探討腺嘌呤抑制脂多醣誘發腸道上皮細胞Caco-2發炎反應及其分子機轉

腸道作為人體中最大的免疫器官之一，具有吸收營養、維持屏障功能與參與免疫調節等多重角色，而發炎性腸道疾病(Inflammatory Bowel Disease，IBD)是以胃腸道為主要發病部位的慢性全身性發炎疾病，包括克隆氏症和潰瘍性結腸炎。近年的研究文獻指出脂多醣(lipopolysaccharide, LPS)為導致IBD發病機制的重要發炎因子，可誘發多種參與發炎相關細胞激素的表現。腺嘌呤(Adenine)為生物體中重要的核酸鹼基，除參與能量代謝及DNA/RNA合成之外，越來越多的研究報導指出腺嘌呤具有抗發炎與抗腫瘤等生物活性。因此，本研究以人類腸道上皮細胞株Caco-2作為細胞模型，探討腺嘌呤對LPS誘導腸道細胞的發炎反應是否具有抑制效果。首先使用MTT assay進行細胞存活分析，結果顯示低濃度腺嘌呤不會對腸道細胞的存活率造成明顯影響。此外，低濃度腺嘌呤還可提升LPS所造成的細胞存活率下降。接著，透過碘化丙啶(Propidium Iodide)染色與流式細胞儀進行細胞週期分析，發現腺嘌呤對細胞週期分布沒有顯著影響。以qPCR分析基因表現的結果顯示，單獨LPS刺激明顯提升TNF-α、IL-1β與IL-8等發炎相關基因的mRNA表現，且合併使用腺嘌呤可有效降低上述基因的mRNA表現量，顯示其具抗發炎潛力。西方墨點法分析結果也顯示合併腺嘌呤處理可提升因LPS刺激而降低的緊密連接蛋白Occludin、Claudin-1和ZO-1的表現，同時降低發炎訊息因子NF-κB、JNK和ERK的活化，以及誘導AMPK的活化。最後，前處理Compound C抑制AMPK 的實驗結果顯示，adenine 之抗發炎效果與提升腸道屏障蛋白表現的作用會因AMPK被抑制而減弱。保護作用可能部分透過 AMPK pathway 進行調控。總結來說，以上結果顯示腺嘌呤具有抑制LPS誘導之Caco-2細胞發炎反應以及提升重要腸道屏障蛋白表現的效用，且AMPK在腺嘌呤對減緩腸道發炎及改善腸道屏障的作用中扮演重要的角色，推測腺嘌呤具有作為輔助治療以及改善IBD的應用潛力。

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is a chronic inflammatory disorder primarily affecting the gastrointestinal tract. As one of the largest immune organs in the human body, the intestine plays critical roles in nutrient absorption, maintenance of epithelial barrier integrity, and immune regulation. Recent studies have demonstrated that lipopolysaccharide (LPS) is an important pro-inflammatory factor involved in the pathogenesis of IBD through the induction of inflammatory cytokine expression. Adenine, an essential purine nucleobase involved in energy metabolism and nucleic acid synthesis, has recently attracted increasing attention due to its diverse biological activities, including anti-inflammatory and anticancer effects. However, the effects of adenine on LPS-induced intestinal inflammation remain unclear. Therefore, this study aimed to investigate whether adenine could attenuate LPS-induced inflammatory responses in intestinal epithelial cells using the human colorectal adenocarcinoma cell line Caco-2 as an in vitro model. Cell viability was evaluated using the MTT assay, cell cycle progression and signaling pathways were evaluated by flow cytometric analysis and western blotting. Gene expression was determined by reverse-transcription quantitative real-time PCR (qPCR). The results revealed that low concentrations of adenine did not significantly affect cell viability. Moreover, adenine treatment partially restored the reduction in cell viability induced by LPS. Flow cytometric analysis demonstrated that adenine treatment had no significant effect on cell cycle distribution. qPCR analysis demonstrated that LPS stimulation markedly increased the mRNA expression levels of inflammatory cytokines, including TNF-α, IL-1β, and IL-8, whereas co-treatment with adenine significantly suppressed the expression of these inflammatory genes. Western blotting analysis showed that co-treatment with adenine increased the expression levels of tight junction proteins, including Occludin, Claudin-1, and ZO-1, while reducing the expression of inflammation-related signaling proteins NF-κB, JNK, and ERK. Finally, pretreatment with AMPK inhibitor compound c diminished adenine’s anti-inflammatory activity and promoting effect on tight junction molecules. In conclusion, these findings indicate that adenine suppresses LPS-induced inflammatory responses and restores tight junction molecules in Caco-2 cells, suggesting that adenine may serve as a potential anti-inflammatory adjuvant to improve IBD.