Ntcp在异烟肼所致肝损伤中的作用及机制

The hepatotoxicity resulted from long term use of drugs for anti-tuberculosis(anti-TB) chemotherapy accounted for 43.78% of drug-induced liver injury(DILI), ranking first in the etiology of a variety of DILI. Despite isoniazid (INH) is the most effective front line drugs for anti-TB chemotherapy, the hepatotoxicity resulted from long term use of INH remains a significant problem in clinical. Our previous studies have shown that isoniazid liver injury in rats, mice, and has significantly increased the expression of transporter Ntcp liver-specific distribution. Therefore, the project intends to study isoniazid by liver cell membrane transporter Ntcp adjustment mechanism, through the in vivo and in vitro experiments of sandwich-cultured hepatocyte and rat, on the one hand to clearly the effect relationship between Ntcp and Ntcp mediated the concentration changes of taurocholate acid in the liver cells of isoniazid-induced liver injury, on the other hand to explore the mechanism of isoniazid Ntcp raised by HNF4α/ PGC1α path, so as to further clarify the mechanism of isoniazid-induced liver injury, in order to provide new ideas for clinical prevention and treatment of isoniazid induced hepatotoxicity.

抗结核药引起的肝损伤占药源性肝损伤的43.78%，居各种药源性肝损伤（Drug induced liver injury, DILI）病因之首。异烟肼是一线基础抗结核药具有明显的肝毒性。课题组前期研究表明，异烟肼肝损伤大鼠、小鼠模型中，具有肝脏特异性分布的转运体Na+-牛磺胆酸共转运多肽（Na+-taurcholate co-transporting polypeptede, Ntcp）表达显著升高。因此，本项目拟将通过研究异烟肼对肝细胞膜转运体Ntcp的调节机制，通过“三明治”结构肝细胞的体外实验及大鼠体内实验，一方面明确经Ntcp介导的结合型牛磺胆酸在肝细胞内的浓度变化与异烟肼诱导肝损伤的量效关系，另一方面通过HNF4α/PGC1α路径探究异烟肼上调Ntcp的机制，从而为进一步阐明异烟肼致肝损伤机制，以期为临床预防和治疗异烟肼肝毒性提供新的思路。

Na+-牛磺胆酸共转运多肽（Ntcp）的表达在胆汁酸吸收入肝的过程中发挥了重要作用，同时其在异烟肼导致的肝损伤过程中也起到了关键作用。本课题申请人建立了异烟肼导致的药物性肝损伤模型，并考察了在此过程总Ntcp的表达请况，以及其调控通路HNF4α和PGC1α的表达。采用雄性大鼠口服灌胃给予异烟肼350 mg/kg，分别给药7、14和21天，以肝脏病理改变及血清生化指标评价异烟肼诱导的肝损伤程度，免疫组化和Western Blot技术考察Ntcp的定位及表达。采用HPLC-MS/MS测定血清及肝脏中的牛磺胆酸钠含量。在细胞水平，我们建立了异烟肼诱导的HepG2细胞凋亡模型，并考察了Ntcp在此过程中的作用。大鼠肝功能及肝脏病理改变结果显示，异烟肼给药后诱导的肝损伤与Ntcp的上调均在第七天最为明显，而异烟肼给药后第14天和第21天肝损伤程度呈好转趋势，Ntcp的表达水平也随之下调。通过对血清中及肝内的牛磺胆酸钠含量测定结果显示，随着Ntcp表达的上调，可引起肝内牛磺胆酸钠的含量增加，该变化反映了胆汁酸可能在肝内淤积。细胞水平实验我们也发现，给予6.5、13、26、52和104 mM的异烟肼孵育HepG2细胞24小时后，细胞活力呈浓度依赖性的下降，Western Blot结果表明其Ntcp的表达除了给予异烟肼104 mM浓度外也逐渐升高。当给予Ntcp的抑制剂环孢素A后，可逆转异烟肼诱导的HepG2细胞活力下降，同时我们还发现细胞凋亡的相关蛋白如Caspase-3、Bcl-2/Bax等的表达也与Ntcp的过表达有关系。最后，我们发现Ntcp的可能调控通路HNF4α和PGC1α的表达也相应上调。因此，通过本课题发现，Ntcp的高表达在异烟肼诱导的肝损伤和HepG2细胞凋亡过程中发挥了重要的作用，且其可能是通过HNF4α/PGC1α通路调控Ntcp的过表达。