紫檀芪及 3'-羥基紫檀芪對 AOM/DSS 誘導且受生理時鐘紊亂惡化之小鼠結直腸癌的改善功效

Abstract

結直腸癌 (colorectal cancer, CRC) 為目前全球罹癌率高居第三的癌症，許多研究指出 Wnt/β-catenin 訊號路徑參與結直腸癌的各個發展階段，為影響結直腸癌起始的重要分子路徑之一，目前也有許多開發中藥物針對此路徑進行調控抑制。另一方面，全球目前也在面臨結直腸癌患病年齡在不斷下降的問題，各方學者認為年輕化趨勢的出現可能與肥胖、少運動和輪班性質工作等因子高度相關。現代人基於生活型態改變，全球約有 15-20% 的工作者長時間從事輪班性質工作，可能因此導致身體出現生理時鐘紊亂 (circadian rhythm disorder, CRD)。隨著時間的推移，諸多流行病學研究揭示，輪班工作與罹患結直腸癌的風險具有正相關。因此本研究旨在探討生理時鐘紊亂是否加劇結直腸癌的嚴重性，同時評估紫檀芪 (pterostilbene, PSB) 和3'-羥基紫檀芪 (3'-hydroxypterostilbene, 3'-HPSB) 對於 AOM/DSS 誘導合併 CRD 的結直腸癌的改善效果，並探究其背後機制是否經由調節生理時鐘相關蛋白表現或抑制 Wnt/β-catenin 路徑。實驗結果顯示，生理時鐘紊亂下除了會加劇小鼠疾病活動指數 (disease activity index, DAI)、腸道縮短、腸道癮窩構造紊亂及腫瘤數目等表型，提升促發炎細胞激素 IL-1β、IL-6、TNF-α 和 MCP-1 含量加劇發炎反應，亦會降低生理時鐘重要蛋白 BMAL1、CLOCK 和 PER2 表現量，並且透過增加輔助受器 LRP6 蛋白磷酸化、降低破壞複合體組成元件蛋白 APC 和 GSK3β 表現量及提升 β-catenin 穩定性，進而活化 Wnt/β-catenin 訊號路徑，增加下游目標蛋白 c-Myc 和 Cyclin D1 表現量，促進腸道上皮細胞的增生；當介入樣品 PSB 和 3'-HPSB 後，除了改善小鼠各項腸癌表型，降低腸道促發炎細胞激素，亦可恢復生理時鐘相關蛋白的表現，並且抑制 Wnt/β-catenin 訊號路徑，藉由降低LRP6 蛋白磷酸化、增加破壞複合體穩定性、促使 β-catenin 及其下游蛋白表現量下降，進而減少腸道上皮細胞的過度增生，此外，根據腸道菌相分析結果，PSB 和 3'-HPSB 可調節小鼠腸道菌相組成，減少疾病有害菌 Dubosiella 的含量，同時提升 Clostridium、Erysipelatoclostridium、Duncaniella 及 Akkermansia 等有益菌的相對豐富度，增加短鏈脂肪酸的產生，進而改善結直腸癌進程。綜合以上結果，紫檀芪和 3'-羥基紫檀芪對於改善 AOM/DSS 誘導且受 CRD 惡化的結直腸癌方面具有相當大的潛力。

Colorectal cancer (CRC) currently ranks third globally in cancer incidence. Numerous studies have indicated the involvement of the Wnt/β-catenin signaling pathway in various stages of CRC development, making it one of the crucial molecular pathways influencing the onset of CRC. Currently, many drugs in development targeting this pathway for regulation and inhibition. On the other hand, the global trend of decreasing age at diagnosis for CRC poses a challenge. Researches have attributed this to certain factors highly correlated with modern lifestyles such as obesity, lack of exercise, and shift work. Approximately 15-20% of the global workforce engages in shift work, which may lead to circadian rhythm disrorder (CRD) in the body. Epidemiological studies have revealed a positive correlation between shift work and the risk of developing CRC. Therefore, this study aims to investigate whether circadian rhythm disorder exacerbates the severity of CRC and to evaluate the potential effects of pterostilbene (PSB) and 3'-hydroxypterostilbene (3'-HPSB) on AOM/DSS-induced and CRD-exacerbated CRC. Additionally, the study aims to explore whether their mechanisms operate through regulating the expression of circadian clock-related proteins or inhibiting the Wnt/β-catenin pathway. Results demonstrated that under circadian rhythm disorder, various CRC phenotypes in mice were exacerbated. These included increased disease activity index (DAI), colon shortening, disrupted colon crypt structure, and increased number of tumors. Additionally, CRD enhanced the inflammatory response by elevating pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and MCP-1, while reducing the expression of key circadian rhythm-related proteins BMAL1, CLOCK, and PER2. Furthermore, CRD activated the Wnt/β-catenin signaling pathway by increasing the phosphorylation of co-receptor LRP6, decreasing the expression of the destruction complex components APC and GSK3β, and enhancing the stability of β-catenin, thereby promoting the proliferation of intestinal epithelial cells. However, treatment with PSB and 3'-HPSB not only improved various CRC phenotypes in mice and reduced intestinal pro-inflammatory cytokines but also restored the expression of circadian-related proteins. Moreover, they could inhibit the Wnt/β-catenin signaling pathway by reducing the phosphorylation of LRP6 and increasing the stability of the destruction complex component, leading to a decrease expression of β-catenin and its target genes c-Myc and Cyclin D1, therefore reducing excessive proliferation of intestinal epithelial cells. Based on intestinal microbiota analysis, PSB and 3'-HPSB modulated the composition of microbiota in mice by reducing the content of the pathogenic bacteria Dubosiella while increasing the relative abundance of beneficial bacteria such as Clostridium, Erysipelatoclostridium, Duncaniella, and Akkermansia, thereby increasing the gut microbial SCFA level, resulting in suppressing the progression of CRC. In conclusion, pterostilbene and 3'-hydroxypterostilbene show great potential for improving AOM/DSS-induced CRD-exacerbated CRC.