餵食Lactobacillus casei 01及其胞外多醣對1,2-二甲基聯胺誘導F344大鼠結腸癌前病變之影響

Effects of Lactobacillus casei 01 and its exopolysaccharide on 1,2-dimethylhydrazine induced colon precancerous lesions in F344 rats

劉曲婷(Chu-Ting Liu)

指導教授 ： 游若篍

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.03268

摘要

結腸直腸癌在台灣是主要死亡原因之一。大約有70%的結腸直腸癌成因與飲食有關。乳酸菌會被用於癌症化學預防的研究，可能是因為它具有抗癌與抗腫瘤的活性。而乳酸菌對宿主有益之功效，部分原因應與其胞外多醣 (exopolysaccharides, EPS) 的生理活性有關。吾人以Lactobacillus casei 01的EPS為素材，從體外抗癌細胞增生、抗基因毒性試驗，探討其抗細胞毒性的潛力；再利用化學致癌劑-1,2-二甲基聯胺 (1,2-dimethylhydrazine, DMH) 誘導體內動物模式，探討其對結腸癌前病變的影響。最後研究餵食L. casei 01及EPS對DMH誘導大鼠結腸癌前病變之基因表現圖譜影響。
首先研究L. casei 01細胞區分物 (熱致死細胞、粗細胞壁及胞內液) 與EPS對人類結腸癌細胞株HT-29增生及腸道上皮細胞intestine 407細胞生長的影響。結果顯示EPS抗結腸癌細胞HT-29增生較細胞區分物顯著，且不會影響腸道上皮細胞intestine 407的生長。進一步研究，發現50和100 μg/mL的EPS可抑制結腸癌細胞HT-29增生，低濃度 (5-50 μg/mL) 的EPS不會抑制人類正常腸道細胞intestine 407的生長。再經由分段預培養試驗 (section-preincubation test) 和彗星分析法去探討EPS可能的抗細胞毒性機制，並評估EPS抑制4-硝基喹啉氧化物 (4-Nitroquinoline 1-oxide, 4NQO) 造成人類腸道細胞intestine 407 DNA損傷的情形。結果發現10-50 μg/mL的EPS可提升intestine 407細胞存活率。EPS可降低4NQO造成的細胞DNA損傷情形，其中以50 μg/mL的濃度效果最顯著 (p < 0.05)。由結果顯示L. casei 01的EPS具有抗細胞毒性，其主要抗4NQO之機制為阻擋效 (blocking effect) 應，此EPS亦有生物抗細胞毒性作用 (bioanticytotoxic effect)。
在體內動物結腸癌模式中，探討L. casei 01及EPS對大鼠糞便菌相、血清脂質濃度、抗氧化酵素與組織脂質過氧化物例如組織硫巴比妥酸反應物質 (thiobarbituric acid reactive substances, TBARS) 的影響以及它們抑制F344大鼠產生結腸癌前病變的能力。大鼠分為4組:第1組為控制組，第2組、第3組和第4組每週皮下注射1,2-二甲基聯胺 (1,2-dimethylhydrazine, DMH)，1週1次 (20 mg/kg b w)，共進行12週。此外，在實驗期間，第3組大鼠管餵L. casei 01懸浮液 (1010 cfu/10 mL/ kg bw/2 d)，第4組大鼠則管餵EPS (100 mg/10 mL/ kg bw/2 d)。大鼠在12週後犧牲，結果發現攝食L. casei 01會使DMH組大鼠減少丙胺酸轉胺酵素 (alanine aminotransferase, ALT) 活性及三酸甘油酯濃度。DMH組大鼠攝食L. casei 01可提升超氧歧化酶 (superoxide dismutase, SOD) 活性並降低麩胱甘肽還原酶 (glutathione reductase, GRd) 和麩胱甘肽過氧化酶 (glutathione peroxidase, GPx) 的濃度。此外，DMH+L. casei 01組大鼠的糞便中staphylococci、Escherichia coli和total coliforms的菌數皆低於控制組。注射DMH又同步餵食L. casei 01或EPS可以顯著提升糞便中乳酸桿菌和雙叉桿菌菌數。再者，DMH會導致大鼠血清及組織中TBARS的增加，而L. casei 01和EPS可抑制血清、肝臟、腎臟和結腸中TBARS的生成。組織切片結果顯示L. casei 01及EPS可減少DMH誘導而產生的結腸黏膜層病灶。
最後，利用基因組技術去探討餵食L. casei 01及EPS 和對DMH誘導之大鼠結腸癌前病變的基因表現和基因組改變的影響。將DMH、DMH+EPS和DMH+L. casei 01組的RNA和控制組的雜交在4×44K全大鼠微陣列 (whole rat microarray)，並且以即時定量聚合酶連鎖反應 (real-time quantitative PCR) 確認所選擇的基因。以IPA和DAVID 軟體分析完成微陣列數據的功能性分析。微陣列基因表現分析結果顯示在DMH、DMH+EPS和DMH+L. casei 01組中皆有複雜的基因表現變化情形，涉及許多生化路徑的調控。功能性分析顯示這些基因調控對刺激物的反應、轉錄作用、發炎反應、調節免疫、細胞程序和細胞凋亡。由基因表現量分析的結果顯示經DMH誘導的大鼠餵食EPS或L. casei 01可能會提升抑癌基因表現量以及抑制致癌基因表現量。SGK1的基因表現有助於腸道腫瘤的生成，但在DMH、DMH+EPS和DMH+L. casei 01組中均呈現下降的趨勢，是因為SGK1參與的結腸直腸癌訊息傳遞路徑可能被其他SGK同功異構型或蛋白質激酶B/Akt異構物執行。

關鍵字

Lactobacillus casei 01 ； 胞外多醣 ； 4-硝基喹啉氧化物 ； 1,2-二甲基聯胺 ； 基因表現 ； 微陣列

並列摘要

Colorectal cancer is a major cause of death in Taiwan. Approximately 70% of colorectal cancer is related to diets. Lactic acid bacteria might be used for cancer chemoprevention due to their putative anticarcinogenic and antitumor activities. The benefit effects on human health of lactic acid bacteria might be attributable to positive physiological effect of exopolysaccharides (EPS). In the present study, we employ the Lactobacillus casei 01 and EPS as materials.
The cell fractions (heat-treated cells, crude cell walls and intracellular extracts) and EPS of L. casei 01 were studied for their effects on the growth of human intestinal epithelial cells, intestine 407 and the proliferation of human colon cancer cell, HT-29. The results showed that EPS exhibited the most antiproliferation activity on HT-29 cell. The viability of intestine 407 cells was not affected by EPS. EPS was further used to study the prevention of 4-Nitroquinoline N-oxide (4NQO)-induced DNA damage on intestine 407 cells. The results showed that 50-100 μg/mL of L. casei 01 EPS had antiproliferation on HT-29, and low concentration (5-50 μg/mL) of EPS will not affect the growth of intestine 407 cells. Furthermore, possible anticytotoxic mechanisms of EPS were investigated by section-preincubation test and comet assay to explore the effects of EPS against 4NQO-induced DNA damage on intestine 407. The results indicated that low concentrations (10 and 50 μg/mL) of EPS markedly raised the cell viability of intestine 407, besides, 50 μg/mL of EPS significantly reduced the genetoxicity of 4NQO-induced DNA damage on intestine 407 cells (p < 0.05). These findings suggest that EPS had bioanticytotoxic effects, and the blocking effect was the main mechanism of anticytotoxicity for EPS against 4NQO.
Secondly, the effects of L. casei 01 and EPS on alteration of fecal microflora, serum lipid levels, antioxidant enzymes of blood, and tissue lipid peroxidation, such as tissue thiobarbituricacid substances (TBARS) as well as their inhibitory effect on precancerous colon lesions in male F344 rats were determined. Rats were divided into four groups. Group 1 served as control, group 2, 3 and 4 were given weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) at a dose of 20 mg/kg body weight for 12 weeks. Besides, group 3 and 4 were given a treatment of L. casei 01 (1010 cfu/10 mL/kg bw/2 d, 12 weeks) and EPS (100 mg/10 mL/kg bw/2 d, 12 weeks) orally, respectively. Animals were sacrificed at the end of 12 weeks. It was found that L. casei 01 treatment decreased serum alanine aminotransferase (ALT) activity and triglycerides concentration. However, these rats treated with L. casei 01 enhanced activity of superoxide dismutase (SOD), and reduced the concentrations of glutathione reductase (GRd), and glutathione peroxidase (GPx) when compared with those DMH-treated rats. There was a decrease in the population of staphylococci, Escherichia coli, and total coliforms in the feces of L. casei 01-treated rats compared with the control. Both EPS and L. casei 01 markedly increased the cell numbers of lactobacilli and bifidobacteria for F344 rats treated with DMH. Moreover, the amount of TBARS in serum and tissues were significantly raised in DMH-treated rats, while they were significantly diminished in rats received L. casei 01- or EPS-treatment simultaneously. Histopathological changes in the colon of L. casei 01- and EPS-treated rats showed reduced DMH-induced focal slight cryptic necrosis in the mucosal layer.
Finally, the effects of L. casei 01 and EPS on the gene expression profiles of DMH-induced experimental colon precancerous lesions in rats were investigated. For gene expression analysis, the RNA of DMH-, DMH+EPS-, and DMH+L. casei 01-treated group were hybridized with that of the control on 4×44K whole rat genome oligo microarray and selected genes were validated by quantitative RT-PCR. Microarray gene expression analysis showed complex gene expression alterations in DMH-, DMH+EPS-, and DMH+L. casei 01-treated group, while compared with the control encompassing many biological pathways. Functional analysis showed that pathways controlling response to chemical stimulus, transcription, inflammatory response, immune system response, cell process, and apoptosis. SGK1 expression favors the development of intestinal tumors, but the SGK1 expressions in DMH-, DMH+EPS-, and DMH+L. casei 01-treated group were down-regulated. The gene expression alteration analysis showed that EPS and L. casei 01 may raise the tumor-suppressor gene expression and supper the oncogene expression of DMH-treated rats. These findings suggest that SGK1-dependent colorectal cancer pathway may be accomplished by the other SGK isoforms.

並列關鍵字

Lactobacillus casei 01 ； exopolysaccharide ； 4-Nitroquinoline N-oxide, 1 ； 2-dimethylhydrazine ； gene expression ； microarray

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