氨氮和微囊藻毒素-LR联合作用对斑马鱼肠道免疫和菌群的影响
Effects of Combined Exposure to Ammonia and Microcystin-LR on Intestinal Immunity and Microbial Community in Zebrafish (Danio rerio)
-
摘要: 氨氮和微囊藻毒素-LR(MCLR)是水生环境中普遍存在的污染物。为探讨两者对斑马鱼肠道潜在的协同效应，实验将成年雌性斑马鱼分别暴露于氨氮(30 mg·L-1)、MCLR (10 μg·L-1)以及两者混合(30 mg·L-1 + 10 μg·L-1)的环境中，持续30 d。组织病理学分析显示：氨氮暴露导致肠绒毛面积减少；MCLR暴露导致肠道绒毛破裂，空泡化面积增加；而联合暴露对肠组织损伤更为严重。这些变化伴随着肠道中溶菌酶和β-防御素的含量及相关基因表达的显著降低，表明斑马鱼肠道免疫功能受到抑制。此外，氨氮和MCLR的单独及联合处理还激活NOD1/2和TLR4a/4b信号通路，导致促炎因子IL-1β和TNF-α的表达水平和蛋白含量上升，进而可能诱发肠道炎症反应。肠道菌群分析结果进一步显示，氨氮和MCLR处理显著改变斑马鱼肠道内菌群的平衡，即氨氮增加厚壁菌门(Firmicutes)丰富度，MCLR增加放线菌门(Actinobacteria)丰富度但降低变形菌门(Proteobacteria)丰富度，而氨氮和MCLR联合作用增加肠道致病菌群假单胞菌属(Pseudomonas)和分枝杆菌属(Mycobacterium)丰富度。进一步，两者联合暴露还导致肠道中产生短链脂肪酸的菌群丰度和短链脂肪酸含量显著降低。综上所述，氨氮和MCLR联合处理对斑马鱼肠道免疫及菌群稳态产生了协同的负面影响，其对水生动物和水生态系统的健康构成了不容忽视的潜在风险。Abstract: Ammonia and microcystin-LR (MCLR) are commonly found pollutants in aquatic environments around the world. To explore the potential synergistic effects of ammonia and MCLR on the zebrafish intestine, adult female zebrafish were exposed to total ammonia nitrogen (30 mg·L-1)、MCLR (10 μg·L-1) and a mixture of both (30 mg·L-1+ 10 μg·L-1) for a period of 30 d. Histopathological analysis showed that ammonia exposure led to a decrease in intestinal villis area, while MCLR exposure caused villus rupture and increased the ratio of vacuolization in intestines. The combination of ammonia and MCLR resulted in more serious damage in the intestine. Ammonia alone and in combination with MCLR significantly reduced the contents and related gene expression levels of intestinal lysozyme and β-defensin compared with the control group, indicating suppressed intestinal immunity in zebrafish. Moreover, the single and combined exposure to ammonia and MCLR increased the mRNA expression levels and contents of intestinal inflammatory factors IL-1β and TNF-α by activating NOD1/2 and TLR4a/4b signal pathways, leading to intestinal inflammation. Results of intestinal microbiota showed that both ammonia and MCLR caused changes in diversity. Ammonia nitrogen increased the richness of Firmicutes, while MCLR increased the richness of Actinobacteria and decreased the richness of Proteobacteria. The combined exposure to ammonia and MCLR significantly increased the abundance of Pseudomonas and Mycobacterium. Furthermore, their combined exposure reduced the number of OTUs of short-chain fatty acids producers and the contents of short-chain fatty acids in the intestine. In conclusion, the combination of ammonia and MCLR has a synergistic effect on the intestinal immunity and structure of intestinal flora in zebrafish, and their combined effects on the health of aquatic animals and the water ecosystem’s integrity should not be underestimated.
-
Key words:
- ammonia /
- microcystin-LR /
- zebrafish /
- intestinal flora /
- intestinal immune
-
-
Armstrong D A, Chippendale D, Knight A W, et al. Interaction of ionized and un-ionized ammonia on short-term survival and growth of prawn larvae, Macrobrachium rosenbergh [J]. The Biological Bulletin, 1978, 154(1): 15-31 于畅, 孙雨芹. 南京市马汊河氨氮污染分析及治理方案探讨[J]. 环境保护与循环经济, 2014, 34(6): 26-29 Yu C, Sun Y Q. Analysis of ammonia nitrogen pollution in Macha River in Nanjing and discussion on its control scheme [J]. Environmental Protection and Circular Economy, 2014, 34(6): 26-29(in Chinese)
肖炜, 李大宇, 徐杨, 等. 慢性氨氮胁迫对吉富罗非鱼幼鱼生长、免疫及代谢的影响[J]. 南方水产科学, 2015, 11(4): 81-87 Xiao W, Li D Y, Xu Y, et al. Effects of chronic external ammonia stress on growth, immunity and metabolism of juvenile GIFT tilapia(Oreochromis niloticus) [J]. South China Fisheries Science, 2015, 11(4): 81-87(in Chinese)
Cheng C H, Yang F F, Ling R Z, et al. Effects of ammonia exposure on apoptosis, oxidative stress and immune response in pufferfish (Takifugu obscurus) [J]. Aquatic Toxicology, 2015, 164: 61-71 Guo H H, Chen S Q, Ouyang K, et al. Evaluation of ammonia nitrogen exposure in immune defenses present on spleen and head-kidney of Wuchang bream (Megalobrama amblycephala) [J]. International Journal of Molecular Sciences, 2022, 23(6): 3129 Kathyayani S A, Poornima M, Sukumaran S, et al. Effect of ammonia stress on immune variables of Pacific white shrimp Penaeus vannamei under varying levels of pH and susceptibility to white spot syndrome virus [J]. Ecotoxicology and Environmental Safety, 2019, 184: 109626 Jiang L, Feng J X, Ying R, et al. Individual and combined effects of ammonia-N and sulfide on the immune function and intestinal microbiota of Pacific white shrimp Litopenaeus vannamei [J]. Fish & Shellfish Immunology, 2019, 92: 230-240 Zhang T X, Zhang Y, Xu J Y, et al. Toxic effects of ammonia on the intestine of the Asian clam (Corbicula fluminea) [J]. Environmental Pollution, 2021, 287: 117617 Gong W D, Browne J, Hall N, et al. Molecular insights into a dinoflagellate bloom [J]. The ISME Journal, 2017, 11(2): 439-452 Adiv S, Carmeli S. Protease inhibitors from Microcystis aeruginosa bloom material collected from the Dalton Reservoir, Israel [J]. Journal of Natural Products, 2013, 76(12): 2307-2315 Zhao S J, Yuan C Y, Tuo X, et al. MCLR induces dysregulation of calcium homeostasis and endoplasmic reticulum stress resulting in apoptosis in Sertoli cells [J]. Chemosphere, 2021, 263: 127868 Ling X D, Zuo J L, Pan M Q, et al. The presence of polystyrene nanoplastics enhances the MCLR uptake in zebrafish leading to the exacerbation of oxidative liver damage [J]. The Science of the Total Environment, 2022, 818: 151749 Qiao Q, Liang H L, Zhang X Z. Effect of cyanobacteria on immune function of crucian carp (Carassius auratus) via chronic exposure in diet [J]. Chemosphere, 2013, 90(3): 1167-1176 Lin W, Guo H H, Wang L K, et al. Waterborne microcystin-LR exposure induced chronic inflammatory response via MyD88-dependent toll-like receptor signaling pathway in male zebrafish [J]. The Science of the Total Environment, 2020, 702: 134969 Chen C Y, Liu W J, Wang L, et al. Pathological damage and immunomodulatory effects of zebrafish exposed to microcystin-LR [J]. Toxicon: Official Journal of the International Society on Toxinology, 2016, 118: 13-20 Zhang Y, Li Z Y, Tian X, et al. Acute toxic effects of microcystin-LR on crayfish (Procambarus clarkii): Insights from antioxidant system, histopathology and intestinal flora [J]. Environmental Science and Pollution Research, 2023, 30(19): 56608-56619 Mowat A M, Agace W W. Regional specialization within the intestinal immune system [J]. Nature Reviews Immunology, 2014, 14(10): 667-685 Kumar V, Roy S, Meena D K, et al. Application of probiotics in shrimp aquaculture: Importance, mechanisms of action, and methods of administration [J]. Reviews in Fisheries Science & Aquaculture, 2016, 24(4): 342-368 Daen C I, Pinget G V, Tan J K, et al. Detrimental impact of microbiota-accessible carbohydrate-deprived diet on gut and immune homeostasis: An overview [J]. Frontiers in Immunology, 2017, 8: 548 Goel A, Gupta M, Aggarwal R. Gut microbiota and liver disease [J]. Journal of Gastroenterology and Hepatology, 2014, 29(6): 1139-1148 Parada Venegas D, de la Fuente M K, Landskron G, et al. Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases [J]. Frontiers in Immunology, 2019, 10: 277 Brown R H, Duda G D, Korkes S, et al. A colorimetric micromethod for determination of ammonia; the ammonia content of rat tissues and human plasma [J]. Archives of Biochemistry and Biophysics, 1957, 66(2): 301-309 Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod [J]. Methods, 2001, 25(4): 402-408 国家环境保护局. 渔业水质标准: GB 11607—89[S]. 北京: 国家环境保护局, 1989 Sedan D, Laguens M, Copparoni G, et al. Hepatic and intestine alterations in mice after prolonged exposure to low oral doses of microcystin-LR [J]. Toxicon: Official Journal of the International Society on Toxinology, 2015, 104: 26-33 Qian H F, Zhang M, Liu G F, et al. Effects of different concentrations of Microcystis aeruginosa on the intestinal microbiota and immunity of zebrafish (Danio rerio) [J]. Chemosphere, 2019, 214: 579-586 Guo H H, Lin W, Wu X Y, et al. Survival strategies of Wuchang bream (Megalobrama amblycephala) juveniles for chronic ammonia exposure: Antioxidant defense and the synthesis of urea and glutamine [J]. Comparative Biochemistry and Physiology Toxicology & Pharmacology, 2020, 230: 108707 Zhou Y, Xu X P, Yu B B, et al. Characterization of in vitro effects of microcystin-LR on intestinal epithelial cells [J]. Environmental Toxicology, 2017, 32(5): 1539-1547 Saurabh S, Sahoo P K. Lysozyme: An important defence molecule of fish innate immune system [J]. Aquaculture Research, 2008, 39(3): 223-239 Oehlers S H, Flores M V, Chen T, et al. Topographical distribution of antimicrobial genes in the zebrafish intestine [J]. Developmental and Comparative Immunology, 2011, 35(3): 385-391 熊大林, 段亚飞, 王芸, 等. 氨氮胁迫对凡纳滨对虾肠道免疫相关指标的影响[J]. 海洋渔业, 2020, 42(1): 53-60 Xiong D L, Duan Y F, Wang Y, et al. Response of non-specific immune related indexes in intestines of Litopenaeus vannamei under ammonia-N stress [J]. Marine Fisheries, 2020, 42(1): 53-60(in Chinese)
Li Y J, Li Y L, Cao X C, et al. Pattern recognition receptors in zebrafish provide functional and evolutionary insight into innate immune signaling pathways [J]. Cellular & Molecular Immunology, 2017, 14(1): 80-89 Szebeni B, Veres G, Dezsõfi A, et al. Increased expression of toll-like receptor (TLR) 2 and TLR4 in the colonic mucosa of children with inflammatory bowel disease [J]. Clinical and Experimental Immunology, 2008, 151(1): 34-41 Strober W, Murray P J, Kitani A, et al. Signalling pathways and molecular interactions of NOD1 and NOD2[J]. Nature Reviews Immunology, 2006, 6(1): 9-20 Goyette P, Labbé C, Trinh T T, et al. Molecular pathogenesis of inflammatory bowel disease: Genotypes, phenotypes and personalized medicine [J]. Annals of Medicine, 2007, 39(3): 177-199 Chen S S, He J, Wang H Y, et al. Microbial responses and metabolic pathways reveal the recovery mechanism of an anaerobic digestion system subjected to progressive inhibition by ammonia [J]. Chemical Engineering Journal, 2018, 350: 312-323 Garrett W S, Gordon J I, Glimcher L H. Homeostasis and inflammation in the intestine [J]. Cell, 2010, 140(6): 859-870 Kelly D, Yang L Y, Pei Z H. Gut microbiota, fusobacteria, and colorectal cancer [J]. Diseases, 2018, 6(4): 109 Austin B, Austin D A. Bacterial Fish Pathogens: Disease of Farmed and Wild Fish [M]. Chichester New York: E. Horwood Halsted Press, 1999 Guo J L, Han X, Zhan J C, et al. Vanillin alleviates high fat diet-induced obesity and improves the gut microbiota composition [J]. Frontiers in Microbiology, 2018, 9: 2733 Fattahi Y, Heidari H R, Khosroushahi A Y. Review of short-chain fatty acids effects on the immune system and cancer [J]. Food Bioscience, 2020, 38: 100793 Schulthess J, Pandey S, Capitani M, et al. The short chain fatty acid butyrate imprints an antimicrobial program in macrophages [J]. Immunity, 2019, 50(2): 432-445.e7 Tian Y, Xu Q, Sun L Q, et al. Short-chain fatty acids administration is protective in colitis-associated colorectal cancer development [J]. The Journal of Nutritional Biochemistry, 2018, 57: 103-109 Zhang G L, Zhang P Y, Li W, et al. Reduction of gut microbial diversity and short chain fatty acids in BALB/c mice exposure to microcystin-LR [J]. Ecotoxicology, 2020, 29(9): 1347-1357 -
点击查看大图
计量
- 文章访问数: 1387
- HTML全文浏览数: 1387
- PDF下载数: 170
- 施引文献: 0
下载:
