Loss of thioredoxin reductase function in a mouse stroke model disclosed by a two-photon fluorescent probe

Article abstract of DOI:10.1039/d0cc05900e

Thioredoxin reductase (TrxR) enzymes are critical in regulating redox homeostasis in cells. We report the first two-photon fluorescent probe of mammalian TrxR (TP-TRFS). TP-TRFS retains high specificity in recognizing TrxR. More importantly, the two-photon absorbing character of TP-TRFS enables it to be used in vivo. With the aid of TP-TRFS, a remarkable decline of the TrxR function was observed in the brain of a mouse model of stroke for the first time, providing a mechanistic link of TrxR dysfunction with stroke. This journal is

Full text of DOI:10.1039/d0cc05900e

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Loss of thioredoxin reductase function in a mouse
stroke model disclosed by a two-photon
fluorescent probe†
Cite this:DOI: 10.1039/d0cc05900e
Received 1st September 2020,
Accepted 16th October 2020
a
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ac
a
Jintao Zhao, ‡ Yuan Qu,‡ Hao Gao, Miao Zhong, Xinming Li, Fang Zhang,
ad
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Yaxiong Chen, Lu Gan, Guodong Hu, Hong Zhang,
Shengxiang Zhang*
DOI: 10.1039/d0cc05900e
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and Jianguo Fang
*
rsc.li/chemcomm
Thioredoxin reductase (TrxR) enzymes are critical in regulating excessive production of reactive oxygen species (ROS) is a basic
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0,11
redox homeostasis in cells. We report the first two-photon fluor- mechanism for stroke.
The thioredoxin system, as one of
escent probe of mammalian TrxR (TP-TRFS). TP-TRFS retains high the most important systems for regulating the redox balance in
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specificity in recognizing TrxR. More importantly, the two-photon organisms, is closely related to stroke.
Current findings
absorbing character of TP-TRFS enables it to be used in vivo. With indicate that Trx may represent a potential novel therapeutic
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the aid of TP-TRFS, a remarkable decline of the TrxR function was target for ischemic stroke.
observed in the brain of a mouse model of stroke for the first time, only known physiological reducing enzyme of oxidized Trx,
providing a mechanistic link of TrxR dysfunction with stroke. there are few reports on the relationship between TrxR and
However, although TrxR is the
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stroke. To elucidate the relationship between TrxR and
Thioredoxin reductase (TrxR), a major antioxidant enzyme, is stroke, a reliable tool is needed to accurately track the cerebral
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an important selenoprotein and widely found in mammals.
TrxR, thioredoxin (Trx), and NADPH together form the thior-
TrxR level in the pathology of stroke.
Recently, small molecule fluorescent probes have gradually
edoxin system, which plays an indispensable role in regulating become a powerful tool for studying biological specimens in
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,4
the redox balance.
TrxR reduces the oxidized Trx to the biomedical sciences due to their super sensitivity, high speci-
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reduced Trx by taking electrons from NADPH, and then the ficity, real-time detection and non-invasiveness.
In our
reduced Trx interacts with a variety of downstream proteins previous work, we were inspired by the reduction of lipoic acid
through a general thiol–disulfide exchange reaction to perform by TrxR, and prepared TRFS series small molecule fluorescent
a series of physiological functions. TrxR is currently the only probes of TrxR, including TRFS-green, TRFS-red, Mito-TRFS
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known physiological reducing enzyme for Trx, and loss of its and Fast-TRFS.
activity usually results in oxidative stress.
TRFS series probes not only have extremely
high sensitivity, but also have high selectivity toward TrxR.
Cerebral stroke is one of the most fatal diseases in the world Other TrxR probes have also been reported by different
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,6
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with ischemic or hemorrhagic injury as the main clinical groups.
Although encouraging progress has been made in
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manifestations. In the past few years, there has been a great designing and preparing TrxR fluorescent probes, no high-
deal of evidence that oxidative stress associated with the efficiency fluorescent probe has been reported for direct
fluorescence imaging of TrxR activity in tissues or living organ-
a
b
c
isms, which has limited the research on this key enzyme. To
address this issue, we reported the first two-photon fluorescent
probe of TrxR, TP-TRFS. Compared with traditional one-photon
fluorescence imaging, two-photon fluorescence imaging has
the characteristics of deeper tissue penetration, lower back-
ground fluorescence and higher spatio-temporal resolution,
which make two-photon fluorescence imaging more suitable
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China.
E-mail: fangjg@lzu.edu.cn
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental
Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.
E-mail: sxzhang@lzu.edu.cn
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
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for tissue and living organism imaging.
Like the previous
d
Department of Heavy Ion Radiation Medicine, Institute of Modern Physics,
Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, China
Electronic supplementary information (ESI) available. See DOI: 10.1039/
TRFS series probes, TP-TRFS has high specificity toward TrxR.
More importantly, the two-photon absorbing properties of the
probe provide an additional advantage to image the TrxR
function in zebrafish and mice brain. We further demonstrated
†
d0cc05900e
Contributed equally to this work.
‡
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a severe loss of TrxR function in the brain of ischemic mice,
which suggests that targeting the regulation of TrxR may have
therapeutic potential in interference with stroke.
In our previous studies, we demonstrated that a 1,2-dithiolane
scaffold shows high selectivity toward TrxR with little interference
28,31
from other biological species.
fluorescent probe TP-TRFS was designed via integration of the
,2-dithiolane moiety and the 2-acetyl-6-aminonaphthalene (AN)
Therefore, the two-photon
1
fluorophore via a carbamate linker. In this design, AN was chosen
as an optimal fluorophore because of its excellent two-photon
absorption properties and good quenching properties of the
recognition site, and was synthesized according to the published
39,42
literature.
1,2-Dithiolane-4-ol prepared by our previously pub-
lished procedures was designed as the specific recognition group
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1,43
for TrxR.
The synthetic route of TP-TRFS is shown in Scheme S1
1
(
ESI†) and the probe was fully characterized by H NMR,
C NMR, and HRMS (see the ESI†).
1
3
Before testing the response of the probe toward TrxR, we used
tris(2-carboxyethyl)phosphine (TCEP), which is a strong reducing
agent and can effectively reduce disulfide bonds, to reflect the
initial screening properties of the probe. As shown in Fig. 1A,
TP-TRFS (10 mM) exhibited weak fluorescence (f = 0.57%,
absolute quantum yield), and after TP-TRFS (10 mM) was incu-
bated with TCEP (1 mM), a dramatic fluorescence increment at
Fig. 1 Selective activation of TP-TRFS by TrxR. (A) Response of TP-TRFS
490 nm was observed (f = 22.31%, absolute quantum yield) and
to TCEP (1 mM). (B) Time-dependent emission spectra of TP-TRFS toward
the fluorescence intensity of the test system increased by TrxR. TP-TRFS was incubated with TrxR/NADPH (50 nM/200 mM). (C) Time
course of the fold of fluorescence increment (490 nm) of TP-TRFS with
B40-fold within 3 h. To verify the mechanism shown in
Scheme 1, the detailed process of the reaction between TP-
TRFS (20 mM) and TCEP (1 mM) was monitored by HPLC using
a PDA detector. After the reaction with TCEP for 3 h, the peak at
different concentrations of TrxR in the presence of NADPH (200 mM).
(
(
(
D) Response of TP-TRFS to different biological species. GSH (1 mM), Cys
À1
1 mM), Sec (10 mM), esterase (0.25 U mL ), NADPH (200 mM), GR
À1
0.5 U mL ) with NADPH (200 mM), reduced Trx (10 mM) and TrxR
1
0.34 min belonging to TP-TRFS almost disappeared, and a new (50 nM) with NADPH (200 mM). Sec (10 mM) was generated in situ by
mixing Cys (1 mM) and selenocysteine (5 mM). (E) Inhibition of Hep G2 cell
peak (AN) at 4.28 min appeared (Fig. S1 in the ESI†). Moreover,
the fluorescence spectra of the test system are consistent with
the fluorescence spectra of compound AN (Fig. S7, ESI†). Subse-
quently, a response of TP-TRFS (10 mM) with TrxR (50 nM) was
lysate-mediated TP-TRFS reduction by TrxR inhibitor DNCB (20 mM) or AF
2 mM). The NADPH-prereduced Hep G2 cell lysate (60 mL) was incubated
(
with DNCB (20 mM) or AF (2 mM) at 37 1C for 30 min, and further incubation
with TP-TRFS and NADPH (200 mM) for additional 2 h. (F) Response of
observed, and the fluorescence intensity increased by B15-fold TP-TRFS to different amounts of Hep G2 lysate. All the reactions contain
additional NADPH (200 mM). All fluorescence spectra were obtained under
within 3 h (Fig. 1B). The large increase of the fluorescence
intensity at the first scan is due to the intrinsic background
fluorescence of NADPH under our experimental conditions
excitation at 370 nm in TE buffer. Total protein concentration of the lysate
À1
is 4.4 mg mL determined by the Bradford procedure using BSA as a
standard. The TP-TRFS concentration is 10 mM in all experiments.
(excited at 370 nm). Next, as shown in Fig. 1C, a dose-
dependent fluorescence increment at 490 nm was observed after
adding different concentrations of TrxR (25 nM, 50 nM and
100 nM) to the test system but not in its absence. To investigate
the specificity of TP-TRFS toward TrxR, we investigated the
response of TP-TRFS (10 mM) with other biomolecules, such as
glutathione (GSH, 1 mM), L-cysteine (cys, 1 mM), selenocysteine
À1
(
Sec, 10 mM), NADPH (200 mM), esterase (0.25 U mL ), reduced Scheme 1 Proposed recognition mechanism of TP-TRFS.
À1
Trx (10 mM) and glutathione reductase (GR, 0.5 U mL with
2
00 mM NADPH). The reduced Trx was prepared according to our
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4
published procedure. All the tested species could hardly react the presence of the TrxR inhibitor auranofin (AF) or 2,4-dinitro-1-
with TP-TRFS, indicating that TP-TRFS has high selectivity chlorobenzene (DNCB), the fluorescence increment was remark-
toward TrxR. To further investigate the high selectivity of TP- ably inhibited. Fig. 1F shows that the fluorescence of the test
TRFS toward TrxR, fluorescence signals were determined after system increased, which was dependent on the volumes of cell
TP-TRFS was incubated with Hep G2 cell lysates. As shown in lysates. Taken together, these results demonstrated that TP-TRFS
Fig. 1E, the fluorescence intensity gradually increased when could show the ability to recognize TrxR with high selectivity.
TP-TRFS was incubated with Hep G2 cell lysates. However, in More solid evidence of the selective reduction of the probe by
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Fig. 3 Two-photon fluorescence imaging of the TrxR activity in zebra-
fishes. (A) Bright field image of the whole fish. (B) Fluorescent image of the
whole fish. (C) Separate fluorescent image of the whole fish at different
z-axis depths. The fishes were excited at 750 nm. Imaging depth was
2
50 mm. Scale bar: 500 mm.
Fig. 2 Two-photon fluorescence imaging of the TrxR activity in living
cells. (A) HeLa cells treated with TP-TRFS (10 mM) for 4 h. (B) HeLa cells
pretreated with AF (2 mM) for 1.5 h followed by treatment with TP-TRFS
advantages of two-photon fluorescence imaging. As shown in
Fig. 3A and B, zebrafishes incubated with TP-TRFS (2 mM)
(10 mM) for 4 h. (C) HeLa-shNT cells treated with TP-TRFS (10 mM) for 4 h.
(
D) HeLa-shTrxR1 cells treated with TP-TRFS (10 mM) for 4 h. (E) Efficiency showed a strong fluorescence signal. The fluorescence intensity
of TrxR knockdown in HeLa cells determined by western blotting. The varies obviously in different regions, which indicated that the
fluorescence intensity in individual cells of A–B and C–D was quantified by
level of TrxR in each position is different. We also performed
z-scan on the zebrafishes, from which uneven staining at
different depths was observed (Fig. 3C). To the best of our
knowledge, this is the first report showing the specific distribu-
ImageJ and shown in (F). *p o 0.05, **p o 0.01. Cells were excited at
7
50 nm. Scale bar: 10 mm.
TrxR is shown in Fig. 2C and D, where a significant decrease of tion of TrxR in zebrafishes using two-photon fluorescence
the fluorescence intensity in the TrxR knockdown cells (HeLa- imaging.
shTrxR1) was observed (vide infra).
Inspired by the successful application of TP-TRFS in zebra-
With reliable results in hand, we next imaged TrxR in living fish imaging, we further applied it to image TrxR in the brain of
cells with TP-TRFS. Before two-photon fluorescence imaging, mice to evaluate its practicality. What is more meaningful is
we first evaluated the cytotoxicity of TP-TRFS to HeLa cells with that we performed two-photon fluorescence imaging in the
standard MTT assay. When the concentration of TP-TRFS brain of mice with cerebral ischemia reperfusion injury (CIRI)
reaches 40 mM (24 h), the cells had over 90% cell viability, to examine the changes of the TrxR function. Cerebral ische-
indicating that TP-TRFS has low toxicity and good biocompat- mia, an acute brain injury, is a typical stroke model. The CIRI
ibility (Fig. S3 in the ESI†). As shown in Fig. 2A, when HeLa cells mice were established based on the literature and our pub-
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were incubated with TP-TRFS (10 mM) for 4 h, the fluorescence lished protocols.
As shown in Fig. 4B and C, we observed
signal became very strong. However, the fluorescence signal that the brain of both the CIRI mouse and the control mouse
was significantly suppressed when HeLa cells were pretreated showed fluorescence signals, but the fluorescence in the con-
with a selective TrxR inhibitor AF (2 mM) (Fig. 2B). The more trol mouse was significantly brighter than that in the CIRI
convincing result is the significant difference of the fluores- mouse, indicating that the TrxR function decreased after CIRI.
cence intensity between HeLa-shNT cells and HeLa-shTrxR1 Decreased TrxR mRNA in the ischemic cerebral cortex was
1
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cells. As shown in Fig. 2C and D, the fluorescence signal in the reported, while our results demonstrated loss of TrxR func-
HeLa-shNT cells was significantly stronger than that in the tion in CIRI for the first time. These data demonstrated that
HeLa-shTrxR1 cells, which further indicated that TP-TRFS can TP-TRFS has the ability to visualize the TrxR activity in vivo, and
specifically be activated by TrxR. The knockdown efficiency was also provides a mechanistic link between stroke and the loss of
confirmed by western blotting (Fig. 2E). Quantification of the TrxR function, which may open up a new therapeutic window of
fluorescence intensity in individual cells is shown in Fig. 2F. In stroke via the regulation of TrxR function.
addition, we also successfully imaged TrxR in Hep G2 cells with
In conclusion, we constructed the first two-photon fluores-
TP-TRFS. Similarly, in the presence of AF, the fluorescence cent probe, TP-TRFS, for imaging the TrxR activity in living cells
signal of Hep G2 cells also was significantly inhibited (Fig. S2 in and living organisms. TP-TRFS exhibits high selectivity toward
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the ESI†). Combining the results of pharmacological inhibition TrxR as TRFS series probes do.
In addition, the distribu-
and genetic knockdown of the enzyme that inhibited the tion of TrxR in zebrafishes was first imaged by utilizing two-
fluorescence intensity in living cells, we demonstrated that photon fluorescence imaging, demonstrating the practical
TP-TRFS can detect TrxR specifically.
application of TP-TRFS in imaging TrxR in living organisms.
After TP-TRFS was demonstrated to selectively recognize More significantly, we observed a decreased TrxR function in
TrxR in vitro, we further evaluated the imaging of TrxR in living the brain of mice after CIRI for the first time by using two-
organisms in view of the good performance of TP-TRFS and the photon fluorescence imaging, linking the loss of function of
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Fig. 4 Two-photon fluorescence imaging of the TrxR activity in the brain of mice under stroke. (A) Schematic diagram of brain imaging. (B) Fluorescence
signal of the TrxR activity in mice under CIRI. (C) Fluorescence signal of the TrxR activity in control mice not subjected to CIRI. Mice were excited at
7
50 nm. Imaging depth was 50 mm. Scale bar: 100 mm.
TrxR with stroke and suggesting that TrxR is a therapeutic 22 W. Chen, T. Matsunaga, D. L. Neill, C. T. Yang, T. Akaike and
M. Xian, Angew. Chem., Int. Ed., 2019, 58, 16067–16070.
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3 H. D. Li, Q. C. Yao, F. Xu, Y. Q. Li, D. Y. Kim, J. W. Chung, G. Baek,
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The financial support from the National Natural Science
Foundation of China (21778028 and 22077055) is greatly
acknowledged.
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2
2
4 L. Shi, Y. H. Liu, K. Li, A. Sharma, K. K. Yu, M. S. Ji, L. L. Li, Q. Zhou,
H. Zhang, J. S. Kim and X. Q. Yu, Angew. Chem., Int. Ed., 2020, 59,
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5 Y. M. Hu, X. Y. Li, Y. Fang, W. Shi, X. H. Li, W. Chen, M. Xian and
H. M. Ma, Chem. Sci., 2019, 10, 7690–7694.
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7 Z. Luo, T. Lv, K. Zhu, Y. Li, L. Wang, J. J. Gooding, G. Liu and B. Liu,
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