原花青素基于miR-21/NFκB通路拮抗砷诱导的肝脏炎性损伤及机制研究

Arseniasis is one of the major global public health problems. Liver is the principal target organ involved. It was reported that overexpression of miR-21 induced by arsenic could inhibit IκB and activate the expression of NFκB, which would induce the significant hepatic inflammatory injury. Proanthocyanidins, a kind of polyphenolic substances found abundantly in plants, have extremely anti-inflammatory effect. However, a relationship for the use of Proanthocyanidins in alleviating liver inflammation caused by arsenic cannot be ascertained. So the questions of whether these substances are useful in arsenic-induced inflammation and whether NFκB pathway can be involved in their anti-inflammatory properties have not been completely answered. Moreover, a further query of whether miR-21 is the key target in the process of proanthocyanidins inhibiting NFκB pathway must be considered. Apropos the above, a study is designed to testify the aforementioned questions through experiments both in vitro and in vivo. We plan to use human liver cell lines and human liver cell lines with NFκB being knocked out using CRISPR/Cas9 plasimid transfection, along with mice and mice with NFκB in liver being knocked out. The objects mentioned above will be treated with different doses of proanthocyanidins based on inflammatory injury models induced by arsenic. Afterwards, we will detect indexes of inflammatory factors, viz. oxidative stress, the mRNA and protein expression of the key molecules of NFκB pathway and the expression of miR-21. The results of this study will be helpful to validate the relationship between the anti-inflammatory effect of proanthocyanidins and NFκB signal pathway , and the target role of miR-21 in this process. The research will try to provide an evidence for prevention and treatment of arseniasis, which can be helpful in developing a reference for utilization of the native plant resources rich in proanthocyanidins.

砷中毒是全球性公共卫生问题，严重威胁居民健康。肝脏是砷毒性作用的重要靶器官。研究发现，砷可以促进miR-21表达，抑制IκB，激活NFκB通路，诱发显著的肝脏炎性损伤。原花青素是植物富含的多酚化合物，具有极强的抗炎作用。关于原花青素能否抑制NFκB信号通路拮抗砷诱导的肝脏炎性损伤以及miR-21是否为原花青素抑制NFκB的作用靶点尚不明确。本研究拟采用析因实验设计，以人肝细胞系、小鼠为研究对象，采用CRISPR/Cas9质粒构建敲除NFкB的肝细胞系，引入肝脏敲除NFкB的小鼠；用亚砷酸钠诱导肝脏炎性损伤模型，再施以不同剂量原花青素干预，检测炎性因子和氧化应激相关指标、NFκB通路关键基因的mRNA与蛋白表达以及miR-21的表达，揭示原花青素拮抗砷致肝脏炎性损伤的效果与NFκB通路的关系及miR-21的靶点作用，为防治地方性砷中毒提供参考，为开发新疆富含原花青素的特色植物资源提供依据。

砷中毒已成为世界性公共卫生问题，砷可以诱导显著的肝脏炎性损伤，NF-κB信号通路参与了砷致炎性损伤，砷可以激活NF-κB调节miR-21表达。miRNA作为广泛参与基因表达调控的分子已成为药物靶点研究的重要对象，原花青素具有显著的抗炎作用。然而，原花青素是否能够拮抗砷诱导的肝脏炎性损伤及其抗炎性作用是否与抑制 NF-κB 信号通路有关？miR-21 是否为原花青素抗炎作用靶点？问题的研究未见报道。本研究通过体内和体外实验给予砷及原花青素干预，联合使用NF-κB抑制剂、miR-21抑制剂分析NF-κB、miR-21在原花青素拮抗砷致肝脏炎性损伤过程中的作用。研究发现，砷可以促进HSCs、BRL-3A 、BASE-2B细胞活性降低、凋亡，致使细胞炎性损伤、氧化损伤及线粒体凋亡，诱导小鼠肺组织病理性受损，原花青素具有缓解砷对细胞、小鼠肺组织的损伤作用。当使用不同浓度砷染毒L-02、WRL68、BRL-3A、BASE-2B细胞及小鼠时，细胞及肺组织内p65 mRNA、p-p65和p-IκBα蛋白的表达水平明显增加，NF-κB信号通路被激活，miR-21表达水平较对照组显著提高，当使用原花青素与砷联合干预时，砷诱导NF-κB信号通路及miR-21的表达提高被抑制。利用NF-κB抑制剂BAY 11-7082抑制砷激活NF-κB信号通路时，与砷组比较，miR-21表达降低，细胞内ROS水平降低、GSH水平增加、线粒体膜电位增加，砷致炎性损伤、氧化损伤及线粒体损伤被抑制并参与砷调节miR-21，当NF-κB 抑制剂BAY 11-7082作用于砷联合原花青素染毒细胞时，与联合处理组比较，施加BAY 11-7082后细胞内miR-21的表达降低，GSH水平增加，线粒体膜电位增强，提高了原花青素抑制砷促进miR-21的能力以及拮抗砷毒性的作用。此外，使用miR-21抑制剂干预砷染毒细胞抑制miR-21表达时，细胞炎性损伤及线粒体凋亡被缓解，当干预砷及原花青素联合处理细胞时，原花青素拮抗砷毒性的作用被加强。以上说明，砷可以诱发肝细胞炎性损伤，原花青素具有缓解肝细胞炎性损伤作用，其机制可能是原花青素通过抑制NF-κB信号通路拮抗砷激活miR-21，进而抑制促炎因子的水平，缓解砷诱发的肝损伤，为防治地方性砷中毒提供参考。