鑑定與解析咸豐草中的免疫調節物質

Abstract

咸豐草是一種民間常用來治療感染以及調節免疫的草藥植物，為了鑑定並解析咸豐草中的生物活性物質，首先，本研究利用以Jurkat T細胞為主的平台，篩選咸豐草萃取物中具有調節細胞激素IFN-□轉錄活性的有效成份；依循以生物活性為導向的分離純化原則(BGFI)，結果顯示，咸豐草中的兩個黃酮類純物質(centaurein與centaureidin)，具有提升IFN-r的轉錄活性的能力，進一步以centaurein進行分子機制的探討，發現centaurein對IFN-r上游的轉錄因子T-bet，具有提升其基因轉錄活性以及基因表達的能力。由於已知IFN-r能活化巨噬細胞並幫助胞內菌(例如：李斯特菌)的清除，進一步將centaurein以腹腔注射投予C57BL老鼠，結果發現centaurein可提升C57BL老鼠血液中IFN-r的表現量，而藉由IFN-r的提升也可有效幫助巨噬細胞清除李斯特菌。在預防胞內菌的感染方面，實驗結果顯示centaurein可有效避免李斯特菌感染所造成的老鼠死亡情形。另外在治療老鼠受到李斯特菌感染的動物模式上，則發現不論單獨投予centaurein或將centaurein結合低劑量的抗生素(ampicillin)使用，都具有提升老鼠存活率的效果。 此外，本研究也利用體外誘導輔助型T細胞分化的平台，篩選咸豐草中對第一型輔助型T細胞(Th1)與第二型輔助型T細胞(Th2)的細胞分化具有調節能力的有效成份。依循以生物活性為導向的分離純化原則(BGFI)，實驗結果顯示，咸豐草的丁醇分層萃取物以及兩個聚乙炔類純物質(2-ß-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne (1) and 3-ß-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne (2))，具有抑制第一型輔助型T細胞分化並促進第二型輔助型T細胞分化的能力。進一步以第一型輔助型T細胞(Th1)所主導的非肥胖型自體免疫糖尿病老鼠(NOD)為動物模型進行研究，結果發現，咸豐草的丁醇分層萃取物以及兩個聚乙炔類純物質，可明顯抑制NOD老鼠的發病情形，除了可有效避免尿糖以及血糖的升高，並且可幫助血中胰島素的正常分泌。 藉由本論文研究結果，除了建立以T細胞為主的模型，藉以篩選能調控細胞激素表達或具有調節第一型輔助型T細胞(Th1)與第二型輔助型T細胞(Th2)細胞分化的植物有效成份，更以咸豐草為例，成功鑑定並解析出咸豐草中具有免疫調節功能的純物質，本研究除了對民間使用咸豐草來治療感染以及糖尿病提供科學證據，更對植物藥的研究提供一套藥物開發平台。

Abstract B. pilosa has been claimed as an anti-infectious or immunomodulatory folk medicine. However, the pharmacological evidences for the traditional use of this plant are still poor. To characterize the immunomodulatory compounds from this plant, firstly, we screened plant constituents of B. pilosa which able to modulate IFN-r production in T cells using luciferase reporter assays. Our data showed that two flavonoids from B. pilosa, centaurein and centaureidin, with the ability to up-regulate IFN-r transcription, could be isolated using a BGFI procedure. Centaurein increased the IFN-r expression in primary T and NK cells and the IFN-r level in mice serum. Centaurein elevated the transcription of T-bet but not GATA-3, which is consistent with its effect on that of IFN-r but not IL-4. The sera with elevated IFN-r levels from the centaurein-treated mice could clear Listeria in macrophages. In vivo studies showed that centaurein protected mice against Listeria infection. Moreover, centaurein per se or in combination with antibiotics could treat Listeria infection. We also aimed to identify the bioactive compounds with the ability to modulate T cell differentiation. By using T cell differentiation assays, two polyacetylenic compounds, 2-ß-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne (1) and 3-ß-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne (2), were identified from B. pilosa based on a BGFI fashion. These two polyacetylenic compounds could suppress the differentiation of naïve T helper (Th0) cells into Th1 cells but promote the differentiation of Th0 cells into Th2 cells. Furthermore, in vivo results showed that treatment with these compounds significantly prevented the diabetes progression in NOD mice. These results suggest that the bioactive compounds from B. pilosa were identified with the screening methods and functionally characterized with in vivo mouse models. This approach may directly contribute to the ethnopharmacological effects of B. pilosa and furthermore, to facilitate drug discovery from plant sources.