| 摘要: | 目標 潰瘍性結腸炎 (ulcerative colitis, UC) 是為自體免疫發炎性腸道疾病(inflammatory bowel diseases, IBD)之一，常好發於乙狀結腸中。UC患者常介入多種藥物以疾病症狀，但另受困於藥物所帶來的副作用，因此找尋一個有效且安全的治療方法極具重要。先前研究顯示枸杞多醣 (Lycium barbarum polysaccharides, LBP)具有抗發炎與免疫調節功效，然而其對UC功效尚未明確，因此本研究探討LBP，以及其搭配複方如辣椒素 (capsaicin, CAP)或電漿激活水 (plasmon activated water, PAW)對抗UC的功效。
方法 研究(一) 五十隻Sprague-Dawley (SD) 大鼠分為正常控制組、UC組、UC給予 100 mg LBP/kg bw組、UC給予12 mg CAP/kg bw 組， UC給予50 mg LBP/kg bw 與6 mg/kg bw 組。LBP或CAP連續4週以管餵形式給予大鼠補充，並於第三週提供5% DSS飲用水連續6天以誘導UC症狀，誘導一週後進行犧牲並採取大腸組織和盲腸內容物供後續分析。研究(二) 人類大腸癌細胞(Caco-2)於介入前利用10 ng/mL interferon-γ (IFN-γ)以提升tumor necrosis factor receptor 2 (TNFR2)後，誘導前介入100~1000 μg/mL LBP 溶於一般細胞培養液或PAW配置之細胞培養液連續3小時後，隨之添加TNF-α至 50 ng/mL連續24小時以誘導細胞發炎，並收集細胞液與細胞蛋白供後續分析。
結果 研究(一) 大鼠補充LBP或/與CAP改善UC 疾病程度、腸道損傷以及腸道萎縮，並抑制 IFN-γ、interleukin-17A (IL-17A)、IL-22的分泌。UC盲腸菌相富含 Turicibacter和Lachnospira菌屬。LBP和CAP的給予個別提升在盲腸中Ruminiclostridium_9 和 Ruminiclostridium_1菌屬的豐富度。
研究(二) Caco-2細胞介入500 μg/mL LBP或100 μg/mL LBP搭配PAW培養液抑制IL-6與IL-8分泌，而所有LBP劑量在一般或PAW培養液皆降低cyclooxygenase-2 (COX-2)和inducible nitric oxide synthase (iNOS)蛋白質表現量。NLRP3發炎小體相關蛋白質表現量。LBP介入顯著抑制 p-ERK蛋白質表現量，而與PAW培養液的搭配反之提升。細胞凋亡指標caspase-3/procaspase-3於250與500 μg/mL LBP在有無搭配PAW培養液皆顯著下降。
結論 給予DSS誘導大鼠UC管餵LBP可透過抗發炎與菌相調節達到改善UC疾病症狀，另於發炎細胞給予LBP介入可以抑制促發炎細胞激素的分泌，抑制NLRP3發炎小體相關蛋白並且抑制細胞凋亡作用，並且LBP與利用PAW配置細胞液的合併於抑制細胞激素有更顯著的效果，顯示LBP與PAW的結合可能具有加成/協同作用達到改善UC的效果。
Objective Ulcerative colitis (UC) is characterized as one of the sub-forms in inflammatory bowel diseases (IBD) that occurred in the sigmoid colon, which is identified as an autoimmune disease, and its triggers remains unknown. Patients were commonly given with several drugs for symptom controls and were not free from the adverse effects, hence searching for an effective, safer, and natural therapeutic method remains focus of interested. Lycium barbarum polysaccharides (LBP) was well known for its immunomodulatory and anti-inflammatory effects, but its effects against UC is not commonly investigated, hence the study of LBP in against UC, and the combination with capsaicin (CAP) or plasmon-activated water (PAW) was investigated.
Methods Study 1: Fifty Sprague-Dawley (SD) rats were divided in to normal, colitis, and colitis treated with 100 mg LBP/kg bw, 12 mg CAP kg bw, or combined 50 mg LBP/kg bw and 6 mg CAP/kg bw. Rats were continuously gavage fed with LBP or CAP for 4 weeks, and colitis symptoms was induced by providing 5% dextran sulfate sodium (DSS) in rats drinking water for 6 days in week 3. Rats were sacrificed a week after induction, and rats colon tissue and cecum content were collected for further analysis.
Study 2: Caco-2 cells will be treated with 10 ng/mL of IFN-γ for 24h to up-regulate tumor necrosis factor receptor-2 (TNFR2), then cells will be pretreated with 100~1000 μg/mL of LBP with normal or PAW medium for 3 hours and further provoke with 50 ng/mL of TNF-α for 24 h to induce inflammation. Cell medium and cell pellet will be collected for further analysis.
Results Study 1: Rats pretreated with LBP and/or CAP shown to improve disease activity index scores, severity of colon distortion, and colon length shrinkage, and further inhibit secretion of IFN-γ, IL-17A, and IL-22 levels. Cecal microbiota in colitis rats were enriched with the genus Turicibacter and Lachnospira. The relative abundance of genus Rumin52 iclostridium_9 and Ruminoclostridium_1 was increased by LBP and CAP treatment, respectively.
Study 2: Caco-2 cells pretreated 500 μg/mL LBP alone and 100 μg/mL LBP with PAW medium alleviate IL-6 and IL-8 cytokine secretion, protein expression of COX-2 and iNOS was decreased in all LBP groups either with or without PAW-medium. Using 500μg/mL of LBP significantly inhibit NLRP inflammasome and PYCARD protein expression. p-ERK expression was significantly inhibit in LBP treatment but increased of expression was found in combination with PAW. Apoptosis marker caspase-3/procaspase-3 expression was inhibited in 250 and 500 μg/mL of LBP either with or without PAW.
Conclusion: Our animal study demonstrated that the treatment of LBP could ameliorate the severity of DSS induced colitis symptoms via anti-inflammatory and modulate colonic microbiota, and LBP could inhibit secretion of pro-inflammatory cytokines in inflamed cells, inhibit NLRP3 inflammasome protein and anti-apoptotic effect, mixture with PAW medium potentially show better effects in cytokine secretion, suggesting mixture of LBP and PAW may exerted additive/synergistic effects in against colitis. |
