奈米氧化鋅對秀麗隱桿線蟲造成之免疫反應機制分析

Abstract

背景：奈米氧化鋅 (zinc oxide nanoparticles, ZnO-NPs) 被廣泛應用於醫藥產業以及日常生活用品中，因此ZnO-NPs的潛在毒性逐漸受到重視。有許多研究指出ZnO-NPs可能會引起細胞死亡、干擾細胞訊息傳遞並引起不正常發炎反應進而影響免疫系統，然而並沒有完整的研究探討長期暴露低濃度ZnO-NPs對宿主-病原菌 (host-pathogen) 關係之影響。 方法：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，評估長期暴露低濃度ZnO-NPs所造成之整體免疫能力影響。並且利用基因轉殖C. elegans、突變種以及即時定量聚合酶鏈鎖反應 (Q-PCR)，探討免疫以及氧化壓力相關的調控路徑，研究ZnO-NPs引起免疫毒性之可能毒理機制。 結果：研究結果指出長期暴露低濃度500 μg/L ZnO-NPs以及ZnCl2會顯著減少C. elegans在遭受病原菌感染時的存活時間，相較於控制組LT50分別減少了11.4 (12.6%) 與12.4 (13.8%) 小時。在進一步的溶菌酶LYS-7::GFP實驗中，發現僅500 μg/L ZnO-NPs暴露會抑制溶菌酶LYS-7的表達 (0.8倍)。相同的是，在C. elegans腸道活菌數的分析中，也僅有長期暴露500 μg/L ZnO-NPs才可看到腸道內的菌落數與控制組相較顯著提升約3倍。此結果代表ZnO-NPs可能會抑制抗菌相關物質，而讓病原菌滋生而讓宿主更容易死於感染。在免疫以及壓力反應相關的轉錄因子SKN-1分析中，利用基因轉殖C. elegans LD1發現，僅於長期暴露500 μg/L ZnO-NPs後可觀察到顯著抑制SKN-1轉移入核的現象。進一步在SKN-1下游基因GCS-1::GFP實驗中發現，ZnO-NPs以及ZnCl2皆具有抑制GCS-1表達的毒性 (約0.8倍)。此結果建議ZnO-NPs和ZnCl2可能受不同的毒理機制調控。接著本研究更深入探討ZnO-NPs是否會抑制SKN-1的上游調控路徑p38 MAPK pathway。於loss of function突變種的實驗中，所有的暴露組與控制組皆無顯著差異，ZnO-NPs無法於p38 MAPK pathway 相關突變種中更進一步抑制免疫能力，代表ZnO-NPs抑制免疫能力可能與抑制p38 MAPK pathway相關。最後，本研究利用Q-PCR，針對C. elegans中免疫相關基因以及p38 MAPK pathway基因進行分析。基因表達實驗結果指出ZnO-NPs與ZnCl2會顯著抑制gcs-1，與前述GCS-1::GFP實驗的結果相符。此外，ZnO-NPs與ZnCl2亦會以不同比例抑制不同的免疫相關基因 (lys-1、lys-7以及spp-1)；但於p38 MAPK pathway的基因中 (如pmk-1, sek-1, nsy-1)，僅有ZnO-NPs具有抑制現象。因SKN-1為p38 MAPK下游調控之轉錄因子，所以ZnO-NPs抑制SKN-1入核的現象可能與抑制p38 MAPK pathaway有關。 結論：綜合本研究之實驗結果顯示，長期暴露500 μg/L ZnO-NPs會透過抑制p38 MAPK pathway而干擾下游免疫以及壓力反應相關轉錄因子如SKN-1之運作，影響免疫調控並造成ROS失衡；並進而於C. elegans與P. aeruginosa PA14之host-pathogen模式中，ZnO-NPs顯著降低C. elegans抵禦病原菌的免疫能力。長期暴露低濃度ZnO-NPs可能會抑制個體的整體免疫能力，而更容易遭受病原菌感染而死亡。

Background: Zinc oxide nanoparticles (ZnO-NPs) are used extensively in medical industry and consumer products. Thus, the potential toxicity of ZnO-NPs has emerged as an important research topic. Several studies showed that ZnO-NPs induce cell death, interfere proinflammatory cytokines production and thus exert abnormal inflammation. However, the detailed interaction of host and pathogen by ZnO-NPs remains uncleared. Methods: This study used Caenorhabditis elegans and Pseudomonas aeruginosa PA14 as a host-pathogen model to study the immunotoxicity. Additionally, transgenic C. elegans, mutants and real-time quantification polymerase chain reaction (Q-PCR) were implemented to investigate its possible underlying mechanisms including immunity and ROS related regulation pathways by prolonged ZnO-NPs exposure. Results: The results showed that prolonged exposure to 500 μg/L ZnO-NPs and ZnCl2 significantly reduced the survival of C. elegans against the infection of P. aeruginosa PA14. Comparing to control, time to kill half of population (LT50) of prolonged exposure to 500 μg/L ZnO-NPs and ZnCl2 were reduced for 11.4 (12.6%) and 12.4 (13.8%) h, respectively. However, the reduction of lysozyme, LYS-7, expression was only observed in 500 μg/L ZnO-NPs treatment (0.8-fold). Similary, only prolonged exposure to 500 μg/L ZnO-NPs increased P. aeruginosa PA14 colonies about 3-fold in the intestine of C. elegans. This indicated that ZnO-NPs might inhibit antimicrobial factors and promote the accumulation of pathogen leading to death. Present study further investigated the relationship between ZnO-NPs and stress-responsive transcription factor SKN-1. By using the transgenic strain LD1, only 500 μg/L ZnO-NPs inhibited SKN-1 from translocating to nucleus. In GCS-1::GFP strain, both 500 μg/L ZnO-NPs and ZnCl2 decreased the expression of GCS-1 by 0.8-fold, which is a phase-II enzyme under SKN-1 regulation. This suggests that ZnO-NPs and ZnCl2 possibly possess different toxicity mechanisms. This study also investigated whether the toxicity of ZnO-NPs is associated with p38 MAPK pathway, which is a conserved regulation pathway of innate immunity acting upstream of SKN-1. By using loss of function p38 MAPK pathway mutants, the results showed that the survival of worms against P. aeruginosa PA14 infection among all examined mutants were comparable in the absence or presence of ZnO-NPs. This indicates that the immunosuppression effects of ZnO-NPs might be related to p38 MAPK pathway. Finally, Q-PCR was used to assess the effects of ZnO-NPs on the expression of immune and p38 MAPK pathway related genes. The downregulation of expression of gcs-1 by both ZnO-NPs and ZnCl2 was agreed with that of GCS-1::GFP assay. Moreover, both 500 μg/L ZnO-NPs and ZnCl2 reduced the expression of lys-1, lys-7 and spp-1. Interestingly, only 500 μg/L ZnO-NPs decreased the expression of p38 MAPK pathway genes (pmk-1, sek-1, nsy-1). As SKN-1 acts downstream of p38 MAPK, the inhibition of SKN-1 translocation might be related to the suppression of p38 MAPK pathway. The interference of p38 MAPK pathway not only affect immunity regulation but also leads to ROS imbalance. Conclusions: To sum up, present study used a host-pathogen model to provide evidences that prolonged exposure to low concentration of ZnO-NPs (500 μg/L) suppresses innate immunity and affects oxidative stress regulation via suppression of p38 MAPK pathway and downstream transcription factor SKN-1. Prolonged exposure to ZnO-NPs might suppress the immunity of an individual and, therefore, makes individual more susceptible to pathogen infection.