Garcinol 及 3'-hydroxypterostilbene 在高脂飲食誘導的肥胖、腸炎及腸炎相關癌化的抑制作用

Abstract

大腸直腸癌 (colorectal cancer, CRC) 為各個國家普遍盛行的癌症，在台灣地區的統計中癌症發生率排名第一，流行病學調查顯示肥胖及西化的飲食習慣會提升罹患腸癌風險，發炎性腸道疾病 (inflammatory bowel disease) 患者亦會增加併發腸癌機會。越來越多的證據指出，在致癌過程中肥胖、腸道菌相及腸炎與飲食和發炎引起之改變息息相關，肥胖與大腸直腸致癌過程密切相關且已成為新興議題，然其分子機制仍待釐清，有鑒於此，就癌症化學預防 (chemoprevention) 的立場觀之，藉由抑制肥胖誘導的發炎反應、調節腸道菌相與分子機制，進而抑制大腸直腸癌形成應為更適切的預防策略。因此本研究以三部分延續性實驗探討植化素如何抑制高脂飲食誘導肥胖促進之潰瘍性腸癌發展及腸道菌相組成。本研究使用 garcinol 及 stilbenoid 化合物這兩類植化素進行實驗，在第一部分，利用高脂飲食 (high-hat-diet, HFD) 誘導肥胖之小鼠模式，探討 garcinol 抑制肥胖之機制，結果指出 garcinol 可改善高脂飲食誘導的小鼠肥胖，且呈劑量效應，並可逆轉高脂飲食誘導的腸道菌相失調，而參與脂肪細胞脂質新生的 AMP-activated protein kinase α (AMPKα) 路徑也受到 garcinol 的影響。顯示 garcinol 可作為一新型腸道菌相調節劑以預防高脂飲食誘導的腸道菌相失衡和肥胖相關的代謝疾病。在第二部分，先以細胞實驗從stilbenoids 化合物中選出抗發炎及抗脂質新生能力最佳的 3'-hydroxypterostilbene (HPSB)，再以高脂飲食促進 dextran sodium sulfate (DSS) 誘導腸潰瘍動物模式探討 HPSB 在肥胖誘導的發炎反應及腸炎之保護作用與相關之分子機制。結果顯示 HFD+DSS 誘導組的腸炎較 DSS 組更為嚴重，而在不同時間點給予 0.025％ 的 HPSB 可以改善高脂飲食誘導肥胖促進之小鼠腸潰瘍，並顯著降低 cyclooxygenase-2 (COX-2) 及 plasmalemma vesicle-associated protein-1 (PV-1) 的蛋白表現和 signal transducer and activator of ranscription 3 (STAT3) 的磷酸化作用，進而達到化學預防的效果。結果證實 HPSB 為一種能夠預防高脂飲食誘導肥胖促進之腸潰瘍的良好化學預防試劑。第三部分則以高脂飲食促進 azomethane (AOM)/DSS 誘導潰瘍性腸癌動物模式探討 garcinol 在肥胖誘導的腫瘤發展機制化學預防功效及腸道菌相組成。結果顯示，HFD+AOM/DSS 誘導組與給予 AOM/DSS 的組別相比其腸癌情形較嚴重，而給予 0.05% 的 garcinol 可顯著改善高脂飲食促進之潰瘍性腸癌發展。此外，以細胞激素評估小鼠體內發炎程度，經 HFD+AOM/DSS 誘導後，小鼠血清中 tumor necrosis factor alpha (TNF-α) 及 monocyte chemoattractant protein-1 (MCP-1) 受誘導而上升，給予 garcinol 則可減少促發炎細胞激素生成並調節失衡的血清生化值。在腸道菌相的部分，從優勢菌種氣泡圖及主成分分析 (principal component analysis, PCA) 結果可知，各組腸道菌相組成皆有顯著差異並分群，其差異在 Alistipes、Romboutsia 及 Ruminococcus 這些菌屬中最具有代表性。進一步使用典範對應分析 (canonical correspondence analysis, CCA) 找出影響菌相組成的重要驅動因子，結果顯示腫瘤數目、TNF-α、MCP-1 及糞便 pH 值皆顯著影響。為了瞭解分子機制如何調控，使用 RNA 定序 (RNA sequencing) 比較不同實驗條件下轉錄體 (transcriptome) 的變化，以火山圖找出差異基因後，接著利用 GSEA (gene set enrichment analysis) 找出差異較大的基因集 (gene sets)，並以 KEGG (kyoto encyclopedia of genes and genomes) 資料庫進行比對分析，分析後的基因結果皆顯示給予 garcinol 可調節基因並改善高脂飲食促進之潰瘍性腸癌。綜合上述，植化素具有預防肥胖促進之潰瘍性腸癌發展的潛力，期以此研究結果為基礎，在科學上提供植化素在癌症化學預防與保健的機制，在食品的角度上則提供不同飲食攝取方式對生理的影響及功效，並可於經濟層面應用於開發植化素相關機能保健產品或輔助治療試劑。

Colorectal cancer (CRC) is one of the major causes of cancer-related mortality in both men and women worldwide. The incidence of cancer currently ranks first in Taiwan. Epidemiological surveys have shown that obesity and westernized diet can increase the risk of colorectal cancer. Obesity and inflammatory bowel disease are closely related to the colorectal carcinogenesis process. The obesity -related colorectal cancer has become an emerging issue. However, its molecular mechanism remains unclear. Based on the principles of cancer chemoprevention, targeting on suppression of obesity-induced inflammation and modulation of microbiota were known to be an approprite strategy for CRC prevention. Therefore, a three-part continual experiment was conducted to investigate the chemopreventive efficacy of phytochemicals on obesity-promoting colitis and colitis-induced colon tumorigenesis. The phytochemicals chosen in the studies were garcinol and stilbenoids. In the first part, the results showed that garcinol reduced high-fat-diet (HFD)-fed mice obesity in a dose-dependent manner. Furthermore, the 16S rRNA gene sequence data indicated that garcinol not only reversed HFD-induced gut dysbiosis but also attenuated inflammation by increasing the intestinal commensal bacteria, Akkermansia. In addition, the AMP-activated protein kinase α signaling pathway involved in adipocyte adipogenesis was also regulated by garcinol. Taken together, these results demonstrate for the first time that garcinol can prevent HFD-induced obesity and may act as a novel gut microbiota modulator to prevent HFD-induced gut dysbiosis and obesity-related metabolic disorders. In the second part, this study confirmed that the HFD could promote the deterioration of colitis in mice. In compared, the results also showed that 3′-hydroxypterostilbene (HPSB) could be an effective inhibitor for purpose of anti-adipogenesis and anti-inflammation. Therefore, HPSB was used to investigate the protective effect and related molecular mechanisms of HPSB on HFD-promoting dextran sodium sulfate (DSS)-induced colitis in mice. Our results indicate that colitis in the HFD+DSS group tends to be more severe than the DSS-only group, while feeding 0.025% of HPSB at different stages could improve the colitis induced by HFD+DSS. Moreover, HPSB significantly downregulated the expression of cyclooxygenase-2 (COX-2) and plasmalemma vesicle-associated protein-1 (PV-1), and inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in HFD+DSS treated mice. Presented results reveal that HPSB can be a novel functional agent capable of prevent HFD exacerbated colitis. In the last part of the experiment, an animal model to investigate the effect of HFD-induced obesity on promoting colitis-associated colon cancer (AOM (azomethane)/DSS-induced) was designed. The results evidenced that HFD promoted colitis-associated colon cancer as compared to AOM/DSS group without intervention of obesity, meanwhile supplementing 0.05% of garcinol in diet could significantly ameliorate obesity promoted colon carcinogenesis. Besides, the severity of inflammation was evaluated by cytokines status, the results show that the level of tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were increased in HFD+AOM/DSS group, while taking garcinol could effectively reduced the upregulation of pro-inflammatory cytokines. According to the dominant species bubble chart and principal component analysis (PCA) results, microbiota composition of each group was significantly different and clustered. The most representative genus are Alistipes, Romboutsia, and Ruminococcus. Canonical correspondence analysis (CCA) is commonly used to find the important driving factors affecting the distribution of the microbiota. The results showed that the number of tumors, TNF-α, MCP-1, and stool pH significantly affected the experimental results, in terms of microbiota composition. In order to understand the underlying molecular mechanism, RNA sequencing was employed to compare the changes of the transcriptome under different experimental conditions, and to find out the differential expression of genes by the volcano plot. And use of GSEA (gene set enrichment analysis) to find out the specific gene sets. Followed by that, the KEGG (kyoto encyclopedia of genes and genomes) database can be used for comparison and analysis. The RNA- sequencing results showed that administration of garcinol could regulate genes and improve obesity-promoting colitis-associated colon carcinogenesis. In conclusion, our results suggest phytochemicals having the potential to prevent obesity promoted-colorectal carcinogenesis. In addition, we provided the scientific evidence for elucidation of the underlying mechanisms of phytochemicals in cancer chemoprevention. From the perspective of food, it can also provide the effects of different dietary intake on physiology, and can also be applied to the development of phytochemical-related functional foods or adjuvant therapeutic agents.