Near-infrared fluorescent probe for evaluating the acetylcholinesterase effect in the aging process and dietary restrictionviafluorescence imaging

Article abstract of DOI:10.1039/d0tb02833a

Dietary restriction (DR), as a natural intervention, not only benefits the neuroendocrine system, but also has an antiaging action. Acetylcholinesterase (AChE) is one of the most important bioactive substances and plays a major part in choline changes in the aging process. Thus, we aim to evaluate the effect of DR on AChE in the brains of aging animals. In this study, we synthesize a NIR fluorescent probe BD-AChE for the real-time andin situmonitoring of AChE level changes in living cells and living mice, notably in brains.In situvisualization with BD-AChE verified a decrease in the AchE level in the brains of mice aging models. Evidently, the prepared probe has the excellent capability of measuring AChE variation in the brains of aging mice with DRviaNIR fluorescence bioimaging, indicating that long-term DR can effectively affect AChE levels in the brain. The attenuation of AChE level in the brain of aging mice after DR could be helpful in infering the advantageous impact of DR on age-related neurodegenerative disease, as a better treatment alternative in the future.

Full text of DOI:10.1039/d0tb02833a

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Near-infrared fluorescent probe for evaluating the
acetylcholinesterase effect in the aging process
and dietary restriction via fluorescence imaging†
b
Cite this: J. Mater. Chem. B, 2021,
9, 2623
Na He,a Lei Yu,
Lingxin Chen
Minghua Xu,c Yan Huang,de Xiaoyan Wang,de
and Shouwei Yue*a
def
*
Dietary restriction (DR), as a natural intervention, not only benefits the neuroendocrine system, but also
has an antiaging action. Acetylcholinesterase (AChE) is one of the most important bioactive substances
and plays a major part in choline changes in the aging process. Thus, we aim to evaluate the effect
of DR on AChE in the brains of aging animals. In this study, we synthesize a NIR fluorescent probe
BD-AChE for the real-time and in situ monitoring of AChE level changes in living cells and living mice,
notably in brains. In situ visualization with BD-AChE verified a decrease in the AchE level in the brains of
mice aging models. Evidently, the prepared probe has the excellent capability of measuring AChE
variation in the brains of aging mice with DR via NIR fluorescence bioimaging, indicating that long-term
DR can effectively affect AChE levels in the brain. The attenuation of AChE level in the brain of aging
mice after DR could be helpful in infering the advantageous impact of DR on age-related
neurodegenerative disease, as a better treatment alternative in the future.
Received 6th December 2020,
Accepted 3rd February 2021
DOI: 10.1039/d0tb02833a
rsc.li/materials-b
in the cholinergic activity in the brains of patients with demen-
tia and healthy older people and that these functional disorders
1. Introduction
The world’s population is aging rapidly. Aging, an inescapable play a vital role in related cognitive problems and memory loss.
biological process, has been defined as the gradual accumula- Therefore, recovery of cholinergic function may relieve the
tion of various harmful changes with time which increases the cognitive loss.3,4 This hypothesis has been strongly supported
chance of illness and death. Many neurotransmitter systems by the fact that acetylcholinesterase (AChE) inhibitors
appear to be compromised during human aging.1 The choli- show positive therapeutic effects on cognition in people with
nergic system is the main neurotransmitter system, which is Alzheimer’s disease (AD). Since the initial proposal, many
responsible for cognitive symptoms in normal aging. The studies on the clinical development of cholinergic agents have
aging-related decline in cholinergic function of the central followed, and though the overall clinical efficacy is limited,
nervous system is considered to be one of the causes for cognitive enhancers for modulating cholinergic function are
short-term memory impairment during the aging process.2 still one of the most widely used drugs which have been
The cholinergic hypothesis of senile memory dysfunction approved for AD.5 Due to its ability to rapidly hydrolyze the
raised by Bartus assumes that there are functional disorders neurotransmitter acetylcholine into choline and acetate, AChE
makes it possible to control the precise time of synaptic
a Rehabilitation Center, Qilu Hospital, Cheelo College of Medicine,
Shandong University, Jinan 250100, China. E-mail: shouweiy@sdu.edu.cn
b Shandong Peninsula Engineering Research Center of Comprehensive Brine
Utilization, Weifang University of Science and Technology, Weifang 262700, China
c Yantai Central Blood Station, Yantai 264003, China
activation. Therefore, the biological activity of AChE is the key
marker for cholinergic metabolism. AChE is also closely related
to brain growth, learning, memory and nerve injury, besides
participating in cholinergic transmission.6
Dietary restriction (DR), as a method of reducing calorie
intake without malnutrition, has been widely believed to affect
multiple physiological events, involving the neuroendocrine
signalling systems.7 It modifies protein turnover, changes the
expression of numerous genes, and reduces the glycosylation of
macromolecules and has been proved to enhance the function
of synaptic plasticity, including learning ability and long-term
potentiation.8,9 In previous studies, the rats that maintained
d CAS Key Laboratory of Coastal Environmental Processes and Ecological
Remediation, Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of
Sciences (CAS), Shandong Key Laboratory of Coastal Environmental Processes,
YICCAS, Yantai 264003, China. E-mail: lxchen@yic.ac.cn
e School of Pharmacy, Binzhou Medical University, Yantai, 264003, China
f Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071,
China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tb02833a
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 2623–2630 | 2623

View Article Online
Paper
Journal of Materials Chemistry B
DR performed better on memory and learning tasks than the 2. Experimental
age-matched rats that were fed ad libitum.10 Relying on the
capability of prompting the effective expression of neurotrophic
2.1. Synthesis of BD-AChE
Cesium carbonate (0.326 g, 1 mM) and compound 4 (0.529 g,
1 mmol) were mixed in dichloromethane. The mixture was
stirred at room temperature under argon for 30 min; then, we
added dimethylcarbamoyl chloride to the compound (400 mL).
Next, the compound was stirred for 3 days and dimethyl
methotrexate (200 L) was added twice a day. By performing
column chromatography on silica with CH2Cl2/CH3OH (10 : 1)
as the eluent, the compound was purified, and we obtained a
yellow product (0.395 g, 59%). 1H NMR (500 MHz, CD3OD-D4) d
(ppm): 8.16–8.14 (m, 1H), 8.06–8.04 (m, 2H), 7.88–7.80 (m, 2H),
7.51–6.92 (m, 13H), 4.85 (s, 12H), 2.97–2.84 (m, 2H). 13C NMR
(125 MHz, CD3OD-D4) d (ppm): 166.1, 163.5, 158.6, 157.7, 157.4,
150.3, 139.5, 138.7, 130.8, 129.5, 129.0, 128.9, 128.8, 128.7,
128.6, 128.3, 128.2, 128.1, 127.9, 127.6, 126.8, 121.7, 115.8,
115.3, 115.1, 37.5. LC–MS (ESI+): m/z C38H32BF2N5O4, calcd
671.2515, [M + H]+ 672.2468.
factors and particular gene encoded proteins which facilitate
synaptic plasticity and neuronal survival, DR has great benefits
for the nervous system.11 Importantly, it has anti-aging action;
the mild nutritional stress stimulated by DR might effectively
induce metabolic response to provide better survivability and
extend lifespan.12
From what has been discussed above, AChE plays a very
significant part in the choline changes in the aging process.
DR, as a mild intervention, not only has beneficial effects on
the neuroendocrine system, but also has antiaging action. We
aim at assessing the underlying effect of DR on AChE in the
brains of aging animals. Thus, an efficient tool for detecting the
AChE level can be more helpful in exploring the connection
between DR and AChE. Until now, there have been several
methods for detecting AChE, such as the colorimetric Ellman
method or the detection of hydrogen peroxide generated by
choline oxidation.13 In addition, most of them detect AChE
according to other biomolecules. Due to the low sensitivity, and
poor indirectness and specificity of these methods, they cannot
be well applied to the analysis of biological samples.14–17 The
bioimaging technology coupled with small molecule fluorescent
probes has the outstanding advantages of high spatio-temporal
resolution, in situ and real-time detection, high sensitivity and
specificity, as well as non-invasive and rapid analysis, and has been
developed into an excellent technology for examining biological
species in biosystems.18–21 Thus, small molecule fluorescent probes
can accurately assess the level of a biological enzyme while
sustaining the activity of the biological enzyme in living
systems.22 Since AChE plays a significant role in the pathological
and physiological processes, a number of researchers have been
committed to designing and synthesizing fluorescent probes for
the detection of AChE in biological systems.23–33 However, in view
of the fact that the excitation and emission wavelengths of these
probes are located in the visible region, bioimaging is frequently
hindered by background fluorescence, which makes them not
suitable for imaging deep tissues. Therefore, it is urgently necessary
to design and synthesize an ideal and appropriate NIR fluorescent
probe for the detection and imaging of AChE in living cells and
animals.
2.2. Animals and diet
In this research, we utilized female Swiss albino (Balb/C) mice
as the research objects. The mice kept in polycarbonate cages
were reared under normal laboratory conditions (25 Æ 2 1C;
12 h light/dark cycle), and fed with normative water and pellet
feed according to the established experimental plan. To deter-
mine the potential relationship between normal activity of
AChE and age, we selected four distinct age groups (1, 6, 12,
and 18 months). The laboratory procedures of animals were
performed in accordance with the institutional regulations for
laboratory animal use and care. All mice experimental proce-
dures were performed in conformity with the institutional
regulations and guidelines for the use and care of laboratory
animals, and the related protocols were approved by the
Institutional Animal Care and Use Committee in Binzhou
Medical University, Yantai, China. Approval Number: No.
BZ2014-102R.
2.3. Fasting regimen
We divided the 18 month-old mice in this study into two groups
according to the experimental protocol. The first group was the
control group. The mice in the first group were fed freely, and
they were provided with a continuous food supply. The mice
in the second group were made to fast for 24 hours. It should
be noted that water was periodically provided to all
experimental mice.
In this study, we generated a desirable probe BD-AChE as
a chemical bioimaging tool to image and detect AChE in
living cells and in vivo. BD-AChE was capable of providing
excellent selectivity and sensitivity to AChE in a biological
environment. The experimental results validated that the
AChE levels decreased in the brain of mice aging models.
2.4. Dietary restriction regimen
We also indicated that the AChE levels in the brains of aging We divided the 18 month-old mice in this study into four
mice significantly decreased after DR. To the best of our distinct groups according to the experimental plan. The first
knowledge, BD-AChE is the first fluorescent probe for the group was the control group. The mice in control group were
imaging of AChE in old DR mice, and provides clear evidence fed freely, and they were provided with a continuous food
on the connection between the AChE level and DR in aging supply. The mice in the DR regimen group were given food
mice. These results help to infer the beneficial effects of DR on alternate days for 1, 2 and 3 months, separately. It should
on age-related neurodegenerative diseases as a better treat- be noted that water was periodically provided to all
ment option in the future.
experimental mice.
This journal is © The Royal Society of Chemistry 2021
2624 | J. Mater. Chem. B, 2021, 9, 2623–2630

View Article Online
Journal of Materials Chemistry B
Paper
Scheme 1 Detection mechanism of BD-AChE towards AChE.
3. Results and discussion
3.1. Design of probe BD-AChE
region, optical bioimaging with an NIR fluorophore exhibits better
penetration of deep tissues and higher sensitivity. In this study, in
order to acquire an excellent probe for detecting AChE, the
particular recognition moiety of the dimethyl carbamate group
was merged into the fluorophore unit. The dimethyl carbamate
group can bind covalently to a serine residue in the active site of
AChE, then cause the carbamylation of serine in the active site of
AChE, resulting in the deactivation of AChE. The carbonyl group
of BD-AChE is bound to the oxygen atom, the emission of
BODIPY was quenched by the dimethyl carbamate group due
The chemical synthetic procedure for BD-AChE is clearly exhibited
in Schemes S1 and S2 (ESI†). The details of chemical synthesis of
various compounds are described in the ESI† of this article. The
reaction mechanism of BD-AChE is displayed in Scheme 1. The
various compounds shown in this article were characterized using
1H NMR, 13C NMR and HRMS. We used a BODIPY dye to serve as
a fluorophore unit for emitting a fluorescence signal, and its
emission wavelength was located in the NIR range. Given that
tissue auto-fluorescence and absorption are very low in the NIR
Fig. 1 Spectral properties and enzymatic properties of BD-AChE. (a) Variations in the fluorescence emission spectra of BD-AChE (10 mM) with a wide
range of concentrations of AChE (0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0 and 20.0 U mLÀ1). (b) The plot of the linear relationship between the
relative fluorescence intensity at 740 nm and AChE concentration (0–20.0 U mLÀ1). (c) Michaelis–Menten plot of the reaction between different
concentrations of BD-AChE (0.05–100 mM) and 20.0 U mLÀ1 AChE. (d) Plot of fluorescence intensity of BD-AChE (10 mM) against the reaction time with
various AChE concentrations (0, 2, and 20 U mLÀ1). (e) Fluorescence responses of BD-AChE (10 mM) towards various enzymes 2–10 (1.0 mg mLÀ1), 11–17
biospecies (0.1 mM) and AChE (20.0 U mLÀ1) after being mixed for 15 min: (1). Blank, (2). butyrylcholine esterase, (3). pepsin, (4). peroxidase,
(5).horseradish, (6). glutathione S-transferases, (7). nitroreductase, (8). monoamine oxidase A, (9). monoamine oxidase B, (10) alkaline phosphatase,
(11). BSA, (12). glucose, (13). cysteine, (14). alanine, (15). glycine, (16). lysine, (17). arginine and (18). AChE. (f) Fluorescence responses of BD-AChE (10 mM),
towards 100 mM of a variety of anions and metal ions (1). Blank, (2). Na+, (3). K+, (4). Zn2+, (5). Mg2+, (6). Ca2+, (7). ClÀ, (8). BrÀ, (9). HSO3À, (10). SO42À, (11). SO23À, (12).
S2O23À, (13). CO23À, (14). H2PO42À, and (15). AChE. lex = 680 nm, lem = 740 nm. The tests were carried out in HEPES (10 mM, pH 7.4, 37 1C).
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 2623–2630
| 2625

View Article Online
Paper
Journal of Materials Chemistry B
to the photoninduced electron-transfer (PET) mechanism; thus, detection sensitivity. In the range of 0–20.0 U mLÀ1, the
BD-AChE displays faint fluorescence. Then, the dimethyl carba- fluorescence response of BD-AChE and AChE was linearly
mate in BD-AChE is recognized by AChE, and the ester bond is concentration dependent (Fig. 1b). The equation gained from
cleaved, resulting in BD-AChE being transformed to free BODIPY the calibration curve was F740 nm = 19.73 [AChE] U mLÀ1 + 19.92
with fluorescence enhancement. The reaction mechanism is (r = 0.9940). The detection limit toward AChE was calculated to
displayed in Scheme 1. Utilizing BD-AChE, we monitored the be 0.21 U mLÀ1 (3s/k), which demonstrated that the probe BD-
change of AChE in the brains of mice aging models in real time. AChE had great sensitivity towards AChE under simulated
Furthermore, in association with BD-AChE, the physiological environments. The ability of our probe as an effective chemical
function of AChE in the brains of aging mice with DR was tool to qualitatively and quantitatively detect AChE has been
investigated.
demonstrated by the above results.
3.2. Spectroscopic properties
3.3. Kinetic investigation of BD-AChE toward AChE
Under analogous physiological conditions (10 mM HEPES With the assistance of the synthesized probe, we assessed the
buffer, pH 7.4, and 0.5% Tween 80), we measured the absorp- response of BD-AChE toward AChE under analogous experi-
tion and fluorescence spectra of BD-AChE (10 mM). As displayed mental conditions. In Fig. 1c, different concentrations of
in Fig. S1 (ESI†), the maximum absorption wavelength of BD- BD-AChE (0.05, 0.10, 1.0, 5.0, 10.0, 20.0, 40.0, 50.0 and 100.0 mM)
AChE was at 693 nm (e693 nm = 3.42 Â 104 MÀ1 cmÀ1). After were added into the AChE solution (20.0 U mLÀ1). With the
adding AChE (20.0 U mLÀ1), the maximum absorption peak increase in BD-AChE concentration, the reaction rate was acceler-
changes to 710 nm (e710 nm = 4.79 Â 104 MÀ1 cmÀ1). We ated, demonstrating that enzyme activity was the factor which
performed the fluorescence titration of BD-AChE in the limited the reaction rate. The fluorescence intensity at 1 min
presence of AChE in a concentration range of 0–20.0 U mLÀ1
.
was chosen as the basis to quantify the initial rate of reaction.
In Fig. 1a, the corresponding fluorescence emission profiles The kinetic parameters, including the Michaelis constant (Km)
increased with peaks centered at 740 nm. The quantum yield of and maximum initial reaction rate (Vmax), for the enzymatic
BD-AChE increased, ranging from 0.03 to 0.27. We detected the cleavage reaction of BD-AChE were computed to be 85 mM and
excitation and emission wavelengths of BD-AChE in the NIR 5.78 mM minÀ1 (Fig. 1c), separately. The results proved that the
range, indicating that BD-AChE had the advantage of reducing probe has a good response towards AChE. The catalytic activity of
background interference, which could greatly accelerate the AChE towards BD-AChE was also evaluated. Higher concentrations
Fig. 2 Confocal microscopy images and flow cytometry assay of PC12 cells for measuring AChE levels in living cells. (a) AChE imaging with BD-AChE in
PC12 cells at different time points: 0, 10, 20, and 30 min via confocal laser-scanning microscope as the control. (b) Prior to being treated as described for
the control, PC12 cells were performed with pretreatment of 10 mM neostigmine for 30 minutes. (c) PC12 cells were treated as described for the control
after AChE transfection. (d) Flow cytometry analysis of (a–c). (e) Mean fluorescence intensity of PC12 cells in (a–c). (f) Western Blot analysis of AChE
changes in PC12 cells. We employed b-actin as the internal control. (g) Quantitative analysis of AChE expression in (f). Red channel: lex = 680 nm,
lem = 720–780 nm. The data are displayed as mean (s.d.). Scale bars: 20 mm.
This journal is © The Royal Society of Chemistry 2021
2626 | J. Mater. Chem. B, 2021, 9, 2623–2630

View Article Online
Journal of Materials Chemistry B
Paper
of AChE induced a faster response and a greater fluorescence BD-AChE had excellent selectivity towards AChE and it is an
increase, which was proved by studying the fluorescence kinetic
curves of BD-AChE with different concentrations of AChE (0, 2.0,
and 20.0 U mLÀ1) for 30 min (Fig. 1d). When the AChE concen-
tration was 20.0 U mLÀ1, the fluorescence intensity could reach the
plateau within 15 min. These results clarified that BD-AChE
responded very quickly to AChE. For further exploring AChE levels
in complex biological environments, the real-time imaging cap-
ability of BD-AChE for AChE is indispensable.
ideal and effective tool for sensitively detecting AChE levels
under sophisticated biological systems.
3.5. Imaging of AChE in living cells
Since BD-AChE displayed excellent selectivity towards AChE, we
next utilized BD-AChE to detect AChE in living neural cells. The
representative PC12 cells were chosen as the testing models. Before
the various cell experiments, we applied MTT assay to assess the
cytotoxicity of BD-AChE in PC12 cells. The experimental results
broadly illustrated that BD-AChE showed less cytotoxicity to PC12
cells under physiological environments (Fig. S2, ESI†).
3.4. Selectivity of BD-AChE towards AChE
Next, we measured the fluorescence response of BD-AChE
toward other biologically relevant species. As illustrated in
Fig. 1e, the probe was incubated with many enzymes (butyr-
ylcholine esterase, pepsin, peroxidase, horseradish, glutathione
S-transferases, nitroreductase, monoamine oxidase A, mono-
amine oxidase B, and alkaline phosphatase), BSA, glucose, and
biospecies (cysteine, alanine, glycine, lysine and arginine). The
experimental results obviously revealed that a significant
increase in fluorescence emission can only be observed in the
presence of AChE, and neither common metal ions (Na+, Ca2+,
Using laser scanning confocal microscopy, we performed
cell imaging experiments with BD-AChE. Prior to fluores-
cence imaging, all tested PC12 cells were incubated with BD-
AChE at 37 1C for 30 min. We then employed flow cytometry
to reveal the variation in fluorescence intensity. The tested
PC12 cells were divided into three groups separately, as
displayed in Fig. 2. The PC12 cells without any treatment as
the control are shown in Fig. 2a. Fig. 2b shows the PC12 cells
that were pretreated with neostigmine, which is a high-
efficiency AChE inhibitor and is used in clinics at present.
The PC12 cells shown in Fig. 2c were transfected with AChE-
RNA to overexpress AChE. Within the detection time of
K+, Mg2+, and Zn2+) nor anions (ClÀ, BrÀ, HSO3À, SO42À, SO23À
S2O32À, CO23À, H2PO42À) triggered conspicuous fluorescence
,
signals (Fig. 1f). In conclusion, these results validated that 30 minutes, the fluorescence of the control group gradually
Fig. 3 Imaging of AChE levels in the brains of mice aging models. (a) Imaging of AChE levels in mice brains according to distinct ages: 1, 6, 12 and 18
months old. The mice in the four groups were pretreated with intracranial injection of BD-AChE for 30 min. (b) Imaging of brain slices of mice aging models.
(c) Western blot analysis of AChE variation of brain tissue in distinct ages. (d) Quantitative analysis of AChE expression in (c). (e) Mean fluorescence intensity of
the images in (a). Fluorescence imaging channel: lex = 680 nm, and lem = 720–780 nm. Data are displayed as mean Æ SD (n = 5).
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 2623–2630
| 2627

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 4 Effects of DR on the AChE levels in the brains of aging mice. (a) Imaging of AChE levels in the mice brains after short-term 24 h fasting. The aging
mice were treated with a continuous supply of food as a control, while the mice in the 24 h fasting group were fasted for 24 hours (24 h-fasted). All the
aging mice were treated with BD-AChE by intracranial injection for 30 min. (b) Imaging of the brain slices of (a). (c) Western Blot analysis of AChE variation
in the brain tissue of (a). (d) Quantitative analysis of AChE expression in (c). (e) Mean fluorescence intensity of the images in (a). (f) Imaging of AChE levels in
the mice brains after long-term DR. Aging mice treated with a continuous supply of food were used as the control group, while the mice under DR were
only fed on alternate days for 1, 2 and 3 months, respectively. (g) Imaging of the brain slices of (f). (h) Western blot analysis of AChE variation of the brain
tissue in (f). (i) Quantitative analysis of AChE expression in (h). (j) Mean fluorescence intensity of the images in (f). (k) The body weight changes of the aging
mice in the DR 3 months group. Fluorescence imaging channel: lex = 680 nm, and lem = 720–780 nm. Data are presented as mean Æ SD (n = 5).
increased. Compared to Fig. 2a, the fluorescence observed in explore the fluctuation of AChE levels in living mice,
Fig. 2b is much weaker, illustrating that the AChE level of PC12 particularly in the brain. Before exploring the potential
cells in Fig. 2b is lower. Due to the higher AChE level in PC12 cells, relationship between the level of AChE and DR in old mice,
a stronger fluorescence is observed in Fig. 2c. The bright field of we first examined the changes in the level of AChE in cell
Fig. 2a–c are displayed in Fig. S3 (ESI†). To further verify the aging models. Based on the difference in age: 1, 6, 12 and
fluorescence variation of the three groups of cells, a flow cytome- 18 months old, we divided the mice models into four
try assay was employed as shown in Fig. 2d. Fig. 2e displays the groups. Before imaging, the mice in the four groups were
mean fluorescence intensity of PC12 cells. These results displayed pretreated by intracranial injection of BD-AChE. The
satisfactory consistency to the fluorescence image results. Besides, fluorescence intensity of the brain became weaker as the
we checked the AChE levels in the three groups of PC12 cells age increased (Fig. 3a), clarifying that the AChE level
using western blotting (Fig. 2f and g). All results confirmed that decreased with age. The mean fluorescence intensity is
BD-AChE was capable of being utilized as an effective and displayed in Fig. 4e. The fluorescence imaging of the brain
promising tool for the rapid detection of AChE in living cells.
slices (Fig. 3b) and western blot (Fig. 3c and d) were
performed for further evaluating the fluctuation in AChE
levels. These results not only demonstrated that BD-AChE
can be potentially utilized to detect the AChE level in vivo,
3.6. Level variation of AChE in the brains of mice aging
models
Having confirmed that the level of AChE in cells could be but also revealed the fact that the AChE level decreased
tracked using BD-AChE, we next employed BD-AChE to with increase in age.
This journal is © The Royal Society of Chemistry 2021
2628 | J. Mater. Chem. B, 2021, 9, 2623–2630

View Article Online
Journal of Materials Chemistry B
Paper
3.7. Effect of DR on the level of AChE
decreased after DR. With the assistance of BD-AChE, in vivo
visualization for the first time displayed the significant impact
of DR on AChE in the brains of aging mice, which helps to provide
a natural alternative to treating a variety of age-related neurode-
generative diseases without the use of exogenous drugs.
On the basis of the theory of hormesis mechanism, iterative
slight stress has a positive impact from the cell responses and
the repair and maintenance processes stimulated by repeated
exposure to stress results in aging-retardant action.34–36 DR is a
cumulative slight stressor which improves the body’s capability
in coping with serious stressors by increasing cellular meta-
bolic adjustments, stress resistance and adaptability.37 Recent
Conflicts of interest
studies focused on the mechanism by which DR provides its There are no conflicts to declare.
benefits. Research studies confirm that DR is an intervention
that protects the body against protein misfolding diseases, and
ER hormesis is used as a major mechanism of DR function.38
Encouraged by the satisfying function of BD-AChE in speci-
fically detecting and imaging endogenous AChE in cells and
in vivo, we next attempted to explore the effect of DR on AChE
levels in the brains of aging mice. Before detecting the effects of
long-term DR on AChE, we first detect how AChE changes after
a short 24 h fasting. We divided the old (18 months) mice in our
studies into two groups. The first group is the ad libitum fed
control group with a continuous supply of food, while the mice
in the second group were fasted for 24 hours (24 h-fasted).
However, we regularly supplied water to all the groups. Before
imaging, all the tested mice were pretreated with intracranial
Acknowledgements
We thank the National Nature Science Foundation of China (No.
81573393, 21804010, 21976209, 22007005), the Science and Tech-
nology Innovation Development Plan of Yantai of China (No.
2020MSGY113), the Research Initiation Fund of Binzhou Medical
University (Grant No. BY2019KYQD39, BY2020KYQD01), Taishan
Scholar Project Special Funding (No. ts20190962), the Shandong
Peninsula Engineering Research Center of Comprehensive Brine
Utilization (2018LS014), and Weifang Science and Technology
Development Project (2019GX076).
injection of BD-AChE. As shown in Fig. 4a, no significant References
fluorescence fluctuation in the brain was observed in the two
1 E. Perry, J. Court, R. Goodchild, M. Griffiths, E. Jaros,
groups. Fig. 4e displays the mean fluorescence intensity of
Fig. 4a. The fluorescence imaging of brain slices was performed
to further confirm the level of AChE, as was western blot
(Fig. 4b–d). These results indicated that a 24 h short-term
fasting had no obvious effect on AChE in the brain of aging
mice. Then, we investigated whether the level of AChE changed
after long-term DR. The first group was the control. In the other
group, aging mice under the DR regimen were only fed on
alternate days for 1, 2 and 3 months, respectively. In Fig. 4f, the
fluorescence intensity in the DR groups was much weaker than
that in the control group, and the fluorescence degrees in the
DR groups was as follows: 1 months 42 months 43 months.
We also acquired the fluorescence imaging of the brain slices,
as well as western blot data (Fig. 4g–i). In the data we obtained,
we noticed that the body weight of aging mice decreased during
long-term DR (3 months) (Fig. 4k). These results revealed the
intimate connection between DR and AChE, and the level of
AChE decreased after long-term DR. Our research displayed
that DR had a more pronounced and long-lasting effect on
AChE compared to 24 h fasting.
M. Johnson, S. Lloyd, M. Piggott, D. Spurden, C. Ballard,
I. McKeith and R. Perry, J. Neural Transm., 1998, 105,
915–933.
2 Y. G. Prall, K. K. Gambhir and F. R. Ampy, Life Sci., 1998, 63,
177–184.
3 J. A. Dumas and P. A. Newhouse, Pharmacol., Biochem.
Behav., 2011, 99, 254–261.
4 R. T. Bartus, R. R. Dean, B. Beer and A. S. Lippa, Science,
1982, 217, 408–414.
5 P. Anand and B. Singh, Arch. Pharmacal Res., 2013, 36,
375–399.
6 A. J. Franjesevic, S. B. Sillart, J. M. Beck, S. Vyas, C. S. Callam
and C. M. Hadad, Chemistry, 2019, 25, 5337–5371.
7 E. J. Masoro, Interdiscip. Top. Gerontol., 2007, 35, 1–17.
8 S. Ribaric, Oxid. Med. Cell. Longevity, 2012, 2012, 741468.
9 J. R. Speakman and S. E. Mitchell, Mol. Aspects Med., 2011,
32, 159–221.
10 N. Pitsikas and S. Algeri, Neurobiol. Aging, 1992, 13, 369–373.
11 Z. F. Yu and M. P. Mattson, J. Neurosci. Res., 1999, 57,
830–839.
12 C. M. Hine and J. R. Mitchell, J. Clin. Exp. Psychopathol.,
2012, S4.
13 J. Su, H. Liu, K. Guo, L. Chen, M. Yang and Q. Chen,
Molecules, 2017, 1, 176–199.
4. Conclusions
In conclusion, by concentrating on the part of DR in the
regulation of AChE in the brains of aging animals, we designed 14 G. L. Ellman, K. D. Courtney, V. J. Andres and R. M. Feather-
a probe BD-AChE for detecting AChE variation in living cells Stone, Biochem. Pharmacol., 1961, 7, 88–95.
and in vivo with excellent selectivity and sensitivity, particularly 15 X. Ren, J. Wei, J. Ren, L. Qiang, F. Tang and X. Meng,
in the brain. Our experimental results validated that the AChE Colloids Surf., B, 2015, 125, 90–95.
levels decreased in the brains of mice aging models. Notably, 16 S. Hou, Z. Ou, Q. Chen and B. Wu, Biosens. Bioelectron.,
the AChE levels in the brains of aging mice significantly
2012, 33, 44–49.
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 2623–2630 | 2629

View Article Online
Paper
Journal of Materials Chemistry B
17 V. Pavlov, Y. Xiao and I. Willner, Nano Lett., 2005, 5, 29 S. Liao, W. Han, H. Ding, D. Xie, H. Tan, S. Yang,
649–653.
Z. Wu, G. Shen and R. Yu, Anal. Chem., 2013, 85,
18 V. Marx, Nat. Methods, 2014, 11, 717–720.
4968–4973.
19 R. Wang, C. Yu, F. Yu and L. Chen, TrAC, Trends Anal. 30 Y. Zhang, Y. Cai, Z. Qi, L. Lu and Y. Qian, Anal. Chem., 2013,
Chem., 2010, 29, 1004–1013. 85, 8455–8461.
20 Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2012, 113, 31 H. Tong, Y. Hong, Y. Dong, M. Haussler, J. W. Lam, Z. Li,
192–270.
Z. Guo, Z. Guo and B. Z. Tang, Chem. Commun., 2006,
3705–3707.
21 X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2013, 114,
590–659.
32 X. Wang, P. Li, Q. Ding, C. Wu, W. Zhang and B. Tang, J. Am.
Chem. Soc., 2019, 141, 2061–2068.
22 X. Wu, H. Li, E. Lee and J. Yoon, Chemistry, 2020, 11,
2893–2901.
33 X. Wu, J. M. An, J. Shang, E. Huh, S. Qi, E. Lee, H. Li, G. Kim,
H. Ma, M. S. Oh, D. Kim and J. Yoon, Chem. Sci., 2020, 11,
11285–11292.
23 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120–2135.
24 D. Wu, A. C. Sedgwick, T. Gunnlaugsson, E. U. Akkaya, 34 G. E. Guo, L. W. Ma, B. Jiang, J. Yi, T. J. Tong and
J. Yoon and T. D. James, Chem. Soc. Rev., 2017, 46,
7105–7123.
25 J. Fan, M. Hu, P. Zhan and X. Peng, Chem. Soc. Rev., 2013,
42, 29–43.
W. G. Wang, J. Cell. Biochem., 2010, 109, 1000–1005.
35 S. Haider, S. Saleem, T. Perveen, S. Tabassum,
Z. Batool, S. Sadir, L. Liaquat and S. Madiha, Age, 2014,
36, 9653.
26 J. T. Hou, W. X. Ren, K. Li, J. Seo, A. Sharma, X. Q. Yu and 36 S. I. Rattan, J. Gerontol., Ser. A, 2004, 59, 705–709.
J. S. Kim, Chem. Soc. Rev., 2017, 46, 2076–2090. 37 M. P. Mattson, Ageing Res. Rev., 2008, 7, 43–48.
27 F. Feng, Y. Tang, S. Wang, Y. Li and D. Zhu, Angew. Chem., 38 L. Matai, G. C. Sarkar, M. Chamoli, Y. Malik, S. S. Kumar,
Int. Ed., 2007, 46, 7882–7886.
28 D. Liao, J. Chen, H. Zhou, Y. Wang, Y. Li and C. Yu,
Anal. Chem., 2013, 85, 2667–2672.
U. Rautela, N. R. Jana, K. Chakraborty and
A. Mukhopadhyay, Proc. Natl. Acad. Sci. U. S. A., 2019, 116,
17383–17392.
This journal is © The Royal Society of Chemistry 2021
2630 | J. Mater. Chem. B, 2021, 9, 2623–2630