Abstract
Posttraumatic neuroinflammation is a key driver of secondary injury
after traumatic brain injury (TBI). Pyroptosis, a proinflammatory form
of programmed cell death, considerably activates strong
neuroinflammation and amplifies the inflammatory response by releasing
inflammatory contents. Therefore, treatments targeting pyroptosis may
have beneficial effects on the treatment of secondary brain damage
after TBI. Here, a cysteine-alanine-glutamine-lysine peptide–modified
β-lactoglobulin (β-LG) nanoparticle was constructed to deliver
disulfiram (DSF), C-β-LG/DSF, to inhibit pyroptosis and decrease
neuroinflammation, thereby preventing TBI-induced secondary injury. In
the post-TBI mice model, C-β-LG/DSF selectively targets the injured
brain, increases DSF accumulation, and extends the time of the systemic
circulation of DSF. C-β-LG/DSF can alleviate brain edema and
inflammatory response, inhibit secondary brain injury, promote
learning, and improve memory recovery in mice after trauma. Therefore,
this study likely provided a potential approach for reducing the
secondary spread of TBI.
__________________________________________________________________
C-β-LG/DSF inhibits pyroptosis and limits the release of inflammatory
cytokines in treating secondary brain damage.
INTRODUCTION
Traumatic brain injury (TBI) is a series of injuries caused by external
trauma to the head, such as intracranial hematoma, which can lead to
disability or even death ([36]1). With the development of the economy
and technology, the incidence, disability, and mortality of TBI are
increasing during transportation, construction, sports injuries, and
war ([37]2). There are more than 50 million patients with TBI worldwide
yearly, and the incidence is increasing by 4.67% annually ([38]3). In
addition to high mortality, patients with severe TBI exhibit varying
degrees of cognitive function and behavioral dysfunction, leading to
lifelong disability, bringing severe economic and health burdens to
families and society ([39]2, [40]4–[41]6). The main reason for the
aforementioned losses is secondary brain injury; however, there is no
effective pharmacotherapy available to prevent the secondary spread of
damage.
TBI is categorized into primary and secondary brain injuries. Secondary
brain injury occurs over a period of hours or days after the primary
injury and involves changes in the brain’s cells, chemicals, tissues,
and blood vessels, which can further damage brain tissue ([42]7–[43]9).
TBI-induced neuroinflammation is the leading cause of secondary brain
injury. Recent studies have highlighted that pyroptosis, a type of
programmed cell death, is a key driver of TBI-induced inflammation and
plays an important role in the pathogenesis of TBI injury
([44]10–[45]12). This process causes cells to swell and eventually
burst, releasing inflammatory cytokines [such as interleukin-18 (IL-18)
and IL-1β] and triggering a strong inflammatory response
([46]13–[47]15). Many studies have speculated that inhibiting
TBI-induced pyroptosis could be an effective way of treating TBI
([48]16–[49]18). However, effective clinical therapeutic strategies for
pyroptosis remain scarce. To date, there are scant data regarding the
regulation of pyroptosis during the acute phase of TBI.
Disulfiram (DSF), a Food and Drug Administration (FDA)–approved drug
for the treatment of alcohol addiction, has been found to inhibit
pyroptosis by blocking gasdermin D (GSDMD) pore formation ([50]19).
However, insufficient drug targeting and retention in the brain injury
area have led to the failure of drugs used for TBI therapies.
Therefore, there is an urgent need to develop a delivery platform that
can target and enhance drug accumulation in the brain injury area.
Here, we developed a cysteine-alanine-glutamine-lysine (CAQK)
peptide–modified β-lactoglobulin (β-LG) nanoparticle to deliver DSF
(C-β-LG/DSF) for TBI therapy ([51]Fig. 1). C-β-LG/DSF represents a
potential approach to inhibit pyroptosis and limit the release of
inflammatory cytokines in treating secondary brain damage. C-β-LG/DSF
consists of two components with different functions: (i) C-β-LG, a
modified CAQK peptide, can penetrate the blood–brain barrier (BBB) to
accumulate in the brain injury area, which increases the therapeutic
concentration of DSF. (ii) DSF inhibits pyroptosis to block the release
of proinflammatory factors by eliminating the formation of GSDMD-N
plasma membrane pores. Therefore, C-β-LG/DSF effectively inhibits
pyroptosis and exerts a neuroprotective effect on the TBI mice model.
Our findings open an exciting angle to inhibit pyroptosis by blocking
the release of inflammatory cytokines as a potential therapeutic
strategy for TBI.
Fig. 1. Schematic of C-β-LG/DSF for TBI treatment.
[52]Fig. 1.
[53]Open in a new tab
(A) Schematic representation of the preparation process of C-β-LG/DSF.
(B) After TBI, without C-β-LG/DSF treatment, damage-related molecular
patterns (DAMPs) increase, which activate the inflammasome by binding
to pattern recognition receptors (PRRs) to trigger pyroptosis, leading
to the release of inflammatory cytokines (such as IL-18 and IL-1β),
which triggers severe neuroinflammation and exacerbates secondary
injury. (C) After TBI, C-β-LG/DSF treatment is given. DSF inhibits
pyroptosis by blocking the formation of GSDMD-N pores on the cell
membrane. Furthermore, it can also inhibit the release of inflammatory
cytokines, thereby reducing neuroinflammation and alleviating secondary
brain injury.
RESULTS
Preparation and characterization of C-β-LG/DSF
The β-LG/DSF was prepared using the reverse solvent precipitation
method. The CAQK peptide selectively binds to a proteoglycan complex
that is up-regulated during brain injuries and coupled with drug
molecules and nanoparticles to increase their accumulation at the
injury site ([54]20, [55]21). Thus, the CAQK peptide was further
modified into β-LG/DSF to form C-β-LG/DSF and increase the DSF
concentration at the injury site. The diameters of C-β-LG/DSF and
β-LG/DSF were 156.54 ± 4.52 nm and 144.91 ± 2.21 nm, respectively,
which was consistent with the particle size shown by transmission
electron microscopy (TEM) ([56]Fig. 2, A and B). The zeta potentials of
C-β-LG/DSF and β-LG/DSF were −28.15 ± 7.93 mV and −19.18 ± 9.13 mV,
respectively ([57]Fig. 2C). Scanning electron microscopy (SEM) showed
that C-β-LG/DSF ([58]Fig. 2D) and β-LG/DSF ([59]Fig. 2E) were spherical
with a uniform size distribution. The DSF-loading capacity and
encapsulation efficiency of C-β-LG/DSF were 44.80 ± 1.14% and 90.25 ±
2.28%, respectively. The stability of C-β-LG/DSF in the blood
circulation was assessed by conducting serum stability tests. The
results showed that C-β-LG/DSF and β-LG/DSF remained stable in 10%
fetal bovine serum–containing phosphate-buffered saline (PBS) for 7
days without obvious changes in hydrodynamic diameter ([60]Fig. 2F).
β-LG has been widely used in the development of drug delivery systems,
due to its excellent biocompatibility ([61]22, [62]23). Nanocarriers
using β-LG as a shell have desirable attributes of safety, stability,
and biodegradability ([63]24). To simulate a realistic traumatic
environment, cerebrospinal fluid (CSF) samples were collected from
patients with brain trauma for drug release experiments. The release of
DSF from C-β-LG/DSF and β-LG/DSF was evaluated after incubation in
TBI-CSF and PBS (pH 7.4), respectively. The results indicated that
29.16 ± 3.54% and 73.89 ± 2.03% of DSF were released from C-β-LG/DSF in
PBS and TBI-CSF after 24 hours, respectively ([64]Fig. 2G). These
results demonstrated that C-β-LG/DSF was responsive to the post-TBI
environment and could release DSF rapidly. The hydrodynamic diameters
C-β-LG/DSF and β-LG/DSF under TBI-CSF conditions were observed to
increase ([65]Fig. 2H). These results demonstrated that C-β-LG/DSF was
successfully prepared and was degraded in TBI-CSF to release DSF.
Fig. 2. Preparation and characterization of C-β-LG/DSF.
[66]Fig. 2.
[67]Open in a new tab
(A) Size distribution and TEM image of C-β-LG/DSF. (B) Size
distribution and TEM image of β-LG/DSF. (C) The zeta potential of
C-β-LG/DSF and β-LG/DSF. (D) SEM image of C-β-LG/DSF. Scale bar, 200
nm. (E) SEM image of β-LG/DSF. Scale bar, 200 nm. (F) Serum stability
of C-β-LG/DSF and β-LG/DSF. (G) In vitro drug release profiles under
TBI-CSF or pH 7.4 PBS conditions. (H) Size distribution of C-β-LG/DSF
and β-LG/DSF at TBI-CSF at 12 hours. Data were expressed as means ± SD,
(n = 3).
Targeted delivery of C-β-LG/DSF to the TBI site
To validate the selectively targeted ability of CAQK-modified β-LG/DSF,
fluorescein isothiocyanate (FITC)–labeled β-LG was synthesized to
prepare C-β-LG/DSF/FITC nanoparticles. Neuron-ICR (Institute of Cancer
Research) cells, used in cell experiments, are primary neurons
extracted from the frontal cortex tissue of the brain of ICR mouse
embryos with a gestational age of 18 to 19 days ([68]Fig. 3A). First,
the neuron-ICR cell uptake and the intracellular fate of
C-β-LG/DSF/FITC were evaluated. Endocytosis inhibitors [chlorpromazine
hydrochloride, 5-(n-ethyl-n-isopropyl) amiloride (EIPA), genistein,
methyl-β-cyclodextrin (mβCD), and dynasore] were incubated with neurons
([69]25). Chlorpromazine hydrochloride was used to inhibit
clathrin-mediated endocytosis ([70]26). mβCD and genistein inhibit
caveolae-mediated endocytosis by disrupting specific signaling pathways
and lipid composition, respectively ([71]27). EIPA is an inhibitor of
micropinocytosis by inhibiting the function of the Na^+/H^+ exchanger
([72]28). Dynasore is an effective inhibitor that targets
dynamin-dependent endocytic pathways ([73]29). As shown in [74]Fig. 3B,
for C-β-LG/DSF, ~75% reduction in the uptake level was observed at
mβCD. Genistein (~65%), EIPA (~27%), and dynasore (~14%) inhibited the
neuron cell uptake process. Furthermore, the cell uptake level was
decreased in the presence of mβCD and dynasore, which inhibited both
the clathrin- and caveolar-mediated pathways. Collectively, the data
indicated that C-β-LG/DSF entered the neuron-ICR cell mainly via
caveolae-mediated endocytosis, clathrin-mediated endocytosis, and
micropinocytosis.
Fig. 3. The C-β-LG/DSF targeted delivery DSF into TBI site.
[75]Fig. 3.
[76]Open in a new tab
(A) Scheme of primary neuron extraction. (B) Flow cytometry was used to
assess the levels of C-β-LG/DSF uptake by neuron-ICR cells after
treatment with various inhibitors. (C) The CLSM images were performed
to test the ability of C-β-LG/DSF/FITC to penetrate BBB and target
neurons. Red: Dil-labeled BBB layer cell membrane; blue: DAPI-labeled
nuclei; green: C-β-LG/DSF/FITC (FITC signal). Scale bar, 25 μm. (D)
Penetration of β-LG/DSF/FITC and C-β-LG/DSF/FITC through the cell
monolayer. (E) Fluorescent image of β-LG/DSF/Cy5.5 and C-β-LG/DSF/Cy5.5
brain distribution in vivo was observed with small animal imaging
system at post-injection 6, 12, 24, and 48 hours. (F) Statistical
analysis of fluorescence content of C-β-LG/DSF in mice brains with
imaging in vivo. (G) Statistical analysis of the FITC fluorescence
intensity of C-β-LG/DSF/FITC in mice brains with a microplate reader.
(H) Statistical analysis of the DSF concentration of C-β-LG/DSF/FITC in
mice brains with UPLC-MS/MS. (I) Blood retention kinetics of free DSF
and C-β-LG/DSF (DSF concentration of 10 mg/kg) in ICR mice. Data were
expressed as means ± SD (n = 3). For (B), statistical analysis was
calculated via one-way ANOVA test. **P < 0.01, **** P < 0.0001, and ns
P > 0.05. For (D), (F), (G), and (H), statistical analysis was
calculated via Student’s t test. *P < 0.05, **P < 0.01, and ***P <
0.001.
The BBB is a complex structure that comprises not only the tight
junctions between endothelial cells but also the presence of other
cells that form the neurovascular unit and contribute to the tight
regulation of the BBB. The effective penetration of drugs through the
BBB is crucial for the treatment of TBI. An in vitro BBB model was
established using a Transwell system to verify that C-β-LG/DSF could
penetrate the BBB and precisely target traumatized neurons. Human brain
microvascular endothelial cells were cultured at the bottom of the
upper chamber, and neuron-ICR cells were cultured at the bottom of the
lower chamber. When the transendothelial electrical resistance reached
150 ohms•cm^2, the in vitro BBB model was successfully prepared. A
neuron-ICR cell TBI model was prepared in the lower chamber of the
Transwell system through scratch-induced injury. C-β-LG/DSF/FITC and
β-LG/DSF/FITC were separately added to the upper chamber of the
Transwell system, and their cellular uptake in the upper and lower
chambers was observed using confocal laser scanning microscopy (CLSM).
The intensity of FITC fluorescence was detected using an enzyme-linked
immunosorbent assay (ELISA) reader after 4 hours of incubation. The
mean fluorescence intensity of FITC in C-β-LG/DSF/FITC–treated
neuron-ICR cells was significantly higher than β-LG/DSF/FITC–treated
cells, with a penetration rate of 69.26 ± 5.08% versus 17.06 ± 1.59%,
suggesting that CAQK modification effectively improves the BBB
penetration and targeting ability of β-LG/DSF ([77]Fig. 3, C and D).
According to previous literature research, CAQK is a tetrapeptide that
targets the chondroitin sulfate proteoglycan at the injury site
following TBI and carry drugs to the brain injury site ([78]20,
[79]21). However, the in vitro BBB model has limitations and cannot
fully replicate the BBB’s complexity and functionality in real time,
particularly in cases where the BBB has been disrupted after TBI.
Therefore, to assess the targeting efficacy, cyanine5.5
(Cy5.5)–conjugated C-β-LG/DSF was injected into TBI-bearing mice via
the tail vein. The C-β-LG/DSF/Cy5.5 group yielded a higher intensity of
Cy5.5 fluorescence at the brain injury site compared with that yielded
by the β-LG/DSF/Cy5.5 group at 6, 12, 24, and 48 hours after injection
owing to the targeting effect of CAQK and the enhanced permeability and
retention effect ([80]Fig. 3, E and F). Notably, at 6 hours after
injection, notable accumulation of C-β-LG/DSF/Cy5.5 at the brain injury
site was observed. To further assess the targeting efficacy, we
prepared FITC-conjugated C-β-LG/DSF (C-β-LG/DSF/FITC) and measured the
FITC intensity and DSF concentration in the brain following injection
of C-β-LG/DSF/FITC and β-LG/DSF/FITC injection. Six hours after
injection, the FITC intensity and DSF concentration were observed to be
higher compared to 12, 24, and 48 hours after injection ([81]Fig. 3, G
and H). Furthermore, the distribution of C-β-LG/DSF/Cy5.5 in the heart,
liver, spleen, lung, and kidneys was examined at 6, 12, 24, and 48
hours after injection (figs. S1 to S3). Both C-β-LG/DSF/Cy5.5 and
β-LG/DSF/Cy5.5 accumulated mostly in the liver, lungs, and kidneys at 6
and 12 hours after injection. After 24 hours of injection, both
C-β-LG/DSF/Cy5.5 and β-LG/DSF/Cy5.5 demonstrated high accumulation in
the liver and kidneys, and at 48 hours, only few C-β-LG/DSF/Cy5.5 and
β-LG/DSF/Cy5.5 were found in the liver and kidneys (fig. S1, A and B).
The quantification of FITC content further confirmed the predominant
accumulation of C-β-LG/DSF/FITC and β-LG/DSF/FITC in the liver and
kidneys after 24 hours of injection (fig. S2). In addition, we
extracted DSF from the tissues after C-β-LG/DSF/FITC and β-LG/DSF/FITC
injection and found that DSF was also concentrated in the liver and
kidneys after 24 hours of injection, consistent with the Cy5.5 and FITC
fluorescence data (fig. S3). Together, these results suggested that the
C-β-LG/DSF nanoparticles are cleared by the liver and kidneys. The DSF
plasma levels showed rapid clearance from the blood, with a half-life
(t[1/2]) of ~4.83 hours ([82]Fig. 3I). The half-life (t[1/2]) of
C-β-LG/DSF/Cy5.5 was ~8.28 hours. Therefore, C-β-LG/DSF prolonged the
retention time of DSF in the blood, providing a greater chance of
accumulation at the injury site.
Inhibitory effect of C-β-LG/DSF on pyroptosis after TBI
TBI-induced pyroptosis is a type of cell death triggered by
proinflammatory signals and associated with inflammation, which occurs
after TBI. We established an in vitro TBI model using neuron-ICR cells
through scratch-induced injury to investigate the inhibitory effect of
DSF on pyroptosis caused by TBI. First, the toxicity of DSF to glial
cells and neuron-ICR cells was tested through the Cell Counting Kit-8
assays. As shown in figs. S4 and S5, there was no apparent toxicity
within the 0.1 to 0.5 μM range of DSF in free or nanoparticle states.
Drugs are trapped in endosomal vesicles and degraded in the lysosomal
lumen, which leads to failure in the treatment of diseases. Therefore,
the endosomal escape ability of C-β-LG/DSF/FITC was tested. As shown in
fig. S6 (A to C), the separation between the green and red fluorescence
spots was more pronounced, and the colocalization ratio of labeled
endosomes/lysosome and C-β-LG/DSF/FITC was 40.67 ± 5.69% at 8 hours,
indicating that C-β-LG/DSF/FITC could effectively escape from the
endosomes for ensuring the therapeutic efficacy of DSF. Caspase
cleavage of GSDMD, the executioner of pyroptosis, liberates an
N-terminal p30 fragment (GSDMD-N), which oligomerizes and forms pores
in the plasma membrane. These pores serve as a conduit for the release
of IL-1β and IL-18 and, ultimately, the demise of the cell ([83]30).
However, DSF can prevent pyroptosis by inhibiting the formation of
GSDMD N-terminal pores on the cell membrane surface, which stops the
release of inflammatory cytokines ([84]Fig. 4A). As shown in [85]Fig.
4B, the results demonstrated that the expression of GSDMD, GSDMD-N, and
caspase-1 p10 (key indicators of cell pyroptosis) in neuron-ICR cells
significantly increased after scratch-induced injury compared to the
sham group, suggesting that neuron-ICR cells undergo pyroptosis. DSF
and C-β-LG/DSF substantially inhibited the expression of GSDMD,
GSDMD-N, caspase-1, caspase-1p20, and caspase-1 p10 (fig. S7, A to E,
and [86]Fig. 4B). The extracellular release of IL-1β, IL-18, and
lactate dehydrogenase (LDH) increased in neuron-ICR cells after
scratch-induced injury while decreasing after DSF and C-β-LG/DSF
treatment, suggesting that DSF effectively inhibited the occurrence of
pyroptosis after injury ([87]Fig. 4, C to E). The immunohistochemical
test results of GSDMD in TBI mice treated with C-β-LG/DSF further
confirmed that GSDMD expression was obviously decreased compared with
that in other treatment groups ([88]Fig. 4, F and G). When pyroptosis
occurs, pores form in the cell membrane and cell lysis occurs,
releasing a large amount of cytoplasmic content, triggering an
inflammatory response, and further exacerbating secondary damage.
Therefore, to verify whether C-β-LG/DSF can inhibit pyroptosis-mediated
inflammation, ELISA was performed to detect the release of IL-1β,
IL-18, and LDH from brain tissue on the third day after TBI ([89]Fig.
4, H to J). The results revealed that compared with the sham group, the
release of IL-1β, IL-18, and LDH in the brain of the TBI group
significantly increased, further indicating that cell pyroptosis occurs
in brain tissue following TBI and released a large number of
inflammatory factors, leading to secondary damage to brain tissue.
Compared with the TBI and DSF groups, the release of these inflammatory
factors was remarkably reduced in the C-β-LG/DSF groups. Thus,
collectively, C-β-LG/DSF can substantially inhibit pyroptosis
occurrence following TBI. To further confirm pyroptosis production in
mice following TBI, brain tissue from the trauma site was collected for
Western blot analysis. Compared with the sham group, the expression of
GSDMD, GSDMD-N, caspase-1 p20, and caspase-1 p10 in the brain of TBI
mice significantly increased, confirming that cell pyroptosis occurs in
brain tissue following TBI ([90]Fig. 4K). Moreover, compared with the
TBI and DSF groups, the expression levels of the GSDMD, GSDMD-N,
caspase-1 p20, and caspase-1 p10 proteins were significantly reduced in
the C-β-LG/DSF groups (fig. S8, A to E), indicating that DSF inhibited
pyroptosis following TBI.
Fig. 4. Inhibitory effect of C-β-LG/DSF on pyroptosis after TBI.
[91]Fig. 4.
[92]Open in a new tab
(A) The schematic diagram illustrates the inhibition of pyroptosis by
DSF. After the uptake of C-β-LG/DSF by cells, DSF inhibits the
aggregation of GSDMD N-terminal pores on the cell membrane surface.
Furthermore, experimental results demonstrated that the expression of
GSDMD, GSDMD-N, caspase-1, and other related proteins was reduced after
treatment with C-β-LG/DSF. (B) Immunoblotting of GSDMD, GSDMD-N, and
caspase-1, caspase-1 p10, caspase-1 p20 in neuron-ICR cells with sham,
TBI, DSF, and C-β-LG/DSF for 24 hours. (C to E) Levels of (C) IL-1β,
(D) IL-18, and (E) LDH in injured neuron-ICR as detected by ELISA 12
hours after TBI. (F) Immunohistochemical staining was used to observe
the expression of GSDMD in injured tissues of TBI model mice 3 days
after different drugs treatment. Scale bars, 50 μm. (G) Quantitative
analysis of GSDMD-positive cells. (H to J) Levels of (H) IL-1β, (I)
IL-18, and (J) LDH in injured tissues as detected by ELISA 3 days after
TBI. (K) Immunoblotting of GSDMD, GSDMD-N, caspase-1, caspase-1 p20,
and caspase-1 p10 in injured tissue with sham, TBI, DSF, β-LG/DSF, and
C-β-LG/DSF for 24 hours. Data were expressed as means ± SD (n = 5). For
(C) to (E) and (G) to (J), statistical analysis was calculated via
one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P <
0.0001.
Effects of C-β-LG/DSF on BBB permeability and cerebral edema after TBI
After TBI, the BBB is damaged, resulting in increased permeability and
severe brain edema ([93]31–[94]33). Previous studies have demonstrated
that the posttraumatic inflammation cascade promotes TBI-induced BBB
disruption ([95]10). Therefore, we performed several tests to determine
whether C-β-LG/DSF can effectively inhibit BBB disruption. The diagram
and schedule are shown in [96]Fig. 5A. C-β-LG/DSF (6.64 ± 1.13 μg/g)
significantly reduced the Evans blue content per gram of brain tissue
compared with other treatments (DSF: 23.04 ± 1.51 μg/g; β-LG/DSF: 12.61
± 0.98 μg/g), suggesting that inhibiting pyroptosis in brain tissue
following TBI can effectively alleviate brain edema ([97]Fig. 5, B and
C). The brain tissue water content assay further confirmed that
C-β-LG/DSF-treated TBI mice exhibited the lowest water content
([98]Fig. 5D). To further investigate the ability of C-β-LG/DSF to
improve secondary injuries following TBI in mice, brain tissue edema
was examined using magnetic resonance imaging (MRI) with T2-weighted
images on the third day after TBI ([99]Fig. 5E, a). Brain tissue edema
was evident in the right hemisphere surrounding the TBI and DSF groups,
with volumes of 15.56 ± 1.22 mm^3 and 14.92 ± 0.96 mm^3, respectively.
However, the brain tissue edema area was noticeably reduced in the
β-LG/DSF (10.92 ± 1.33 mm^3) and C-β-LG/DSF groups (7.32 ± 1.06 mm^3).
Moreover, TBI mice treated with C-β-LG/DSF exhibited significantly less
brain edema than in mice treated with other treatments, with a
decreased volume of 7.32 ± 1.06 mm^3 ([100]Fig. 5F). The apparent
diffusion coefficient (ADC) value obtained from diffusion-weighted
imaging (DWI) was further used to evaluate the dispersion of water
molecules on the surface of the cerebral cortex to determine the type
and trend of brain tissue edema ([101]Fig. 5E, b). Lower ADC values are
generally associated with cytotoxic brain edema, which reflects the
gradual occurrence and intensification of cytotoxic edema following the
release of neurotoxic substances. The ADC value of DWI in the TBI group
was 0.29 ± 0.05 × 10^−3 mm^2/s, which was considerably lower than that
in the sham group (0.90 ± 0.06 × 10^−3 mm^2/s), indicating that
cytotoxic brain edema was dominant on day 3 following TBI in mice. The
ADC values in the β-LG/DSF and C-β-LG/DSF groups were significantly
higher than those in the TBI group, with ADC values of 0.64 ± 0.05 ×
10^−3 and 0.77 ± 0.05 × 10^−3 mm^2/s, respectively ([102]Fig. 5G),
indicating a specific therapeutic effect of β-LG/DSF and C-β-LG/DSF in
alleviating cytotoxic brain edema in mice, consistent with the trend of
brain edema volume shown in the T2-weighted images.
Fig. 5. Effects of C-β-LG/DSF on BBB permeability and cerebral edema after
TBI.
[103]Fig. 5.
[104]Open in a new tab
(A) Schematic diagram of TBI model experiments. (B) Images of Evans
blue leakage from brain capillary vessels in the right brain of mice
for each group 24 hours after TBI. (C) Quantitative analysis of Evans
blue leakage. (D) Water content in brain tissue of each group. (E)
Representative (a) T2-weighted and (b) DWI MRI images in each group 3
days after TBI. (F) Quantification of the hyperintensity volume around
the injured tissue 3 days after TBI. (G) Quantification of the ADC
signal from the injured tissue 3 days after TBI. For (B) to (D), data
were expressed as means ± SD (n = 5). For (E) to (G), data were
expressed as means ± SD (n = 6). For (C), (D), (F), and (G),
statistical analysis was calculated via one-way ANOVA test. *P < 0.05,
**P < 0.01, ***P < 0.001, and ****P < 0.0001.
Anti-inflammatory effect of C-β-LG/DSF
Pyroptosis can activate strong inflammation and immune responses. To
further validate the neuroprotective effect of C-β-LG/DSF, we assessed
neuronal necrosis, apoptosis, and brain tissue damage in mice on day 7
after TBI. Hematoxylin and eosin (H&E) staining revealed significantly
reduced brain tissue damage in the C-β-LG/DSF group compared with the
other treatments ([105]Fig. 6, A and B). Furthermore, Nissl staining
revealed an obvious decrease in neuronal survival rate in mice
following TBI compared with the sham group, with positive staining cell
numbers of 121.8 ± 11.32 and 259.8 ± 34.57, respectively. Compared with
the other treatments, C-β-LG/DSF treatment significantly increased
neuronal survival with a positive staining cell number of 215.8 ± 22.9
([106]Fig. 6, C to E). To assess the level of neuroinflammation in
brain tissue after TBI, we assessed the expression of glial fibrillary
acidic protein (GFAP) and ionized calcium-binding adaptor-1 (Iba-1),
which are indices of astrocyte and microglia activation, respectively.
After brain tissue damage, microglia and astrocytes are rapidly
activated with swollen and branched cells. Microglia are resident
immune cells in the central nervous system with sensing, caretaking,
and defensive functions. In neurodegenerative diseases, microglia
dysfunction exacerbates neuronal damage, while numerous inflammatory
factors are released in activated astrocytes. Therefore, we examined
the expression of cells through immunohistochemistry to determine the
level of neuroinflammation in brain tissue after TBI ([107]Fig. 6, F
and I). Furthermore, the hippocampus is closely related to learning and
spatial memory, while its degeneration is closely related to TBI
prognosis. We found that GFAP- and Iba-1–positive cells were
substantially reduced in the cortex surrounding the brain injury area
([108]Fig. 6, G and J) and the hippocampus ([109]Fig. 6, H and K) in
the C-β-LG/DSF groups on day 7 after TBI. These results suggest that
C-β-LG/DSF reduces inflammation by inhibiting pyroptosis.
Fig. 6. Anti-inflammatory effect of C-β-LG/DSF.
[110]Fig. 6.
[111]Open in a new tab
(A) Representative H&E staining of brain tissue 7 days after TBI. (B)
Quantitative analysis of injury area ratio of H&E staining. (C)
Representative images of Nissl-positive cells 7 days after TBI. (D)
Enlarged image of Nissl staining, Scale bar, 50 μm. (E) Quantitative
analysis of Nissl-positive cells. (F) Immunohistochemical staining was
used to observe the expression of GFAP in the damaged cortex area of
each group of TBI model mice 7 days after different drug treatment.
Scale bars, 50 μm. (G) Quantitative analysis of GFAP-positive cells in
the damaged cortex area of each group of TBI model mice 7 days after
different drug treatment. (H) Quantitative analysis of GFAP-positive
cells in the hippocampus ipsilateral of each group of TBI model mice 7
days after different drug treatment. (I) Immunohistochemical staining
was used to observe the expression of Iba-1 in the damaged cortex area
of each group of TBI model mice 7 days after different drug treatment.
Scale bar, 50 μm. (J) Quantitative analysis of Iba-1–positive cells in
the damaged cortex area of each group of TBI model mice 7 days after
different drug treatment. (K) Quantitative analysis of Iba-1–positive
cells in the hippocampus ipsilateral of each group of TBI model mice 7
days after different drug treatment. Data were expressed as means ± SD
(n = 5). For (B), (E), (G), (H), (J), and (K), statistical analysis was
calculated via one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001,
and ****P < 0.0001.
RNA-seq analysis of inflammasome-mediated inflammation changes following
C-β-LG/DSF treatment
TBI is a critical public health and socioeconomic concern worldwide.
Inflammation-induced secondary injury is a vital pathogenic parameter
of TBI. The molecular signaling cascades of pyroptosis, a specific type
of cellular necrosis, are key drivers of TBI-induced inflammation.
RNA-sequencing (RNA-seq) analysis revealed significant up-regulation of
GSDMD, CASP1, and other inflammasome-related genes in the TBI group
compared with the sham group ([112]Fig. 7A). However, following
C-β-LG/DSF treatment, compared with the TBI group, the expression of
pyroptosis-related genes, such as GSDMD, CASP1, and NOD-like receptor
thermal protein domain associated protein 3 (NLRP3), was significantly
down-regulated ([113]Fig. 7B). We also mapped a gene heatmap of 17
genes closely related to pyroptosis in brain tissue after TBI, and the
results were consistent with those of the volcanic map ([114]Fig. 7C).
The expression levels of CASP1, nucleotide-binding oligomerization
domain (NOD), NLRP3, and GSDMD increased after TBI but decreased after
C-β-LG/DSF treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis revealed notable activation of apoptosis,
necroptosis, NOD-like receptor, and Toll-like receptor signaling
pathways after TBI. Among them, the NOD-like and Toll-like receptor
signaling pathways were closely related to pyroptosis and involved in
its occurrence. The significant activation of the inflammatory pathway
through leukocyte transendothelial migration suggests that concurrent
with pyroptosis, inflammatory cells infiltrate the damaged brain
tissue, leading to further secondary damage ([115]Fig. 7D) (P < 0.05).
In addition, Gene Ontology (GO) analysis revealed that pore formation
on the cell membrane and the expression of IL-1, IL-1β, and NLRP3
inflammasome complex, which are closely related to pyroptosis, were
obviously up-regulated alongside inflammation-related immune receptors
after TBI ([116]Fig. 7E) (P < 0.05). However, compared with the TBI
group, pore formation on the cell membrane, pyroptosis-related cell
death, leukocyte migration and proliferation, and inflammasome complex
were significantly down-regulated following C-β-LG/DSF treatment
([117]Fig. 7F) (P < 0.05). Collectively, these results suggest that
C-β-LG/DSF can regulate inflammatory responses.
Fig. 7. RNA-seq analysis reveals that C-β-LG/DSF inhibits
inflammasome-mediated inflammation following TBI.
[118]Fig. 7.
[119]Open in a new tab
(A) Analysis of differences in expression of pyroptosis-related genes
between the TBI group and the sham group (n = 3). (B) Analysis of
differences in expression of pyroptosis-related genes between the TBI
group and the C-β-LG/DSF group (n = 3). (C) Sham, TBI, and C-β-LG/DSF
groups’ gene heatmaps (n = 3). (D) Enrichment analysis of KEGG pathway
in the TBI group versus the sham group (n = 3). (E) GO analysis in the
TBI group versus the sham group (n = 3). (F) GO analysis in the
C-β-LG/DSF group versus the TBI group (n = 3).
Effect of C-β-LG/DSF on neural function recovery after TBI in mice
We conducted a Morris water maze (MWM) test on mice to evaluate their
learning and spatial memory abilities after TBI. The test was conducted
from day 21 to day 29 after injury, with the recovery of learning
ability assessed during the first 7 days and the recovery of spatial
memory tested on day 8 and day 9. A schematic timeline of experiments
is displayed in [120]Fig. 8A. Mice treated with C-β-LG/DSF exhibited
significantly better recovery of learning and spatial memory abilities
than those treated with the other treatments ([121]Fig. 8B). During the
first 4 days, the average swimming distance of the sham group mice was
shorter than that of the TBI and other experimental group mice
([122]Fig. 8B). Compared with the C-β-LG/DSF group, the TBI and DSF
groups demonstrated significantly longer latency period and swimming
distances before reaching the platform ([123]Fig. 8C) (P < 0.05). On
day 28 after TBI, the 8th day of the water maze test, the memory of the
mice was evaluated by removing the platform and measuring the time
spent swimming in the quadrant where the platform used to be located as
well as determining the number of crossings over the platform area.
Compared with the TBI group, mice treated with C-β-LG/DSF spent
significantly more time and traveled longer distances within the target
quadrant ([124]Fig. 8, D and E), exhibited increased frequency of
platform crossings ([125]Fig. 8F), and stayed for a longer duration in
the target quadrant ([126]Fig. 8G) (P < 0.05), indicating more
purposeful searching for the platform. These results suggest that
C-β-LG/DSF treatment promotes spatial memory and learning ability
recovery in mice following TBI. We evaluated the recovery of motor
function in mice treated with C-β-LG/DSF using a wire suspension test
([127]Fig. 8H). Compared with the sham group, mice in all the other
groups exhibited significantly longer hanging times. In addition,
compared with the TBI group, mice treated with C-β-LG/DSF demonstrated
obviously longer hanging times on the wire (P < 0.05), indicating
improved motor function recovery. To assess the recovery of
neurological function in mice following TBI, we used the modified
neurological severity score (mNSS) to evaluate mice on days 3, 7, and
14 after the injuries using a blinded scoring method. Mice treated with
C-β-LG/DSF exhibited significantly improved neurological function
compared with those in the TBI group, whereas mice treated with DSF did
not show significant improvement ([128]Fig. 8I). These results further
demonstrate that C-β-LG/DSF promotes neurological function recovery in
mice following TBI by inhibiting neuronal pyroptosis, preventing
further neuroinflammation, and notably alleviating secondary brain
injury.
Fig. 8. Effect of C-β-LG/DSF on neural function recovery after TBI in mice.
[129]Fig. 8.
[130]Open in a new tab
(A) mNSS motor function score, MWM, and suspension test analysis
schedule. (B) Computer printouts of the swimming trajectories during
the learning phase and memory phase. (C) Swimming distance to the
platform and (D) searching time for the platform at the last trial for
each of the 7 days of training. During the 2-day memory experiment, (E)
the duration and (F) distance of swimming in the platform area of each
group of mice. (G) Comparison of crossing platform times among
different groups in the memory stage. a.u., arbitrary units. (H)
Comparison of the duration of stay in the target quadrant among
different groups in the memory stage. (I) Motor function was evaluated
by the hanging wire grip test 14 days after TBI. (J) The mNSS scores
were calculated 3, 7, and 14 days after TBI. Data were expressed as
means ± SD (n = 5). For (E) to (I), statistical analysis was calculated
via one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P
< 0.0001.
In vivo compatibility of C-β-LG/DSF
To verify whether C-β-LG/DSF had toxic side effects in mice, we
administered consecutive doses to mice for 4 days and monitored their
body weight every 2 days after TBI. The weight curve of the mice did
not exhibit any substantial decline in body weight (fig. S9). In
addition, the mice were euthanized in each group 21 days after TBI and
administration and the major organs were taken out for tissue paraffin
sectioning and H&E staining. No obvious tissue damage or morphological
changes were observed in the heart, liver, spleen, lungs, and kidneys
tissue sections of the β-LG/DSF and C-β-LG/DSF groups compared with the
sham group, indicating good biocompatibility and high safety in mice
(fig. S10). The routine hematological examination and biochemical tests
were also performed by taking blood samples via the tail vein on the
7th day after administration. The results showed no significant
difference in the hematology markers and serum alkaline phosphatase,
alanine aminotransferase, aspartate transaminase, creatinine, and blood
urea nitrogen levels between the C-β-LG/DSF groups and the sham group
(P > 0.05). Therefore, C-β-LG/DSF is not harmful to the liver and
kidneys of mice and is biologically safe (fig. S11).
DISCUSSION
With increasing use of transportation and rapid development of the
construction industry owing to socioeconomic growth, TBI has become a
frequent occurrence, leading to a significant economic burden for
patients’ families and society ([131]2, [132]4–[133]6). TBI is one of
the leading causes of disability and death among young adults
worldwide, with long-term effects on the survivors’ quality of life
([134]34–[135]36). The pathological mechanisms underlying TBI are
complex and often accompanied by secondary brain injury, which can
result in more severe consequences such as inflammation and neural
damage. However, currently, there is no effective treatment for
secondary brain injury in clinical practice ([136]37–[137]39).
Pyroptosis is a specific type of cellular necrosis that is a key driver
of inflammation in TBI ([138]40–[139]42) and can be activated by
damage-associated molecular patterns and pathogen-associated molecular
patterns recognized by Toll-like receptors and NOD-like receptors on
the membranes of neurons and glial cells, which activates
inflammasomes, cleaves GSDMD proteins, forms pores on the cell membrane
surface, promotes the release of cell contents into the intracellular
environment, and causes severe inflammation and cytotoxic edema,
thereby exacerbating the inflammatory response and brain tissue edema
of neurons and other brain tissue cells ([140]43–[141]46). Our study
showed that the expression of GSDMD, GSDMD-N, caspase-1 p20, and
caspase-1 p10 increased and the levels of IL-1β, IL-18, and LDH
improved in the brain of TBI mice, suggesting that cell pyroptosis
occurred in brain tissue following TBI ([142]Fig. 4). Furthermore,
RNA-seq analysis revealed notable up-regulation of GSDMD, caspase-1,
IL-1β, IL-18, and other inflammasome-related genes in TBI mice
([143]Fig. 7A). The outcomes of our study suggest that pyroptosis
increases in the damaged brain following TBI, consistent with the
results of numerous studies ([144]30, [145]47, [146]48). Therefore,
inhibiting pyroptosis in neurons and glial cells following TBI is a
potential target for treating secondary brain injury. However,
effective clinical therapeutic strategies for pyroptosis remain scarce.
In addition, there is debate over the optimal time window for
inflammation treatment following TBI. The therapeutic time window is
complex, as TBI is both acute and chronic ([147]49). For acute brain
injuries, the optimal treatment time window is typically within a few
hours after the injury. For subacute and chronic brain injuries, the
optimal treatment time window may be longer, depending on the type and
severity of the injury in specific cases. It is generally recommended
to initiate treatment within 24 hours of the trauma to minimize
subsequent damage and promote recovery. Early intervention can help
control the inflammatory response caused by the injury, thereby
reducing secondary damage ([148]50). However, initiating treatment too
early may interfere with the body’s own stress response and repair
processes ([149]51).
DSF is an FDA-approved drug for alcohol addiction and can inhibit
pyroptosis by blocking GSDMD pore formation. However, DSF is rapidly
metabolized, hydrophobic, and less stable, which limits its systemic
application. Therefore, there is an imperative demand to develop an
effective delivery system of DSF for treating TBI. Here, we fabricated
CAQK peptide–modified β-LG to deliver DSF (C-β-LG/DSF) to enhance DSF
accumulation in the TBI area and inhibit pyroptosis from blocking the
release of proinflammatory cytokines. β-LG is an edible whey protein
and CAQK is a tetrapeptide (cysteine-alanine-glutamine-lysine) with
targeting capability for traumatic brain tissue, which are all safe
materials. C-β-LG/DSF could effectively penetrate the BBB and target
injured brain tissue in vitro and in vivo ([150]Fig. 3). C-β-LG/DSF
with a half-life (t[1/2]) of 8.28 hours prolonged the retention time of
DSF in the blood and increased its chances of accumulating at the
injury site. Furthermore, we observed obvious accumulation of
C-β-LG/DSF at the TBI site 6 hours after injection, and this high
concentration was maintained even at 24 hours after injection.
C-β-LG/DSF was administered three times every other day. Upon entering
the TBI site, 73.89 ± 2.03% of DSF was released within 24 hours,
whereas only 29.16 ± 3.54% of DSF was released from C-β-LG/DSF in PBS.
These results suggested that C-β-LG/DSF is responsive to the post-TBI
environment and meets a specific time window for anti-inflammatory
treatment of TBI.
Here, we investigated the efficacy of C-β-LG/DSF to inhibit pyroptosis
by blocking the release of proinflammatory cytokine following TBI.
Results from an in vitro traumatic model experiment on neuron-ICR
primary neurons from fetal mice revealed that C-β-LG/DSF inhibited cell
pyroptosis as well as the extracellular release of IL-1β, IL-18, and
LDH. In vivo TBI mouse model studies further supported that C-β-LG/DSF
effectively blocks the extracellular release of IL-1β, IL-18, and LDH,
and down-regulated the expression of GSDMD, GSDMD-N, caspase-1 p20, and
caspase-1 p10 in the brain of TBI mice. Neuroinflammation and BBB
disruption following TBI are interrelated ([151]52–[152]54). The
posttraumatic dysfunction of the BBB causes BBB permeability and brain
edema. C-β-LG/DSF effectively protected BBB and reduced brain edema, as
demonstrated by the Evans blue extravasation experiment, brain water
content measure, and MRI assay ([153]Fig. 5). Diffusion-weighted images
and ADC coefficients showed that C-β-LG/DSF significantly reduced
cytotoxic edema in brain tissue following TBI. These findings indicate
that C-β-LG/DSF can effectively reduce cerebral edema, possibly through
inhibiting cell pyroptosis after TBI.
In addition, Nissl staining revealed that C-β-LG/DSF significantly
reduced neuronal damage following TBI. Chronic nonresolving
inflammation activated by microglia is a typical feature of secondary
injury following TBI. Early-activated microglia can clear cell debris,
which is beneficial for TBI recovery ([154]55, [155]56). However,
microglia still maintain an activated phenotype during the chronic
stage of TBI, which is usually considered harmful. To verify the
regulatory effect of C-β-LG/DSF on neuroinflammation, we detected the
expression of GFAP and Iba-1, activation markers for astrocytes and
microglia, respectively, on day 7 following injury to elucidate the
strength of the neuroinflammatory responses among the different groups.
The immunohistochemical quantification analysis of GFAP and Iba-1 in
the C-β-LG/DSF group revealed a significant reduction in positive
cells, indicating that C-β-LG/DSF can effectively inhibit
neuroinflammation.
In a behavioral experiment, mNSS was used to evaluate the motor,
sensory, reflex, and balance abilities of mice following TBI.
C-β-LG/DSF exhibited neurological function closer to that of normal
mice. The MWM test was used to assess learning and memory in TBI mice.
The results revealed that C-β-LG/DSF exhibited significantly improved
learning ability and could find the platform faster. Memory testing 2
days after removing the platform showed that C-β-LG/DSF exhibited
significantly improved spatial memory following TBI. The above results
suggest that C-β-LG/DSF significantly enhances DSF accumulation in
brain injury areas and inhibits cell pyroptosis and inflammatory
cytokine release in neurons and mouse brain tissue following TBI to
alleviate neuroinflammation, resulting in an effective therapeutic
effect on neurological function.
Pyroptosis is a relatively rare therapeutic target for TBI due to a
lack of clear understanding of the mechanisms of pyroptosis in TBI. In
addition, the duration and intensity of inflammatory responses may vary
among individuals, making the optimal treatment time window different
for each person. Personalized treatment plans may need to take into
account factors such as the type and severity of the trauma, individual
patient characteristics, and other relevant factors. Further research
is needed to determine the optimal timing for treatment and develop
personalized treatment strategies.
We developed a CAQK peptide–modified β-LG nanoparticle for the delivery
of DSF to inhibit pyroptosis and block the release of inflammatory
cytokines, thereby alleviating neuroinflammation and preventing
secondary damage spread. C-β-LG/DSF effectively accumulates in the
target TBI wound cavity and enhances DSF concentration. C-β-LG/DSF
significantly inhibits pyroptosis and relieves neuroinflammation in
vivo. Furthermore, C-β-LG/DSF effectively protected the BBB and
alleviated brain edema. Last, TBI mice exhibited behavioral function
recovery following C-β-LG/DSF treatment. Therefore, C-β-LG/DSF is a
potential therapeutic candidate as it inhibits the occurrence of
pyroptosis following TBI, thereby reducing secondary injury.
MATERIALS AND METHODS
Materials
The following were the materials used in this study: DSF
(Sigma-Aldrich, PHR1690, USA), β-LG (Sigma-Aldrich, L3908, USA), CAQK
(GL Biochem, Shanghai, China), succinimidyl-4-(N-maleimidomethyl
cyclohexane)-1-carboxylate) (SMCC) (Aladdin, M123456, Shanghai, China),
FITC (Sigma-Aldrich, 46950, USA), Cy5.5 N-hydroxysuccinimide (NHS)
ester (Cy5.5 NHS) (Ruixi, Xi’an, China), Dil (Beyotime, C1036,
Shanghai, China), Evans blue (TCI, E0197, Japan), anti–caspase-1 p20
(Santa Cruz Biotechnology, sc-398715, USA), anti-cleaved GSDMD (Cell
Signaling Technology, #10137S, USA), anti–caspase-1 p10 (GeneTex,
GTX134551, USA), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(Proteintech, 60004-1-Ig, Wuhan, China), anti-GFAP (Cell Signaling
Technology, #45946, USA), anti–Iba-1 (Sigma-Aldrich, SAB2702364, USA),
Dulbecco’s modified Eagle’s medium (DMEM; WISENT, 319-015-CL, Canada),
Neurobasal (Gibco, 21103-049, USA), extracellular matrix (ScienCell,
1001, USA), 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, C1005,
China), Cell Counting Kit-8 (Meilunbio MA0218, Dalian, China),
poly-l-lysine hydrobromide (Sigma-Aldrich, P6282, USA), LysoTraker
(Meilunbio MB604-1, Dalian, China), B-27 (Gibco, 17504-044, USA),
epidermal growth factor (Peprotech, AF-100-15, USA), fibroblast growth
factor (Peprotech, AF-100-188, USA), IL-1β ELISA Kit (Jonln, JL18442,
Shanghai, China), IL-18 ELISA Kit (Jonln, JL20253, Shanghai, China),
LDH ELISA Kit (Solarbio, BC0685, Beijing, China), and sesame oil
(Macklin, S905724, Shanghai, China).
Preparation and characterization of C-β-LG/DSF
C-β-LG/DSFs were prepared via an antisolvent precipitation method. A
β-LG solution (1 mg/ml) was prepared by adding 10 mg of β-LG to 10 ml
of distilled water, stirring the mixture at room temperature for 1
hour, adjusting pH to 7.0 with 0.1 M HCl or NaOH, and heating the
protein solution to 90°C or above for 30 min. A DSF solution was
prepared by dissolving 15 mg of DSF in 200 μl of acetone, which was
then slowly added to the β-LG solution (under ice bath conditions) and
subjected to ultrasonic shock for 10 min. The resulting β-LG/DSF
solution was vaporized under pressure at 25°C for 30 min to remove
residual acetone. One milligram of CAQK and 1 mg of SMCC were dissolved
in 1 ml of water to obtain CAQK solution (1 mg/ml) and SMCC solution (1
mg/ml), respectively. Add 1 ml of water to a 2-ml centrifuge tube and
add 100 μl of CAQK solution, then stir vigorously. At the same time,
CAQK-SMCC solution was prepared by adding 100 μl of SMCC solution into
the above solution and stirring for 3 hours under ice bath conditions
at 1500 rpm. Absorb 100 μl of CAQK-SMCC solution and slowly add it to
β-LG/DSF solution and stir it violently for 2 hours under ice bath
conditions to prepare C-β-LG/DSF.
The particle size, zeta potential, and its stability in serum (10%)
were measured by a laser particle size analyzer (Zetasizer NanoZS90,
Malvern, UK). The morphology of the delivery system was observed by TEM
Tecnai G2 Spirit Twin (FEI, USA). The samples dispersed in PBS were
dropped onto 200-mesh C-coated Cu grid before recording the TEM images.
The drug loading (DL) and encapsulation efficiency (EE) of DSF were
analyzed by ultraperformance liquid chromatography (UPLC)–tandem mass
spectroscopy (MS/MS). The column was an advanced Waters Acquity UPLC
BEH C18 100 × 2.1 mm, and the mobile phase (1 ml/min) consisted of
water with 0.1% methanoic acid:acetonitrile with 0.1% methanoic acid
(1:1). Mass spectrometer parameters were as follows: capillary, 0.5 kV;
cone voltage, 18 V; collision energy, 10 V; ion source temperature,
150°C; desolvation temperature, 500°C; cone gas flow, 800 liters/hour;
cone gas flow, 20 liters/hour; mass-charge ratio: 297 to 116.0633. The
prepared C-β-LG/DSF was freeze-dried to obtain dry-powder C-β-LG/DSF
nanoparticles. The assay was repeated three times by dissolving 1 mg of
lyophilized nanoprodrug powder in 1 ml of water containing 0.1%
methanoic acid and mixing an equal volume of acetonitrile with 0.1%
methanoic acid, 1 μl per injection. The experiment was repeated three
times. A standard curve of DSF (0 to 100 ng ml^−1) in aqueous solution
was tested to determine the concentration. Y = 437.38 × X - 86.5239,
R^2 = 0.9916.
[MATH: EE (%)=(weight of drug loaded)/(weight of drug in feed)×100%
:MATH]
[MATH: DL (%)=(weight of drug loaded)/(weight of nanoparticles)×100%
:MATH]
[MATH: EE (%) of DSF=90.25±2.28%
:MATH]
[MATH: DL (%) of DSF=44.80±1.14%
:MATH]
Drug release
C-β-LG/DSF was prepared as mentioned in the “Preparation and
characterization of C-β-LG/DSF” section. C-β-LG/DSF nanoparticles (1
ml) were transferred into dialysis bags with 3500 molecular weights,
and then immersed in 100 ml of PBS (pH 7.4) and CSF of TBI patients,
respectively. Drug release was detected at 37°C. Samples of 200 μl were
taken at different time periods and stored at 4°C. At the same time,
the corresponding volume of PBS or CSF of TBI patients was added for
volume supplement. Released DSF was detected by UPLC-MS/MS. The
detection methods and chromatographic and MS conditions were the same
as those in the “Preparation and characterization of C-β-LG/DSF”
section. This study was performed after informing patients and their
families with fully informed consent, and it was approved by the Ethics
Committee of the Affiliated Hospital of Xuzhou Medical University
(ethics number: XYFY2020-KL208-01).
Cell culture
The human brain microvascular endothelial cells (HBMECs) were obtained
from the Shanghai Cell Bank, Type Culture Collection Committee, Chinese
Academy of Sciences, and were routinely cultured in ECM endothelial
cell medium containing 5% fetal bovine serum and 0.1% Endothelial Cell
Growth Supplement. Neuron-ICR was extracted from the frontal cortex of
embryonic mice aged 18 to 19 days during pregnancy. Pregnant ICR mice
were anesthetized with isoflurane gas and euthanized by decapitation,
and their abdomen was disinfected with alcohol. The abdominal cavity
was opened with scissors and forceps, and embryos were removed from the
uteri. The brains of the embryos were dissected and transferred to
prechilled Hanks’ balanced salt solution (HBSS) without phenol red and
kept on ice. The frontal cortex tissues were isolated from the brain
tissues using ophthalmic scissors and forceps under ice bath
conditions. The isolated frontal cortex tissues were transferred to
HBSS with phenol red, minced with ophthalmic scissors, and transferred
to a sterile 15-ml Eppendorf tube for centrifugation at 1500 rpm for 5
min. The supernatant was removed, and 2 ml of 0.25% trypsin-EDTA was
added. The sample was pipetted up and down, placed in a cell culture
incubator for 15 min, and then pipetted in 5 ml of DMEM+++ (10% fetal
bovine serum, 1% glutamine, and 1% penicillin-streptomycin solution) to
remove the trypsin. After mixing, the cells were washed three times,
and the supernatant was discarded after centrifugation. Subsequently, 5
ml of DMEM+++ was added and vigorously pipetted 20 to 30 times. The
processed cell suspension was then passed through a cell strainer,
counted, and seeded in 10-cm dishes with 2 million cells in 7 ml of
DMEM+++. For six-well plates, 300,000 cells were seeded in 2 ml of
DMEM+++, while for 96-well plates, 6000 cells were seeded in 200 μl of
DMEM+++. The plates were kept in a cell culture incubator for 6 hours
and observed under a microscope. If the cells were adherent, the medium
was replaced with Neurobasal+++ (2% B27, 1% glutamine, and 1%
penicillin-streptomycin solution). The medium was replaced every 7
days, and the cells were discarded after 21 days ([156]57).
Cell Counting Kit-8
Neuron-ICR cells were plated with poly-l-lysine (Sigma-Aldrich) in
96-well plates. HBMEC cells were plated in 96-well plates. After 24
hours, the cells were treated with different concentrations of DSF,
β-LG/DSF, and C-β-LG/DSF, and the culture was continued for 24 hours.
Last, 10 μl of Cell Counting Kit-8 solution was added to each well and
incubated for 30 min. The absorbance value was detected at 450-nm
wavelength.
TBI cell model
Neuron-ICR cells cultured in 10-cm dishes were divided into four
experimental groups: sham group (no scratch injury model), TBI group
(scratch injury without drug treatment), DSF group (scratch injury with
free DSF drug treatment), and C-β-LG/DSF group (scratch injury with
β-LG–encapsulated DSF targeted peptide–modified nanomedicine
treatment). The cells were placed in a laminar flow hood, and sterile
pipette tips were used to create vertical and horizontal scratches
(except for the sham group) on the bottom of the dish, with five
scratches in each direction and a distance of 0.5 cm between each
scratch. After 1 hour, the medium was replaced with Neurobasal+++
medium containing the respective group drugs (except for the TBI
group), with a DSF concentration of 0.5 μM.
Cellular uptake of C-β-LG/DSF
Neuron-ICR cells were seeded in a six-well plate under cell culture
conditions. After 48 hours of cell growth, the cells were treated at 4°
or 37°C for 1 hour with 25 μM chlorpromazine hydrochloride, 100 μM
EIPA, 5 mM mβCD, 740 μM genistein, and 80 μM dynasore, respectively
([157]25–[158]29). Subsequently, FITC-labeled C-β-LG/DSF nanoparticles
were added to the wells and incubated for 2 hours. The cells were then
washed twice with PBS, and the collected cells were subjected to flow
cytometry.
Endosomal escape of C-β-LG/DSF
Neuron-ICR cells were seeded in a six-well plate under cell culture
conditions. FITC-labeled C-β-LG/DSF nanoparticles were added to the
wells and incubated for 1, 4, and 8 hours, respectively. The cells were
then washed twice with PBS and incubated with Neurobasal+++ medium
containing Lysotracker for 30 min, and the collected cells were
subjected to CLSM. ImageJ (version 2.9.0) software was used for
fluorescent colocalization analysis.
Detection of C-β-LG/DSF to cross BBB
The TBI cell model was prepared as mentioned in the “TBI cell model”
section. After preparing the injury model for 12 hours, neuron-ICR
cells were cultured in the lower compartment of the culture plate.
HBMEC cells were cultured in Transwell. When the trans-endothelial
electrical resistance reached 150 ohms•cm^2, BBB formation was
successful. C-β-LG/DSF/FITC and β-LG/DSF/FITC were added to the upper
chamber of Transwell. The fluorescence intensity of FITC (green) in
C-β-LG/DSF/FITC–incubated HBMEC and neuron-ICR cells was detected by
CLSM after 4 hours. Cell membranes of HBMEC were labeled with Dil
fluorescent dye (red). Neuron-ICR nuclei were labeled with DAPI (blue).
The fluorescence intensity of the upper and lower chamber of each group
was detected by a microplate reader after a 4-hour incubation. When red
and green were double-labeled in images, red was replaced by purple
pseudo-color. The fraction of C-β-LG/DSF/FITC and β-LG/DSF/FITC
penetrating the BBB was determined by calculating the ratio of the
lower chamber fluorescence intensity to the sum of the upper and lower
chamber fluorescence intensity in each group. The calculation formula
is as follows: Penetration rate (%) = Upper chamber fluorescence
intensity/(Upper chamber fluorescence intensity + Lower chamber
fluorescence intensity) × 100%.
Animal
Six-week-old male ICR mice were purchased from Zhejiang Weitonglihua
Company (Zhejiang, China), with certificate number
20220826Abzz0619000492. They were housed in individually ventilated
cages at a controlled environment temperature of approximately 24°C and
provided with sufficient food and water. After 1 to 2 weeks of
adaptation, TBI mouse model experiments were performed. All animal
experimental procedures were carried out in accordance with the
relevant regulations of the “Jiangsu Provincial Experimental Animal
Management Measures” and have been approved by the Animal Use Committee
of Xuzhou Medical University (no. 202208S110). The TBI mouse model was
randomly divided into five groups: sham group (sham operation group,
only the right parietal bone was opened without inducing injury), TBI
group (control group, without treatment after injury), DSF group
(intraperitoneal administration of free DSF drug after injury),
β-LG/DSF group (intravenous administration of β-LG–encapsulated DSF
nanomedicine after injury), and C-β-LG/DSF group (intravenous
administration of CAQK-modified β-LG–encapsulated DSF nanomedicine
after injury), with five mice in each group.
TBI animal model
ICR mice were randomly divided into sham, TBI, DSF, β-LG/DSF, and
C-β-LG/DSF groups. We modified the mature technique of Feeney’s TBI
model. ICR mice were anesthetized with isoflurane gas (induction
concentration: 3%, maintenance concentration: 2%, and gas flow rate:
400 ml/min). After complete anesthesia, the ICR mice were fixed on a
stereotaxic instrument with the nose close to the gas anesthesia
machine mask. The scalp hair on the cranial vertex was trimmed with
scissors, followed by routine disinfection with iodine and deiodination
with alcohol. The scalp was incised along the midline of the frontal
vertex of the ICR mouse for approximately 1.5 cm to expose the skull
and remove the periosteum. A hole was drilled on the right side of the
sagittal suture of the skull using a handheld skull drill, with a
diameter of approximately 0.5 cm (careful operation to ensure that the
dura mater was not injured). A mouse TBI impactor (Zhenghua Biological
Instrument Co. Ltd., Anhui) was used to induce TBI, with a hammer mass
of 40 g and a fall height of 7.5 cm. After successful preparation of
the TBI model, the injured brain tissue and dura mater were promptly
handled. In case of brain bleeding, sterile cotton balls were used to
compress and stop bleeding. The scalp was carefully sutured and
disinfected. PBS, DSF (DSF dissolved in sterile edible sesame oil and
injected intraperitoneally), β-LG/DSF, and C-β-LG/DSF were administered
at a dose based on 10 mg/kg of DSF. The sham group received no
treatment. All groups were administered once a day for three
consecutive days and transferred to cages with group labels affixed,
with five mice in each group. After 1 hour of TBI, mice status was
observed. Mice were carefully monitored 2 hours after TBI surgery in
the cages after their waking up. If a mouse died after TBI
administration but before the end of the 2-hour observation period (2
hours after TBI administration), the data were not included for
analysis.
Pharmacokinetic detection
The TBI animal model was prepared as mentioned in the “Detection of
C-β-LG/DSF to cross BBB” section. DSF (intraperitoneal injection),
β-LG/DSF, and C-β-LG/DSF were injected through the caudal vein at the
dosage of 10 mg/kg DSF. One hundred microliters of blood was collected
from tail vein at different time points (5 min, 10 min, 20 min, 30 min,
1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours), and
transferred to a medical anticoagulant tube. Ten microliters of 10%
phosphoric acid was added and centrifuged at 15,000 rpm. Fifty
microliters of plasma was carefully absorbed and transferred to a new
1.5-ml centrifuge tube. Fifty microliters of protein precipitator was
added (acetonitrile:10% trichloroacetic acid = 2:3). The plasma was
thoroughly mixed and centrifuged at 15,000 rpm for 10 min. The
supernatant was absorbed with pipetting gun, filtered, and 1 μl was
injected. The concentration of DSF in each tissue was determined by the
UPLC-MS/MS method. The experimental conditions and parameters of
UPLC-MS/MS were the same as those in the “Preparation and
characterization of C-β-LG/DSF” section, and the experiment was
repeated three times.
In vivo tissue targeting distribution analysis
The TBI animal model was prepared as mentioned in the “Detection of
C-β-LG/DSF to cross BBB” section. C-β-LG/DSF and β-LG/DSF were labeled
with Cy5.5 NHS and injected into mice. After 6, 12, 24, and 48 hours
after injection, the brain, heart, liver, spleen, lungs, and kidneys of
mice were dissected out and imaged by a small animal imaging system.
C-β-LG/DSF and β-LG/DSF were labeled with FITC and injected into mice.
After 6, 12, 24, and 48 hours after injection, the brain, heart, liver,
spleen, lung, and kidneys of mice were dissected out, homogenized, and
weighed to detect the intensity of FITC fluorescence and DSF
concentration in each organ by a microplate reader. One milliliter of
tissue homogenate was collected and transferred to a centrifuge tube.
One hundred microliters of 10% phosphoric acid was added and
centrifuged at 15,000 rpm. Five hundred microliters of supernatant
fluid was carefully absorbed and transferred to a new centrifuge tube.
Five hundred microliters of protein precipitator was added
(acetonitrile:10% trichloroacetic acid = 2:3). The tissue homogenate
was thoroughly mixed and centrifuged at 15,000 rpm for 10 min. The
supernatant was absorbed with pipetting gun, filtered, and 1 μl was
injected. The concentration of DSF in each tissue was determined by the
UPLC-MS/MS method. The experimental conditions and parameters of
UPLC-MS/MS were the same as those in the “Preparation and
characterization of C-β-LG/DSF” section, and the experiment was
repeated three times.
Enzyme-linked immunosorbent assay
The TBI cell model was prepared as mentioned in the “TBI cell model”
section. After preparing the TBI cell model for 12 hours, the culture
medium of each cell group was transferred to sterile centrifuge tubes
and centrifuged at 3000 rpm, 4°C for 20 min, and then the supernatant
of each group was collected. ELISA experiments were performed to detect
the cellular release levels of IL-1β, IL-18, and LDH. Twenty-four hours
after the preparation of the TBI animal model, the mice were injected
intraperitoneally with 1% pentobarbital sodium and were deeply
anesthetized. The mice were killed by decapitating, and the brain
tissue was removed. The brain was separated along the longitudinal
cerebral fissure, and the quality of the brain tissue on the striking
side was accurately weighed and recorded. The tissue grinder was used
for full grinding (ice water bath), and after full homogenization, the
grinding liquid was transferred to a centrifuge tube with a pipette.
After centrifugation (4°C, 12,000 rpm, 20 min), the supernatant was
taken to detect the release of the above three cytokines in the brain
tissue. The specific experimental procedures were strictly carried out
according to the instructions of the IL-1β and IL-18 ELISA assay kits
(Jonln, JL20253, Shanghai, China) and the LDH ELISA assay kit
(Solarbio, BC0685, Beijing, China).
Immunoblot assay
The TBI cell model was prepared as mentioned in the “TBI cell model”
section. After preparing the TBI cell model for 12 hours, neuron-ICR
cells were collected. For immunoblot assay, collected cells were lysed
in the radioimmunoprecipitation assay lysis buffer containing 50 mM
tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40, 0.1%
SDS, 1 mM sodium fluoride, and 1 mM sodium orthovanadate. Twenty-four
hours after the preparation of the TBI animal model, the mice were
injected intraperitoneally with 1% pentobarbital sodium and were deeply
anesthetized. The mice were killed by decapitating, and the brain
tissue was removed. The brain was separated along the longitudinal
cerebral fissure, and the quality of the brain tissue on the striking
side was accurately weighed and recorded. The proper amount of tissue
cell lysate was added and fully ground with a tissue grinder (on ice),
the grinding liquid was transferred to a centrifuge tube with pipette
after full homogenization and centrifuged (4°C, 10,000 rpm, 10 min),
and the supernatant, that is, total protein, was taken. Protein sample
concentration was determined and stored at −80°C. Proteins were boiled
in the loading buffer (Beyotime, P0015L, Shanghai, China). Primary
antibodies used for immunoblot were listed as follows: anti–caspase-1
p20 (Santa Cruz Biotechnology, sc-398715; 1:1000), anti–cleaved GSDMD
(Cell Signaling Technology, #10137S; 1:1000), anti–caspase-1 p10
(GeneTex, GTX134551; 1:1000), and anti-GAPDH (Proteintech, 60004-1-Ig;
1:1000). Representative uncropped immunoblots were presented in the
Supplementary Materials. All immunoblot results were independently
repeated for at least three times.
Brain water content assay
The water content of mouse brain tissue was determined by measuring wet
weight and dry weight. On the third day of the preparation of the TBI
mouse model (as mentioned in the “TBI cell model” section), 1%
pentobarbital sodium was injected intraperitoneally, the mice were
deeply anesthetized, and the mice were sacrificed by decapitation.
After the brain tissue was removed, the cerebellum and brainstem were
removed. The brain was separated along the longitudinal cerebral
fissure, the healthy side brain tissue was discarded, and the wet
weight of the affected side brain tissue was accurately weighed. Then,
the brain tissue of the striking side was placed in a 90°C oven for 3
days, and its mass was accurately weighed as the brain tissue dry
weight. The experiment was repeated five times. The formula for
calculating water content is as follows: Water content of brain tissue
(%) = (Wet weight − Dry weight)/Wet weight × 100%.
Evans blue tests
The TBI mouse model was prepared using the same method mentioned in the
“Detection of C-β-LG/DSF to cross BBB” section). On day 3, Evans blue
dye was injected into the tail vein (dissolved normal saline,
administered at 2%, at a dose of 2 ml/kg). After 3 hours, the Evans
blue dye is fully circulated in the blood system. Mice were deeply
anesthetized by intraperitoneal injection of 1% pentobarbital sodium.
The mouse chest was cut open by tissue scissors, and 20 ml of normal
saline and 20 ml of 4% paraformaldehyde were injected into the apex of
the mouse heart (left ventricle) through a catheter. The brain tissue
was carefully peeled off, so that it does not get damaged. The brain
was separated along the longitudinal cerebral fissure, and the quality
of the brain tissue on the striking side was accurately weighed and
recorded. The brain tissue was transferred to a centrifuge tube
containing 1 ml of PBS, and the tissue grinder was fully homogenized
for centrifugation at 4°C at a rotational speed of 10,000 rpm for 10
min. Tissue homogenate supernatant (300 μl) was added to 700 μl of
acetone, incubated at room temperature for 24 hours, and centrifuged at
4°C, at a rotational speed of 10,000 rpm, for 10 min. The absorbance
value was measured at 620 nm by enzyme label. The corresponding Evans
blue content was calculated by plotting a standard curve with known
different concentration gradients.
MRI test
After 3 days of preparation of the mouse TBI model (using the same
method as that mentioned in the “Detection of C-β-LG/DSF to cross BBB”
section), T2-weighted sequences and DWI-weighted sequences were
detected by a small-animal MRI apparatus (Bruker, Bio Spin MRI Pharma
Scan 7.0 T). Isoflurane gas was inhaled into anesthetized mice
(induction concentration: 3%, maintenance concentration: 2%, and gas
flow: 400 ml/min). After induced anesthesia, mice were fixed on an MRI
scanner operating bed. The inhalation flow of isoflurane gas was
adjusted according to the data during the scan so that the mice would
not die from overanesthesia. If a mouse dies during the scan, it will
be excluded. T2 parameters were as follows: repetition time: 2500 ms,
echo time: 36 ms, layer thickness: 1 mm, field of view: 20 × 20 mm, and
image matrix: 256 × 256. DWI parameters were as follows: repeat time:
3000 ms, echo time: 20 ms, section thickness: 1 mm, and field of view:
100 × 110 mm. All MRIs were evaluated by a skilled MRI technician;
ImageJ software was used to calculate the volume of cerebral edema
around the injury site in T2-weighted images. The ADC value was
automatically calculated using Paravision 6.1 software of an MRI
scanner based on diffusion-weighted images. The diffusion-weighted
images were processed by Paravision 6.1 software with false color.
Section staining and immunohistochemistry
The brain tissue of mice was taken out after anesthesia and dealt with
10% formalin and ethanol dehydration and then embedded in paraffin.
Later sections were stained with Nissl, H&E, GSDMD, GFAP, and Iba-1.
Then, sections were observed with a fluorescence microscope (Leica,
Germany), and pictures were analyzed with Image-Pro Plus 16.0.
RNA-seq analysis
At 48 hours after TBI, cortical tissue samples proximal to or located
at the injury site were sent to Haiteng Century Biotechnology Co. Ltd.
(Beijing, China) for RNA-seq library preparation. After clustering, we
performed transcriptome sequencing on the DNBSEQ-T7 platform that
generated the raw reads. Data quality was assessed by the FastQC tool
after removal of adaptor sequences, ambiguous “N” nucleotides
(proportion of “N” > 5%), and low-quality sequences (quality score <
10). The ggplotR package was used to identify StringTie genes.
Difference was statistically significant threshold for |log[2] fold
change| ≥ 1 or P < 0.05. GO enrichment, KEGG pathway analysis of genes
and genomes, and heatmap and volcanic figure analysis were carried out
at [159]https://www.omicshare.com (Gene Denovo, Guangzhou, China).
Morris water maze
On the 21st day after the preparation of the TBI animal model (using
the same method as that mentioned in the “Detection of C-β-LG/DSF to
cross BBB” section), the MWM experiment was conducted, and the water
temperature was set at 24°C. In the first 7 days, mice were randomly
placed into water from a certain quadrant. These mice could swim freely
in the swimming pool until they found a resting platform, which was
hidden 1 cm below the water surface. The time (t) and swimming distance
(s) were recorded. The swimming time of each mouse was 120 s. The mice
that could not find the platform by the end of time were guided to the
platform with the guide stick and stayed for 30 s. On the 8th day, the
platform was removed, and quadrants were randomly selected to allow
mice to find the original location of the platform. Each mouse swam for
120 s, and the time in the original location and quadrant of the
platform as well as the crossing times of the platform were recorded.
All data during the test period were recorded and analyzed by a
video-tracking system (Anhui Zhenghua Biological Instrument Equipment
Co. Ltd., Huaibei, China). Swimming tests were performed on each mouse
before the experiment, and those mice that could not swim were
excluded. There were five mice in each group.
mNSS score
On the 3rd, 7th, and 14th day after the preparation of the TBI animal
model (using the same method as that mentioned in the “Detection of
C-β-LG/DSF to cross BBB” section), the mNSS was performed on the trauma
model mice by the double-blind method, and the score was recorded. mNSS
scores range from 0 (normal) to 18 (most severe) for movement,
sensation, balance, and reflexes.
Hanging wire grip test
To test the strength, balance, and endurance of forelimbs after brain
trauma in rats, on the 14th day after the preparation of the TBI animal
model (using the same method as that mentioned in the “Detection of
C-β-LG/DSF to cross BBB” section), the hanging wire grip test was
conducted on the TBI model mice by the double-blind method. A piece of
wire 50 cm above the table top was hung. The hind legs of the mice were
wrapped with tape (to prevent the mice from climbing), and the front
legs were placed on the wire. The hanging time of mice was recorded,
five mice in each group.
Toxicity test
After 14 days of TBI animal model preparation (using the same method as
that mentioned in the “Detection of C-β-LG/DSF to cross BBB” section),
mice were deeply anesthetized by intraperitoneal injection of 1%
pentobarbital sodium. Twenty milliliters of normal saline and 20 ml of
4% paraformaldehyde were injected into the apex of the heart (left
ventricle) through the catheter. The heart, liver, spleen, lungs, and
kidneys were dissected and fixed in a centrifuge tube filled with 4%
paraformaldehyde solution. Paraffin blocks were prepared, sliced,
dewaxed with xylene, dehydrated with alcohol, and stained with H&E
staining. The experiment was repeated three times.
Blood biochemical examination
On day 7 of TBI animal model preparation (using the same method as that
mentioned in the “Detection of C-β-LG/DSF to cross BBB” section), mice
were lightly anesthetized by intraperitoneal injection of 1%
pentobarbital sodium. The blood routine and blood biochemical indexes
of each treatment group were detected, and the physiological indexes
and liver and kidney function were evaluated by the blood extraction
method of inner canthus, which was transferred to the anticoagulant
tube and the coagulant promoting tube, respectively.
Statistical analyses
Each experiment was independently repeated more than three times. All
data were included for statistical analyses using GraphPad Prism 9.0.
Unpaired Student’s t test (two-tailed) was used for the comparison
between unpaired two-groups, and one-way analysis of variance (ANOVA)
test was applied for multigroup data comparison. Bar graphs were
expressed as means ± SD, with statistical significance at *P < 0.05,
**P < 0.01, ***P < 0.001, or ****P < 0.0001.
Acknowledgments