内质网应激在利福平致胆汁淤积性肝损伤适应现象的作用和机制研究

The adaption to antituberculosis drugs-induced liver injury is an interesting research direction in the field of drug-induced liver diseases. The research team led by the applicant had previously observed that rifampicin-induced cholestasis model had the phenomenon of adaptive changes,but the molecular mechanism of this phenomenon remains to be clarified. In this project, we propose "while rifampicin induce cholestasis, it initiates the unfolded protein response of endoplasmic reticulum stress, increases synthesis and folding of bile acid transporters and tight junction proteins, results in adaptation phenomenon" as a hypothesis.It is designed that the rat model of rifampicin-induced cholestasis adaptation phenomenon and 3D cultured human primary hepatocytes are acted as experimental subjects.In the experimental scheme by GRP78 overexpression and pretreatment with endoplasmic reticulum chemical molecular chaperone PBA,RT-PCR、Western Blot and immunofluorescence confocal microscopy are used to detect the gene transcription, protein expression and cellular localization of bile acid transporters and tight junction proteins and the molecular changes of unfolded protein response signaling pathways.It is expected that the effects of unfolded protein response in adaptive phenomenon of rifampicin-induced cholestasis are clarified. These results will ravel the molecular mechanism of adaptive response of rifampicin-indueced cholestasis,and provide theoretical and experimental basis for reasonably treatment measures of antituberculosis drug-induced cholestasis. This study has not been reported until now.

抗结核药物导致药物性肝损伤适应现象是药物性肝损伤领域中令人感兴趣的研究方向。本课题组先期研究发现，利福平所致胆汁淤积模型有适应性变化的现象，但分子机制尚有待阐明。本项目以"利福平导致胆汁淤积，同时启动内质网应激的非折叠蛋白反应，增加相应胆汁酸转运体和紧密连接蛋白的合成与折叠，从而产生胆汁淤积适应现象"为假说，以利福平胆汁淤积适应现象的大鼠模型和3D培养的人原代肝细胞为实验对象，通过以内质网化学小分子伴侣PBA预处理以及GRP78过表达的实验方案，采用RT-PCR、Western Blot和免疫荧光共聚焦显微镜，检测胆汁酸转运体和紧密结合蛋白基因转录、蛋白表达和细胞定位以及内质网应激的非折叠蛋白反应信号通路的分子变化，阐明非折叠蛋白反应在利福平胆汁淤积适应现象中的作用。此研究将为深入认识利福平胆汁淤积的适应机制，及合理治疗提供新的理论和实验依据。此项研究国内外未见报道。

抗结核药物导致药物性肝损伤适应现象是药物性肝损伤领域中令人感兴趣的研究方向。适应现象约占全部抗结核药所致药物性肝损伤患者总数的10%-15%，识别适应现象的意义在于避免不必要的停药，而目前对于适应的机制尚知之甚少。因此，需要建立合适的模型开展研究，以深入了解抗结核药诱导适应现象的变化规律及其机制，为科学合理地指导临床用药提供依据。.课题组先期研究发现，利福平所致胆汁淤积模型有适应性变化的现象，但分子机制尚不清楚。本课题进一步研究发现，利福平可在体内外诱导胆汁淤积性生化改变，同时诱导胆汁酸转运体表达适应性增加，表现为胆汁酸盐输出泵（BSEP）、多药耐药蛋白l（MDR1）、多药耐药相关蛋白2（MRP2）、多药耐药相关蛋白4（MRP4）、有机阴离子转运蛋白2（OATP2）、有机溶质转运体（OSTβ）、Na+依赖性的牛磺酸共转运蛋白（NTCP）、Cl-／HCO3-阴离子交换蛋白（AE2）等胆汁酸转运体蛋白表达及基因表达的增加，同时增加非折叠蛋白反应（UPR）中蛋白激酶R样内质网激酶（PERK）通路下游分子核因子E2相关因子2（Nrf2）的表达。在此基础上，课题组进一步发现Nrf2具有增加上述胆汁酸转运体表达的作用，并且PERK-Nrf2通路是一种促进细胞生存的信号通路。课题组在体外研究中发现，利福平还能诱导另一条PERK-ATF4-CHOP通路的激活从而增加细胞凋亡，4-苯基丁酸（4-PBA）通过抑制PERK-ATF4-CHOP通路激活减轻利福平诱导的L02细胞损伤。牛磺熊去氧胆酸（TUDCA）通过增加Nrf2和胆汁酸转运体的表达、抑制PERK-ATF4-CHOP通路激活、抑制氧化应激而减轻利福平诱导的HepG2细胞损伤。我们的研究结果提示利福平诱导胆汁淤积，同时启动UPR、激活Nrf2，增加相应胆汁酸转运体的合成，从而产生胆汁淤积适应现象。