基于ROS/NF-κB信号通路的天南星凝集素致炎机制及炮制对蛋白的影响

Pro-inflammatory mechanism of Arisaema erubescens lectin based on ROS/NF-κB signaling pathway and processing effect on protein

目的:研究天南星科有毒中药天南星凝集素蛋白(AEL)的致炎机制及炮制对蛋白的影响。方法:通过体外培养RAW264.7巨噬细胞,经不同浓度AEL刺激1h后,ELISA法检测炎性因子肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)的释放,Western blot法检测p-IκB、IκB、p-p65、p65蛋白的表达,倒置荧光显微镜及荧光酶标仪检测细胞内活性氧(ROS)、线粒体膜电位及胞内游离钙离子水平;加入ROS的阻断剂N-乙酰半胱氨酸(NAC)后,采用ELISA和Western blot法检测TNF-α、IL-1β及p-IκB、IκB、p-p65、p65蛋白表达的变化。采用8%白矾溶液炮制AEL,ELISA法检测炮制前后炎性因子TNF-α、IL-1β释放的变化。结果:AEL刺激巨噬细胞可导致炎性因子TNF-α、IL-1β大量释放,促进p-IκB、p-p65水平升高,导致巨噬细胞中ROS水平升高,线粒体膜电位降低以及胞内游离钙离子水平升高。当加入ROS的阻断剂NAC后,可明显降低ROS的生成,同时抑制了IκB、p65的磷酸化以及炎性因子TNF-α、IL-1β的大量释放。AEL经8%白矾溶液炮制后,能显著降低巨噬细胞炎性因子TNF-α、IL-1β的大量释放。结论:AEL刺激巨噬细胞后可诱导氧化应激,生成过量ROS,进而激活NF-κB炎性信号通路并导致炎性因子大量释放。AEL经白矾炮制后毒性降低,推测凝集素蛋白在白矾溶液中发生变性或降解,这可能是矾制天南星减毒的机制之一。

Objective： To investigate the pro-inflammatory mechanism of Arisaema erubescens lectin（AEL） and its processing effect on protein. Methods： RAW 264.7 macrophage cells were cultured in vitro, and they were stimulated by different concentrations of AEL for one hour. ELISA assay was used for the detection of TNF-α, IL-1β. Western blot assay was used to detect the levels of p-IκB, IκB, p-p65 and p65, and fluorescent inverted microscope and microplate reader were applied for ROS, mitochondrial membrane potential（MMP） and cytosolic free Ca2＋. N-acetylcysteine（NAC）, an ROS blocking agent, was added to reduce ROS production, and the expression of TNF-α, IL-1β and p-IκB, IκB, p-p65, p65 were detected by ELISA and Western blot. AEL was processed by 8%alum solution, and ELISA assay was used for the detection of TNF-α, IL-1β before and after processing. Results： AEL could stimulate macrophage and cause the release of inflammation TNF-α, IL-1β and increase the level of p-IκB, p-p65, and up-regulation of ROS and cytosolic free Ca2＋, but down-regulation of MMP. When NAC was added to the ROS, the production of ROS could be significantly reduced, and the phosphorylation of IκB and p65 and the release of inflammatory cytokines were inhibited. AEL which processed by 8%alum solution could inhibit the production of TNF-α and IL-1β. Conclusion： AEL could cause oxidative stress and generate excess ROS and stimulate the NF-κB signaling pathway, and the overproduction of pro-inflammatory factors. 8%alum solution could inhibit the toxicity of AEL, so it is speculated that the lectin hydrolyzed in alum solution denatured or dissolved under the action of ions, which may be one of the mechanisms of processing by alum.