Highly Selective NIR Probe for Intestinal β-Glucuronidase and High

Aug 28, 2018 - College of Integrative Medicine, College of Pharmacy, the National ..... This evidence fully indicated that our probe possessed the goo...
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Highly selective NIR probe for intestinal #-Glucuronidase and High-throughput screening inhibitors to therapy intestinal damage Lei Feng, Yongliang Yang, Xiaokui Huo, Xiangge Tian, Yujie Feng, Hanwen Yuan, Lijian Zhao, Chao Wang, Peng Chu, Feida Long, Wei Wang, and Xiaochi Ma ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00471 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Highly selective NIR probe for intestinal β-Glucuronidase and Highthroughput screening inhibitors to therapy intestinal damage Lei Feng,†,§,# Yongliang Yang,§,# Xiaokui Huo,†,# Xiangge Tian,†,# Yujie Feng,† Hanwen Yuan,‡ Lijian Zhao,⊥ Chao Wang,†,§,* Peng Chu,§ Feida Long,§ Wei Wang,‡,* Xiaochi Ma†,* †

College of Integrative Medicine, College of Pharmacy, the National & Local Joint Engineering Research Center for Drug Development of Neurodegenerative Disease, Dalian Medical University, Dalian 116044, China ‡ TCM and Ethnomedicine Innovation & Development International Laboratory, Sino-Pakistan TCM and Ethnomedicine Research Center, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China § Center for Molecular Medicine, School of Life Science and Biotechnology, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China ⊥

Bio/Medical Experiment Center, College of Biology, Hunan University, Changsha 410082, China

ABSTRACT: β-Glucuronidase (GLU) as a vital factor in enterohepatic circulation and drug-inducing enteropathy was paid more and more attention in recent years. In this study, an off-on near-infrared (NIR) fluorescent probe (DDAO-glu) for selectively and sensitively sensing GLU was developed on the basis of its substrate preference. DDAO-glu can rapidly and selectively respond to bacterial GLU under physiological conditions for detecting the real-time intestinal GLU bioactivity of complex biological systems such as human feces in clinic. Meantime, DDAO-glu has been successfully applied for visualization of endogenous GLU in bacterial biofilm, thallus and even in vivo. Using this NIR probe, we successfully visualized the real distribution of intestinal GLU in the enterohepatic circulation. Furthermore, a high-throughput screening method was successfully established by our probe, and a potent natural inhibitor of GLU was identified as (−)-epicatechin-3-gallate (ECG) for effectively preventing NSAIDs-inducing enteropathy in vivo. DDAO-glu could serve as a powerful tool for exploring real physical functions of intestinal GLU in enterohepatic circulation, under physiological and pathological contexts, and developing the novel inhibitor of GLU to therapy acute drug-inducing enteropathy in clinic. KEY WORDS: fluorescent probe; β-glucuronidase; intestinal bacteria; high-throughput screening; intestinal damage

β-Glucuronidase (EC 3.2.1.31, GLU) as a lysosomal enzyme, plays a vital role in the degradation of glucuronic acid from glycosaminoglycans.1-5 Over-expression of GLU is observed in different pathological conditions, such as infection of urinary tract, renal disease rejection of transplantation, various malignant tumors and rheumatoid arthritis.6-8 Nowadays, the most widely prescribed nonsteroidal antiinflammatory drugs (NSAIDs) leads to an increased clinical risk of serious gastrointestinal adverse effects such as bleeding, ulceration, and even perforation of intestines. In the small intestine, the glucuronide of NSAIDs is usually hydrolyzed to free NSAIDs via GLU, leading to enterohepatic circulation.9-11 It is generally accepted that enterohepatic circulation plays an important role in NSAIDs-induced enteropathy, which is closely related to enterocyte exposure of aglycon released from phenol glucuronides of NSAIDs via GLU. When taken long-term, the complications associated with NSAIDs-induced enteropathy (bleeding, protein loss, strictures, and rare perforations) may become very serious and even fatal.12-14 Therefore, the detection of intestinal GLU activity using the highly sensitive assay, even the further block of the enterohepatic circulation mediated by intestinal GLU is regarded as an important approach to alleviate fatal NSAIDs-induced enteropathy. In recent years, enzyme-activated fluorescent probes have been developed to study human pathogens,15-17 due to their noninvasiveness, superb spatiotemporal resolution in visualiz-

ing reactive species, as well as the capability of being applicable for the high-throughput screening.18,19 Several sensitive fluorescent probes have been reported to measure the enzymatic activity of GLU merely in cells, tissue samples, and animals.20-24 Previously, we have developed a fluorescent probe based on hemicyanine for the real-time detection and imaging of endogenous GLU in various hepatoma carcinoma cells, tumor tissues, and tumor-bearing mouse models, for cancer diagnosis and therapy.22 However, intestinal bacteria as the important resource of GLU, which could induce some diseases, have been paid little attention. Therefore, the development of an off-on NIR fluorescent probe for rapid, selective, and sensitive detection of GLU in bacteria, and intestine is urgently demanded to further reveal the biological function and distribution of intestinal GLU, and to rapidly screen its effective inhibitor for treatments of drug-inducing enteropathy. Although the previously reported hemicyanine derivate (HC-glu) was used as the NIR fluorescent probe for GLU,22 a NIR fluorescent probe with good biocompatibility such as little cytotoxic sensing product was developed in the present work. Based on the catalytic properties of GLU, DDAO-glu has been designed and synthesized as a NIR probe for realtime detecting bioactivity of GLU in human gastrointestinal microbiota. 1) Using DDAO-glu as the highly sensitive and specific probe, we accurately determined the GLU bioactivities of human feces in clinic. 2) DDAO-glu could be success-

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fully applied for in situ and in vivo visualization of GLU activity, and systemically elucidating its intestinal distribution. 3) Further using this versatile probe, we revealed the great importance of GLU in drug-inducing intestinal damage, and further to develop a natural inhibitor of GLU using our sensitive and high-throughput screening method, to effectively prevent and therapy the serious drug-inducing enteropathy. EXPERIMENTAL SECTION Incubation conditions and analysis method. The incubation reaction was conducted in the system which consisted of 100 mM potassium phosphate buffer (pH = 7.0), and GLU with a total volume of 200 µL. Next, DDAO-glu (10 µM) was added for starting, organic solvent (DMSO) was not more than 1% (v/v). After 30 min incubation at 37 °C, the reaction was terminated by adding 100 µL ice-cold acetonitrile, followed by centrifugation at 20,000 g for 20 min at 4 °C. The supernatant was subjected to Synergy neo Microplate Reader (Bio-Tek) for analysis. The selectivity of DDAO-glu towards GLU. In order to confirm the selectivity for GLU, DDAO-glu was incubated with various enzymes such as α-Glucosidase, Lysozyme, Nacetyl glucosaminidase, Carbonic anhydrase, Proteinase K, CE1b, CE1c, CE2, BSA, β-Galactosidase, β-Glucosidase, and β-Glucuronidase in the standard incubation system at 37 °C for 30 min with the concentration of 10 µg/mL, respectively. Furthermore, the influence of several amino acids and endogenous substance (Glu, Gln, Gly, Ser, GSH, Arg, Lys, Cys, Trp, Glucose, bilirubin, Tyr, myristic acid), and metal ions (Ca2+, Zn2+, Mn2+, Mg2+, Fe3+, Cu2+, K+, Ni+, Ba2+, Sn4+, Na+) were performed in the standard incubation system. To further confirm the selectivity of DDAO-glu, DDAOglu (10 µM) was incubated with GLU in the presence or absence of selective or general inhibitors: Bis-pnitrophenyl phosphate (BNPP, general for CEs), loperamide (LPA, selective for CE2), α-Galatose, and Baicaline (1, 10, 100 µM). All incubations were performed at 37 °C for 30 min. Fluorescence imaging of DDAO-glu in human intestinal microbiota. After the plate streaking culture of twelve bacterial strains (Enterobacter cloacae, Escherichia coli, Escherichia coli ETEC, Escherichia coli EPEC, Escherichia coli EAEC, Enterococcus faecalis, Klebsiella oxytoca, Klebsiella Pneumoniae, Monilia albican, Pseudomonas Aeruginosa, Staphyloccocus aureus Rosenbach, Streptococcus agalactiae) on Luria-Bertani (LB) agar medium for 24 h, the bacterial colonies were used for the fluorescence imaging using in vivo imaging system. After the blank experiments were conducted for the culture plates, DDAO-glu (20 µM) was incubated with the bacterial colonies for 1 h. Then, the fluorescence intensities of these bacterial colonies were measured using with the excitation wavelength of 605 nm and emission wavelength at 660 nm. Similarly, for confocal microscopic bacterial imaging, the fluorescent DDAO-glu (20 µM) was added and incubated for 1 h. After washing LB culture medium with PBS, the bacterial cells were re-suspended in 0.1 M PBS, pH 7.4 and spotted on glass slides and immobilized with coverslips. Bacterial imaging tests were conducted with Leica Confocal Microscope with the excitation wavelength at 633 nm and emission wavelength at 645 - 690 nm, respectively. To study the inhibitory effect of baicalin on GLU, the bacteria were pre-treated with baicalin (200 µM) for 1 h before DDAO-glu (20 µM) was added. In vivo imaging of GLU in mouse. Mice (male, 20 g) were housed under specific pathogen-free conditions and received

food and water ad libitum. Depletion of gut microbiota was achieved by administering mice broad-spectrum antibiotics. The antibiotic regimen mainly included ampicillin, metronidazole, neomycin and vancomycin. Ampicillin was added into the tap drinking water at a final concentration of 1 g/L. Metronidazole (100 mg/kg), neomycin (100 mg/kg) and vancomycin (50 mg/kg) were oral administrated by gavage twice a day. Metronidazole, neomycin and vancomycin were oral administrated by gavage twice a day at concentration of 1 and 0.5 g/L, respectively. Mice in control group received an equivalent volume of physiological saline every day. After a week, mice were anesthetized and then administration with DDAO-glu for imaging in vivo.

Scheme 1. Structure of DDAO-glu and proposed sensing mechanism for GLU enzymatic activation of DDAO-glu.

RESULTS AND DISCUSSION Spectral properties of DDAO-glu. As well-known about NIR-active fluorophores, DDAO derivatives have favorable ICT (intramolecular charge transfer) properties for probe design, with a phenolic group for turning electrondonating capability, of which the fluorescence emission wavelength could be distinctly changed by removing the substituted group. Herein, we designed a NIR fluorescent probe DDAO-glu by grafting a GLU activatable unit onto the OH moiety, which was a conjugate of β-glucuronic acid and DDAO as a chromogenic and water- soluble substrate of intestinal GLU (Scheme 1, Figure S1-S7). It is also water-soluble to reach the maximum concentration of 800 µM in PBS containing 1% methanol, due to the introduction of glucuronic acid as the polar moiety. After adding GLU in the incubation system, the maximum absorption peak is red-shifted 185 nm, with a distinct color variation from light yellow to blue (Figure S8). And a remarkable fluorescence enhancement (more than 1000-folds) at 660 nm indicated that DDAO-glu could be used as a “nakeeye” probe for detecting GLU activity. Furthermore, HPLC analysis and electrospray ionization (ESI) mass spectra of the reaction product further verified the probe hydrolysis by GLU to yield DDAO (Figure S9, S10). Meantime, the cytotoxicities of DDAO and HC as a sensing product previously reported a NIR fluorescent probe for GLU, were compared. And the results indicated that HC exhibited the stronger cytotoxic effect than DDAO, which would limit the application of HC-glu at high concentration. (Figure S11) In addition, the fluorescence intensity was found linearly increased when GLU was progressively added from 0 to 10 µg/mL (R2 = 0.989, Figure S12). Such high sensitivity could be attributed to its NIR emission with little interference by matrix auto-fluorescence. We also evaluated the pH dependence of the emission profiles of DDAO and biotransformation rate of GLU (Figure S13). The excellent fluorescence response for DDAO were confirmed during pH range from 6.0 − 12.0, and the high GLU activity was remained at pH range from 4.0 − 7.0. Additionally, the detection of GLU by DDAO-glu could

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ACS Sensors not be influenced by some common metallic ions or endogenous substances including amino acids and bilirubin in biological systems (Figure S14). And DDAO-glu was stable in acid solution including gastric acid. All of these results fully exhibited that DDAO-glu could be served as an off-on NIR fluorescent probe for rapidly detecting GLU activity in aqueous solution under physiological conditions. Selectivity of DDAO-glu. To explore the applicability of DDAO-glu for detecting endogenous GLU in complex biological samples, the selectivity of this probe further needed to be evaluated systemically. The remarkable fluorescence enhancements of DDAO-glu (10 µM) after incubation of GLU (10 µg/mL) were detected (Figure S15A), while no significant fluorescence variation was observed after adding other hydrolases such as α-Glucosidase (α-GLC), β-Glucosidase (βGLC), β-Galactosidase (β-GLA), Lysozyme (LS), N-acetyl glucosaminidase (NAG), Carbonic anhydrase I (CAS), Proteinase K (PAK), Carboxylesterases (CE1b, CE1c, CE2) and Bovine serum albumin (BSA), respectively. To further investigate the selectivity of DDAO-glu towards GLU, chemical inhibition assays in GLU incubation system were also carried out, using the different types of selective inhibitors against hydrolases, including Bis-pnitrophenyl phosphate (BNPP), loperamide (LPA), α-galactose, and baicalin (Figure S15B). Among them, baicalin regarded as a selective inhibitor of GLU, could significantly inhibit the deglycosylation of DDAO-glu mediated by GLU from E. coli with IC50 value of 16.59 ± 0.98 µM (Figure S16), while the selective inhibitors of other hydrolysis enzymes did not display the positive inhibitory effects towards GLU activity. These results fully demonstrated that DDAO-glu could be selectively catalyzed by GLU to produce DDAO, and it could be used as an efficient molecular tool for detecting the real-time bioactivity of GLU in complex biological system. In the process of biological application, the measurement of enzymatic parameters was crucial for the accurate quantification of the activity-based probes. Herein, kinetic parameters of different GLUs from E. coli, E. coli K 12, E. coli Type IX-A, and E. coli Type VII-A, were determined using DDAO-glu (Table S1). As shown in Figure S17, all the GLUs from E. coli Type IX-A, E. coli Type VII-A, E. coli K 12, and E. coli, exhibited biphasic kinetics behaviors. The activity of GLU from E.coli-K12 indicated the much higher CLint and Vmax than that of other GLUs. And GLU activity from different bacterial resources exhibited the significantly variant, which implying that GLU activity should exist in the remarkable individual differences inducing intestinal functional variation. Meantime, our results indicated that DDAO-glu exhibited excellent capability to determine the GLU activity in intestinal microbiota. Quantification of GLU in human feces. GLU is mainly responsible for the regeneration and consequent toxicity of NSAIDs in human intestine. Therefore, it is very necessary to real-time detecting the GLU activities in human feces to predict the potential risk of intestinal toxicity induced by diclofenac and other NSAIDs in human. In present work, DDAOglu was used to quantitatively determine GLU activity in human feces from different individuals and further compared with commercial kit. The reaction rates of GLU ranged from 0.448 to 392 pmol/mg protein/min in feces samples from 24 volunteers by DDAO-glu (Figure S15C), which indicating a significant individual differences of GLU in human. Meantime, GLU activity derived from DDAO-glu assays was well consistent with the results of commercial kits, with a correla-

tion coefficient (R) of 0.9662 (Figure S15D), further suggesting that DDAO-glu could be successfully applied for real-time determination of human fecal GLU in clinic. Therefore, our bioassay is great significance for accurately determining the human intestinal GLU to further predict and prevent the potential risk of NSAIDs-induced enteropathy in clinic. Fluorescence biofilm and morphology imaging of DDAO-glu in human intestinal microbiota. Encouraged by excellent detection capability mentioned above, we continue to evaluate application of DDAO-glu in real-time visual imaging of intestinal bacteria. Totally twelve common microbes from human intestinal tract were selected to detect the bacterial GLU using DDAO-glu. (E. coli, E. coli ETEC, E. coli EPEC, S. agalactiae, E. coli EAEC; E. faecalis; S. aureus Rosenbach; K. oxytoca; P. aeruginosa; K. Pneumoniae; E. cloacae and M. albican) Therefore, we performed the visual and selective detection of endogenous GLU in plate culture medium under the appropriate cultivation conditions. After incubation of DDAO-glu (20 µM) for 1h, all bacterial colonies on plate medium were tightly localized, as shown by red-green fluorescence signal, and were readily distinguished from the blank colonies, which showed no fluorescence signals under exciting wavelength of 630 nm. This evidence fully indicated our probe possessed the good cell permeability and reaction activity with bacterial GLU. Comparing with control bacteria, the positive bacteria such as E. coli, E. coli ETEC, E. coli EPEC, S. agalactiae, E. coli EAEC, E. faecalis, K. oxytoca, P. aeruginosa, and M. albican, exhibited the remarkable fluorescence signals at 660 nm, especially E. coli, E. coli ETEC, E. coli EPEC and S. agalactiae (Figure 1A, S18), which suggesting these strains expressed high level of endogenous GLU. Baicalin as the inhibitor of GLU was applied to pretreat these bacteria before the incubation of DDAO-glu. It was obvious that little fluorescence emission was observed for these bacterial colonies (Figure 1A), which also indicated the special fluorescence response of DDAO-glu depending on the endogenous GLU.

Figure 1. The fluorescence images of intestinal bacteria in plate medium (A, λex 605 nm, λem 660 nm) and the confocal fluorescence images of intestinal bacteria (B, λex 633 nm, λem 645 − 690 nm, Scale bar 25 µm) staining with DDAO-glu (20 µM) for 1h at 37 °C to determine intracellular GLU activity. Baicalin (200 µM) as the inhibitor of GLU was pretreated for bacteria before DDAOglu incubation. (E. coli; E. coli ETEC; E. coli EPEC; S. agalactiae).

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Our findings fully suggested that DDAO-glu as a sensitive fluorescent probe to rapidly detect the endogenous GLU in the biofilm of human intestinal bacteria, and to accurately distinguish the expressed levels of GLU in the various strains. On the basis of plate imaging, E. coli, E. coli ETEC, E. coli EPEC, and S. agalactiae were selected for further imaging endogenous GLU using DDAO-glu in bacterial cells. After incubation with DDAO-glu (20 µM) at 37 °C for 1 h, these microorganisms were subjected to image capture excited at 633 nm. And these intestinal bacteria including E. coli, E. coli ETEC, E. coli EPEC and S. agalactiae, all exhibited a specific fluorescent signal in red channel (645 − 690 nm) of laser confocal microscope, which fully indicated that DDAO-glu had the excellent cell permeability and capability of intracellular GLU (Figure 1B). Additionally, after pretreating baicalin as a chemical inhibitor of GLU, all bacteria exhibited a sharply suppressed fluorescence as compared to the control group, under the same imaging conditions (Figure 1B). These evidences strongly suggested that GLU are mainly responsible for this fluorescence variation in bacterial cells, and DDAO-glu exhibited preferred capability for sensitively detecting the endogenous GLU in the intestinal bacteria, with satisfactory cell permeability and optical stability, low toxicity and autofluorescence. High-throughput screening of GLU inhibitor by using DDAO-glu from herbal medicines. As mentioned above, bacterial GLU could be a key factor in the initiation of NSAIDs enteropathy. Hence, highly selective inhibition of bacterial GLU maybe protect against intestinal injury induced by NSAIDs in clinic. Using the fluorescent probe DDAO-glu, the activity assay of GLU has been successfully established, which was also could be applied for high throughput screening of novel inhibitors of GLU. It is well known that natural products were the important resources for discovering bioactive substances, which provide a large amount of new medicinal candidates helping for human health. In present work, 50 common medicinal herbs were chosen for the inhibitory evaluation towards GLU by using the fluorescence assay of DDAO-glu. Totally, the ethanol extracts of 50 herbal medicines were obtained and added into the GLU enzymatic system with the final concentration of 10 µg/mL. The fluorescence intensities of these samples were measured using in vivo imaging system and Microplate reader, respectively. Wells C5, D7, and E2 corresponding to Rheum palmatum L., Smilax china L. and Radix Saposhnikoviae, displayed weak fluorescence emission at 665 – 735 nm, which indicated their strong inhibitory effects towards GLU activity, and the corresponding fluorescence intensities were recorded by Microplate reader to calculate the remaining activity of GLU as 17.05%, 27.98% and 64.96% in wells C5, D7, and E2, respectively (Figure S22). And, it was deduced that there were some potential inhibitors of GLU in the ethanol extract of Rheum palmatum L. Thus, DDAO-glu has been successfully applied for the GLU inhibitor screening with high anti-interference capability in the complex system. Identification of the inhibitory chemical constituents of R. palmatum towards GLU activity. On the basis of primary screening results, the GLU inhibitors were isolated from R. palmatum under the guidance of fluorescent bioassay for GLU activity. The HPLC fractions (1−23) of ethanol extract of R. palmatum L. were obtained using preparative HPLC (Figure 2A). Based on the fluorescent activity assay, the inhibitory effects of fractions 1−23 towards GLU activity were rapidly

measured in 96-well plates, by visualization imaging system and Microplate reader, respectively. As shown in Figure 2B, the fluorescence intensities of each well indicated that the inhibitory effects of these fractions could be well visualized in convenient model. Our results strongly indicated that fractions 4, 6 and 7 − 13 displayed significant inhibitory effects on GLU at the concentration of 10 µg/mL. The fluorescence intensities of 96-well plates gave the similar values corresponding to imaging results (Figure 2C). On the other hand, the remaining GLU activities of each well were also determined using our fluorescent method. It was obvious that the remaining enzyme activity and fluorescence intensities displayed the excellent correlation, which confirmed the dependability of our fluorescent probe for GLU activity assay. On the basis of these data mentioned above, we established the corresponding relationship between HPLC chromatogram and inhibitory activity of GLU for guiding the real-time isolation of bioactive compounds. Furthermore, using chromatographic techniques, totally three compounds DH-1, DH-2 and DH-3 were isolated and purified from the extract of R. palmatum L., which corresponded to fractions 6, 11 and 13, respectively. On the basis of spectroscopic data, three isolated compounds were identified to be (−)-epicatechin3-gallate (DH-1),25 6-hydroxymusizin-8β-D-glucopyranoside (DH-2),26 and 1,2-di-O-galloyl-6cinnamoyl-β-D-glucose (DH-3),27 respectively, reported as the inhibitor of GLU for the first time. The inhibitory effects of three compounds (DH 1-3) and IC50 values toward GLU were calculated (Figure S23, 24). Furthermore, the inhibition kinetics was performed for analyzing the metabolism of DDAO-glu by (−)-epicatechin 3-gallate (ECG, DH-1). The dosedependent inhibition behavior of ECG towards metabolism of DDAO-glu was observed with the IC50 value of 2.78 ± 0.25 µM. Furthermore, the mixed inhibition model of ECG was observed for the metabolism of DDAO-glu in GLU system. The inhibition kinetic parameter (Ki) of ECG was calculated to be 2.72 µM. Computer simulation study of DH-1 with bacterial βGlucuronidase (GLU). Furthermore, we wanted to dissect the binding mechanism of DH-1 complexed with GLU (PDB code: 3LPF) using molecular docking coupled with molecular dynamics (MD) simulation and MM/PBSA free energy calculation. Our MD results revealed that DH-1 is bound deeply in the active site of GLU which is composed by two neighboring monomers (monomer-1 and monomer 3 of the GLU homotetramer structure (Figure S25). The detailed analysis about the interaction between DH-1 and GLU was submitted in the supporting information. In vivo imaging and intestinal distribution of GLU in mouse. The prominent features of DDAO-glu encouraged us to investigate the in vivo visualization for detecting endogenous GLU in living animals. In the present experiment, male mice were selected as the animal model and intraduodenally administered with DDAO-glu (100 µM, 200 µL). After administration of 30 min, the animals were observed by an in vivo fluorescence imaging device. Remarkable fluorescence images were acquired from small intestine of mice (Figure 3A). In sharp contrast, the mouse injected with PBS did not exhibit any red fluorescence under the same imaging conditions. Then, the mice were pretreated for 3 days by ciprofloxacin as a fluoroquinolone antimicrobial agent that has a broad spectrum of activity, with the aims to destroy enterobacteria. And then the mice eliminated the intestinal GLU bioactivity, were used to image the endogenous GLU bioactivity. Almost no

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Figure 2. The inhibitory effects of the pre-HPLC fractions from the extracts of R. palmatum L. towards GLU. (A) The total HPLC chromatogram and prepared fraction profiles of R. palmatum L. (B) Fluorescence images response to the inhibition effects of fractions 1 − 23 against intestinal GLU after incubated with DDAO-glu in 96-well plates for 30 min (λex 630 nm, λem 665 − 735 nm). (C) The corresponding inhibitory effects of fractions 1−23 on intestinal GLU measured using the present fluorescent assay by microplate reader (blue, λex 600 nm, λem 660 nm) and the remaining GLU activity (red) based on the fluorescence images, respectively.

Figure 3. In vivo real time image of GLU in intestinal tracts after administration of DDAO-glu for 30 min. The fluorescence variation of GLU from duodenum injection of DDAO-glu (100 µM, 200 µL) in the normal mouse (A), destroying enterobacteria mouse (B) and normal mouse combined with ECG (100 µM) (C). The bright (D) and fluorescence fields (E) of intestinal distribution of GLU after incubation with DDAO-glu (100 µM) for 30 min at 37 oC. The intestinal tissues of mouse including duodenum (1), jejunum (2-4), ileum (5 and 6) and colon (7), respectively. (λex 630 nm, λem 665 − 735 nm).

fluorescence signals were monitored in the intestinal tract after the mouse pretreated with ciprofloxacin (Figure 3B), all of which strongly suggesting that GLU in enterobacteria should be mainly responsible for metabolism of DDAO-glu. In addition, ECG as a potential and selective inhibitor of GLU mentioned above, was used to effectively inhibit the intestinal GLU bio activity. And in the groups of the mice treated together with ECG and DDAO-glu, the significant decrease of red fluorescence intensity was observed compared with that of experimental group treated by DDAO-glu, and vehicle controls of ECG did not exhibit any apparent pathological lesions and fluorescent background (Figure 3C). Furthermore, in order to confirm the real distribution of GLU in intestinal tract, the mice were anesthetized by chloral hydrate and the abdomens were opened to obtain the whole intestinal tract. According to physiological structure, intestinal tract of mice were separated into several sections including duodenum (1), jejunum (2-4), ileum (5 and 6) and colon (7) and then stained with DDAO-glu (100 µM) for 30min at 37 o C. Our results fully exhibited that the significant fluorescence signals were observed in ileum (5-6) and final jejunum (4) without the autofluorescence background, while no fluorescence signals of DDAO in duodenum (1), upper jejunum (2) and colon (7) was observed (Figure 3D, E). In addition, the middle section of jejunum (3) exhibited the weak fluorescent

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intensity. These evidences strongly suggested that main distribution of GLU should be in the ileum (5 - 6) and final jejunum (4), which is well consisted with the NSAIDs- induced enteropathy location in clinic.28,29 It is first time to accurately reveal distribution of GLU in intestinal tract. All these findings indicated that GLU were abundant in final section of small intestine including ileum and final jejunum, and our probe was good capable of real-time detecting endogenous GLU in living animals to reveal the biological function of intestinal GLU. ECG blocked enterohepatic circulation and alleviated diclofenac-induced intestinal injury in rats. NSAIDs, despite being generally safe drugs, have widespread gastrointestinal adverse drug reactions including gastroduodenal injury, mucosal erosions and ulceration in the small intestine.30-32 Diclofenac, one of the most widely prescribed nonsteroidal NSAIDs, leads to an increased risk of serious gastrointestinal adverse events.33 To determine whether the potent inhibitor screened by our probe, could disturb enterohepatic circulation in vivo though GLU inhibition, diclofenac was used as a model agent. As shown in Table S2 and Figure 4A, coadministration of ECG could decrease the Cmax2 of diclofenac (0.57 ± 0.061 mg/L in co-administered ECG vs 1.87 ± 0.31 mg/L in diclofenac alone, P