長期暴露黃麴毒素B1影響秀麗隱桿線蟲的老化及先天免疫

Chronic exposure to aflatoxin B1 adversely affects aging and innate immunity in Caenorhabditis elegans

張慈庭(Tzu-Ting Chang)

指導教授 ： 廖秀娟

共同指導教授 ： 劉貞佑

國立臺灣大學/公共衛生學院/食品安全與健康研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903362

摘要

黃麴毒素B1 (aflatoxin B1) 是已知的人類一級致癌物，也是麴菌毒素中毒性最強的毒素。Aflatoxin B1常存在於保存不良的農作物產品，尤其常在米、花生和玉米中發現。Aflatoxin B1會造成肝臟毒性及和免疫毒性等不量反應，然而，鮮少研究探討長期暴露aflatoxin B1如何影響老化及先天免疫。本研究利用秀麗隱桿線蟲Caenorhabditis elegans以及綠膿桿菌 (Pseudomonas aeruginosa PA14) 之host-pathogen模式，探討長期暴露aflatoxin B1所造成之老化及先天免疫能力的影響；並利用C. elegans食物Escherichia coli OP50的活性和暴露aflatoxin B1對C. elegans老化相關指標的影響，探討aflatoxin B1對免疫老化的調節和其分子機制。研究結果指出C. elegans從幼蟲 (L1) 暴露5與10 µM aflatoxin B1到成蟲第0天會延遲C. elegans的成長，並在2.5與5 µM的暴露下會顯著降低C. elegans的繁殖能力；而在較高濃度 (2.5及5 µM) 暴露的情況下也會縮短C. elegans的壽命長度。在先天免疫方面，長期暴露2.5 μM aflatoxin B1後會顯著降低C. elegans在遭受P. aeruginosa PA14感染後的存活。結果亦顯示，長期曝露aflatoxin B1 2.5 µM至成蟲第4天及第6天，野生種N2 C. elegans腸道體內累積的live E. coli OP50顯著多於未暴露aflatoxin B1的野生種N2 C. elegans；然而並未在餵食以經UV去活性dead E. coli OP50 觀察到此現象。暴露aflatoxin B1並以live E. coli OP50餵養之C. elegans的老化指標 (body bends, head thrashes, defecation cycle, lipofuscin) 都受到影響，但以dead E. coli OP50餵養的C. elegans則沒有統計上顯著差異。進一步利用two-way AVONA進行統計分析，顯示E. coli OP50的活性和aflatoxin B1在C. elegans有交互作用。利用基因轉殖C. elegans LD1發現，暴露於aflatoxin B1 2.5 μM會抑制SKN-1轉移入細胞核的現象，而在skn-1突變種的實驗中，有無暴露aflatoxin B1和以live及dead E. coli OP50餵養之C. elegans之間皆無顯著差異，推論aflatoxin B1抑制先天免疫和老化與抑制SKN-1及其下游基因表達相關。最後，在基因表達量的結果顯示，aflatoxin B1會抑制gst-4、gcs-1、hsp-16.1、hsp-16.49及hsp-70的mRNA表達量。綜合本研究之實驗結果顯示，長期暴露2.5 µM aflatoxin B1會加速老化和抑制先天免疫能力，並和其食物E. coli OP50的病源性有交互作用。

關鍵字

黃麴毒素B1 ； 長期暴露 ； 秀麗隱桿線蟲 ； 先天免疫 ； SKN-1 ； 老化

並列摘要

Aflatoxin B1, a human carcinogen, is the most toxic secondary metabolite in aflatoxins. It is usually found in poor storage of agricultural products, especially in rice, peanut, and maize. Aflatoxin B1 exposure causes several adverse effects such as hepatotoxicity and immunotoxicity. However, studies regarding effects of aging and innate immunity by aflatoxin B1 are limited. This study used Caenorhabditis elegans and Pseudomonas aeruginosa PA14 as a host-pathogen model to study how aflatoxin B1 affects aging and innate immunity. Moreover, live Escherichia coli OP50 and UV-treated dead E. coli OP50 were used to test aging-related indicators by chronic aflatoxin B1 exposure in C. elegans and the underlying molecular mechanisms were investigated. The results showed that prolonged exposure to aflatoxin B1 from L1 larvae delayed the growth stage (5, 10 μM), decreased reproduction, and reduced longevity (2.5, 5 μM) of C. elegans. Moreover, exposure to 2.5 µM aflatoxin B1 also significantly reduced the survival of C. elegans against the infection of P. aeruginosa PA14. Furthermore, after chronic aflatoxin B1 exposure (2.5 µM), there was a significantly increased accumulation of E. coli OP50 in the intestine of wild-type N2 C. elegans in day-4 adulthood compared with the untreated ones while feeding with live E. coli OP50. In contrast, the phenomena was absent while feeding with dead E. coli OP50. In addition, an increased body bends and head thrashes as well as a decreased defecation cycle and lipofuscin were observed in wild-type N2 C. elegans fed with dead E. coli OP50 in day-4 and day-6 adulthood in the presence of aflatoxin B1. Moreover, two-way ANOVA analysis revealed that aflatoxin B1 and the pathogenic activity of E. coli OP50 interact together affecting aging indicators in C. elegans. By using the transgenic strain LD1, 2.5 µM aflatoxin B1 inhibited the nuclear translocation of SKN-1. In skn-1 mutant, chronic exposure to 2.5 µM aflatoxin B1 didn’t affect lipofuscin at day-4 adulthood fed with either live or dead E. coli OP50. This suggests that aflatoxin B1 suppresses innate immunity as well as adversely affect aging indicators mediated by SKN-1. Finally, chronic exposure to 2.5 µM aflatoxin B1 suppressed the mRNA levels of gst-4, gcs-1, hsp-16.1, hsp-16.49 and hsp-70. Taken togethetr, results from this study demonstrate that chronic aflatoxin B1 exposure enhances aging decline and suppresses innate immunity and has an interaction with the pathogenic activity of E. coli OP50.

並列關鍵字

Aflatoxin B1 ； chronic exposure ； Caenorhabditis elegans ； innate immunity ； SKN-1 ； aging

參考文獻

Aballay, A., and Ausubel, F.M. (2002). Caenorhabditis elegans as a host for the study of host-pathogen interactions. Curr Opin Microbiol 5, 97-101.

Alegado, R.A., Campbell, M.C., Chen, W.C., Slutz, S.S., and Tan, M.W. (2003). Characterization of mediators of microbial virulence and innate immunity using the Caenorhabditis elegans host-pathogen model. Cell Microbiol 5, 435-444.

Amaike, S., and Keller, N.P. (2011). Aspergillus flavus. Annu Rev Phytopathol 49, 107-133.

Anderson, O.A., Finkelstein, A., and Shima, D.T. (2013). A2E induces IL-1β production in retinal pigment epithelial cells via the NLRP3 inflammasome. PLoS One 8, e67263.

Anukul, N., Vangnai, K., and Mahakarnchanakul, W. (2013). Significance of regulation limits in mycotoxin contamination in Asia and risk management programs at the national level. J Food Drug Anal 21, 227-241.