慈姑多糖激活Nrf2-ARE通路对INH/RFP肝损伤的预防性保护作用及机制研究

It is an urgent problem to be solved in the antituberculosis chemotherapy program that co-administration of isoniazid (INH) and rifampin (RFP) can induce significant liver toxicity. Based on the important role of Nrf2 in protection of liver cells, it became a hot spot to hunt for its agonists as a new recuperative target against liver injury. Sagittaria sagittifolia, a food also used as herbal medicine, has high medicinal and nutritional value and can fight against chemical-induced liver injury. In our preliminary work, it confirmed that Sagittaria sagittifolia has apparent protective effect on liver injury caused by co-administration of INH and RFP, whose mechanism may be positively correlated with the activation of Nrf2, and the liver protection active ingredients of Sagittaria sagittifolin polysaccharide(SSP) has been screened and successfully prepared. By applying liver injured mice induced by INH/RFP as an animal model and HepG2 cell model, it was firstly designed to observe the activation role of SSP to Nrf2-ARE combined with the technique of Nrf2 interfered by Si-RNA. It was secondly designed to analyze the preventive hepatoprotective effects of SSP from the aspect of hepatotoxicity, oxidative damage and CYP450 metabolism via metabolomics and other techniques. It was thirdly designed to comprehensive analyze the hepatoprotective mechanism of SSP from the aspect of Nrf2 which is a key link in the hepatoprotective role activation procedure, such as Nrf2-ARE pathway activation, mitochondria injury protection and apoptosis inhibition. The objective of this study is to elucidate the molecular mechanism of protective effect of SSP against liver injury induced by INH/RFP and also to provide the experimental basis for the development of SSP as liver protection products.

异烟肼和利福平联用(INH/RFP)引起明显肝损伤是目前抗结核化疗亟待解决的重要问题。鉴于Nrf2在保护肝细胞中发挥重要作用，寻找天然植物Nrf2激活剂作为肝损伤调治的新靶点已成为热点。慈姑药食兼用，药用和营养保健价值较高，保肝作用肯定。前期工作证实慈姑对INH/RFP肝损伤保护作用明显，其机制可能与Nrf2的激活呈正相关，并已筛选出主要活性成分慈姑多糖(SSP)，成功制备。本研究拟利用INH/RFP肝损伤小鼠和HepG2细胞模型，首先考察SSP对Nrf2-ARE的激活作用，siRNA干扰Nrf2表达进行验证；然后从肝脏毒性、氧化损伤、代谢物谱分析、CYP450代谢探究SSP的预防性保肝作用；最后从Nrf2发挥保肝作用的重要环节(激活Nrf2-ARE抗氧化、保护线粒体和抑制细胞凋亡)综合分析SSP保肝机制，旨在阐明SSP对抗INH/RFP肝损伤的分子机理，为将其开发为保肝产品提供实验依据。

异烟肼和利福平联用(INH/RFP)引起明显肝损伤是目前抗结核化疗亟待解决的重要问题。鉴于Nrf2在保护肝细胞中发挥重要作用，寻找天然植物Nrf2激活剂作为肝损伤调治的新靶点已成为热点。慈姑药食兼用，药用和营养保健价值较高，保肝作用肯定。前期工作证实慈姑对INH/RFP肝损伤保护作用明显，其机制可能与Nrf2的激活呈正相关，并已筛选出主要活性成分慈姑多糖(SSP)，成功制备。本研究首先利用苯酚-硫酸法测定所制备的SSP中多糖含量为34.31%，该方法灵敏、重现性好，制备足量SSP精制进一步实验。然后利用INH/RFP肝损伤小鼠和HepG2细胞模型，从肝脏毒性和氧化损伤两方面考察了SSP对INH/RFP所诱导的肝损伤具有的保护作用。综合肝细胞存活率，ALT、AST和LDH活性，MDA、SOD、CAT和GSH含量，以及HE染色肝组织病理观察，SSP具有明显抗氧化作用，在体外浓度0.5mg/ml、作用24h时，对肝损伤保护效果最好；体内其低、中、高剂量(0.2/0.4/0.8g/kg)均对肝损伤具有一定的保护作用，且0.8g/kg为最佳剂量，其保肝效果接近阳性对照水飞蓟素(0.1g/kg)。进而利用RT-PCR、Western blot技术、Elisa法、免疫组化法进行体内外实验，并利用siRNA干扰Nrf2表达进行体外验证，证实了SSP保肝机制与激活Nrf2/ARE信号通路、抑制CYP2E1和CYP3A4酶表达、抑制肝细胞凋亡相关；最后采用基于超高效液相色谱-高分辨率质谱(UPLC-HRMS)的非靶向代谢谱分析方法，分析了小鼠血浆代谢物谱，发现SSP能引起14种代谢物表达量改变，SSP保肝机制还与脂肪酸代谢、三羧酸循环、氨基酸代谢、牛磺酸代谢和鸟氨酸循环有关。本项目揭示了SSP对抗INH/RFP肝损伤的保护作用及其分子机理，为将其开发为保肝产品提供了实验依据。