透過您的圖書館登入
IP:34.229.50.161
  • 學位論文

蒟蒻纖維對高脂肪飲食相關大腸癌危險因子之調節作用-臨床、動物及初步細胞研究

The modulatory effects of konjac glucomannan on high fat-related risk factors of colon carcinogenesis - clinical, animal and cell line studies

指導教授 : 陳曉鈴

摘要


黏稠性的蒟蒻纖維(konjac glucomannan)藉由促進腸道中益菌生長及短鏈脂肪酸的產生,改善人體及動物腸道健康。本研究目的是探討蒟蒻纖維於臨床及動物實驗上,其預防大腸癌發生之調節作用;另外,以人類腺癌細胞株探討蒟蒻纖維可能的抗癌作用。 第一部分的實驗,以雙盲、安慰劑及飲食控制之實驗設計,探討於低纖維飲食中,給予受試者蒟蒻纖維補充劑後,對大腸癌前相關指標的調節作用。結果顯示,給予蒟蒻纖維補充劑4週後(4.5克/天)顯著降低糞便β-glucuronidase活性及二級膽酸濃度。此外,蒟蒻纖維也降低糞便水單獨或合併H2O2處理後,對Caco-2細胞毒性及DNA傷害的程度。這些蒟蒻纖維的保護作用可能與其代謝產物:丁酸,和促進益菌生長有關。因此,低纖維飲食中給予蒟蒻纖維補充劑,可能降低大腸癌前病變的危險因子。 纖維攝取不足之外,飲食西化攝取高油高熱量的飲食習慣,亦是發生大腸癌的另一個主要危險因子。具黏稠性的水溶性膳食纖維已被證實可減少脂肪的吸收,但卻增加膽酸的排出,因此可能抵銷了蒟蒻纖維促進腸道環境健康的效益。因此,第二部分的實驗在探討高玉米油飼料中,添加蒟蒻纖維調節大腸癌發生相關危險因子,包括:糞便中細菌酵素活性和二級膽酸濃度;以及相關保護因子,包括:短鏈脂肪酸及益菌生長的作用。32隻雄性Sprague-Dawley大鼠隨機分配至正常脂肪(5%玉米油)無纖維組、高脂肪(25%玉米油)無纖維組、以及高脂肪無纖維飼料中添加5%蒟蒻或菊醣纖維,共餵食四週。結果顯示,蒟蒻或菊醣纖維組明顯降低糞便中β-glucuronidase、mucinase活性及二級膽酸lithocholic acid濃度,因此可能減少大腸細胞接觸致癌物質;此外,蒟蒻纖維促進糞便中bifidobacteria和lactobacilli數目及提高盲腸內容物短鏈脂肪酸含量。 高脂肪的攝取常伴隨著活性氧物質的生成,而大腸組織是一個經常接觸腸道菌叢及腸道內容物的環境,因此,第三部分的實驗在探討餵食高脂肪飼料對動物體內抗氧化防禦系統之調節作用。24隻雄性Sprague-Dawley大鼠餵食高脂肪(25%玉米油)飼料,分別為無纖維、或者添加5%蒟蒻或菊醣纖維三組。餵食四週後結果顯示,於高脂肪無纖維飼料中添加蒟蒻或菊醣纖維,有效降低大腸與肝臟組織脂質過氧化產物(malondialdehyde)濃度,並顯著降低週邊血液單核球DNA傷害。此外,蒟蒻和菊醣纖維顯著提高大腸黏膜麩胱苷肽過氧化酶和過氧化氫酶的基因表現、肝臟中超氧歧化酶和過氧化氫酶的基因表現、及血漿維生素E的濃度,並且增加糞便中乙酸及丁酸的排出量。這些結果顯示,餵食蒟蒻及菊醣纖維可提升動物體內局部和全身性抗氧化防禦系統。 上述的實驗結果顯示,蒟蒻纖維可減少糞便水造成的DNA傷害,並可促進大腸黏膜抗氧化酵素的基因表現。因此,蒟蒻纖維可能減少致癌物質(azoxymethane, AOM)所引起的DNA傷害。第四部分實驗目的是探討蒟蒻、菊醣及纖維素對於單劑AOM注射後,C57BL/6J小鼠大腸上皮細胞的急性期反應。72隻六週齡雄性小鼠隨機分配至高脂肪(20%)低纖維(1%纖維素)控制組,或額外補充5%蒟蒻、菊醣或纖維素三組,餵食三週後於第22天腹腔注射一次AOM (10 mg/kg BW),於注射第0、 6及24小時後犧牲動物。結果顯示,高脂肪飼料中添加纖維可調節AOM注射前(0小時)大腸細胞抗氧化狀態;相較於高脂低纖維組,菊醣可降低0小時的大腸細胞DNA傷害,可能與其盲腸內容物中丁酸濃度較高有關;補充纖維的三組在0小時都顯著增加大腸細胞glutathione S-transferase π (GST-π)的基因表現;然而僅蒟蒻及菊醣纖維顯著增加大腸細胞麩胱苷肽過氧化酶基因表現。此外,補充纖維的三組促進近端大腸上皮細胞汰換率,但不影響遠端結腸細胞。相較於控制組,菊醣纖維組於AOM注射24小時之內,持續提升GST-π基因表現。然而,在AOM注射6小時後,控制組的大腸細胞麩胱苷肽過氧化酶、超氧歧化酶和過氧化氫酶的基因表現都比菊醣纖維組高。另外,AOM注射24小時後,蒟蒻及菊醣纖維組大腸細胞DNA傷害明顯較控制組低,其超氧歧化酶和過氧化氫酶的基因表現也分別提升,此外,蒟蒻纖維組顯著降低遠端大腸細胞增生作用,並顯著提升細胞凋亡,推測可能與其盲腸中含高量短鏈脂肪酸有關。因此,於高脂肪低纖維飼料中添加蒟蒻和菊醣纖維可降低大腸細胞因AOM引起的DNA傷害,推測與其發酵能力及促進抗氧化酵素基因表現有關。 體內實驗顯示蒟蒻纖維具有抗癌潛力,但其中可能的機制目前仍不明,且比較不同纖維對細胞機制的研究也很少,因此,第五部份研究目的是探討蒟蒻、菊醣、纖維素體外發酵上清液對於大腸腺癌細胞生長及凋亡的影響。體外發酵48小時後,蒟蒻纖維發酵上清液中短鏈脂肪酸含量為糞便控制組(無纖維添加)的1.5倍,其中正丁酸更高達3.4倍。各種纖維發酵上清液抑制細胞生長具時間及劑量效應;以15%蒟蒻纖維發酵上清液處理24小時後顯著增加大腸腺癌細胞凋亡比例。此外,模擬蒟蒻發酵上清液所合成的短鏈脂肪酸混合液也同樣具有抑制細胞生長及促進凋亡效果。 綜合以上結果顯示,攝取蒟蒻纖維可有效:(1)降低餵食高脂肪飼料動物或合併致癌物後其週邊血液單核球及大腸細胞的DNA傷害,及人體糞便水對Caco-2細胞的傷害;(2)調節有害細菌酵素活性和二級膽酸濃度來降低大腸環境中致突變物質的負荷;(3)降低高脂肪飼料誘發之大鼠組織中脂質過氧化產物;(4)促進動物大腸黏膜細胞及肝臟中抗氧化基因的表現。實驗結果推測補充蒟蒻纖維促進上述的作用皆與其發酵性及促進益菌生長相關。此外,蒟蒻纖維對於AOM注射後的急性反應則是透過調節細胞增生與凋亡的表現。在細胞實驗的結果顯示,蒟蒻纖維發酵上清液中含有較高量的正丁酸可能造成抑制人類大腸腺癌細胞生長與促進其凋亡作用。

並列摘要


Konjac glucomannan (KGM), a viscous dietary fiber, has been shown to improve the health of colonic environment by promoting the growth of probiotics and production of short-chain fatty acids (SCFA) in humans and mice. The aim of my doctoral research was to investigate the roles of KGM in prevention risk factors of colon cancer in clinical and animals studies, and to determine its potential anti-cancer effects in human adenocarcinoma cell line model. In the first study, the effects of KGM supplementation into a low-fiber diet on precancerous markers of colon cancer was determined in a double-blind, placebo- and diet-controlled study. Results indicated the fecal β-glucuronidase activity and secondary bile acid levels were significantly decreased in volunteers after taking KGM (4.5 g/d) supplement for 4 weeks. KGM also ameliorated fecal water or fecal water combined with H2O2-induced cytotoxicity and DNA-damaging effects toward Caco-2 cells. These protective effects of KGM may be related to its metabolite, butyrate, and its probiotic effect. Therefore, the supplementation of KGM into a low-fiber diet in adults may reduce the precancerous risk factors of human colon cancer. In addition to low-fiber intake, the westernized diet characterized by high fat and calorie intakes is another main risk of colon carcinogenesis. Soluble viscous dietary fibers have been shown to reduce dietary fat absorption but promote the bile acid excretion that could counteract the beneficial effect of KGM in the colonic ecology. Therefore, the second study was aimed to determine effects of KGM on the colon cancer biochemical markers including fecal bacteria enzymes and secondary bile acid, and on preventive factors such as SCFA and probiotics in rats fed a high-fat low-fiber diet. Male Sprague-Dawley rats (n = 32) were fed a normal-fat (5% corn oil, w/w) fiber-free, or high-fat (25%, w/w) diets containing no fiber, 5% (w/w) of KGM or inulin for 4 weeks and the risk and preventive factors of colon carcinogenesis were determined. Incorporation of KGM, as well as inulin, into the high-fat fiber-free diet in rats beneficially reduced the fecal β-glucuronidase and mucinase activities, and lithocholic acid (secondary bile acid) concentration, which may reduce the carcinogenic load to colonocytes. In addition, KGM promoted the fecal bifidobacteria and lactobacilli, and cecal SCFA contents. The high-fat intake usually accompanies generation of reactive oxidative substances (ROS). The colon, the interface between the large population of microflora and the colonic contents, constantly contact with challenge. Therefore, the third study was aimed to determine effects of KGM on the balance between pro-oxidative status and antioxidative defense systems in rats fed a high-fat diet. Male Sprague-Dawley rats (n = 24) were fed a high-fat (25% corn oil, w/w) fiber-free diet or that supplemented with KGM or inulin fiber (5%, w/w) for 4 weeks. Results indicated that incorporation of KGM and inulin into the high-fat fiber-free diet beneficially reduced the malondialdehyde (MDA) levels in the colon and liver and DNA damage in peripheral blood mononuclear cells (PBMC). KGM and inulin also enhanced the gene expressions of glutathione peroxidase and catalase in the colonic mucosa, and the superoxide dismutase and catalase in the liver. Furthermore, KGM and inulin promoted the elevation of plasma α-tocopherol level and increased the concentration and daily excretion of fecal acetate and butyrate as well. These results suggested that in vivo utilization of KGM and inulin stimulated both local and systemic antioxidative defense systems in rats. KGM has been shown to reduce DNA damage caused by fecal water and upregulate the colonic antioxidant enzyme gene expressions in the above studies. Therefore, KGM could potentially reduce the DNA damage effect caused by azoxymethane (AOM), a carcinogen commonly used to initiate colon carcinogenesis in rodents. The fourth study was aimed to determine the effects of KGM, inulin and cellulose on the acute responses of colonic epithelium in C57BL/6J mice after an AOM injection. Six-week-old mice (n =72) were fed a high-fat (20%, w/w) low-fiber (1% cellulose, w/w) control diet or that supplemented with 5% of KGM, inulin or cellulose for 3 weeks. Mice were administered with a single AOM (10 mg/kg BW, i.p.) injection on day 22th and were killed at 0, 6, or 24 h afterwards. Results indicated that fiber modulated the basal antioxidant status of colonocytes before the act of AOM. Inulin beneficially reduced the basal DNA damage in colonocytes, as compared with that of the control group, which is likely to be related to the higher cecal butyrate level. All fibers significantly enhanced while only KGM and inulin enhanced the gene expression of glutathione peroxidase 2 (GPx). In addition, all dietary fibers enhanced the turnover of epithelium cells in the proximal colon, but did not change in the distal colon. After the AOM challenge, inulin persistently promoted the GST-π expression during the 24-h period. The expression of GPx, superoxide dismutase 1 (SOD), and catalase (CAT) in the colonocytes was increased in the control diet group at 6 h, as compared with inulin counterpart. The colonic DNA damages were significantly decreased in the KGM and inulin groups, while the gene expressions of SOD and CAT elevated simultaneously at 24 h after AOM injection, respectively. Besides, KGM group showed the lowest distally proliferation and the highest apoptosis in the colon which may related to significantly increase cecal SCFA production at 24 h after AOM injection. Therefore, addition of KGM or inulin into the high-fat low-fiber diet beneficially protects the colonocytes from DNA damage induced by AOM, which may be mediated by their fermentation and antioxidant enzymes transcription. With all the evidence obtained from the in vivo studies that indicate the anti-cancer potential of KGM, the mechanisms whereby KGM exerts these effects have never been examined. In addition, the cellular mechanisms of various fibers have not been compared. Therefore, the fifth study was designed to initially examine the effects of fermentation supernatants of KGM, inulin or cellulose on cell growth and apoptosis in WiDr adenocarcinoma cells. The fermentation supernatant of KGM contained 1.5-fold higher amount of SCFA and 3.4-fold higher n-butyrate, as compared with those of fecal blank after 48 h of fermentation in vitro. The fermentation supernatants affected the cell growth in a time- and dose-dependent manner. The 15 % fermentation supernatant of KGM significantly increased apoptosis analyzed by flow cytometer. In addition, the synthetic SCFA composition of fermentation supernatant of KGM also exerted these effects. In summary, this dissertation research demonstrated that KGM intake effectively caused (1) reduction of DNA damage in the PBMC and colonocytes of animals fed either in the presence of high-fat or that combined with carcinogen injection, and that in Caco-2 cells while incubated with fecal water obtained from humans; (2) decreased mutagen load in the colon by modulating deleterious bacteria enzyme activities and secondary bile acid profile in humans and rats those fed a high-fat diet; (3) reduced MDA level in tissues of rats fed a high-fat diet; (4) up-regulating gene expression of antioxidant enzymes in the colonic mucosa and liver of rats and that in the colonocytes of mice after a carcinogen injection. We suggested that the effects mentioned above of KGM were associated with the ability of fermentation and the growth of probiotics. Besides, KGM can regulate the balance between proliferation and apoptosis and of mice subjected to a high-fat diet and carcinogen. The preliminary cell line study suggests that butyrate in the fermentation supernatant of KGM is a potential modulator that inhibits cell growth and induce apoptosis of human adenocarcinoma cells.

參考文獻


(1) Hill, M. J. Metabolic epidemiology of dietary factors in large bowel cancer. Cancer Res. 1975, 35, 3398-3402.
(2) Reddy, B. S. Dietary fat and colon cancer: animal model studies. Lipids 1992, 27, 807-813.
(3) Giovannucci, E. and Willett, W. C. Dietary factors and risk of colon cancer. Ann. Med. 1994, 26, 443-452.
(4) Bingham, S. A.; Day, N. E.; Luben, R. et al. Dietary fibre in food and protection against colorectal cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC): an observational study. Lancet 2003, 361, 1496-1501.
(5) Trowell, H. Editorial: Definitions of fibre. Lancet 1974, 1, 503.

延伸閱讀