Higuchi Aina, Wakai Eri, Tada Tomoko, Koiwa Junko, Adachi Yuka, Shiromizu Takashi, Goto Hidemasa, Tanaka Toshio, Nishimura Yuhei
Department of Integrative Pharmacology, Mie University Graduate School of Medicine, Tsu 514-8507, Mie, Japan.
Ise Red Cross Hospital, Ise 516-8512, Mie, Japan.
Pharmaceuticals (Basel). 2021 Oct 31;14(11):1117. doi: 10.3390/ph14111117.
Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.
肝凋亡参与肝脏的多种病理生理状况，包括肝炎、脂肪变性和药物性肝损伤。因此，开发易于操作且可靠的体内检测方法将极大地促进对肝脏疾病的理解以及相关基因和潜在药物的鉴定。在本研究中，我们开发了一种转基因斑马鱼品系，该品系适用于通过体内荧光成像评估肝脏中的半胱天冬酶3活性。转基因斑马鱼幼虫在磷酸烯醇丙酮酸羧激酶1基因启动子的控制下在肝脏中主要表达Casper3GR。Casper3GR由两种荧光蛋白tagGFP和tagRFP组成，它们通过一个可被活化的半胱天冬酶3切割的肽接头连接。在暴露于特征明确的肝毒性药物异烟肼的斑马鱼中，在tagGFP激发条件下，我们分别检测到与tagGFP和tagRFP相关的荧光增加和减少。这一结果表明，异烟肼激活了斑马鱼肝脏中的半胱天冬酶3，其消化了tagGFP和tagRFP之间的接头，导致在tagGFP激发时向tagRFP的荧光共振能量转移减少。我们还检测到在使用肝保护剂熊去氧胆酸和奥贝胆酸处理的斑马鱼中，异烟肼诱导的半胱天冬酶3活性受到抑制。本研究中开发的转基因斑马鱼可能是一种强大的工具，可用于鉴定肝毒性和肝保护药物，以及分析感兴趣的基因对肝凋亡的影响。
