cecal contents. Serum creatinine (Cr), uric acid (UA) and urea nitrogen (BUN) were respectively measured by modified picric acid method, enzyme colorimetric method and urease glutamate dehydrogenase method. RT qPCR and Western blot were respectively used to detect the mRNA and protein expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor (MyD88), nuclear transcription factor-κB (NF-κB), interleukin-1β (IL-1β), tumor necrosis factor-ɑ (TNF-ɑ) and interleukin-6 (IL-6). Lastly, the gut microbiota was transplanted into sterile mice for the verification test. The results showed that: 1) Compared with healthy goslings, the number of operational taxonomic units (OTUs), Chao 1 index and Shannon index of gut microbiota in gout goslings were significantly decreased (P<0.01), and the abundance of Proteobacteria, Escherichia, Proteus and Enterococcus increased significantly. The gut microbiota of gout goslings had higher participation in the immune system, bacterial infection, membrane transport and nucleotide metabolism. 2) Compared with healthy goslings, the mRNA and protein expressions of TLR4, MyD88, NF-κB, IL-1β, TNF-α and IL-6 in kidney tissue of gout goslings were significantly increased (P<0.05 or 0.01). The mRNA expression levels of these molecules were positively correlated with Cr, BUN and UA, and strongly positively correlated with the abundance of Escherichia, Proteus and Enterococcus. 3) The mRNA expression of TLR4, MyD88, NF-κB in kidney tissue and the levels of Cr, BUN and KIM-1 in blood of mice transplanted gut microbiota of gout gosling were significantly higher than those of mice transplanted gut microbiota of healthy gosling. These changes could be reversed by TAK-242. In summary, gut microbiota changed in gout gosling can activate LTR4/MyD88/NF-κB signaling pathway, induce inflammatory response, and promote kidney injury.

Key words: gout gosling, gut bacteria, 16S rDRNA sequencing, LTR4/MyD88/NF-κB signaling pathway, kidney injury

中图分类号:

S856.5

朱道仙, 吴植, 陆江, 黄涛, 刘莉. 痛风雏鹅肠道菌群通过TLR4/MyD88/NF-κB通路促进肾损伤的研究[J]. 畜牧兽医学报, 2021, 52(2): 543-552.

ZHU Daoxian, WU Zhi, LU Jiang, HUANG Tao, LIU Li. Gut Bacteria of Gout Gosling Promoted Kidney Injury Through LTR4/MyD88/NF-κB Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 543-552.

参考文献 23

[1]	JIN M L, WANG X Y, NING K, et al. Genetic characterization of a new astrovirus in goslings suffering from gout[J]. Arch Virol, 2018, 163(10):2865-2869.
[2]	李曼曼, 丁雪东, 荣雪路, 等. 高蛋白饲粮致雏鹅痛风的临床病理学研究[J]. 华南农业大学学报, 2019, 40(1):46-52.LI M M, DING X D, RONG X L, et al. Clinical pathological study of gosling gout induced by high protein diet[J]. Journal of South China Agricultural University, 2019, 40(1):46-52. (in Chinese)
[3]	GUO X, HUANG K, TANG J. Clinicopathology of gout in growing layers induced by high calcium and high protein diets[J]. Br Poul Sci, 2005, 46(5):641-646.
[4]	ZHANG Q S, CAO Y X, WANG J, et al. Isolation and characterization of an astrovirus causing fatal visceral gout in domestic goslings[J]. Emerg Microbes Infec, 2018, 7(1):71.
[5]	张玉杰, 孙宁, 刘东, 等. 鹅星状病毒的分离鉴定及全基因组序列分析[J].畜牧兽医学报,2020,51(11):2765-2777.ZHANG Y J, SONG N, LIU D, et al. Isolation, identification and complete genomic sequence analysis of goose astrovirus[J].Acta Veterinaria et Zootechnica Sinica,2020,51(11):2765-2777. (in Chinese)
[6]	张清水. 新发肾致病型鹅星状病毒的分离鉴定及弱毒株选育[D]. 北京:中国农业大学, 2018.ZHANG Q S. Characterization of a new nephropathogenic goose astrovirus and development of an attenuated vaccine candidate[D]. Beijing:China Agricultural University, 2018. (in Chinese)
[7]	LEHTO M, GROOP P H. The gut-kidney axis:putative interconnections between gastrointestinal and renal disorders[J]. Front Endocrinol, 2018, 9:553.
[8]	NISHIYAMA K, AONO K, FUJIMOTO Y, et al. Chronic kidney disease after 5/6 nephrectomy disturbs the intestinal microbiota and alters intestinal motility[J]. J Cell Physiol, 2019, 234(5):6667-6678.
[9]	COPPO R. The gut-renal connection in IgA nephropathy[J]. Semin Nephrol, 2018, 38(5):504-512.
[10]	YUE R Z, ZUO C, ZENG J, et al. Atorvastatin attenuates experimental contrast-induced acute kidney injury:a role for TLR4/MyD88 signaling pathway[J]. Ren Fail, 2017, 39(1):643-651.
[11]	XI Y M, YAN J S, LI M Y, et al. Gut microbiota dysbiosis increases the risk of visceral gout in goslings through translocation of gut-derived lipopolysaccharide[J]. Poultry Sci, 2019, 98(11):5361-5371.
[12]	奚雨萌, 闫俊书, 应诗家, 等. 高蛋白质高钙饲粮对雏鹅内脏型痛风发生、肾脏功能及肠道微生物区系的影响[J]. 动物营养学报, 2019, 31(2):612-621.XI Y M, YAN J S, YING S J, et al. Effects of high protein and calcium diets on visceral gout development, kidney function and intestinal microbial community of goslings[J]. Chinese Journal of Animal Nutrition, 2019, 31(2):612-621. (in Chinese)
[13]	CAPORASO J G, KUCZYNSKI J, STOMBAUGH J, et al. QⅡME allows analysis of high-throughput community sequencing data[J]. Nat Methods, 2010, 7(5):335-336.
[14]	MORRIS C. Multivariate analysis of ecological data using canoco 5, 2nd edition[J]. Afr J Range For Sci, 2015, 32(4):289-290.
[15]	SEGATA N, IZARD J, WALDRON L, et al. Metagenomic biomarker discovery and explanation[J]. Genome Biol, 2011, 12(6):R60.
[16]	DOUGLAS G M, BEIKO R G, LANGILLE M G I. Predicting the functional potential of the microbiome from marker genes using PICRUSt[J]. Methods Mol Biol, 2018, 1849:169-177.
[17]	ZHAO L P, ZHANG F, DING X Y, et al. Gut bacteria selectively promoted by dietary fibers alleviate type 2 diabetes[J]. Science, 2018, 359(6380):1151-1156.
[18]	陈双双, 司华哲, 李光玉, 等. 动物肠道菌群与营养物质代谢的研究进展[J]. 饲料工业, 2018, 39(2):33-36.CHEN S S, SI H Z, LI G Y, et al. Research progress of animal gut microbiota and nutrient metabolism[J]. Feed Industry, 2018, 39(2):33-36. (in Chinese)
[19]	KHOURY T, TZUKERT K, ABEL R, et al. The gut-kidney axis in chronic renal failure:a new potential target for therapy[J]. Hemodial Int, 2017, 21(3):323-334.
[20]	GUO Z, ZHANG J C, WANG Z L, et al. Intestinal microbiota distinguish gout patients from healthy humans[J]. Sci Rep, 2016, 6:20602.
[21]	刘会芳. 高尿酸通过TLR4/NF-κB信号通路诱导肾小管上皮细胞上皮-间充质转化的作用研究[D]. 贵阳:贵州医科大学, 2018.LIU H F. Uric acid induces epithelial-mesenchymal transition of renal tubular epithelial cells via TLR4/NF-κB signaling pathway[D]. Guiyang:Guizhou Medical University, 2018. (in Chinese)
[22]	吴乙偲, 陈星华, 丁国华. TLR4与急性肾损伤的关系研究进展[J]. 国际泌尿系统杂志, 2016, 36(5):788-791.WU Y S, CHEN X H, DING G H. Research progress on the relationship between TLR4 and acute kidney injury[J]. International Journal of Urology and Nephrology, 2016, 36(5):788-791. (in Chinese)
[23]	张弢, 隋淼, 于扬, 等. 痛风患者尿KIM-1、NGAL、IL-18表达及与肾损伤的关系[J]. 中国中西医结合肾病杂志, 2020, 21(1):25-28.ZHANG T, SUI M, YU Y, et al. Expressions of urinary KIM-1, NGAL and IL-18 in gout patients and their relationships with renal injury[J]. Chinese Journal of Integrated Traditional and Western Medicine, 2020, 21(1):25-28. (in Chinese)