探討臺灣水鹿和紅鹿鹿茸水萃物在改善發炎性腸道疾病之潛力

Abstract

發炎性腸道疾病 (Inflammatory bowel disease, IBD) 是一種小腸和結腸的發炎疾病，在過去數十年間罹病患者逐年增加。現階段的治療途徑為給予廣效性的抗生素和抑制免疫反應的類固醇。然而，有限的療效常伴隨腸炎反覆復發。本研究室先前之研究中已證實，鹿茸水萃物具有抗發炎和促進傷口癒合之生物活性，對於腸道受損的腸炎患者中或許具有改善之潛力。因此，本研究目的旨在評估不同的臺灣國產鹿種水萃物在改善腸道發炎疾病之效果，並進一步以發炎反應之程度、腸道上皮完整性和腸道菌相分群，分析減緩腸炎之可能生物途徑，並嘗試找尋萃取物中之有效成分。 首先，我們以人類結腸癌上皮細胞株之模式挑選潛力鹿種水萃物。試驗結果發現，臺灣水鹿和紅鹿鹿茸水萃物可減緩葡聚糖硫酸鈉損傷所造成的跨膜電阻值下降，兩種鹿茸水萃物皆可劑量回饋的增加趨化素CCL20分泌，顯示鹿茸萃取物對於細胞修復和完整性有正向作用。 接著我們以葡聚糖硫酸鈉的腸炎小鼠模型評估鹿茸萃取物的預防功效。試驗選自九至十週齡的C57BL/6母鼠，依據組別每日給予磷酸鹽緩衝生理食鹽水、不同劑量之臺灣水鹿和紅鹿鹿茸水萃液 (100 mg/kg、200 mg/kg) 並持續兩週，第三週給予含有2.5% 葡聚糖硫酸鈉之飲用水誘導急性腸炎。試驗發現兩種鹿茸萃取物皆可顯著減緩糞便潛血程度，在核磁共振影像和各階段腸道組織之組織切片圖中可觀察到較完整的上皮組織排列，其中十二指腸、迴腸和結腸段之病理切片評分和負控制組比較達顯著差異，然而兩種鹿茸水萃物的處理組別在結腸長度測定上和負控制組比較並無顯著差異。另外，我們以酵素結合免疫吸附分析法和流式細胞儀測定脾臟、血清和腸繫膜淋巴結和發炎反應相關之細胞激素，兩種鹿茸水萃物的組別皆有下降的趨勢，顯示全身性和局部性的發炎反應較趨緩，其中高劑量紅鹿鹿茸水萃物組在脾臟腫瘤壞死因子-α、白細胞介素-6、干擾素-γ之測定達顯著下降。腸道上皮細胞的完整性的評估，我們以西方墨點術測定迴腸和結腸段上皮細胞間的緊密連結蛋白。試驗結果發現，和負控制組相比，兩種鹿茸組別在封閉蛋白和水閘蛋白蛋白質表現量皆較高，其中高、低劑量的紅鹿處理組皆有達顯著差異，代表上皮細胞之間空隙較少、破損程度較輕微。 腸道菌相分析以盲腸內容物進行次世代基因定序，結果發現鹿茸水萃物並無法提升組內菌群豐富度，但在菌群歧異度上，紅鹿鹿茸水萃物有提高之趨勢，透過最小平方法判別分析法，鹿茸水萃物仍有部分序列群組操作單元和誘導腸炎組重疊。由於複雜之菌相組成和消長作用，以線性回歸判別分析分數為依據，進行線性判別分析效力鑑別分析，找出各組具有顯著差異之菌群物種，進一步和免疫反應和上皮細胞完整性之指標進行斯皮爾曼關聯性分析，釐清並驗證之間可能機轉和影響因子。 初步的試驗結果證實鹿茸萃取物有預防腸炎之潛力，另外我們以結腸癌細胞株模式找尋可能有效成分，試驗結果發現小於3kDa的萃取液分子可顯著誘使趨化素CCL20和血管內皮生長因子分泌，顯示水萃物中的有效成分應落在小於3kDa片段。接著，我們以液相和氣相串聯質譜儀之代謝體學，搭配蛋白質和資料庫比對，找尋其中的有效活性成分。 綜上所述，本研究於細胞和動物實驗證實臺灣水鹿和紅鹿鹿茸具有維持上皮細胞完整性、抗腸道發炎之潛力。未來我們將進一步鑑定其中的活性成分，期盼能率先發現具有潛力之鹿茸萃取物活性，為我國養鹿產業提供一新出路。

Inflammatory bowel disease (IBD) is a chronic debilitating inflammation of intestinal tract and colon. In the past decades, the prevalence of IBD are gradually increasing. The mainly current therapies are antibiotic and corticosteroids. However, the efficacy is limited. Our previous study has demonstrated that velvet antler extracts has the anti-inflammatory and the wound healing functionalities. These features have been reported to associate with alleviation of the injury in colitis. Hence, the aim of the study is to evaluate the effect of amelioration on different velvet antler water extracts in Taiwan. The possible pathways and potential components were also investigated. Velvet antler water extracts were cultured with human colonic epithelial cell line (Caco-2) for a week to select the potential deer species by monitoring the trans-epithelial electrical resistance (TEER). In vitro study indicated that the velvet antler extract from Formosan sambar deer (SVAE) and red deer (RAVE) could reduce the decline of TEER value and elevate the CCL20 production, an intestinal epithelial restitution and migration chemokine, under dextran sulfate sodium (DSS) injury in a dose-dependent manner (p < 0.05), suggesting that both extracts could enhance the cell repair effect of epithelial cell. For the further animal study, specific-pathogen-free female C57BL/6 mice from 9 to 10-week old were studied in DSS induced colitis model. We found that both treatments (SVAE and RVAE) could significantly relieve the degree of bloody feces (p<0.05). Besides, The mice fed with SVAE and RVAE showed a better tightly arrayed epithelial cell structures in intestine and colon by H&E staining and magnetic resonance imaging. Compared to the injury group, the histological scores of the section of duodenum, ileum and the colon have the significant difference (p < 0.05). However, no significant improvement in colon length was observed in the treatment group. Since the degree of inflammation is crucial biomarker of colitis, the cytokines in spleen, serum and mesenteric lymph nodes were determined by ELISA to represent the systemic inflammatory response. Results showed that the level of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) were reduced under extract treatments. Hence, compared to the colitis group, the high dosage of RVAE group show the significant difference. In addition, the expression of tight junction protein were analyzed via western blotting of colon tissue. Besides, the result of RVAE showed the statistical difference in the protein expression of occludin and claudin1, which represent more integrity of epithelial mucus. For the next generation sequencing study was analyzed with cecum content. There were no significant difference between all groups of Chao1 index. However, RAVE could enhance in Shannon index, which represent more diversity under RAVE effect. Results of partial least squares discrimination analysis (PLS-DA) showed that the OTU of the velvet antler extract treated groups could not be clearly separated from the colitis group. Further analyzing by linear discriminant analysis effect size analysis (LEfSe) with linear discriminant analysis (LDA) scores, several potential biomarkers in genus and species levels were identified. Those biomarkers were investigated by Spearman’s correlation coefficient with pro-inflammatory cytokine and tight junction related protein to clarify the possible mechanism and pathways. In the present study, we demonstrated that the velvet antler extracts from Formosan sambar deer (SVAE) and red deer (RAVE) could prevent the inflammatory bowel disease. To further identify which components probably involving the effect, we used in vitro model to separate the extracts with different molecular weight and found that extracts under 3 kDa have the similar effect in CCL20 and vascular endothelial growth factor (VEGF) production, showing that the potential compound in the water extracts fall below the 3 kDa fragment. Next, we used metabolomics with liquid and gas phase tandem mass spectrometer, matching the identified compound with online database to figure out the effective active components. In summary, our study proves that the velvet antler extracts from Formosan sambar deer and red deer have the potential on amelioration inflammatory bowel disease in various aspects in vitro and in vivo. Further identification of potential active compounds are necessary to confirm the anti-colitis effect in the future. We hope to develop new application in deer livestock industry.