行政院國家科學委員會補助專題研究計畫
□ 成 果 報 告
□期中進度報告
右歸丸對氣喘動物模式蛋白質體學研究及其藥物組合之
分析探討
計畫類別：█ 個別型計畫
□ 整合型計畫
計畫編號：NSC 97－2320－B －039 －014－MY3
執行期間：
2008 年
8 月
1 日至
2011 年 7 月
31 日
計畫主持人：
高尚德共同主持人：
陳玉芳、鄭如茜、葉家舟計畫參與人員：
林志哲、黃薇頻、賴正邦成果報告類型(依經費核定清單規定繳交)：█精簡報告
□完整報告
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
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□國際合作研究計畫國外研究報告書一份
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執行單位：中國醫藥大學 中醫系
中
華
民
國
99
年
5
月
31
日
中、英文摘要及關鍵詞
關鍵詞：右歸丸、塵蟎、氣喘、Th1/Th2 平衡、細胞激素、化學趨化因子、趨合分子 、 蛋白質體學 氣喘是全球性的重大公共衛生問題，研究指出，引發氣喘最重要的因子是過敏反 應，其罹患率與致死率在過去二十年中持續增加，氣喘於治療與預防有其重要性。 目前已知中藥對於免疫系統具調節功效，我們先前的研究也發現中藥方劑可以調節 Th1 與 Th2 細胞平衡，小青龍湯源自東漢‧張仲景《傷寒論》，是治療氣喘的名方。我們研究發現小青龍湯能夠降低塵蟎(Dermatophagoides pteronyssinus, Der p)所誘發之氣喘 老鼠肺泡沖洗液的發炎細胞總數及嗜酸性白血球浸潤，調降過敏原誘發肺內淋巴球反 應，及降低肺泡沖洗液和血清中前趨發炎細胞激素(proinflammatory cytokine)並可抑制肺 組織中轉錄因子、細胞激素與化學驅化因子之表現。去年本研究室探討小青龍湯蛋白質 體層次之研究，我們得到了更進一步的進展。
右歸丸是明 張景岳《景岳全書》氣喘緩解期調理氣喘的名方，我們發現右歸丸能 夠降低塵蟎(Dermatophagoides pteronyssinus, Der p)誘發氣喘天竺鼠立即性與遲發性氣喘 反應，亦能減少塵蟎所誘發之氣喘老鼠肺泡沖洗液的發炎細胞總數及嗜酸性白血球浸潤 及降低肺泡沖洗液中 CD4+ T 細胞族群。探討右歸丸在蛋白質體學層次的作用，並與右 歸丸全方作用相比，了解各藥物在方中的作用與所扮演的角色，我門取老鼠肺泡沖洗液 及肺組織進行蛋白質學研究，了解氣喘動物與正常鼠間右歸丸與各藥物治療氣喘蛋白質 之變化，並從其中找尋更佳更有效的組合，進一步分析探討，以利未來新藥或新方配法 之開發。
Asthma is a major public health problem worldwide, and asthma morbidity and mortality have increased over the last two decades.
There are detailed description of the clinical experiences and prescriptions of asthma in traditional Chinese medicine; Yo-Qui-Wang(YQW) is one of the Chinese herbal medicines used to treat bronchial asthma for the thousand-year clinical practice. In our previous research, we found YQW has bronchodilator and immunomodulatory effects on reducing bronchial inflammation in the allergen-induced murine model. Now, we want to evaluate the YQW in proteomic change in allergic-challenged mice and the subgroups effect of YQW on changes of the inflammatory cell infiltration、cytokines、chemokines、serum Ag-specific antibodies 、 Th1/Th2 balance 、 transcription factor and proteomic change in allergic-challenged mice to further understand the possible mechanisms on immune response. Those herbs maybe provide a good database to develop new formulae or drug in future.
報告內容
近年來許多研究傳統中醫藥經由調節免疫系統(例如 T 輔助型細胞[Th]中 Th1/Th2 細
胞之調控等)，而達到“改善體質”之作用來達到預防與治療氣喘的目的，我們研究發現，
中藥方劑可以降低氣喘天竺鼠呼吸道過度反應性、立即性與遲發性呼吸道阻力，調節發 炎細胞浸潤與氣喘老鼠肺周邊淋巴結(draining lymph node ; DLN)中 Th2 細胞之百分比 [1-6]。
氣喘致病機轉中，T-淋巴球在產生、調控及氣喘呼吸道慢性發炎上扮演重要角色， 其暴露於過敏原後，肥大細胞(mast cell)開始了發炎之過程，淋巴球很可能提供氣喘活化 繼而建立慢性且持續性發炎之訊息。我們的研究發現右歸丸可調控 T 淋巴球之浸潤[4]， 而動物模式亦顯示由塵蟎(Dermatophagoides pteronyssinus, Der p)所激發之氣喘模型中， Th2 細胞(IL-4+/CD3+/CD4+)之百分比亦明顯增加[4]。由前人之研究中可看出輔助性 T 輔 助型淋巴球依所分泌淋巴激素不同可分成 Th1 及 Th2，Th1 細胞產生 IL-2、IL-3、GM-CSF， INF-γ、IL-10、TNF-β等細胞激素，Th2 細胞產生 IL-3、GM-CSF、IL-4、IL-5、IL-6、IL-10、 IL-13 細胞激素，這二種次分類的 Th 細胞參與不同的免疫反應，Th1 細胞參與遲發性過 敏 反 應 (delayed type hypersensitivity) 而 Th2 細 胞 參 與 過 敏 性 發 炎 反 應 (allergic inflammation)。
在 Th1 和 Th2 細胞之間的平衡關係中，研究顯示調節 Th1/Th2 細胞的平衡以使 Th2 比例減少而 Th1 比例增加，被認為是治療由 Th2 細胞為主的過敏疾病，因而 Th1/Th2 之免疫調節劑之發展乃為近年來研究對過敏氣喘預防與治療之重點之一，不少運用重組 細胞激素(recombinant cytokine)或細胞激素拮抗劑(cytokine antagonist)，調節 Th1/Th2 平 衡而改變疾病癒後的研究中發現，IL-12 在某些情況下可將已建立之 Th2 反應轉變為 Th1 反應優勢，被認為其可能用於治療過敏[7-8]，IFN-γ或抗 IL-4 抗體或抗 IL-5 抗體會抑制 過敏性老鼠肺內嗜酸性白血球症[9]，但是直接給予此類的細胞激素會造成令人憂心的副 作用，此外由於它們缺少口服活性因而限制了療效。另一種調控細胞激素之方法是給予 小分子如 AS-101 (tellurium-based compound) [10-11]和 OK-432 (streptococcal preparation) [12]來操作內部的 Th1/Th2 平衡，其因能調節 IL-10 或 IL-12 的產生而使得傾向 Th1 反應， 但是它們口服無效。因此尋找能調節 Th1/Th2 細胞平衡的口服藥物是值得開發的方向。 過敏性氣喘是一種呼吸道疾病，是探討其成因、發展過程或尋找診斷指標和治療標 的，毫無疑問的從肺組織直接取樣是最理想的方式。肺泡沖洗液是一種容易取得和能反 映肺部病態的樣本，從肺泡沖洗液中不單可收集呼吸道肺泡中細胞，作細胞學檢查[13]， 肺泡沖洗液中含有多種源自血液或由局部上皮或發炎細胞所釋放的蛋白，可以定性和定 量分析[14]。許多肺部疾病的研究如氣喘和急性呼吸緊迫症候肺泡沖洗液中蛋白組成皆出 現異常。Wathez 等利用 2-DE 分析人類肺泡沖洗液，研究 sarcoidosis、idiopathic pulmonary fibrosis 和 hypersensitivity pneumonitis[15]。另外有研究發表利用 2-DE 分析吸入性α 1-protease inhibitor 對 cystic fibrosis 的療效評估[16]。
目前 2-DE 與氣喘相關研究並不多，我們試圖透過塵蟎誘發呼吸道發炎及過敏反應， 來了解中藥對呼吸道微環境蛋白質的表現，進一步鑑定中藥在治療塵蟎過敏中涉及那些 主要蛋白分子調控。 過敏性氣喘病理生理機轉非常複雜，包含很多細胞表現不同功能[17-18]，病理機轉 之研究需深入了解不同蛋白質在正常肺狀態及特殊疾病過程中的功能角色，肺泡沖洗液 包含不同細胞及源自於血液或上皮及發炎細胞所釋放出來之蛋白質[19] ，由於肺泡沖洗 液中蛋白質有不同來源，分析肺泡沖洗液可得知重要的病理性介質及很多肺疾病在分子 層次上更正確的特點，過敏原誘發氣喘動物模式，我們可了解正常老鼠及氣喘老鼠在過 敏原激發與發炎及應之間關係。 目前已知中藥對於免疫系統具調節功效，研究中醫藥是否能調整 Th1/Th2 平衡乃為 近年來研究重心之一。我們先前的研究也發現中藥方劑可以調節 Th1 與 Th2 細胞平衡， 臨床上右歸常用於氣喘病人，其治療機轉本實驗室已獲得初步成果，本計劃將進一步探 討右歸丸對氣喘老鼠呼吸道蛋白質體方面之變化，並依中藥配伍理論將右歸丸做不同的 組合與右歸丸全方作用相比，了解各藥物在方中的作用與所扮演的角色，我們取老鼠肺 泡沖洗液及肺組織進行蛋白質學研究，了解氣喘動物與正常鼠間與右歸丸全方及各藥物 治療氣喘蛋白質之變化，並從其中找尋更佳更有效的組合，進一步分析探討，以利未來 新藥或新方配法之開發。
四、研究方法：
一、藥品製備與購買
1. YQW preparationYQW ( KODA pharmaceutical Co., Ltd. in Taiwan) is composed of ten medicinal plants . Six hundred gram of fifteen components is soaked in 6 L of spring water with for 30 min at room temperature and then boiled for 50 min. Repeat two times of boiled extraction and harvest these two times of supernatant. This supernatant will be sequentially passed through No.1 filter paper for remove insoluble ingredients. The supernatant was concentrated to 600 ml. Hesperidin was purchased from Sigma.
二、
Table 1.實驗分組：
組別 處理
Naïve 組 6-8 週 BALB/c 雄鼠不激發、不餵藥
Der p 組 BABL/c 小鼠，以 50μl Der p（1mg/ml）氣管內接種激發 5 次，
Der p＋右歸丸組 BABL/c 小鼠，以 50μl Der p（1mg/ml）氣管內接種激發 5 次， 每次間隔一週，且於每次激發前 30 分鐘給 0.3ml 藥物，
最後一次激發後 3 天犧牲。
三、氣喘動物模式建立簡圖：
Drug Drug Drug Drug Drug 72hr
Day0 Day7 Day14 Day21 Day28 Day31
Der-p/PBS Der-p/PBS Der-p/PBS Der-p/PBS Der-p/PBS Sacrifice
IT IT IT IT IT
四、實驗方法：
1. Mice and reagents
Specific pathogen-free, male, 6-to 8-wk-old BALB/c mice（Laboratory Animal Center ） were used in this study. The mice were housed in microisolator cages（Laboratory Products, Inc., Maywood, NJ, USA） fed sterile food and water ad libitum. All experimental animal care and treatment followed the guidelines set up by the National Science Council of the Republic of China. Lyophilized house-dust mite （Dermatophagoides pteronyssinus [Der p]） was purchased from Allergon （Engelholm, Sweden）. The crude mite preparation was extracted with ether. After dialysis with deionized water, the mite extract was lyophilized and stored at -80℃before use. LPS concentration of the Der p preparation was ＜0.96 EU/mg of Der p （Limulus amebocyte lysate test; E-Toxate; Sigma-Aldrich）.
2. Allergen challenge, assessment of blood eosinophilia and airway inflammation
Groups of five BALB/c mice were i.t. inoculated with five doses of Derp（1 mg/ml, 50μl）in phosphate-buffered saline（PBS） at 1-wk intervals. At three days after the last challenge, the number of blood eosinophils was determined using diagnotic reagent system（Unopette test 5877; BD Biosciences, Rutherford, NJ）with blood samples collected via the orbital sinus. Mice were then killed by i.p. injection of xylazine （200μg/mice）and ketamine（2 mg/mice）, serum samples were collected and stored at -80℃ until assay. BAL was performed （two washes of 1 ml of ice –cold endotxin-free PBS） according to the previously described procedure[7]. The BAL fluids were separated （1500 rmp, 10 min, 4 ℃）and stored at -80 ℃. After total leukocyte counting, differential counts were performed on cytospin preparations （1 × 105
cells/100 μl of BALF）stained with Liu stain（Biotech, Taiwan）in a blind manner. For TCM study, mice were given 1g/kg YGW at 1-wk intervals before each Der p inoculation and naïve mice were also included in the experiment for purposes of comparison.
3. Two-dimensional electrophoresis
Protein concentrations of the BAL fluid were determined using a commercial protein assay kit (Micro BCA, Pierce, Rockford, IL, USA) with bovine serum albumin as standard. The proteins were precipitated with TCA (10% final concentration) in an ice bath for 20 min, and subsequently centrifuged at 3500 rpm for 15 min and 4℃. The pellet was suspended in ice-cold acetone using a sonicator and centrifuged as described above. The pellet was
air-dried for a few minutes and, finally, resuspended in the sample solution (9 M urea, 0.5% v/v Triton X-100, 2% v/v Pharmalyte 3-10 and 65 mM dithioerythritol (DTT)) by sonication.
In thefirstdimension,250 μlofeach samplewere subjected to isoelectricfocusing ina Pharmacia IPG strip (pH 3-10 L, 7 cm). The gels were rehydrated overnight in rehydration solution (8 M urea, 0.5% v/v Triton X-100, 2 mM acetic acid and 9.7 mM DTE). The first phase was set at 500 V for 5 h, the second phase was a linear gradient spanning from 500 V to 3500 V in 5 h, and the final phase was set at 3500 V for 14 h. After electrophoresis the strips were kept at -80oC or prepared directly for the second-dimensional electrophoresis.
4. Silver staining
The gels were fixed in 40% ethanol and 10% acetic acid in water overnight, and then incubated in a buffer solution containing 30% ethanol, 4.1% sodium acetate and 0.2% sodium thiosulfate for 30 min. After washing three times in water for 5 min each, the gels were stained in 0.1% silver nitrate solution containing 0.02% formaldehyde for 40 min. Development was performed for 15 min in a solution consisting of 2.5% sodium carbonate and 0.01% formaldehyde. EDTA solution (1.46%) was used to sto p the development and the stained gels were then washed three times in water for 5 min each.
5. In-gel enzymatic digestion
Protein spots were excised from gel with an Ettan Spot Picker(Version 1.0, Amersham Pharmacia Biotech), destained twice with 30mM potassium ferricyanide and 100mM sodium thiosulfate (1:1 v/v) and then equilibrated in 50mM NH4HCO3 to pH 8.0. After dehydrating
with ACN and drying in N2at 37℃ for 20 min, the gel pieces were rehydrated in 15 μL trypsin
solution (10 μg/mL in 25mM NH4HCO3) at 47℃ for 30 min and then incubated at 37℃
overnight. Peptides were then extracted twice using 0.1% TFA in 50% CAN and dried with N2.
6. Image acquisition and analysis
The stained gels were scanned in an ImageScanner (Amersham Biosciences) operated by the software, LabScan 3.00, also from Amersham Biosciences. Intensity calibration was carried out using an intensity stepwedge prior to gel image capture. Image analysis including spot detection, quantification and normalization was carried out using the ImageMaster 2D Elite software 4.01c (Amersham Biosciences). The relative intensities of spots were used for comparison among the three groups and only those significantly different spots (two-fold
increase or decrease) were selected for analysis by MS.
7. MALDI-TOF-MS/MS identification
Digested peptides were dissolved with 0.8 μL saturated solution of CHCA in 50% v/v ACN/0.1%TFA, applied on a MALDI target plate and air-dried. MS analysis was performed using a 4700 Proteomics Analyzer (Applied Biosystems). Proteolytic peptides of standard Myoglobin were used for internal calibration and the 6 strongest peptides per spot were selected automatically for MS/MS analysis. PMF and sequence data were matched by searching the Swiss-Prot (http://us.expasy.org) database using MASCOTengi
8. Statistical analysis
Results are expressed as mean±SEM. One-way ANOVA was used for multiple group comparisons and the Student’s t-test for others. Differences with P<0.05 were judged to be significant.
參考文獻
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結果與討論
初步取得右歸丸的支氣管肺泡沖洗液，並進行 2-D 的實驗。經由初步比對 native 組、 Der p 組、右歸丸拆方組-X、右歸丸拆方組-R、右歸丸拆方組-K，發現給予右歸丸 不同拆方組的老鼠其支氣管肺泡沖洗液確實有不相同之處。 Result：native 組(4 隻老鼠) N2 N3 N4 N5 7Der p 組 (4 隻老鼠)
右歸丸拆方組-K (4 隻老鼠) 因此本實驗具有深入探討的價值，而此實驗結果將作為日後細部研究右歸丸各成分，在分 子層面機制上，如何達到治療以及預防氣喘效果的重要參考指標。
