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標題: | 以 PhIP 及 DSS 誘導模式探討 S-allylcysteine 對腸癌之化學預防功效 Studying chemopreventive effects of S-allylcysteine on colon carcinogenesis induced by PhIP and DSS model |
作者: | 林偉盛 Wei-Sheng Lin |
指導教授: | 潘敏雄 Min-Hsiung Pan |
關鍵字: | 結腸直腸癌,雜環胺,葡聚醣硫酸鹽,硫-烯丙基半胱胺酸,癌症化學預防,腸道菌相, colorectal cancer,heterocyclic amines,dextran sulfate sodium,S-allylcysteine,cancer chemoprevention,microbiota, |
出版年 : | 2022 |
學位: | 博士 |
摘要: | 結腸直腸癌 (colorectal cancer, CRC) 罹癌率於全世界及我國常年位居前三,其致病機制與飲食有著密不可分之關係,其中雜環胺 (heterocyclic amines, HCAs) 為肉類、水產品等富含蛋白質食物經高溫處理所產生,而 PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) 為人類飲食中暴露量相較高之雜環胺,且毒性資料最為完整,多項流行病學指出其對腸癌之發展有極高的攸關性。值得注意的是,現今藉由飲食預防病症之觀念逐漸重視,如許多膳食調查與流行病學指出大蒜之攝取可減少罹患大腸癌之風險,然而受限於強烈的辛辣味及刺激感不宜直接食用,因此大蒜之加熱熟成、發酵等加工製品近年受到關注,包括黑蒜。黑蒜已被證實較生蒜具有更高之生物活性,如抗氧化、抗發炎及神經保護等,歸因於其具更高含量之植化素,如硫烯丙基半胱胺酸 (S-allylcysteine, SAC),先前的研究顯示 SAC 與其他有機硫化物 (organosulfur compounds) 相比,具有較低之毒性與副作用,並於體內外試驗中發現具調升抗氧化酵素相關因子與抑制發炎受體與下游路經進而減少氧化壓力與抑制發炎反應,此外也發現具減緩癌細胞的增生與促凋亡之活性,擁有癌症化學預防之潛力,因此本研究建立以 PhIP/DSS 誘導小鼠腸癌之模式,探討 SAC 是否具減緩食安汙染物雜環胺所造成之腸癌發展。結果顯示,於飲食中每天給予 0.05% SAC 可抑制 PhIP/DSS 誘導小鼠之結腸長度縮短和結腸瘜肉數目的形成。此外,補充SAC可以顯著抑制 PhIP/DSS 誘導的血漿和結腸組織促炎細胞因子,並調降結腸組織中 iNOS、COX-2 及 MMP-2 蛋白表現,並阻止腸道屏障 E-cadherin 蛋白表現量下降。此外,SAC 可能藉由上調結腸組織中 pERK 1/2/Nrf2信號傳導來增加 HO-1 之表達,進而降低氧化壓力水平,而抑制 PhIP 對起始期細胞之 DNA 損害與促進期發炎反應,減輕 PhIP/DSS 引起的結腸炎與腫瘤形成。此外,透過 16S rRNA 定序分析糞便檢體腸道菌相,結果顯示 PhIP/DSS 組別具有較高腸炎與腸癌相關菌群如 Erysipelotrichaceae、Bifidobacteriaceae 及 Clostridiaceae 與較低抑制發炎相關菌群 Lachnospiraceae;而在有給予 SAC 組別有較低之可能使雜環胺活化菌屬 Bacteriodes,顯示 SAC 可改善腸癌情況之腸道微生物穩態,使其與正常飲食組相似。而為探究 SAC 是否抑制 PhIP 引起的早期基因損傷並減少癌起始細胞形成,以體外模式評估 SAC 對經 PhIP 處理之人類正常結腸粘膜上皮細胞 NCM 460 之影響,顯示 PhIP 誘導會造成氧化壓力失衡與 DNA 損傷,而透過蛋白質體外結合實驗與細胞熱轉移分析法,顯示 SAC與 Keap1 蛋白可能具有親和力關係;此外,SAC 也可能透過抑制 p38 與增加ERK1/2與AKT之磷酸化進而降低 Keap1 與 Nrf2 之結合,使 Nrf2/HO-1 信號上調而增加 GSH與GSH/GSSG 比值,進而抑制PhIP 誘導之 ROS 上升與 DNA 損傷。此外 SAC 可抑制 AhR、ARNT、CYP1A1 及 CYP1B1 蛋白質表現,顯示可能具避免致癌物代謝轉化作用之潛力存在。綜合上述,顯示 SAC 可能抑制 PhIP 誘導正常腸細胞之氧化壓力失衡與 DNA 損傷,並減輕小鼠腸道中之發炎反應,進而減緩 PhIP/DSS 誘導的結直腸癌發生,闡明黑蒜成分中 SAC 的癌症化學預防試劑的理論基礎及影響方式,期可作為輔助腸癌化學預防之方案,並作為未來相關產業功能性食品開發之依據。 Colorectal cancer (CRC) has the third-highest incidence and the second-highest mortality of all cancers worldwide. Worthwhile, CRC is closely related to dietary and environmental factors. In particular, a high intake of processed and red meat increases the risk of CRC, as the high-temperature cooking process induces the formation of mutagenic and carcinogenic compounds, such as polycyclic aromatic hydrocarbons and heterocyclic amines (HCAs) compounds. One of these compounds, namely, 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP), is the most abundant HCAs formed in cooked fish, poultry, and meat. Chemopreventive phytochemicals can block or reverse the premalignant stage in multistep carcinogenesis or retard precancerous cells into malignant cells. Thus, chemoprevention is a promising strategy for preventing cancer. Many dietary surveys and epidemiological studies have pointed out that garlic intake can reduce the risk of CRC. However, due to its strong spicy taste and revolting flavor, it is not suitable to be eaten directly. Therefore, processed products such as aging or fermented product have attracted attention in recent years, including black garlic. Previous studies have demonstrated S-allylcysteine (SAC) is a natural organosulfur compound that has been studied extensively, and SAC is relatively high amounts in black garlic. Numerous recent studies have revealed that SAC has low toxicity and possesses various biofunctional properties, including anti-oxidation, anti-inflammatory, and retard cancer cell proliferation in vivo and in vitro. Therefore, we want to explore whether SAC could prevent carcinogenesis caused by food-contaminants HCAs. The results show that 0.05% SAC dietary supplementation significantly reduced colon shortening and tumor formation by up-regulating pERK1/2/Nrf2/HO-1 antioxidant signaling, which suppressed pro-inflammatory mediators such as iNOS, COX-2, and MMP-2 in the colon mucosa. Moreover, SAC supplementation alleviated intestinal barrier disruption. In addition, 16S rRNA sequencing was used to analyze the gut microbiota of fecal samples, and the results showed that PhIP/DSS group had higher colitis and colon cancer-related flora such as Erysipelotrichaceae, Bifidobacteriaceae, and Clostridiaceae and lower anti-inflammation bacteria Lachnospiraceae. Moreover, the SAC treatment group had lower Bacteriodes levels. These bacteria may lead to a strong increase in the bacterial mutagenicity of HCAs. The principal component analysis plot showed that SAC-supplemented group were closely clustered around that of the control group, suggesting that SAC has a marked direct or indirect effect on the composition of the gut microbial community and also reversed PhIP/DSS-induced gut dysbiosis. Furthermore, we investigate the inhibitory effect of SAC against NCM 460, a normal human colon mucosal epithelial cell line, carcinogenesis caused by PhIP during the initiation stage of carcinogenesis. The result shows that SAC may have an affinity with Keap1 protein using pull-down assay and cellular thermal shift assay. SAC may also reduce the binding of Keap1 and Nrf2 by inhibiting p-p38 and increasing the phosphorylation of ERK1/2 and AKT, thereby increasing the Nrf2/HO-1 signal up-regulated the ratio of GSH to GSH/GSSG, which inhibits the PhIP-induced oxidative stress and DNA damage. In addition, SAC can inhibit the expression of AhR, ARNT1, CYP1A1, and CYP1B1 proteins level, showing that it may potentially avoid the metabolic transformation of carcinogens. Overall, this is the first investigation with evidence of SAC’s ability to mitigate PhIP-induced ROS production and DNA damage and block inflammatory signaling at PhIP/DSS-induced colitis hence retard cancer initiation and promotion. These results indicate the potential application of SAC in colon cancer chemoprevention. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91950 |
DOI: | 10.6342/NTU202200634 |
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顯示於系所單位: | 食品科技研究所 |
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