Real-time imaging of intestinal bacterial β-glucuronidase activity by hydrolysis of a fluorescent probe.
Real-time imaging of intestinal bacterial β-glucuronidase activity by hydrolysis of a fluorescent probe.
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DOI：
10.1038/s41598-017-03252-4
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发表时间：
2017-06-09
影响因子：
4.6
通讯作者：
Chuang KH
中科院分区：
文献类型：
--
作者：
Chen M;Cheng KW;Chen YJ;Wang CH;Cheng TC;Chang KC;Kao AP;Chuang KH
肠道细菌β-葡萄糖醛酸酶（βG）在肠道内将葡萄糖醛酸化代谢产物水解为毒性形式，导致肠道损伤。因此，开发一种抑制βG的方法是重要的，但由于难以直接评估活动物中的酶活性而受到限制。在这里，我们利用荧光探针，荧光素二-β-D-葡萄糖醛酸苷（FDGlcU），在裸鼠肠道细菌βG活性的非侵入性成像。基于细胞的体外测定表明，检测限为104个菌落形成单位/孔的β G表达细菌，并且7.81 ng/mL的FDGlcU足以产生显著的荧光信号。在裸小鼠全身光学图像中，在灌胃FDGlcU后3 h检测到肠中βG活性的最大荧光信号。在用细菌βG抑制剂预处理后，荧光信号在结肠镜和切除的肠图像中显著降低。对于4天的抗生素治疗以消耗肠道细菌，基于FDGlcU的图像显示，βG活性在第4天降低了8.5倍，然后在治疗停止后逐渐增加。结果提示，FDGlcU显像技术可用于肠道细菌βG的体内外活性研究，为特异性βG抑制剂的动物药效学研究奠定基础。
Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit βG is thus important but has been limited by the difficulty of directly assessing enzyme activity in live animals. Here, we utilized a fluorescent probe, fluorescein di-β-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial βG activity in nude mice. In vitro cell-based assays showed that the detection limit is 104 colony-forming units/well of βG-expressing bacteria, and that 7.81 ng/mL of FDGlcU is enough to generate significant fluorescent signal. In whole-body optical images of nude mice, the maximum fluorescence signal for βG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial βG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to deplete intestinal bacteria, the FDGlcU-based images showed that the βG activity was decreased by 8.5-fold on day 4 and then gradually increased after treatment stopped. The results suggested that FDGlcU-based imaging revealed the in vitro and in vivo activity of intestinal bacterial βG, which would facilitate pharmacodynamic studies of specific bacterial βG inhibitors in animal studies.