基于DDIT4-mTOR信号通路探究斑蝥素诱导肝毒性损伤的分子机制

从中药斑蝥中提取的斑蝥素（CTD）对多种肿瘤具有显著的疗效。但因CTD毒性较大会造成严重的药物性肝损伤（DILI），而有关CTD致DILI的分子机制还不清楚。课题组前期的研究发现，CTD暴露后，DDIT4的表达异常，差异基因在mTOR信号通路出现显著富集，肝细胞出现不同程度的凋亡和自噬。以往的研究表明，DDIT4可通过抑制mTOR的磷酸化，导致自噬的发生，也能使凋亡增加。因此，我们推测：CTD可能是通过DDIT4-mTOR信号通路调控细胞自噬和凋亡而导致肝脏的毒性损伤。我们拟通过建立体内、体外的CTD暴露模型，检测小鼠肝脏组织及人L-O2细胞中DDIT4的表达；通过透射电镜、TUNEL和流式细胞仪等实验，检测肝细胞的自噬和凋亡情况；分别通过小干扰RNA片段和自噬抑制剂阻断DDIT4和mTOR通路，检测自噬及凋亡的变化。为解析CTD致肝毒性的机制及临床中降低其肝毒性的治疗方案提供参考。

Cantharidin (CTD), which is extracted from traditional Chinese medicine Mylabris, has a significant effect on many kinds of tumors. However, CTD can cause severe drug-induced liver injury (DILI) due to its hepatotoxicity. The underlying molecular mechanism of CTD-induced DILI is unclear. Our previous research found that after CTD exposure, DDIT4 gene expression was abnormally, differential genes were significantly enriched in the mTOR signaling pathway, and hepatocytes showed varying degrees of apoptosis and autophagy. Previous studies have shown that DDIT4 can lead to autophagy and increase apoptosis by inhibiting the phosphorylation of mTOR. Therefore, we speculate that CTD may regulate autophagy and apoptosis of cells through DDIT4-mTOR signaling pathway, thus causing toxic damage to liver. We intend to establish a model of CTD exposure in vivo and in vitro to detect the expression of DDIT4 in mouse liver tissue and human L-O2 cells. The autophagy and apoptosis of hepatocytes were detected by transmission electron microscopy, TUNEL and flow cytometry. Finally, the expression of the marker gene and protein of autophagy and apoptosis were detected by blocking the signal pathway of DDIT4 and mTOR by small interfering RNA fragment and autophagy inhibitor, respectively. The study can provide a reference for the analysis of the mechanism of CTD hepatotoxicity and the clinical treatment schemes of reducing its hepatotoxicity.