類固醇與抗組織胺對氣管與鼻黏膜的效應

Effects of glucocorticoids and H1-antihistamines on tracheal and nasal mucosa

劉紹正(Shao-Cheng Liu)

指導教授 ： 王興萬

共同指導教授 ： 李飛鵬(Fei-Peng Lee)

臺北醫學大學/醫學院/臨床醫學研究所/博士(2017年)

摘要

動機與目的：類固醇與抗組織胺均為治療慢性呼吸道疾病的常用藥物，但過去抗組織胺被認為只提供短效拮抗組織胺受體活化而引起的細胞組織水腫與腺體分泌，類固醇則以抗發炎為主，少有文獻探討此二類藥物在呼吸道重塑中之直接與間接效應。另一方面「冷」是引起呼吸道疾病的重要因子之一，但冷受體在呼吸道上皮的表現與其病、生理角色仍不明。因此，本研究主要以離體組織與鼻黏膜上皮培養模型來探討類固醇與抗組織胺的基因與非基因之調控模式，我們假設在呼吸道上皮黏膜有冷受體，當冷受體被活化時，呼吸道的張力與分泌物會隨之改變。
研究方法：使用離體大白鼠氣管與張力實驗，我們探討類固醇與抗組織胺對呼吸道的非基因調控模式，分析類固醇與抗組織胺對氣管因methocholine收縮的影響。同時也測試薄荷對離體鼻黏膜張力的影響。另一方面，我們建立鼻黏膜上皮培養模型，並對鼻黏膜切片與培養之上皮施予免疫染色來驗證冷受體的表現。隨後將培養之上皮分組，分別給予類固醇或抗組織胺治療，在治療1週後，對各組染色與使用西方墨點法觀察hismamine-1-receptor (H1R)、muscarinic-1-receptor(M1R)、M3R受體之變化；另給予組織胺與methocholine誘發過敏反應，比較各組黏液分泌量之差異。此外給予冷受體致效劑與拮抗劑，探討細胞分泌黏液的差異性。
研究結果：張力實驗顯示，在以methacholine刺激氣管收縮後，單獨使用類固醇(Kidsolone)或是抗組織胺(Xyzal)對張力變化無顯著影響，但同時給予兩項藥物會產生協同增效作用，張力顯著下降。在基因調控模式之研究中，類固醇(budesonide)與抗組織胺(azelastine)均能抑制上皮增生速度，且azelastine抑制的效果明顯大於budesonide，合併使用此兩項藥物的抑制效果大於單獨使用任一藥物，西方墨點法顯示budesonide可明顯上調H1R、M1R與M3R之表現量；而azelastine則明顯下調；另在給予組織胺或methacholine刺激後，接受budesonide治療的上皮分泌黏液的量明顯比對照組與接受azelastine之組高，接受azelastine組的上皮分泌黏液量則明顯的比對照組低。在鼻黏膜切片中，我們首度發現冷受體在鼻上皮與腺體組織呈現明顯的免疫染色反應。另一方面，薄荷與冷刺激可明顯誘發培養之上皮分泌黏液，且這效應可被冷受體拮抗劑所抑制。而薄荷刺激對鼻黏膜基礎張力沒有明顯影響，但可抑制鼻黏膜對交感神經致效劑的反應而舒張鼻黏膜。
結論：本研究顯示類固醇與抗組織胺對呼吸道重塑性同時存在基因與非基因調控模式，類固醇與抗組織胺會產生協同增效作用以放鬆氣管，抗組織胺則具有下調上皮增長速率與組織胺受體表現的效果，在治療慢性呼吸道疾病中，抗組織胺可減少由於長期使用類固醇所引起的副作用，因此，合併使用此兩個藥物，比單獨使用更可產生明顯的效益。另一方面，我們首次呈現了冷受體在鼻黏膜的分佈與作用，人使用薄荷後會覺得鼻部通暢，但鼻張力無明顯變化；且長期曝露於冷空氣中，冷受體活化後會拮抗交感神經作用，因此造成黏膜舒張與黏液分泌增加，引發過敏或氣喘發作。針對這些呼吸道疾病，冷受體拮抗劑可能有臨床上的療效。

關鍵字

潑尼松龍；抗組織胺；組織胺受體；呼吸道重塑性；冷受體；鼻黏膜張力；呼吸道阻力

並列摘要

Objective: Both glucocorticoids and H1-antihistamines are widely used on patients with chronic airway diseases. H1-antihistamines are well known for their short-term effect, by rapidly inhibiting allergic symptoms. Glucocorticoids work primarily through their anti-inflammatory properties. However, their genomic and non-genomic effects against airway remodeling have not been fully explored. On the other hand, exposure to cold air is a major environmental factor exacerbating chronic inflammatory airway diseases, but the distribution and physiological role of the cold receptor in the nose is not clear. Therefore, the aim of this study is to explore the genomic and non-genomic effects of glucocorticoids and H1-antihistamines and its implications for airway remodeling. Meanwhile, we hypothesize the cold receptor, transient receptor potential channel melastatin 8 (TRPM8), exists in nasal airway epithelial cells. When TRPM8 is activated, the tone of the airway and the secretion productions corresponding to stimulants will be changed.
Methods: Using isometric contraction ex-vivo studies, we tested the genomic effects of glucocorticoids (Kidsolone) and H1-antihistamines (Xyzal) on isolated rat trachea. Changes in contractility in response to the application of parasympathetic mimetic agents were measured. We also tested the effectiveness of the TRPM8 agonist, menthol, on nasal mucosa. The human nasal epithelial cells (HNEpC) primary cultures were established and immunohistology was used to examine the expression of TRPM8, as well as in the nasal mucosa strips. Then, HNEpC were cultivated with/without glucocorticoids (budesonide) and H1-antihistamines (azelastine). The histamine1-receptor (H1R), muscarinic1-receptor (M1R) and M3R were measured using immunocytochemistry and western blotting after 7-days treatment. We used histamine and methacholine to stimulate the mucus secretion from HNEpC and observed the MUC5AC expression in culture supernatants. Meanwhile, we also explored the physiological role of TRPM8 for mucus production in the nose, with/without its agonist and antagonist.
Results: Sole use of Kidsolone or Xyzal elicited no significant effect or only a minor relaxation response on tracheal tension following methacholine treatment. However, a significant synergetic spasmolytic effect was observed following co-administration of Kidsolone and Xyzal. As for the genomic effects of those two drugs, concentration-dependent treatment induced-inhibition of the HNEpC growth rate was observed. Cells incubated with azelastine proliferated significantly slower than that with budesonide and the combined use of those drugs significantly inhibited HNEpC proliferations. Western-blotting revealed budesonide could significantly up-regulate the H1R, M1R and M3R level while azelastine had the opposite effects. Histamine and methacholine stimulated MUC5AC secretion was greater in cells treated with budesonide but less in those treated with azelastine, as compared to controls. On the other hand, for the first time we demonstrated TRPM8 was expressed by the nasal mucosa epithelium and glandular tissue. Both menthol and cold at 24oC induced a statistically significant increased level of mucins production by HNEpC. The TRPM8 antogonist had a statistically significant inhibitory effect in mucins production induced by menthol and cold. Menthol had a negligible effect on the basal tension of the nasal mucosa, but higher doses of menthol had a significant spasmolytic effect on nasal mucosa precontracted with methoxamine.
Conclusions: We confirmed both glucocorticoids and H1-antihistamines exert their effects via both genomic and non-genomic mechanisms, which are beneficial in reducing airway remodeling. Glucocorticoids could be synergized with H1-antihistamines to dramatically relax the trachea smooth muscle. H1-antihistamines offer benefit in reducing airway hyper-reactivity symptoms and reducing mucus productions, by regulating the H1R, M1R and M3R levels, and might offset the side effects resulting from glucocorticoids. Therefore, combined use of glucocorticoids and H1-antihistamines in patients with chronic inflammatory airway diseases is recommended. We also for the first time demonstrated the TRPM8 immunoreactivity and its physiologic role in the human nose. The subjective clear feeling after menthol inhalation might be an illusion because menthol has no direct effect on the basal tension of nasal mucosa. Meanwhile, the prolonged exposure to cold will lead to the activation of TRPM8, mucin hypersecretion, and increase in airway resistance, which is thought to increase the risk of acute exacerbation of nasal allergy and asthma. Therefore, TRPM8 antagonists deserve consideration for treatment of chronic inflammatory airway diseases.

並列關鍵字

glucocorticoid；antihistamine；histamine receptor；airway remodeling；TRPM8；nasal mucosa tension；airway resistance

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