芳香烴受器與第二型轉麩胺酶的表現在巨噬細胞中會受到多環芳香烃化合物與戴奧辛的影響

The effect of Polycyclic aromatic hydrocarbon and Dioxins on the expression of Aryl Hydrocarbon Receptor and Transglutaminase 2 in macrophage

方毓翔(Yu-Hsiang Fang)

指導教授 ： 蔡嘉哲

中山醫學大學/醫學院/微生物免疫研究所/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00045

摘要

目的：
目前自體免疫疾病病因仍然尚未清楚，而先前研究指出自體免疫疾病如類風濕性關節炎可能因為此巨噬細胞活化於是分泌一些發炎性細胞激素 (例如TNF-α, IL-1β)，導致滑膜增生和肥大、膠原酶釋出。最後導致軟骨破壞，硬骨侵蝕，造成關節損傷、變形與關節功能喪失。而也有研究指出在類風濕性關節炎的病患中的滑膜組織有高度芳香烃受器(AHR)表現，並且在吸菸的患者下有更高度的表現。但很少有研究在AHR活化對於巨噬細胞的影響。另外有研究指出在人類樹突細胞以及小鼠胚胎纖維母細胞中可以觀察到AHR以及NF-κB有密切相關。再者，更有文獻報導第二型轉麩胺酶(TG2)的表現是依靠NF-κB路徑活化並且會影響巨噬細胞的吞噬功能。因此，我們的研究想藉此了解在於巨噬細胞中AHR的活化是否與第二型轉麩胺酶有相關性，並進一步了解巨噬細胞中AHR的活化對於自體免疫疾病的影響。

實驗方法：
首先，我們使用小鼠巨噬細胞(Raw264.7)以2,3,7,8-Tetrachlorodibenzodioxin(TCDD)多環芳香烃化合物處理依時間點收取細胞RNA並測其AHR以及AHR下游基因CYP1A1表現量，並選取藥物處理時間。測定時間點後我們以TCDD以及LPS處理小鼠巨噬細胞六小時並收取細胞RNA以及細胞蛋白觀察AHR以及TG2 mRNA與蛋白表現量，再以統計方法分析兩者之間的相關性以及顯著性。

結果：
由實驗結果我們可以觀察到巨噬細胞受到多環芳香烃化合物刺激下活化AHR，雖然AHR的表現沒有顯著性，但在AHR下游基因CYP1A1可以看到有表現的現象並也說明了AHR的活化結果，並且在第六個小時有最高峰的表現。而在以TCDD處理六小時下可以觀察到AHR與CYP1A1的活化與LPS處理下都有顯著效果，這也說明了使用LPS活化NF-κB路徑會影響AHR的活化的可能性；並且在TCDD處理下TG2的表現量與LPS處理也都有顯著性，這也可以推測使用多環芳香烃化合物刺激的結果不只活化AHR也會活化TG2的表現並做進一步研究AHR以及TG2的相關性研究。

結論：
由我們的實驗結果說明了在小鼠巨噬細胞中AHR的表現雖然沒有顯著性，但觀察其下游基因可以了解AHR依然有活化的情形，而對於使用多環芳香烃化合物刺激下TG2的表現也可以做為未來了解AHR活化是否與NF-κB路徑的影響，這些結果讓我們可以進一步研究AHR在巨噬細胞的活化以及影響其功能的研究，並了解巨噬細胞受到環境毒物因子或是吸菸的情形下對於自體免疫疾病的發展性與致病機轉的研究。

關鍵字

芳香烴受器 ； 第二型轉麩胺酶

並列摘要

Objective

Recently, the pathogenesis of autoimmune diseases remain unclear. Previous studies have shown that pathogenesis of rheumatoid arthritis (RA) is activation of macrophages, and then secreting inflammatory cytokines, resulting in proliferate and hypertrophy of synovial tissue and collagenase secreting. These cause destruction of cartilage, erosion of bones and loss of function of joints. Another study has shown that aryl hydrocarbon receptor (AHR) expression is higher in synivol tissue of RA patients who have habit of smoking. However, less of studies indicate activation of AHR in macrophages. In addition, the evidences show activation of AHR and NF-κB pathway in human dendritic cells and mouse embryonic fibroblast. Accumulating articles reveal that expression of transglutaminase (TG2) depends on NF-κB pathway and affect function of macrophages. Therefore, we investigate the cross-link between activation of AHR and expression of TG2 in macrophage, and interest in activation of AHR in autoimmune disease.

Methods
To investigate the roles of AHR and TG2 with TCDD treatment in macrophage, RT-PCR and Western blot were performed. The paired t test and one-way ANOVA were used to analyze for statistical significance. A P value < 0.05 was considered significant.

Results
In the present study, we report mRNA and protein expression of AHR and TG2 are significantly increased in Raw264.7 by TCDD treatment. In addition, we observe protein and mRNA expression of AHR and TG2 are significantly increased in Raw264.7 by LPS and TCDD treatment.

Conclusion
Taken together, our experiment data suggests that the cross-link between AHR and TG2 in macrophage, possibly through the involvement of NF-κB pathway and activation of AHR. These findings could provide evidences in investigating the role of AHR in macrophages and activation of AHR related disease.

並列關鍵字

Aryl Hydrocarbon Receptor ； Transglutaminase 2

參考文獻

1. Nam Trung Nguyen & Taisuke Nakahama & Tadamitsu Kishimoto：Aryl hydrocarbon receptor and experimental autoimmune arthritis；Semin Immunopathol (2013) 35:637–644

2. Christoph F.A. Vogel, Elaine M. Kahna, Patrick S.C. Leung：Cross-talk between Aryl Hydrocarbon Receptor and the inflammatory response: a Role for NF-κB；J. Biol. Chem. published online December 3, 2013

3. Philip B Busbee, Michael Rouse, Mitzi Nagarkatti, and Prakash S Nagarkatti：Use of natural AhR ligands as potential therapeutic modalities against inflammatory disorders； Nutrition ReviewsR Vol. 71(6):353–369

4. Denison MS, Nagy SR (2003). Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43: 309-34.

5. N. Hao, M.L. Whitelaw：The emerging roles of AhR in physiology and immunity；Biochemical Pharmacology 86 (2013) 561–570