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標題: | 聚苯乙烯塑膠微粒誘發小鼠腸道菌群失衡及中樞神經毒性之研究 Gut microbiota dysbiosis and central nervous system toxicity after oral gavage with polystyrene microplastics in mice |
作者: | Han Chang 張涵 |
指導教授: | 鄭尊仁(Tsun-Jen Cheng) 鄭尊仁(Tsun-Jen Cheng | tsunjenc@gmail.com | ), |
關鍵字: | 聚苯乙烯塑膠微粒,胃管灌食,腸道緊密蛋白,腸道菌群,中樞神經系統, polystyrene microplastics,oral gavage,gut tight-junction protein,gut microbiota,central nervous system, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | 塑膠微粒是一種直徑小於 <5毫米塑膠顆粒的新興污染物。塑膠微粒的暴露途徑有攝入、吸入及皮膚接出,其中攝入 (Ingestion) 被認為是人類主要暴露塑膠微粒的途徑。根據過去的研究顯示,承裝食物的塑膠容器、塑膠茶包、嬰兒奶瓶在承裝高溫的水後皆會釋放出大量的塑膠微粒。此外,更有學者指出持續暴露塑膠微粒會導致小鼠腸道菌群失衡,進而引起發炎、腸道失能等生物毒性,後續這些生物毒性可能會發展成不同的疾病如中樞神經系統損傷等,進而影響行為。然而目前相關研究大部分都著重在短期暴露粒徑較大的塑膠微粒,針對次微米粒徑中長期暴露所造成的生物毒理機制結果仍相當有限。因此本研究目的為探討攝入塑膠微粒是否會改變小鼠的腸道菌群組成,並進一步透過腸腦軸線影響中樞神經系統。 本研究使用C57BL/6 雌性小鼠,以胃管灌食方式餵食粒徑5 μm與 0.5 μm Nile Red 螢光聚苯乙烯微粒。每週兩次,每次灌食0.3毫克的微粒。以全代謝籠於暴露期間隔週收集24小時糞便。暴露結束後,對小鼠進行了被動式迴避試驗 (Passive avoidance test) 與曠野實驗 (Open field test),以評估其空間辨識記憶、活動力及焦慮情形,並於行爲試驗結束後進行犧牲。犧牲時,採集盲腸內容物及結腸內容物分別做腸道菌群和短鏈脂肪酸分析。同時採集腦及腸道組織做分析。使用流式細胞儀 (Flow cytometry) 來檢測組織腸道的空腸與結腸、腦消化過後的組織消化液,以偵測單位微粒數目濃度。 此外,在生物檢測方面,部分腦組織會進行石蠟包埋與切片染色,透過蘇木精-伊紅染色 (Hematoxylin and eosin stain, H&E stain) 切片對神經元細胞進行計數,並以免疫組織染色 (Immunohistochemistry stain, IHC stain) 切片對氧化壓力指標丙二醛(Malondialdehyde, MDA)、小膠質細胞活化指標Iba-1及星形膠質細胞活化指標GFAP的陽性細胞比例進行計數。其他腦組織還有部分結腸組織會用於量測血清素 (Serotonin)、多巴胺 (Dopamine) 與乙醯膽鹼 (Acetylcholine) 神經傳遞物質的表現量。剩餘結腸部分則是透過免疫螢光學染色 (Immunofluorescence stain, IF stain) 切片對免疫蛋白受體 TLR-1、AP-1、IRF-5與西方墨點法 (Western Blot) 半定量緊密蛋白如ZO-1、 Claudin-1、Claudin-3去評估腸道免疫功能及屏障功能。後續腸道及腦皆會進行氧化壓力MDA、SOD的濃度檢測及發炎因子 (Cytokine) 的含量檢測,包括 Interferon- γ (IFNγ)、Interleukin- 1β (IL- 1β)、Interleukin-6 (IL-6) 與 Tumor Necrosis Factor- α (TNFα)。 在暴露12週PS-MPs後,被動式迴避試驗結果並沒有發現暴露組與控制組相比有顯著學習記憶差異。在曠野實驗中,0.5 μm 暴露組、5 μm 暴露組與控制組在各項參數中皆無顯著差異。 在微粒累積分析中發現空腸有0.5 μm微粒的累積,其餘腦與結腸組織近乎沒有0.5 μm、5 μm微粒的累積。在神經傳遞物質檢測方面,5 μm 暴露組腦中的血清素顯著下降 (p<0.05),其餘多巴胺及乙醯膽鹼的表現量皆無顯著差異;結腸部分,三種神經傳遞物質表現量無達統計顯著。小腸的Claudin-1在暴露組的表現量顯著低於控制組 (p<0.05)。兩組暴露組的腦中SOD濃度均有顯著增加,但MDA表現量沒有差異;在結腸樣本中,0.5 μm 暴露組、5 μm 暴露組的SOD和MDA均有所下降,而 0.5 μm 暴露組的MDA表現量達統計顯著 (p<0.05)。此外,5 μm暴露組腦中的促發炎因子IL-6顯著高於控制組 (p<0.05),其餘發炎因子表現量在腦與結腸中皆沒有統計顯著。腸道菌群分析顯示兩組暴露組的α 多樣性、β多樣性都有顯著差異 (p<0.05),0.5 μm 暴露組以Actinobacteria和Deferribacteres,5 μm 暴露組以Actinobacteria、Deferribacteres、Bacteroideres、Firmicutes、Proteobacteris、Tenericutes與Verrucomicrobia門類菌種有顯著改變 (p<0.05)。兩組暴露組與控制組相比在各短鏈脂肪酸指標中皆無顯著差異。切片染色分析部分,IHC staining切片顯示MDA、Iba-1及GFAP表現量在腦區皆無顯著差異。IF staining在結腸TLR-1、AP-1、IRF-5表現量無顯著差異。黏液分泌切片染色 (AB-PAS staining) 也無顯著差異。 結果表明,在暴露微粒相比5 μm微粒,0.5 μm微粒更能累積在小腸中,造成腸道緊密蛋白減少。針對腸道菌群部分,5 μm微粒更能造成α 多樣性上升、β多樣性與特定腸道菌種的改變,也引起腦中血清素下降、氧化壓力與發炎因子上升。此外,透過相關性分析發現Tenericutes門類菌種與腦中血清素呈現統計顯著負相關。然而詳細菌群透過腸腦軸線影響中樞神經系統之機制仍須後續研究近依一步探討。 Microplastics (MPs), a new type of environmental pollutant, is a kind of plastic particles which is <5 mm in diameter. The exposure routes of plastic particles are ingestion, inhalation and skin contact. Ingestion is considered the major route of human exposure to microplastics. Plastic food containers, plastic teabags, feeding bottles from previous studies have shown that they will release a large number of microplastics in high-temperature water. Moreover, researchers indicated that continuous exposure to plastic particles can cause gut dysbiosis in mice and lead to biological toxicity such as inflammation, dysfunction of the intestine. These changes may develop into several diseases including central nervous system injury, which will affect animals’ behavior. However, most toxicology studies mainly focused on larger size polystyrene plastic particles. Therefore, the purpose of my research is to investigate whether ingesting microplastics will alter the composition of gut microbiota and further impact on central nervous system through gut-brain axis. In my research, C57BL/6 female mice were exposed to 5 μm and 0.5 μm Nile red fluorescent polystyrene microplastics (PS-MPs) respectively at the dose of 0.3 mg/day via oral gavage. Oral gavage was be performed twice a week for 12 weeks. The stool was also collected during the exposure. After exposure, passive avoidance test and open field were conducted before sacrificing in order to evaluate the spatial recognition memory, locomotor, and anxiety of C57BL/6 female mice. After sacrificing, cecum content and colon content were collected for microbiota and short chain fatty acid analysis respectively. The brain and gut were collected for analysis as well. Flow Cytometer (FCM) was used to detect the number concentration of particles in brain and gut, especially Jejunum and colon. Furthermore, for the biological analysis, part of the brain samples were processed for tissue sections, stained with H&E and IHC for the quantification of neuronal cells, IHC-positive cells (MDA for oxidative stress, Iba1for the activation of microglia and GFAB for the activation of astrocytes), the other brain samples and colon were then utilized for measuring the expression of serotonin, dopamine and acetylcholine (neurotransmitters). As for some gut samples, immunofluorescence staining (TLR-1, AP-1, IRF- 5), the expression level of tight junction proteins such as ZO-1, Claudin-1 and Claudin-2 were carried out to assess intestine’s mucosal immune function, barrier function on colon and ileum respectively. In addition, I also measured the level of IFNγ, IL-1β, IL-6 and TNFα for inflammation, and SOD, MDA level for oxidative stress in both brain and gut tissues through ELISA kits. After oral gavage the polystyrene for 12 weeks, there were no significant changes in spatial recognition memory between 0.5 μm, 5 μm exposure groups and the control group through passive avoidance assessment. As in the open field test, there were also no significant changes in every parameter. In the accumulation analysis, 0.5 μm particles were accumulated in jejunum, and there were nearly no particles been found in brain, colon. The serotonin level in brain was significantly decreased in the 5 μm exposure group (p<0.05), however the concentration of dopamine, acetylcholine were not significantly changed; the neurotransmitters were not statistically significant in colon. There was a significant increase in SOD level in brain in both exposure groups (p<0.0.5); in colon samples, the MDA in the 0.5 μm exposure group has decreased significantly (p<0.0.5). Moreover, the IL-6 cytokine level has decreased in the 0.5 μm exposure group in brain (p<0.0.5). Lastly, the α, β diversity on microbiota have shown a significant difference in both exposure groups, the Actinobacteria, Deferribacteres in the 0.5 μm exposure group and Actinobacteria, Deferribacteres, Bacteroideres, Firmicutes, Proteobacteris, Tenericutes, Verrucomicrobia in the 5 μm exposure group have significantly changed (p<0.0.5). The short chain fatty acid in colon, IHC-positive cells (MDA, Iba-1, GFAP) in brain, IF-positive cells (TLR4, AP-1, IRF-5) in colon and the mucin positive cells from AB-PAS staining were not statistically significant between the control and the two exposure groups. Our study indicated that after 12 weeks of PS-MPs exposure, comparing to the 5 μm particles, the 0.5 μm particles could accumulate in jejunum, leading to the expression of TJ protein decrease and increase the permeability of the intestine. The 5 μm particles could alter the α, β diversity and specific gut microbiome, decrease the serotonin level, oxidative stress increase, inflammation increase in mice brain. Moreover, the Tenericutes phylum microbiota is negative correlated with the serotonin level in brain. However, the mechanism on how microbiota influence central nervous system through gut-brain axis still need to be further investigate. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85142 |
DOI: | 10.6342/NTU202202232 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2025-01-01 |
顯示於系所單位: | 環境與職業健康科學研究所 |
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