Heat Stroke in Cell Tissues Related to Sulfur Dioxide Level Is Precisely Monitored by Light-Controlled Fluorescent Probes

Article abstract of DOI:10.1021/jacs.9b13936

Heat stroke (HS) can cause serious organism damage or even death. Early understanding of the mechanism of heat cytotoxicity can prevent or treat heat stroke related diseases. In this work, probe Ly-NT-SP was synthesized, characterized, and used for sulfur

Full text of DOI:10.1021/jacs.9b13936

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Article
Heat Stroke in Cells Tissues related Sulfur Dioxide level
Precisely Monitored by Light-controlled Fluorescent Probes
Weijie Zhang, Fangjun Huo, Yongkang Yue, Yongbin Zhang, Jian-Bin Chao, fangqin cheng, and Caixia Yin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b13936 • Publication Date (Web): 17 Jan 2020
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Heat Stroke in Cells Tissues related Sulfur Dioxide level
Precisely Monitored by Light-controlled Fluorescent Probes
Weijie Zhang, †,§ Fangjun Huo,‡,§ Yongkang Yue, † Yongbin Zhang, ‡ Jianbin Chao, ‡ Fangqin Cheng,$
Caixia Yin,†,*
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†Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science,
Shanxi University, Taiyuan 030006, China.
‡Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China.
$Institute of Resources and Environmental Engineering, Shanxi University, Taiyuan 030006, China.
ABSTRACT: Heat stroke (HS)can cause serious organism damage or even death. Early understanding of the mechanism of heat
cytotoxicity can prevent or treat heat stroke related diseases. In this work, probe Ly-NT-SP was synthesized, characterized and used for
sulfur dioxide (SO2) detection in lysosomes. PBS solutions of probe Ly-NT-SP at pH 5.0 presents a marked broad emission band in the
green zone (535 nm). After UV irradiation the spiropyran group in Ly-NT-SP isomerizes to the merocyanine form (Ly-NT-MR)
which presented a weak red-shifted emission at 630 nm. Besides, photo-controlled isomerization of Ly-NT-SP to Ly-NT-MR
generated a C=C─C=N+ fragment able to react, through a Michael addition, with SO2 yielding a highly emissive adduct with a marked
fluorescence in the green channel (535 nm). In vitro studies showed a remarkable selectivity of photo-activated Ly-NT-MR to SO2 with
a limit of detection as low as 4.7 µM. Besides, MTT viability assays demonstrated that the Ly-NT-SP is non-toxicity to HeLa cells and
can be used to detect SO2 in lysosomes. Taking advantage of this, the sensor is successfully applied to image increasing SO2 values in
lysosomes during heat shock for the first time. Moreover, we also confirmed that the increased SO2 can protect the small intestine
against damage induced by heat shock through regulating oxidative stress in cells and mice.
apoptosis.15-16 Recently studies have shown that heat stroke is
INTRODUCTION
closely related to central nervous system dysfunction, which
Fluorescent imaging offers great opportunities to monitor and
always results in a higher mortality rate.17-19 Besides, it has been
analyze disease-related bio-molecules in live system with a high
also evidenced that small intestine damage caused by heat stroke
spatiotemporal resolution.1-6 Comparing with the traditional used
is a major factor resulting in heat stroke-related morbidity and
fluorophores, light responsive dyes encountere their best ages
mortality.20 In spite of these facts, however the molecular and
because of their diverse physical and chemical properties
organelle behaviors during a heat shock process is still poorly
changes upon light irradiation.7-9 Spiropyran, as one species of
understood.21 Lysosomes, as one of the vital organelles, store a
light responsive dyes featuring reversibly switch between two
large number of enzymes to regulate cell homeostasis and thus
isomer states with distinct fluorescent signals changes.10-12 Their
protect cells from stress.22-26 In fact, SO2 as an antioxidant plays
readily modified structures bring various derivatives as
crucial roles in maintaining the cell homeostasis or inducing cell
fluorescent probes to detect a various of analytes. Specifically,
apoptosis.27-28 Abnormal SO2 will affect the amount and activities
the merocyanine form of spiropyran hold polarized unsaturated
of lysosomal enzymes in macrophages, which will further reduce
C=C, caged in the spiropyran form, which could act as
impair local immune function.29-30 A question worth considering
nucleophilic reaction site for nucleophilic reagent such as sulfur
is the relationship between lysosome temperature and SO2
dioxide (SO2).13-14 The above unit constructed probes permit
fluctuation? Thus it is highly desirable for researchers to develop
artificially activation in situ during their bio-labeling and imaging
applications which promoted them as supra candidate to be
designed organelle targetable probes being able to eliminate the
effective and reliable tools to study the relevance between SO2
level and lysosome temperature in lysosomes.
In forward-moving work, we found an effective sensing
strategy based on spiropyran group as a specific light-controlled
site to more efficiently target lysosomes SO2 compared with
other no light-controlled sensors. The detailed sensing strategy
was described in Scheme 1. Lysosomes usually displayed an
acidic environment of pH 3.8–5.0.31-33 Morpholine moiety of Ly-
NT-SP was easily protonated, and once probe Ly-NT-SP
entered cells, it would accumulate in the lysosome. However, it
could not response to SO2 due to the sensing activity controlled
by
false signals output before they accumulated in organelle.
Additionally, the light-control over photochromic probes is mild
and non-invasive with high spatiotemporal resolution.
Recently, our group reported a light-controlled fluorescent
probe for highly selective response of SO2 based on spiropyran
group and accurate detection of SO2 in complex biological
systems.14 Thus; it will provide an effective tool to study the
physiology role of SO2 in living system Among all kinds of
diseases, there is an unimpressive one heat stroke (HS) as a heat-
related pathology which invokes a series of cellular responses,
including metabolic disorders, inflammatory responses, and
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Scheme 1. (a) Molecular design of Ly-NT-SP and proposed sensing mechanism towards SO2. (b) Schematic illustration of
lysosome-target Ly-NT-SP and in suit response of SO2 in controlled by UV irradiation.
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UV irradiation. After UV irradiation, the structure of spiropyran
(SP) can be reversibly switched to the merocyanine (MR) state.
And a “red-shift” fluorescence response from green fluorescence
to red fluorescence could be observed owning to Förster
resonance energy transfer (FRET) process from naphthalimide
donor to the MR acceptor. In particular, the UV-activated MR
state contains a special C=C double bonds, which was a
proposed recognition site and attacked by SO2 in situ. In this
regard, this in situ UV-activated strategy would allow us
accurately monitor SO2 in lysosomal with ratiometric manners,
which largely avoided the interference of false positive signals
during the transit of probes in cells. Furthermore, taking
advantage of this, we showed for the first time that the lysosome
SO2 rises with the increase of temperature. Moreover, tissue
imaging suggested that SO2 could protect small intestinal injury
against heat shock. This might have a guiding significance to
deeply study the pathologic pathway of certain diseases and
cause of death initiated by heat stroke.
SO2 after exposure to UV light. Upon the addition of Na2SO3
(as a SO2 donor), the emission band of the probe at 630 nm
shifts forward to 535 nm (Figure 1e), and the absorption band of
Ly-NT-MR at 540 nm gradually decreased concomitantly
(Figure 1f). Moreover, ESI-MS of the final product exhibited a
peak at m/z = 849.2901 corresponding to [Ly-NT-MR-SO32-
+
H]+ (Figure S2). Thus confirming that the Michael addition of
SO2 to Ly-NT-MR probe contributing to the observed optical
changes. Besides, the plot of the fluorescence intensity ratios
between the two emissions at 630 nm and 535 nm (F630/F535) of
the system exhibited a linear relationship with the increased
concentration of Na2SO3 (from 0 to 50 µM). Determined from
10 blank solutions, the detection limit of Ly-NT-MR toward
SO2 was calculated to be 4.7 µM based on IUPAC (CDL =
3Sb/m) (Figure S3).35
To evaluate the selective of probe, some representative
analytes such as thiols (Cys, Hcy, GSH and NAC) and common
biomolecules (SO42-, SCN-, F-, Cl-, Br-, I-, AcO-, NO2-, N3-,
formaldehyde) etc. were treated with Ly-NT-SP under pH 5.0.
It was found that Ly-NT-SP showed negligible fluorescence
response on other tested species (see Figure S4). By contrast,
significant fluorescence intensity ratio change was observed in
the presence of Na2SO3, indicating that Ly-NT-SP showed high
selectivity for SO2 over bio-relevant species. Afterward, the time-
dependent fluorescence emission change of Ly-NT-SP with
Na2SO3 was explored. The fluorescence signal at 535 nm
increased and reached a maximum equilibrium within 5 min
(Figure S5), indicating that the MR state exhibit highly reactivity
towards SO2 derivatives.
RESULTS AND DISCUSSION
Photochemical Properties of Ly-NT-SP in PBS Buffer
Solution. We initially investigated the basic chemical and
photochemical features of probe Ly-NT-SP upon UV/Vis
irradiations in buffer solution. Since the lysosomal pH value is
ranging from 3.8 to 5.5, all these spectra measurement was
carried out at pH 5.0 in this study. As could be seen in Figure 1a,
probe Ly-NT-SP displayed maximum emission at 535 nm in
PBS. Notably, we can find that the emission maxima at 535 nm
shifted to 630 nm with UV irradiation for 120 s. Synchronously,
under UV irradiation, a new band at 540 nm appeared and its
intensity growth progressively with time yielding the maximum
absorbance after 120 s (Figure 1b). Importantly, probe shows
good reversibility between UV and Vis light controlled
absorbance (Figure 1c) and fluorescence switch (Figure 1d).
These observed changes are ascribed to the transformation of
the spiropyran conformation of probe Ly-NT-SP to the
merocyanine form (Ly-NT-MR). As a consequence a FRET
process from naphthalimide donor to the merocyanine acceptor
is active which account for the observed emission band shifts.
And the FRET efficiency of Ly-NT-MR was calculated to be
68.5% (E=1-FDA/FD, Figure S1).34
Subsequently, the pH effect of Ly-NT-MR for SO2 detection
was investigated. As shown in Figure S6, Ly-NT-SP was silent
in a broad pH range (3.0-8.0) after UV irradiation, and an
obvious enhancement of ratiometric signals (F630/F535) was
achieved in both neutral and weak acid pH range (4.0-7.0).
Finally, the influence of temperature (from 37° to 45°C) on the
fluorescence intensity ratio of Ly-NT-MR was also studied
(Figure S7). The results showed that fluorescence ratio
(F630/F535) of PBS solutions (10 mM, pH 5) of Ly-NT-MR in
the absence of Na2SO3 remained unchanged when the
temperature increased from 37° to 45°C. All the above
mentioned results indicated that probe Ly-NT-SP was suitable
for accurate measurement of SO2 concentration changes in
lysosomes.
SO2 detection using Ly-NT-SP probe. After we assessed
the UV light controlled transformation of probe Ly-NT-SP to
Ly-NT-MR, then we tested the fluorescence emission and UV-
visible behaviours of the probe Ly-NT-SP in the presence of
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Figure 1. (a) Emission and (b) absorption spectra changes of PBS (10 mM, pH 5) solutions of probe Ly-NT-SP (10 µM) after irradiation with
UV light for 120 s (for the emission spectra excitation was set at 450 nm). Fluorescence emission (c) and UV-Vis absorbance (d) photo-
switching of Ly-NT-SP in PBS (10 mM, pH 5). a) Absorption and emission spectra change of Ly-NT-MR (10 µM) upon addition of Na2SO3
(0-100 µM) in PBS buffer solution (pH = 5.0, 10 mM). λex = 450 nm, slit: 5 nm/5 nm.
Imaging of exogenous and endogenous SO2 in living
cells. Firstly, we assessed cytotoxicity of Ly-NT-SP via MTT
assays using HeLa cell line. MTT assay showed no significant
cytotoxicity of Ly-NT-SP at the level up to 10 μM (Figure S8).
Next, the cell viability was checked under different UV
irradiations time (0, 50, 100, 200, 300 s), and these results
showed that cells were perfectly viable (＞70 %) upon 200 s UV
irradiation (Figure S9), indicating a low photo-toxicity of UV
irradiation procedure in this experiment.
Then, we tested the intracellular light-controlled manners of
Ly-NT-SP in HeLa cells. For this purpose, HeLa cells were
incubated with Ly-NT-SP (10 μM) for 20 min at 37 °C, then
irradiated with UV light (100 s) and with visible light (10 min)
consecutively. As expected, HeLa cells treated with Ly-NT-SP
showed a significant green fluorescence in the green channel
ascribed to probe internalization. Meanwhile, negligible emission
was observed in the red channel (Figure 2). However, after UV
irradiation, the fluorescence of the green channel vanished and a
marked red emission appeared in the red channel as a
consequence of the transformation of inactive Ly-NT-SP to the
SO2-active Ly-NT-MR. Then, upon irradiation with visible
light, a marked increase in green fluorescence together with a
reduction in the red channel was observed. These results clearly
validated the light-controlled strategy of probe Ly-NT-SP for
intracellular photochromic actions.
Having demonstrated the favorable optical performances of
Ly-NT-SP in living cells, the ability of the Ly-NT-SP for
imaging lysosomes SO2 was carried out in HeLa cells. To
investigate the lysosome-targeting ability of Ly-NT-SP in living
cells, the co-localization study of Ly-NT-SP with commercial
available lysosome red (Lyso-Tracker Red) was conducted in
HeLa cells. HeLa cells was incubated with 10 μM Ly-NT-SP at
37 °C for 20 min, and further incubated with 500 nM Lyso-
Tracker Red for another 20 min. As shown in Figure 3, both the
green and red channels displayed strong emission, and the
fluorescence images overlapped well (Figure S10) with high
Pearson’s colocalization 0.82. These results showed that Ly-NT-
SP can well accumulated in lysosomes of living cells.
Figure 2. Confocal microscopy images of Ly-NT-SP (10 µM) with
alternate UV/Vis irradiation. From top to bottom, probe Ly-NT-
SP, UV irradiation, Vis irradiation, UV irradiation. From left to
right, Red channel, λem = 610 ± 30 nm (λex = 561 nm), Green
channel, λem = 530±20 nm (λex = 458 nm), Bright field, Overlay
images, Scale bar: 20 μm.
Figure 3. Confocal microscopy images of Ly-NT-SP (10 µM) and
Lyso-Tracker in living cells. (a) Confocal microscopy image of Lyso-
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Tracker in the red channel. (b) Confocal microscopy image of Ly-
NT-SP in the green channel. (c) Overlay image. (d) The correlation
of Lyso-Tracker and Ly-NT-SP intensities. Red channel, λem = 610
temperature (Figure 5b). The marked emission changes
(reduction and enhancement in the red and green channel
respectively) are in agreement with the isomerization of Ly-NT-
SP to the merocyanine form Ly-NT-MR and subsequent
reaction with lysosome SO2. For the control experiment, FA
(formaldehyde), which known as an inhibitor of bisulfite, was
used for SO2 imaging during heat shock.36 Ly-NT-SP located
HeLa cells was incubated with FA (200 μM) for 20 min at 37 °C,
and then irradiated with UV light and further incubated at 43 °C
for another 30 min. As expected, the presence of FA enhanced
the fluorescent signals in the red channel, and the green-to-red
emission ratio was significantly reduced. To investigate the role
of SO2 in heat shock, Ly-NT-SP located HeLa cells were
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±30 nm (λex = 561 nm), Green channel, λem = 530±20 nm (λex
458 nm), Scale bars = 10 μm.
=
Furthermore, we investigated the sensing performance of
probe Ly-NT-SP for exogenous and endogenous SO2 detection
in living cells. HeLa cells were pretreated with Ly-NT-SP for 15
min, and then further treated with UV irradiation for two
minutes. As shown in Figure 4a, strong red fluorescence was
observed in red channel, while negligible signal captured in green
channel after UV irradiation. By contrast, after addition of
exogenous SO2 (Na2SO3), the fluorescence signals in green
channel largely increased along with decrease red fluorescence in
the red channel. To evaluate the ability of Ly-NT-SP for
endogenous SO2 detection, HeLa cells were pretreated with 1
μg/mL lipopolysaccharide (LPS), which can induce the
inflammatory response of cells to produce low-level sulfite
endogenously.13 In comparison with cells treated with Ly-NT-
SP and irradiated with UV light, LPS pretreated HeLa cells
exhibited strong green fluorescence accompanied with relative
weak red fluorescence (Figure 4b). Taken together, probe Ly-
NT-SP was capable of tracking both exogenous and
endogenous SO2 in living cells.
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pretreated with NAC (N-acetyl-L-cysteine,
a well-known
antioxidant, 200 μM), and then cultured at 43 °C for another 30
min. Conspicuously, after NAC treatment, the expression levels
of SO2 was down-regulated. Possible reasons are as follows; HS-
treated cells can induce the over-generation of intracellular
reactive oxygen species (ROS), while the presence of NAC can
significantly inhibit ROS generation. As a result, the oxidative
stress-dependent increased of SO2 was suppressed. These results
pointed to a direct relationship between cell temperature and
SO2 concentration in lysosomes, strongly suggested that SO2
plays a crucial role as an antioxidant to regulating redox
homoeostasis, and up-regulation of SO2 in the lysosomes was
dependent on the oxidative stress induced by heat shock.
Figure 4. (a) Confocal microscopy images of Ly-NT-MR with
exogenous Na2SO3 (50 μM). (b) Confocal microscopy images of Ly-
NT-MR with endogenous Na2SO3 (pretreating the cells with 1
μg/mL lipopolysaccharide). Red channel, λem = 610±30 nm (λex
=
561 nm), green channel, λem = 530 ±20 nm (λex = 458 nm), bright
field, overlapped images, ratio images. Scale bar: 20 μm.
Next, as a proof of concept, we tested the ability of probe Ly-
NT-SP for imaging of SO2 concentration changes in HeLa cells
under heat shock. 39 °C, 41 °C and 43 °C were used as model
temperatures of heat stroke, whereas 37 °C was used as control
group. HeLa cells were firstly incubated with Ly-NT-SP (10
µM) for 20 min at 37°C and then irradiated with UV light. Then,
in a second step, cells were further incubated at 37, 39, 41, and
43 °C for another 30 min and confocal microscopy images were
finally taken. As could be seen in Figure 5a, upon temperature
increase the red emission markedly decreased whereas the in the
green channel a moderate fluorescence enhancement was
observed. Also the green-to-red emission ratio increased with
Figure 5. (a) Confocal microscopy images of HeLa cells incubated
with Ly-NT-MR (10 µM), Ly-NT-MR (10 µM) with FA (200 µM)
and Ly-NT-MR (10 µM) with NAC (200 µM) at different
temperatures (37°, 39°, 41° and 43°C) after UV irradiation. Red
channel, λem = 610 ±30 nm (λex = 561 nm), green channel, λem
=
530 ±20 nm (λex = 458 nm), bright field, overlapped images. Scale
bar: 20 μm. (b) Ratio of green/red fluorescence intensity at different
temperatures.
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SO2 upregulation in small intestines of mice, which confirming
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the advantage of Ly-NT-SP for precise SO2 imaging. Apart
from this, reduced heat stroke-induced intestinal damage was
observed in Na2SO3 and NAC pre-treat mice. By contrast,
marked intestinal damage such as villous stroma broadening and
focal necrosis was noticed in HS and HS + HCHO treated mice.
These results suggested the increased SO2 could largely reduce
heat stroke-induced intestinal damage by defense of over-
generated ROS.
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In summary, we have successfully developed a lysosome-
targeted ratiometric fluorescent probe for the evaluation of
changes in the SO2 concentration in lysosomes. Ly-NT-SP
probe displays excellent lysosome-targeting ability and is able to
detect SO2 upon UV irradiation. This UV-activation enables
precision imaging of SO2 in a spatial and temporal manner.
Furthermore, Ly-NT-SP was successfully used for the
visualization of exogenous and endogenous SO2 in HeLa cells.
Additionally, Ly-NT-SP is used for the first time to tack
lysosomal SO2 changes during heat shock. What’s more, cell and
intestinal tissue imaging studies have clearly revealed the
elevation of endogenous SO2 play an important role in regulating
oxidative stress, and increased SO2 could protect small intestine
from heat stroke induced damage. We believe that the present
studies may fuel the development of targeting photo-actable
probes for the detection of small molecules, such as SO2, which
might related to certain pathologies and treatments as for
instance those related with heat shock.
Figure 6. Confocal microscopy images of SO2 in HS pre-treated
intestinal tissues. From top to bottom: control (normal), HS, HS +
NAC, HS + HCHO, HS + Na2SO3 and ratio Green/Red. Red
channel, λem = 610 ±30 nm (λex = 561 nm), green channel, λem
530±20 nm (λex = 458 nm), Scale bar: 200 μm.
=
Heat stress-related oxidative stress has been shown to cause
small intestines damage of both rats and pigs.37-38 Thus, we
further examined the level of SO2 change as well as
theantioxidant effect of SO2 in heats stroke effected mice. HS-
induced small intestines injury was modeled according to
previously reported methods as described in supporting
information. Pathogen-free 6 to 8 week old male BALB/c mice
were randomly assigned to five experimental groups, control, HS,
HS + NAC, HS + HCHO and HS + Na2SO3. Firstly, the UV
irradiation for converting Ly-NT-SP to the Ly-NT-MR was
tested in intestinal tissues. As shown in Figure S11, the ileum
slice incubated with Ly-NT-SP (20 µM) displayed slight red
emission in the red channel. Then, the tissue was irradiated with
UV light in darkroom for 100 s. After that, we observed a clearly
increased red fluorescence in the red channel, and the green
fluorescence decreased simultaneously. These results suggested
the transformed Ly-NT-MR could potentially response to SO2
in organ tissues. Then HS group pre-treated ileum slices were
incubated with Ly-NT-SP (20 µM) for 30 min at 37 °C, and
exposed to UV light for 3 min, followed by culturing another 30
min at 37 °C. As expected, compared with control group (first
row of images in Figure 6), remarkable reduction in the red
channel together with an increased fluorescent signals in the
green fluorescence was noticed in HS treated intestines tissues
(second row of images in Figure 6). And the emission ratio
(green/red) of HS-treated mice increase by 4.1-fold compared
with control group (Figure S12). However, due to fact that
suppressed of SO2 generation, the fluorescent ratio of NAC and
FA pre-treated mice only increased 1.85-fold and 2.78-flod
compared with control group, respectively (Figure S12. As for
Na2SO3 treated mice, the obtained tissues images showed
significant fluorescence enhancement in green channel, and the
image from red channel showed rather weak fluorescent signals
(last row of images in Figure 6). Besides, compared with control
group, we can notice that the injection of Na2SO3 (100 mg/kg)
triggered most obvious fluorescent ratio changes up to 4.6-fold
(Figure S12). These results indicated that heat stroke induced
ASSOCIATED CONTENT
Supporting Information
Generally
information;
experimental
section;
additional
photophysical spectra; NMR and ESI-MS spectra.
AUTHOR INFORMATION
Corresponding Author
* E-mail: yincx@sxu.edu.cn
ORCID
Caixia Yin: 0000-0001-5548-6333
Author Contributions
§Weijie Zhang and Fangjun Huo contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the National Natural Science Foundation of China (No.
21775096), One hundred people plan of Shanxi Province, Shanxi
Province "1331 project" key innovation team construction plan
cultivation team (2018-CT-1), 2018 Xiangyuan County Solid Waste
Comprehensive Utilization Science and Technology Project
(2018XYSDJS-05), Shanxi Province Foundation for Returness
(2017-026), Shanxi Collaborative Innovation Center of High Value-
added Utilization of Coal-related Wastes (2015-10-B3), Shanxi
Province 2019 annual science and technology activities for overseas
students selected funding projects, Innovative Talents of Higher
Education Institutions of Shanxi, Scientific and Technological
Innovation Programs of Higher Education Institutions in Shanxi
(2019L0031), Key R&D Program of Shanxi Province
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