In Situ Observation of mtDNA Damage during Hepatic Ischemia-Reperfusion

Article abstract of DOI:10.1021/acs.analchem.0c05220

Hepatic ischemia-reperfusion (IR) injury is a severe pathophysiological event during liver surgery or transplantation and could lead to liver failure or even death. The energy supply of mitochondria plays an essential role in preventing IR injury. Mitochondrial DNA (mtDNA) is involved in maintaining the balance of energy by participating in an oxidative phosphorylation process. However, the exact relationship between IR and mtDNA remains unclear by reason of the lack of an accurate real-time analysis method. Herein, we fabricated a mitochondria-targeting fluorescent probe (mtDNA-BP) to explore mtDNA stability and supervise the changes in mtDNA in IR liver. By virtue of pyridinium electropositivity and suitable size, mtDNA-BP could accumulate in mitochondria and insert into the mtDNA groove, which made mtDNA-BP fluoresce strongly. This is attributed to the reduction of the intramolecular rotation energy loss that is restricted by DNA. By in situ fluorescence imaging, we observed in real time that mtDNA damage was aggravated by deteriorating IR injury, so the ROS-mtDNA-mediated IR damage signal pathway was speculated. Furthermore, on the basis of mtDNA-BP real-time response capability for mtDNA, we established a drug-screening method for inhibiting IR injury and found superior therapeutic performance of two potential drugs: pioglitazone and salidroside. This work contributes to our understanding of mtDNA-related disease and provides a new drug analysis method.

Full text of DOI:10.1021/acs.analchem.0c05220

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Article
In Situ Observation of mtDNA Damage during Hepatic Ischemia-
Reperfusion
Wen Zhang,# Simin Bi,# Ping Li,* Jihong Liu, Chunmiao Zhou, Xin Wang, Wei Zhang, Hui Wang,
and Bo Tang*
Cite This: Anal. Chem. 2021, 93, 5782−5788
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ABSTRACT: Hepatic ischemia-reperfusion (IR) injury is a severe
pathophysiological event during liver surgery or transplantation and
could lead to liver failure or even death. The energy supply of
mitochondria plays an essential role in preventing IR injury.
Mitochondrial DNA (mtDNA) is involved in maintaining the
balance of energy by participating in an oxidative phosphorylation
process. However, the exact relationship between IR and mtDNA
remains unclear by reason of the lack of an accurate real-time analysis
method. Herein, we fabricated a mitochondria-targeting fluorescent
probe (mtDNA-BP) to explore mtDNA stability and supervise the
changes in mtDNA in IR liver. By virtue of pyridinium electro-
positivity and suitable size, mtDNA-BP could accumulate in
mitochondria and insert into the mtDNA groove, which made
mtDNA-BP fluoresce strongly. This is attributed to the reduction of the intramolecular rotation energy loss that is restricted by
DNA. By in situ fluorescence imaging, we observed in real time that mtDNA damage was aggravated by deteriorating IR injury, so
the ROS-mtDNA-mediated IR damage signal pathway was speculated. Furthermore, on the basis of mtDNA-BP real-time response
capability for mtDNA, we established a drug-screening method for inhibiting IR injury and found superior therapeutic performance
of two potential drugs: pioglitazone and salidroside. This work contributes to our understanding of mtDNA-related disease and
provides a new drug analysis method.
schemia-reperfusion (IR) is a routine operation, but a major
obstacle in liver resection and transplantation surgery.1,2
develop efficient probes and establish a real-time imaging
method of the mtDNA level and distribution.
I
With the intent to resist hepatic IR injury, it ought to provide
stronger support from mitochondria that generate ATP to
maintain the internal stability of the organism and prevent
disease.3 Accordingly, the mitochondrial DNA (mtDNA) is
involved in the oxidative phosphorylation process and closely
related to ATP supply.4 However, the exact relationship
between IR and mtDNA remains unclear because of the lack of
an accurate real-time analysis method. Therefore, to under-
stand the obligatory role of mtDNA in liver IR injury,
developing in situ real-time monitoring tools for mtDNA is
extremely important.
Fluorescence microscopy is an authoritative tool to study
concentration changes and location distributions of essential
molecules.5−8 This instrument contains many advantages
including high resolution, simple operation, and in situ
dynamic monitoring.9−13 To make better use of fluorescence
microscopy, many researchers have developed a variety of
small-molecule fluorescent probes for tracking molecular
events in cells and in vivo.14−19 Although high-resolution
imaging at the subcellular level was performed,20−23 few probes
can detect mtDNA located in the mitochondria of living cells.
To reveal mtDNA-mediated IR damage, it is important to
In this work, we constructed a small molecular mitochon-
drial-targeted probe mtDNA-BP (1-ethyl-2-{3-[3-(4-bromo)-
butyl-2-benzothiazolinylidene]propenyl}pyridine bromide) for
the detection of mtDNA (Figure 1). An appropriate size of
mtDNA-BP was designed to embed in the DNA groove.
Relying on the spatial constraints of DNA, the rotation of the
pyridine ring was limited, and also the nonradiative transition
of the excited state reduced, resulting in lighting up the DNA.
Meanwhile, the unique electronegativity of the mitochondrial
membrane could attract the pyridine cation to tread through
the mitochondrial membrane and realize the mtDNA monitor.
Utilizing mtDNA-BP, we traced the time-dependent damage of
mtDNA during the IR process. Furthermore, a drug-screening
method for protecting IR was devised by means of mtDNA-BP.
Received: December 13, 2020
Accepted: March 18, 2021
Published: March 30, 2021
https://doi.org/10.1021/acs.analchem.0c05220
© 2021 American Chemical Society
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(0.75 g, 1.8 mmol, 60% yield). HRMS data, m/z calculated
for [C21H24N2SBr+], 415.0838; found, 415.0824. 1HNMR
(400 MHz, CDCl3): δ 8.54 (d, 1H), 7.81 (m, 2H), 7.57 (d,
1H), 7.42 (t, 1H), 7.25 (d, 1H), 7.06 (m, 2H), 6.29 (m, 2H),
5.24 (s, 1H), 4.49 (m, 2H), 4.04 (t, 2H), 3.59 (t, 2H), 1.93
(m, 2H), 1.49 (m, 5H). 13CNMR (100 MHz, CDCl3): δ
157.94, 157.85, 151.43, 141.93, 140.52, 139.03, 126.33, 123.44,
122.41, 121.14, 121.10, 120.99, 118.09, 117.96, 103.21, 62.67,
51.34, 43.85, 28.43, 24.96, 20.07.
Figure 1. Design and luminescence mechanism of mtDNA-BP.
Cytotoxicity Assay. We evaluated the cell viability by
conventional MTT analysis. MTT reagent is 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).24
We first grow HL-7702 cells at a density of 106 cells mL−1
into a 96-well plate of volume 200 μL well−1. After 12 hours,
numerous concentrations of mtDNA-BP (0, 1 × 10−9, 1 ×
10−8, 1 × 10−7, 1 × 10−6, and 1 × 10−5 mol·L−1) were added to
the wells, and then the cells were incubated for an additional
24 h. Afterward, the MTT reagent (20 μL, 5 mg mL−1)
dissolved in DMSO was added to each well. After waiting for 4
h, we removed the MTT solution and then added 150 μL of
DMSO to every well. Eventually, we used the Triturus
microplate reader to measure the absorbance of the solution
at 490 nm.
Models of Ischemia-Reperfusion in Cells and Mice.
We constructed hepatic IR cell models by treating cells with
oxygen and glucose deprivation/reperfusion. The ischemic
state of hepatocytes was cultivated in DMEM without glucose
and 0.5 mM deoxygenated sodium dithionite. Half an hour
later, these cells were incubated with DMEM (high level of
glucose) in a CO2/O2 (5/95) atmosphere as the state of
reperfusion.25
Next, the IR mice model was established by 8-week-old wild-
type Kunming female mice. We first used an intraperitoneal
injection of 4% chloral hydrate to anesthetize the mice with a
dosage of 3 mL kg−1. Then we fixed the anesthetized mice onto
the manipulator and cut open the abdomen of the mice to find
the liver. Hepatic ischemia was achieved by gripping the vena
and artery of the center and also the left lateral lobes of the
liver with a microvessel clip, causing 70% liver anemia. And the
reperfusion was performed by opening the vascular clamp. We
have already obtained permission for the animal experiments
from the Shandong Normal University authorities.
EXPERIMENTAL SECTION
■
Synthesis of 1-Ethyl-2-methylpyridine Bromide (1).
The 2-methylpyridine (9.31 g, 0.1 mol) and ethyl bromide
(10.89 g, 0.1 mol) were refluxed at 70 °C for 7 h and then
allowed to cool down to room temperature; the resulting
product was washed several times with ethyl ether and dried in
vacuum to provide the crude product compound 1 (11.11 g,
0.091 mol, 90% crude yield). HRMS data, m/z calculated for
[C8H12N+], 122.0964; found, 122.0956.
Synthesis of 1-Ethyl-2-[2-(phenylamino)ethenyl]-
pyridine Bromide (2). Compound 1 (1.83 g, 15.0 mmol)
and N,N′-diphenylformamidine (2.94 g, 15.0 mmol) were
heated to 160 °C for 90 min. The resulting solid (compound
2) was washed three times with ethyl ether and then dried in
vacuum (0.78 g, 6.45 mmol, 48% yield). HRMS data, m/z
+
calculated for [C15H17N2 ], 225.1386; found, 225.1393.
Synthesis of 1-(4-Bromo)-butyl-2-methylbenzothia-
zolium Bromide (3). The 2-methylbenzothiazole (1.49 g,
10.0 mmol) and 1,4-dibromo-butane (4.32 g, 20.0 mmol) were
dissolved in 10 mL of toluene and heated to 110 °C for 17.5 h.
After cooling, the solid was washed with ethyl ether and dried
in vacuum to provide the crude product compound 3 (3.03 g,
8.3 mmol, 83% crude yield). HRMS data, m/z calculated for
[C12H15NSBr+], 284.0109; found, 284.0115.
Synthesis of mtDNA-BP. The mtDNA-BP was synthe-
sized by a simple and economical synthetic method and well-
characterized [Figure 2, Figures S14−S19 (Supporting
Information)]. The compounds 2 (0.34g, 1.5 mmol) and 3
(0.55g, 1.5 mmol) were dissolved in 15 mL 1:1 (v/v) CH2Cl2/
CH3OH soultion with 1.5 mL of (Ac)2O and 1.5 mL of N(Et)3
and heated to 110 °C for 6.5 h. The black red solid (mtDNA-
BP) was purified by silica gel column chromatography
(CH3OH/CH2Cl2, 1/20) and the product was collected
Extraction of Mitochondria and mtDNA. The mito-
chondria were isolated from liver tissue of normal and IR mice
Figure 2. Synthesis of the probe mtDNA-BP. (i) Reflux, 7 h; (ii) 160 °C, 1.5 h; (iii) Br(CH2)4Br, toluene, 110 °C, 17.5 h; (iv) CH2Cl2, CH3OH,
(Ac)2O, N(Et)3, rt, 6.5 h.
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Figure 3. Fluorescent properties of mtDNA-BP for DNA detection. (A) Fluorescence responses of 5 μM mtDNA-BP toward the addition of DNA
with various concentrations (0, 20, 40, 60, 80, 100, and 120 μg/mL). (B) Fluorescence spectra of 5 μM mtDNA-BP after the addition of 120 μg/
mL DNA with different concentrations of DNase I (0, 5, 10, 15, and 20 U/mL). All spectra were acquired in Tris (pH 7.4) at λex/λem = 520/580
nm.
Figure 4. Fluorescence selectivity of mtDNA-BP for DNA. (A) 5 μM mtDNA-BP was treated with 1−25:10 mM K+, 10 mM Na+, 100 μM Cu+,
100 μM Cu2+, 100 μM Fe3+, 100 μM Fe2+, 1 mM Zn2+, 1 mM Ca2+, 100 μM Mn2+, 10 μM O2• −, 100 μM 1O2, 100 μM •OH, 10 mM H2O2, 100
μM ONOO−, 100 μM NO, 100 μM NaClO, 100 μM L-Tyr, 100 μM L-His, 100 μM Glc, 100 μM Vc, 100 μM Cys, 100 μM GSH, 10 mM ATP, 15
mM RNA, 120 μg/mL DNA. All spectra were acquired in Tris (pH 7.4) at λex/λem = 520/580 nm. (B) 5 μM mtDNA-BP was loaded with (black)
and without (red) 120 μg/mL DNA at varying pH values (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0).
employing a Tissue Mitochondria Isolation Kit (Beyotime
Biotechnology Co., Ltd.) and mtDNA was isolated from liver
tissue of mice using a mtDNA isolation kit consistent with the
kit’s instructions.
We repeated each experiment at least three times to get the
same results.
Quantification of mtDNA Damage. We used the
mtDNA long-range PCR to measure the level of mtDNA
damage in IR-injured cells. The human mtDNA sequence-
specific primers were utilized to amplify a 10 kilo base pair (10
kbp) fragment of mtDNA. The following primers were used to
amplify a 10-kb fragment of mtDNA: sense primer 5′-
TTTCATCATGCGGAGATGTTGGATGG-3′ and antisense
primer 5′-TCTAAGCCTCCTTATTCGAGCCGA-3′.
Flow Cytometry. Hepatocytes seeded on 6-well plates
were incubated continuously for 36−72 h at 37 °C in a CO2
(5%) cell incubator with 2 mL of high-glucose DMEM per
well. Before we performed the flow cytometry experiments, we
washed pretreated hepatocytes with 2 mL of PBS and then
treated them with 400 μL of 0.05% trypsin for 1 min. The
Fluorescence Imaging Experiments. The living cells
were detached and reseeded onto 15 mL glass-bottom dishes
24 h before imaging. Next, we pretreated the cells with
mtDNA-BP for 30 min, removed the medium, and then
washed the cells with 500 μL of PBS three times. Fluorescence
imaging of living cells was then executed. Mitochondrial
localization imaging was performed utilizing confocal fluo-
rescence microscopy with a green fluorescence channel (λex =
514 nm and λem = 540−630 nm) and a red fluorescence
channel (λex = 633 nm and λem = 640−700 nm). For the data
processing, the average fluorescence intensity of each image in
every process was obtained by selecting the areas of interest.
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trypsin was discarded and 1 mL of high-glucose DMEM was
added to each well. Subsequently, the hepatocytes were
centrifuged for 5 min at 1000 rpm and the medium discarded.
Then the hepatocytes were resuspended in 2 mL of PBS and
once more centrifuged for 5 min at 1000 rpm. Afterward, every
pellet was nurtured with Annexin V-FITC binding buffer (195
μL), Annexin V-FITC (5 μL), and propidium iodide (PI) (10
μL) in the dark at room temperature for 20 min. When the
incubation operation was completed, the hepatocytes were
centrifuged for 5 min at 1000 rpm and resuspended in PBS
(200 μL). Lastly, the hepatocytes were subjected to flow
cytometry. The fluorescence signals within the two channels of
FITC and PI were surveyed for 5000 cells on an Image-
StreamX Mark II flow cytometer (Merck). The data was
analyzed by IDEAS software version 6.2.
DNA transiently, which is beneficial for pick up the real-time
information hinted at by DNA changes. Furthermore, the
probe used for biological imaging should not add toxicity or
side effects to the cells. Hence, the cytotoxicity assays were
executed by MTT analysis (Figure S5). The IC50 of mtDNA-
BP is 38.02 mM. It indicates that mtDNA-BP is a qualified
imaging reagent with high velocity and safety.
Binding DNA Properties of mtDNA-BP. Studying the
ability of mtDNA-BP to bind to DNA can provide a basis for
their application in complex life units. Hence, we explored in
detail the binding mode and ability of mtDNA-BP to DNA by
SYBR Green I displacement assay.26,27 SYBR Green I is a
commercial DNA binding dye that indicates the degree of
binding to DNA by changes in its fluorescence intensity.
According to the Figure S6, when the SYBR Green I
encountered the DNA, it exhibited intense fluorescence
emission at 520 nm. Then the fluorescence intensity of
SYBR Green I decreased with the addition of mtDNA-BP, and
the fluorescence intensity decreased linearly with the increase
of the probe concentrations. This indicated that SYBR Green I
was extruded from the DNA groove by mtDNA-BP, and also
suggested that the mtDNA-BP could embed in the DNA
groove and had stronger binding ability to the DNA groove. In
addition, we calculated the binding constants of mtDNA-BP to
DNA based on the following equation (1)28 and binding
constant K = 46773.51 L·mol−1 (Figure S7). Overall, the
mtDNA-BP could be embedded in DNA grooves and have
strong interactions with DNA.
RESULTS AND DISCUSSION
■
Properties of mtDNA-BP. We exploited the mtDNA-BP
spectral property responding to DNA by using UV−vis and
fluorescence spectra in a simulated physiological environment.
As illustrated in Figure S1, upon addition of 120 μg/mL DNA,
the maximum absorption wavelength appeared at 520 nm.
Around the maximum fluorescence emission peak at 580 nm,
the mtDNA-BP fluorescence intensity increased obviously with
the addition of DNA while decreasing significantly with various
DNase treatments (Figure 3). Furthermore, the fluorescence
intensity enhanced linearly with the DNA concentrations rising
(Figure S2). The linear range of DNA detection was 0−120
μg/mL and the limit of detection was 0.2331 μg/mL. These
data prove that mtDNA-BP has a superior ability to detect
DNA.
log[(F − F)/F] = log K + n log[Q ]
(1)
0
where F0 and F are the fluorescence intensity without and with
mtDNA-BP.
A high concentration of reactive oxygen species (ROS) and
reactive nitrogen species exist in mitochondria, and there are
amounts of metal ions in the path of mtDNA-BP entering
mitochondria. All the disruptors may interfere with the
fluorescence response of the probe, so the selectivity of
mtDNA-BP should be investigated efficiently. Figure 4A shows
that there were no significant fluorescence changes of mtDNA-
BP toward intracellular active species except for DNA. It
indicates that mtDNA-BP could detect DNA in cells
exclusively. In addition, to further validate the mtDNA-BP
specificity to DNA in a more realistic life environment, we
performed a complete extraction of the mitochondria within
the hepatocytes. In the mitochondrial internal fluid containing
DNA, whether the mtDNA-BP is affected by other factors was
investigated. As shown in Figure S3, the mtDNA-BP
fluorescent in the authentic mitochondrial environment was
not disturbed by ROS, metal ions, amino acids, and RNA. The
coexistence interference experiment was consistent with the
selectivity experiment in Figure 4A, further illustrating the
distinctive response of the probe for DNA in a complex living
environment. Besides, in view of the unbalanced distribution of
[H+] in the subcellular region, the influence of pH fluctuations
has been estimated. As shown in Figure 4B, the fluorescence
signal of mtDNA-BP was varied imperceptibly in the
physiological pH range 4.0−9.0. The experimental results
show that mtDNA-BP has the potential to perform intra-
cellular imaging without being disturbed by the intracellular
microenvironment.
Mitochondrial Localization of mtDNA-BP. We ex-
ploited the distribution of mtDNA-BP in living cells.29−31
Interestingly, mtDNA-BP prominently accumulated in mito-
chondria relying on the positive charge of the pyridine group.
We employed the mitochondrial commercial dyes (Mito-
Tracker Deep Red, Invitrogen, red channel) to co-stain with
mtDNA-BP (green channel). As shown in Figure 5, all of the
green fluorescence overlaid with the partly red color to exhibit
yellow. Moreover, the distribution of green fluorescence was
characterized by discrete points. This indicates that the probes
could target specific regions within the mitochondria.
Furthermore, we investigated whether mtDNA-BP could
recognize mtDNA. Figure S8 showed that the fluorescence
intensity of mtDNA-BP was significantly enhanced with the
addition of mtDNA and the fluorescence intensity was reduced
with Hind III (mtDNA digested enzyme) pretreated living
cells. These data imply that mtDNA-BP could specifically
image mtDNA in hepatic cells.
Real-Time Fluorescence Imaging of mtDNA-BP in IR
Injury. Mitochondrial DNA plays a significant role in
maintaining normal liver function by participating in the
oxidative phosphorylation process for the supply of energy.
The balance of the energy in the mitochondria is an important
factor to resist IR damage. There seems to be a close link
between mtDNA and IR injury. However, current research has
not been able to determine the definite relationship. So, we
tried to understand the communication between IR damage
and mtDNA with the help of mtDNA-BP. The mtDNA
fluctuations in the IR process were visualized in real time. As
shown in Figure 6, with the time course of IR injury, the
mtDNA-BP fluorescence intensity was gradually decreased for
The faster the probe responds to the object, the easier it is to
capture rapidly changing information about the active
molecule. So, the DNA response rate of mtDNA-BP was
explored. As shown in Figure S4, mtDNA-BP could recognize
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assay kit. As shown in Figure S10, the stronger fluorescence
was exhibited in the mitochondria of the IR group compared to
that of the normal group, indicating that the total
mitochondrial ROS was indeed abnormally elevated in IR.
However, it is still not clear whether ROS also mediates
mtDNA damage from IR. Hence, we explored the behavior of
H2O2 to mtDNA via a simple extraction method of the liver.
As shown in Figure S11, when the mitochondrial DNA was
pretreated with 10 μM H2O2, the mtDNA-BP fluorescence
intensity was reduced significantly. It suggested that over-
loaded ROS could mediate mtDNA damage, resulting in 8-
hydroxydeoxyguanosine formation and mtDNA fragmentation,
also causing the cross-linking of mtDNA to proteins.36
Considering that the location of mtDNA is close to the
respiratory chain complex produced by ROS in large
quantities, and there is a lack of protective histone protein
and some DNA repair activities in mitochondria, oxidative
damage caused by ROS to mtDNA is more likely to occur.37
And the ultimate fate of most IR cells was apoptosis and
necrosis (Figure S12). The survival rate of IR injury cells was
66.45%, which was quite lower than the 87.54% survival rate of
normal cells. This is consistent with the results of MTT
analysis (Figure S13). We speculated on the signal pathway of
ROS-mtDNA mediated IR injury, which provided a new idea
about the IR injury therapeutic targets (Figure 7).
Figure 5. Mitochondrial colocalization experiment. Confocal
fluorescence images of hepatocytes coincubated with 5 μM
mtDNA-BP and 5 μM MitoTracker Red. (A) mtDNA-BP
fluorescence was collected at 540−630 nm with 514 nm excitation.
(B) MitoTracker Red fluorescence was collected at 660−730 nm with
633 nm excitation. (C) Bright-field imaging. (D) Merged image of A
and B. Scale bar = 10 μm.
1 h. It showed mtDNA damage was aggravated by the
deteriorating IR process and finally destroyed the mtDNA. To
further verify the validity of the fluorescence imaging results,
we performed a quantitative study of mtDNA based on a long
patch PCR-based approach.32,33 As shown in Figure S9, the
amount of healthy mtDNA was significantly reduced in the IR
group compared to the control group. And the degree of DNA
damage increased with the duration of IR. These results
demonstrate that IR injury caused the damage in mtDNA. The
consistency of fluorescence imaging with PCR experimental
results indicate that mtDNA-BP is a powerful tool for revealing
the real-time relationship between IR and mtDNA.
Our previous real-time fluorescence imaging experiment
found that superoxide and peroxynitrite anion were important
regulatory factors in IR injury.34,35 It can be seen that the
abnormal change of ROS at the cellular level is an important
factor for IR damage. Here, we further identified the changes in
total ROS levels in IR mitochondria by employing the ROS
Figure 7. Signaling pathway of ROS-mtDNA-mediated IR injury.
Protective Effects of Drugs on IR Hepatocyte. On the
basis of the experimental results we found above, the mtDNA
might be implemented as a potential therapeutic evaluation
target. Therefore, the drug-screening platform for inhibiting IR
injury had been established based on the mtDNA real-time
response capability of the probe. Promising drugs to treat IR
injury were selected from the known literature.38−46 As shown
in Figure 8, precultured only with pioglitazone (100 μM) or
salidroside (100 μM), IR hepatocytes still emitted bright
fluorescence in accordance with the normal group. Obviously,
these two potential drugs can protect mtDNA. To prove the
validity of the probe-based screening method, we evaluated the
therapeutic effect of selected drugs. We performed flow
Figure 6. Time course of IR injury. (A) The hepatocytes incubated with 5 μM mtDNA-BP suffered ischemia for 20 min and then reperfusion for
40 min. (B) Average fluorescence intensity output of A. The fluorescence images were collected at 540−630 nm with an excitation wavelength of
514 nm. Scale bar = 25 μm.
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Figure 8. Protective effects of drugs on IR hepatocyte. (A) 5 μM mtDNA-BP-labeled HL-7702 cells were preincubated with the drugs for only 24
h; the IR group suffered ischemia for 30 min and reperfusion for 30 min. (B) Average fluorescence intensity output of A. Fluorescence images were
collected at 540−630 nm with an excitation wavelength of 514 nm. Scale bar = 10 μm, P = 0.001.
cytometry analysis to estimate the survival rate of liver cells
(Figure S12). Compared to the IR group (66.45%), the
survival rate of the two drugs preconditioning groups were
significantly higher (pioglitazone 90.20% and salidroside
86.24%). These results clearly demonstrate the rationality
and reliability of the drug platform design based on mtDNA-
BP, and the pioglitazone and salidroside show strong
protective effects for the mtDNA to resist IR injury.
Republic of China; orcid.org/0000-0002-2356-0962;
Email: lip@sdnu.edu.cn
Authors
Wen Zhang − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China
Simin Bi − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China
Jihong Liu − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China; orcid.org/0000-0003-3306-7802
Chunmiao Zhou − College of Chemistry, Chemical
Engineering and Materials Science, Key Laboratory of
Molecular and Nano Probes, Ministry of Education,
Collaborative Innovation Center of Functionalized Probes for
Chemical Imaging in Universities of Shandong, Institutes of
Biomedical Sciences, Shandong Normal University, Jinan
250014, People’s Republic of China
Xin Wang − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China
CONCLUSION
■
In summary, we have developed a DNA-targeted fluorescence
probe mtDNA-BP for in situ real-time detections of mtDNA.
The mtDNA-BP was constructed with a proper molecular size
and pyridine cation displaying turn-on fluorescent responses
with mtDNA. This probe exhibited a suitable combination of
high specificity, exclusive mtDNA targeting, and fast response.
With facilitation of mtDNA-BP, we successfully monitored
mtDNA levels in real time in hepatic IR injury and disclosed
the ROS-mtDNA-mediated IR injury signaling pathway. We
also built the drug-screening method for inhibiting IR injury by
relying on mtDNA-BP, and two potential effective drugs were
screened out. Our probe can be expanded to mtDNA-related
disease and be used to support new drug discovery for those
diseases associated with mtDNA disharmony.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.0c05220.
■
sı
Additional experimental data, including experimental
details, photophysical properties, cytotoxicity, and
characterization (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Bo Tang − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China; orcid.org/0000-0002-8712-7025;
Email: tangb@sdnu.edu.cn
Wei Zhang − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China
Hui Wang − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Ping Li − College of Chemistry, Chemical Engineering and
Materials Science, Key Laboratory of Molecular and Nano
Probes, Ministry of Education, Collaborative Innovation
Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
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https://doi.org/10.1021/acs.analchem.0c05220
Anal. Chem. 2021, 93, 5782−5788

Analytical Chemistry
pubs.acs.org/ac
Article
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1606167.
Universities of Shandong, Institutes of Biomedical Sciences,
Shandong Normal University, Jinan 250014, People’s
Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.0c05220
(21) He, L.; Yang, X.; Xu, K.; Lin, W. Chem. Commun. 2017, 53
(29), 4080−4083.
(22) Wu, Z.; Liu, M.; Liu, Z.; Tian, Y. J. Am. Chem. Soc. 2020, 142
(16), 7532−7541.
Author Contributions
#W.Z. and S.B. contributed equally.
(23) Li, H.; Shi, W.; Li, X.; Hu, Y.; Fang, Y.; Ma, H. J. Am. Chem.
Soc. 2019, 141 (45), 18301−18307.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
(25) Liu, R.; Zhang, L.; Lan, X.; Li, L.; Zhang, T. T.; Sun, J. H.; Du,
G. H. Neuroscience 2011, 176, 408−419.
■
This work was supported by the National Natural Science
Foundation of China (21535004, 91753111, 21675105,
22077075, and 21927811), the Key Research and Develop-
ment Program of Shandong Province (2018YFJH0502), and
National Major Scientific and Technological Special Project for
“ S i g n i fi c a n t N e w D r u g s D e v e l o p m e n t ”
(2017ZX09301030004).
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5788
https://doi.org/10.1021/acs.analchem.0c05220
Anal. Chem. 2021, 93, 5782−5788