生防真菌基因异源表达化合物通过TLR4/NF-κB信号通路调控细胞极化状态抑制脂多糖诱导炎症反应

Inhibitory activity of natural products derived from biocontrol fungus gene heterologous expression strain on lipopolysaccharide-induced inflammatory responsethrough the TLR4/NF-κB signaling pathway

摘要

目的 检测生防真菌基因在大肠杆菌中异源表达所产生的具有新奇骨架的氧杂蒽类天然产物21、23和33对脂多糖(lipopolysaccharide, LPS)诱导炎症反应的抑制作用。方法 用LPS诱导小胶质细胞BV-2炎症反应模型对3个化合物的抗炎活性进行筛选。对化合物33的抗炎机制进行进一步分析,使用RT-qPCR和ELISA等方法检测模型组、给药组、阳性对照组及正常组中BV-2细胞的M1极化和M2极化的特征性基因和蛋白的表达水平;使用Western-blot检测模型组和给药组中核转录因子NF-κB在细胞浆和细胞核中的含量。结果 给药组BV-2细胞的M1极化特征性炎症因子IL-1β、IL-6和TNF-α的基因转录和蛋白表达水平、炎性介质NO和PEG₂的产量及其合成相关基因iNOS和COX-2的转录平均显著低于模型组,M2极化特征性性基因Arg-1、Fizz1、Ym1及CD206转录水平明显高于模型组,TLR4/NF-κB信号通路中核转录因子NF-κB在细胞核中的含量明显低于模型组。结论 化合物33通过阻止TLR4/NF-κB信号通路中NF-κB的入核调控BV-2细胞的极化状态抑制LPS诱导的炎症反应,为抗炎新型药物研发奠定了基础。

Abstract

Objective To investigate the inhibitory effects of the novel scaffold-containing xanthore natuary products 21, 23 and 33, generated by heterologous expression of biocontrol fungus genes in Escherichia coli, on lipopolysaccharide (LPS) -induced inflammatory responses. Methods The anti-inflammatory activities of the three compounds were screened using a LPS-induced microglial BV-2 cell inflammation model. The anti-inflammatory mechanism of compound 33 was further analyzed. The expression levels of M1 and M2 polarization characteristic genes and proteins in BV-2 cells in the model group, treatment group, positive control group and normal group were assessed by RT-qPCR and ELISA. The contents of nuclear factor NF-κB in the cytoplasm and nucleus of the model group and the treatment group were detected by Western-blot. Results The levels of gene transcription and protein expression of M1 polarization characteristic inflammatory factors IL-1β, IL-6 and TNF-α, and the levels of the production of inflammatory mediators NO and PEG₂ and the synthesis-related gene transcription of iNOS and COX-2 in treatment group were significantly lower than those in the model group,while the transcriptional levels of M2 polarization characteristic genes Arg-1, Fizz1, Ym1 and CD206 were significantly higher than those in the model group. The content of nuclear factor NF-κB in the TLR4/NF-κB signaling pathway in the cell nucleus was significantly lower than that in the model group. Conclusion Compound 33 inhibits LPS-induced inflammatory responses by preventing the nuclear translocation of NF-κB in the TLR4/NF-κB signaling pathway, thereby regulating the polarization state of BV-2 cells. It laid the foundation for the development of new anti-inflammatory drugs.

关键词

天然产物 / TLR4/NF-κB / 脂多糖 / 炎症反应

Key words

natural product / TLR4/NF-κB / lipopolysaccharide / inflammatory response

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基金资助

云南省科技厅科技计划项目(202401AT070174);云南省教育厅科学研究基金项目(2024J0332)