背景:非甾体抗炎药(NSAIDs)已在临床上用于治疗炎症性疾病。然而,NSAIDs的不良反应不容忽视.因此,找到可以减少草药不良反应的替代抗炎药对我们来说至关重要,比如IristectorumMaxim.,具有治疗作用,可以治疗炎症性疾病和肝脏相关疾病。
目的:本研究旨在从I.tectorum中分离活性化合物,并研究其抗炎作用和作用机制。
方法:采用硅胶柱色谱法从I.tectorum中分离出14种化合物,SephadexLH-20,ODS和高效液相色谱法,它们的结构是通过检查物理化学性质来鉴定的,紫外光谱,红外光谱,质谱,核磁共振波谱.使用脂多糖(LPS)刺激的RAW264.7细胞和大鼠原代腹膜巨噬细胞建立经典的炎症细胞模型,以检查这些化合物的作用。检查行动机制,用Griess试剂检测一氧化氮(NO)水平,用ELISA检测上清液中炎症细胞因子的水平;Westernblotting检测前列腺素E2(PGE2)合成中主要蛋白的表达以及核因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路。通过定量实时聚合酶链反应检测mRNA表达水平;通过高含量成像检查p65的核易位。分子对接用于预测活性化合物与靶蛋白的结合。
结果:我们的发现表明,在LPS诱导的RAW264.7细胞和大鼠腹膜巨噬细胞中,IristectorieninC(IT24)显着抑制NO和PGE2的水平,而不影响环氧合酶(COX)-1/COX-2的表达。此外,IT24可降低LPS诱导的大鼠腹膜巨噬细胞中微粒体前列腺素合成酶-1(mPGES-1)的表达。IT24不抑制NF-κB途径中蛋白质的磷酸化和核易位,但它抑制了LPS刺激的RAW264.7细胞中p38/JNK的磷酸化。此外,分子对接分析表明IT24可能与mPGES-1蛋白直接结合。
结论:IT24可能抑制mPGES-1和p38/JNK通路发挥其抗炎作用,也可能作为mPGES-1的抑制剂,预防和治疗mPGES-1相关疾病,如炎症性疾病,并有望进一步研究和药物开发。
BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used clinically to treat inflammatory diseases clinically. However, the adverse effects of NSAIDs cannot be ignored. Therefore, it is critical for us to find alternative anti-inflammatory drugs that can reduce adverse reactions to herbal medicine, such as Iris tectorum Maxim., which has therapeutic effects and can treat inflammatory diseases and liver-related diseases.
OBJECTIVE: This study aimed to isolate active compounds from I. tectorum and investigate their anti-inflammatory effects and action mechanisms.
METHODS: Fourteen compounds were isolated from I. tectorum using silica gel column chromatography, Sephadex LH-20, ODS and high performance liquid chromatography, and their structures were identified by examining physicochemical properties, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. Classical inflammatory cell models were established using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and rat primary peritoneal macrophages to examine the effect of these compounds. To examine the action mechanisms, the nitric oxide (NO) levels were measured by Griess reagent and the levels of inflammatory cytokines in the supernatant were measured by ELISA; The expressions of major proteins in prostaglandin E2 (PGE2) synthesis and the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were examined by Western blotting, and the mRNA expression levels were measured by quantitative real-time polymerase chain reaction; and the nuclear translocation of p65 was examined by high content imaging. Molecular docking was used to predict the binding of active compound to target protein.
RESULTS: Our findings revealed that Iristectorigenin C (IT24) significantly inhibited the levels of NO and PGE2 without affecting cyclooxygenase (COX)-1/COX-2 expression in LPS-induced RAW264.7 cells and rat peritoneal macrophages. Furthermore, IT24 was shown to decrease the expression of microsomal prostaglandin synthetase-1 (mPGES-1) in LPS-induced rat peritoneal macrophages. IT24 did not suppress the phosphorylation and nuclear translocation of proteins in the NF-κB pathway, but it inhibited the phosphorylation of p38/JNK in LPS-stimulated RAW264.7 cells. Additionally, molecular docking analysis indicated that IT24 may directly bind to the mPGES-1 protein.
CONCLUSIONS: IT24 might inhibit mPGES-1 and the p38/JNK pathway to exert its anti-inflammatory effects and could be also developed as an inhibitor of mPGES-1 to prevent and treat mPGES-1-related diseases, such as inflammatory diseases, and holds promise for further research and drug development.