背景:溃疡性结肠炎(UC),慢性特发性炎症性肠病(IBD),目前药物治疗选择有限。因此,寻找安全有效的UC预防和治疗药物势在必行。我们先前的研究表明,来自Nostoc公社的酚类化合物对羟基苯甲醛(HD),有效缓解肠道炎症。然而,HD抗炎作用的潜在机制尚不清楚.
目的:本研究探讨了HD的药效学及其潜在的抗炎机制。
方法:对于体内实验,建立葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎模型。使用脂多糖(LPS)诱导的RAW264.7和骨髓源性巨噬细胞(BMDMs)建立体外炎症模型。通过监测小鼠的临床症状和组织学形态来确定HD对结肠炎的保护作用。随后用酶联免疫吸附试验(ELISA)和生化试剂盒分析炎症因子和氧化应激标志物的水平。此外,蛋白质印迹(WB),免疫荧光(IF),荧光素酶报告基因,药物亲和反应靶稳定性(DARTS)测定,分子对接,和分子动力学(MD)模拟用于确定HD的潜在靶标和分子机制。
结果:我们的发现表明HD显著减轻了小鼠结肠炎的临床症状和组织学形态,并减少了促炎细胞因子的产生,包括TNF-α,IL-6,IFN-γ,COX-2和iNOS.此外,HD刺激SOD的产生,CAT,和GSH-px,提高总抗氧化能力(T-AOC),和降低MDA水平。机械上,HD增加了Nrf2,HO-1和NQO-1的表达,同时下调了p65,IκBα的磷酸化,c-Jun,c-fosML385和siNrf2在肠炎小鼠和RAW264.7细胞中大大减弱HD的保护作用,以及促进HO-1表达水平。ZnPP介导的HO-1敲除逆转HD诱导的结肠炎症抑制。荧光素酶报告基因测定和IF测定证实了HD对Nrf2的转录激活。DARTS分析,分子对接,MD结果显示高结合强度,Nrf2和HD之间的相互作用效率和显著的稳定性。
结论:这些结果扩展了我们先前的研究结果,即HD可以通过Nrf2/HO-1/NQO-1/NF-κB/AP-1途径抵抗氧化应激,有效缓解结肠炎,并提出HD的新靶点以防止肠屏障损伤。
BACKGROUND: Ulcerative colitis (UC), a chronic idiopathic inflammatory bowel disease (IBD), presents with limited current drug treatment options. Consequently, the search for safe and effective drug for UC prevention and treatment is imperative. Our prior studies have demonstrated that the phenolic compound p-Hydroxybenzaldehyde (HD) from Nostoc commune, effectively mitigates intestinal inflammation. However, the mechanisms underlying HD\'s anti-inflammatory effects remain unclear.
OBJECTIVE: This study delved into the pharmacodynamics of HD and its underlying anti-inflammation mechanisms.
METHODS: For in vivo experiments, dextran sodium sulfate (DSS)-induced colitis mouse model was established. In vitro inflammation model was established using lipopolysaccharide (LPS)-induced RAW264.7 and bone marrow-derived macrophages (BMDMs). The protective effect of HD against colitis was determined by monitoring clinical symptoms and histological morphology in mice. The levels of inflammatory factors and oxidative stress markers were subsequently analyzed with enzyme-linked immunosorbent assay (ELISA) and biochemical kits. Furthermore, western blotting (WB), immunofluorescence (IF), luciferase reporter gene, drug affinity reaction target stability (DARTS) assay, molecular docking, and molecular dynamics (MD) simulation were used to determine the potential target and molecular mechanism of HD.
RESULTS: Our findings indicate that HD significantly alleviated the clinical symptoms and histological morphology of colitis in mice, and curtailed the production of pro-inflammatory cytokines, including TNF-α, IL-6, IFN-γ, COX-2, and iNOS. Furthermore, HD stimulated the production of SOD, CAT, and GSH-px, enhanced total antioxidant capacity (T-AOC), and reduced MDA levels. Mechanically, HD augmented the expression of Nrf2, HO-1, and NQO-1, while concurrently downregulating the phosphorylation of p65, IκBα, c-Jun, and c-Fos. ML385 and siNrf2 largely attenuated the protective effect of HD in enteritis mice and RAW 264.7 cells, as well as the promotion of HO-1 expression levels. ZnPP-mediated HO-1 knockdown reversed HD-induced inhibition of colonic inflammation. Luciferase reporter assay and IF assay confirmed the transcriptional activation of Nrf2 by HD. DARTS analysis, molecular docking, and MD results showed high binding strength, interaction efficiency and remarkable stability between Nrf2 and HD.
CONCLUSIONS: These outcomes extend our previous research results that HD can combat oxidative stress through the Nrf2/HO-1/NQO-1/NF-κB/AP-1 pathways, effectively alleviating colitis, and propose new targets for HD to protect against intestinal barrier damage.