Płoszaj Paulina, Regiec Andrzej, Ryng Stanisław, Piwowar Agnieszka, Kruzel Marian L
a Department of Organic Chemistry , Wrocław Medical University , Wrocław , Poland.
b Department of Toxicology , Wrocław Medical University , Wrocław , Poland.
Immunopharmacol Immunotoxicol. 2016 Dec;38(6):486-494. doi: 10.1080/08923973.2016.1247854.
The 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide (HIX) is a synthetic isoxazole derivative with a potential for development as an anti-inflammatory drug candidate. The goal of this study was to explore in vitro autoimmune and inflammatory gene modulation by HIX in human Caco-2 cultured cells. The effect of low dose of HIX was tested on the expression level of RNA in 24 h Caco-2 cultures using the QIAGEN Th17 for Autoimmunity & Inflammation RT Profiler PCR Array. We choose the PCR technology as the most reliable and sensitive gene expression profiling method for analyzing specific gene regulatory networks. In all experiments, Leflunomide (5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide), an immuno-suppressive disease-modifying antirheumatic drug was used, as a reference to clinical utility of the isoxazole derivatives. Changes in RNA levels were analyzed and differentially expressed genes with at least 2-fold change were identified. For the majority of genes tested, the effects of HIX and Leflunomide were similar, including up-regulation of CX3CL1 and IL-17F, and down-regulation of IL-10 and TLR4. However twelve genes were were differently regulated by the two compounds: interleukins (IL) IL-1B, IL-6 and a chemokine CCL22 were upregulated by HIX and significantly supressed by Leflunomide. In contrary, IL-2 and IL-27 were upregulated by Leflunomide and suppressed by HIX. The network search by Ingenuity Pathway Analysis showed, that majority of differentially expressed genes were involved in cellular inflammatory responses. These results suggest that 5-amino-3-methyl-4-isoxazolecarbohydrazide has a potential for future clinical developments with structure modification as a disease modifying agent in different than Leflunomide applications.
5-氨基-3-甲基-4-异恶唑甲酰肼（HIX）是一种合成异恶唑衍生物，具有开发成为抗炎候选药物的潜力。本研究的目的是探讨HIX对人Caco-2培养细胞的体外自身免疫和炎症基因调控作用。使用QIAGEN自身免疫与炎症RT Profiler PCR芯片检测低剂量HIX对24小时Caco-2培养物中RNA表达水平的影响。我们选择PCR技术作为分析特定基因调控网络最可靠、最灵敏的基因表达谱分析方法。在所有实验中，使用免疫抑制性病情缓解抗风湿药物来氟米特（5-甲基-N-[4-(三氟甲基)苯基]-4-异恶唑甲酰胺）作为异恶唑衍生物临床应用的参考。分析RNA水平的变化，并鉴定出至少有2倍变化的差异表达基因。对于大多数测试基因，HIX和来氟米特的作用相似，包括CX3CL1和IL-17F的上调以及IL-10和TLR4的下调。然而，有12个基因受到这两种化合物的不同调控：白细胞介素（IL）IL-1B、IL-6和趋化因子CCL22被HIX上调并被来氟米特显著抑制。相反，IL-2和IL-27被来氟米特上调并被HIX抑制。通过Ingenuity Pathway Analysis进行的网络搜索显示，大多数差异表达基因参与细胞炎症反应。这些结果表明，5-氨基-3-甲基-4-异恶唑甲酰肼在结构修饰后有作为不同于来氟米特应用的疾病修饰剂用于未来临床开发的潜力。
