基于Keap1-Nrf2/NF-κB通路平衡体系研究广藿香活性成分抗急性肺损伤的作用机制

Pogostemon cablin, a well-known Chinese herbal medicine, possesses remarkable therapeutic effect on acute lung injury (ALI) induced by influenza virus and pathogen. However, its effective components and underlying mechanism remains obscure. Crosstalk between Keap1-Nrf2 and NF-κB pathways is closely associated with the pro-inflammatory/anti-inflammatory imbalance and oxidative stress which play a crucial role in the pathogenesis of ALI. Our previous studies have shown that patchouli alcohol (PA) and pogostone (PO), two major components of Pogostemon cablin, exerted protective effects against ALI induced by lipopolysaccharide (LPS). Treatments with PA and PO could obviously reduce inflammation and oxidative stress in the lungs. In addition, PA and PO could markedly inhibit the NF-κB activation in LPS-stimulated RAW264.7 macrophages. Based on these facts, we propose a hypothesis that PA and PO exert protective effects against ALI by regulation of the crosstalk between the Keap1-Nrf2 and NF-κB pathways. To present evidence to support the novel hypothesis, we will examine the impacts of PA and PO on the activities of Keap1-Nrf2 and NF-κB pathways using in vivo and in vitro (animal and cellular) experimental models of ALI. Molecular biology and biochemistry techniques (such as RT-PCR, western blot, flow cytometry and so on) will be applied to assess the in vivo and in vitro effects of PA and PO on the activities of the Keap1-Nrf2 and NF-κB pathways. Furthermore, to verify the impacts of PA and PO on the crosstalk between Keap1-Nrf2 and NF-κB pathways, we will knockdown the Nrf2 and NF-κB p65 genes in the cell model of ALI using RNA interference (RNAi), and assess whether PA and PO have protective effects against the inflammation, oxidative damage, and apoptosis in the model. This study aims to explore the underlying mechanism of PA and PO in ALI, which contributes to identify the effective anti-ALI components in Pogostemon cablin. Studies with specific agents that can regulate the crosstalk between Keap1-Nrf2 and NF-κB pathways may be one of the prospective strategies for prevention and treatment of ALI. Therefore, this study may aid in finding a new therapeutic target for traditional Chinese medicine in prevention and treatment of ALI.

广藿香可治疗流感病毒、细菌等感染性急性肺损伤（ALI），但其物质基础及作用机制尚不明确。Keap1-Nrf2与NF-κB通路平衡体系的失衡，与促炎/抗炎失衡、氧化应激反应等ALI的关键病理机制密切相关。前期研究发现，广藿香的主要成分广藿香醇、广藿香酮对LPS诱导的ALI小鼠模型有显著疗效，且可明显减少肺组织炎症、氧化应激水平；此外，二者可抑制LPS诱导的NF-κB信号通路的激活。本课题组提出“广藿香的活性成分广藿香醇、广藿香酮可能通过调节Keap1-Nrf2与NF-κB平衡体系以发挥抑制ALI的作用”的研究假说。拟采用ALI动物、细胞模型，比较广藿香醇、广藿香酮抗ALI活性；明确二者对Keap1-Nrf2与NF-κB平衡体系的作用；利用RNAi技术阻断该平衡体系，反向验证上述作用机制。本研究将进一步阐明广藿香抗ALI作用的物质基础及机制，并为中药抗ALI作用机理研究提供新的靶点。

广藿香可治疗流感病毒、细菌等感染性急性肺损伤（ALI），但其物质基础及作用机制尚不明确。Keap1-Nrf2与NF-κB通路平衡体系的失衡，与促炎/抗炎失衡、氧化应激反应等ALI的关键病理机制密切相关。前期研究发现，广藿香的主要成分广藿香醇、广藿香酮对LPS诱导的ALI小鼠模型有显著疗效，且可明显减少肺组织炎症、氧化应激水平；此外，二者可抑制LPS诱导的NF-κB信号通路的激活。本报告提出“广藿香的活性成分广藿香醇、广藿香酮可能通过调节Keap1-Nrf2与NF-κB平衡体系发挥抑制ALI的作用”的研究假说。本项目采用气管内注入LPS 诱导的小鼠ALI 模型和TNF-α 诱导肺泡Ⅱ型上皮细胞的ALI 细胞模型，以肺组织匀浆液和肺泡Ⅱ型上皮细胞中Keap1-Nrf2 与NF-κB 通路的关键转录因子和相关蛋白的磷酸化水平、下游靶基因的表达等为指标进行检验。结果显示，广藿香醇和广藿香酮体内体外给药均能有效提高Nrf2蛋白以及相关基因（GCLC, HO-1, NQO-1）的表达，同时抑制p-p65蛋白以及相关基因（IL-8, IL-6, IL-1β）的表达。采用siRNA转染技术构建Nrf2与NF-κB p65基因沉默的细胞模型，考察转染前后广藿香醇、广藿香酮干预细胞炎症介质产量、活性氧水平、细胞凋亡的变化。结果显示，转染前，广藿香醇和广藿香酮均可有效降低炎症介质产量、活性氧水平以及细胞凋亡率，转染后，高剂量的广藿香醇和广藿香酮反而增加了炎症介质产量、活性氧水平以及细胞凋亡率。以上结果反向验证了广藿香醇、广藿香酮是通过调控Keap1-Nrf2 与NF-κB 通路平衡体系发挥抗ALI作用。本项目研究结果阐明了广藿香活性成分广藿香醇、广藿香酮抗ALI作用的物质基础及机制，并为中药抗ALI作用机理研究提供新的靶点。