Abstract
Oxidative stress is associated with many acute and chronic inflammatory
diseases, yet limited treatment is currently available clinically. The
development of enzyme-mimicking nanomaterials (nanozymes) with good
reactive oxygen species (ROS) scavenging ability and biocompatibility
is a promising way for the treatment of ROS-related inflammation.
Herein we report a simple and efficient one-step development of
ultrasmall Cu[5.4]O nanoparticles (Cu[5.4]O USNPs) with multiple
enzyme-mimicking and broad-spectrum ROS scavenging ability for the
treatment of ROS-related diseases. Cu[5.4]O USNPs simultaneously
possessing catalase-, superoxide dismutase-, and glutathione
peroxidase-mimicking enzyme properties exhibit cytoprotective effects
against ROS-mediated damage at extremely low dosage and significantly
improve treatment outcomes in acute kidney injury, acute liver injury
and wound healing. Meanwhile, the ultrasmall size of Cu[5.4]O USNPs
enables rapid renal clearance of the nanomaterial, guaranteeing the
biocompatibility. The protective effect and good biocompatibility of
Cu[5.4]O USNPs will facilitate clinical treatment of ROS-related
diseases and enable the development of next-generation nanozymes.
Subject terms: Acute kidney injury, Bioinspired materials, Nanomedicine
__________________________________________________________________
Oxidative stress is involved in several diseases and is a target for
intervention. Here, the authors report on the synthesis of ultrasmall
copper-based nanozymes as reactive oxygen species scavengers and
demonstrate improved treatment outcomes in acute liver and kidney
injury and wound healing in vivo.
Introduction
Inflammation is a natural defensive response to stimulating factors
such as infection, injury, and toxins^[45]1,[46]2. However, excessive
and uncontrolled inflammation has been demonstrated to cause numerous
diseases, such as cardiovascular disease, hepatitis, nephritis, and
delayed wound healing^[47]3,[48]4. It has been widely acknowledged that
inflammation is intimately associated with oxidative
stress^[49]5,[50]6. Reactive oxygen species (ROS) in excessive
inflammatory response could aggravate localized tissue injury and lead
to chronic inflammation^[51]7,[52]8. Consequently, scavenging ROS with
broad-spectrum antioxidants such as N-acetyl cysteine and
acetyl-l-carnitine has been regarded as a feasible strategy for the
treatment of various inflammatory diseases such as acute liver injury,
liver fibrosis, acute or chronic wounds, acute kidney injury
(AKI)^[53]9,[54]10. However, poor bioavailability, low stability and
efficacy of these drugs limit their clinical application in the
treatment of ROS-related diseases^[55]11,[56]12.
Advances in nanomedicine have enabled new ways of ROS clearance and
thus treatment of ROS-related diseases using various functional
nanomaterials^[57]13, such as carbon^[58]14, ceria^[59]15,
platinum^[60]16, redox polymer^[61]17, and polyphenol nanoparticles
(NPs)^[62]18. Among them, one promising strategy is to develop
nanozymes to maintain natural redox balance in biological system,
including catalase (CAT), superoxide dismutase (SOD), and glutathione
peroxidase (GPx)^[63]19. Such nanozymes shall have high ROS scavenging
ability comparable to native enzymes, broad-spectrum ROS scavenging
activities against various toxic ROS species, high stability in harsh
disease environment, and rapid clearance from the body to guarantee
excellent biocompatibility. Therefore we believe nanomaterials with
ultrasmall size (hydrodynamic diameter <5.5 nm) shall be designed
because this kind of nanomaterials will have high catalytic activity
due to the relatively large surface to volume ratios^[64]20 and rapid
renal clearance because they can pass through the glomerular capillary
wall in kidney^[65]21,[66]22.
Some ultrasmall ROS scavenging nanomaterials have been developed
recently. For example, Cai and co-workers reported the synthesis of
ultrasmall melanin-based^[67]23, molybdenum-based^[68]10, and DNA
origami-based^[69]24 antioxidative NPs with preferential renal uptake
for AKI treatment. Besides, Qu and co-workers^[70]25 synthesized
Cu-TCPP MOF nanodots with SOD mimic property. However, the relatively
low catalytic activity and/or high cost of these nanomaterials could
impair their clinical translation. It remains a challenge to develop a
nanomaterial with simple structure and excellent ROS scavenging ability
to enable potential large-scale production and subsequently clinical
applications.
Copper (Cu), an essential trace element in humans, plays an important
role in many enzymes, such as tyrosinase and Cu–Zn SOD^[71]26,[72]27.
Therefore, it is reasonable to infer that copper-based nanomaterials
can be used to scavenge ROS. For example, Cu NPs have excellent
catalytic activity to scavenge H[2]O[2] and O[2]^− due to their strong
quantum confinement of electrons in the ultrasmall size regime^[73]26,
but cannot eliminate OH· simultaneously^[74]27. Cuprous oxide (Cu[2]O)
NPs possess good catalytic activity and can promote electron transfer
reactions to inactivate H[2]O[2] or OH·, thereby partially mimicking
peroxidase^[75]13,[76]28. Therefore, we presume that broader-spectrum
enzymatic catalytic properties and antioxidant activities could be
concurrently achieved by combining Cu[2]O and Cu nanocrystals. Besides,
the stability of Cu[2]O coating on Cu NPs can be largely enhanced owing
to the effective electron–hole separation between Cu[2]O and Cu, which
is also of benefit to improve the overall ROS scavenging
ability^[77]29,[78]30.
Herein, we report a strategy to synthesize ultrasmall Cu[5.4]O NPs
(Cu[5.4]O USNPs) with excellent biocompatibility, enzymatic ROS
scavenging abilities, and high renal clearance properties, in order to
tackle broad ROS-related diseases (Fig. [79]1). Cu[5.4]O USNPs exhibit
remarkable antioxidant efficiency, with a working concentration of
approximately 25 ng mL^−1 in vitro and 2 μg kg^−1 for AKI in vivo. This
dosage is at least two orders magnitude lower than all the other
reported nanomaterials to treat ROS-related diseases. Furthermore, they
possess broad-spectrum ROS scavenging activities and function as CAT,
GPx, and SOD analogs. Moreover, the ultrasmall NPs show high renal
clearance and achieve an outstanding therapeutic effect against broad
ROS-related diseases without any noticeable toxicity. This study
provides an attractive strategy to develop ultrasmall copper-based
nanozyme systems, which may serve as a blueprint for next-generation
nanomedicines used in ROS-related diseases treatment and prevention.
Fig. 1. Schematic illustration of Cu[5.4]O ultrasmall nanoparticles in the
treatment of ROS-related diseases.
[80]Fig. 1
[81]Open in a new tab
Cu[5.4]O ultrasmall nanoparticles with multiple enzyme-mimicking and
broad-spectrum ROS scavenging ability are synthesized by a simple and
green method. Due to the robust ROS scavenging ability in vivo,
Cu[5.4]O ultrasmall nanoparticles exhibit therapeutic effect against
broad ROS-related diseases, including acute kidney injury, acute liver
injury and diabetic wound healing.
Results and Discussion
Synthesis and characterization of Cu[5.4]O USNPs
USNPs were synthesized by a green, rapid, and cost-effective method
(Fig. [82]2a). The ratio of Cu^2+ to l-ascorbic acid (AA), reaction
temperature, and time were tuned to determine their impact on the
particle size and catalytic activity of obtained NPs (Supplementary
Figs. [83]1–[84]3). A relatively long reaction time is required for
obtaining uniform USNPs due to Ostwald ripening (Supplementary
Fig. [85]2). Too low and too high temperatures are not feasible to
control the reaction (Supplementary Fig. [86]3). The catalytic activity
of the obtained copper-based USNPs was almost the same with the feeding
ratio of Cu^2+ to AA in the range of 1:10 to 1:40. Therefore, the molar
ratio of Cu^2+ to AA was fixed at 1:10 for the following studies.
Fig. 2. Preparation and characterization of Cu[5.4]O USNPs.
[87]Fig. 2
[88]Open in a new tab
a Schematic preparation of Cu[5.4]O USNPs. b TEM image of Cu[5.4]O
USNPs. Inset is the statistical chart of particle size distribution.
The cross-sectional area of each particle was measured by using ImageJ
Software from the TEM images, with at least 500 particles counted per
sample. c Hydrodynamic diameter distribution of the Cu[5.4]O USNPs. d
X-ray diffraction (XRD) pattern of the Cu[5.4]O USNPs, the rhombus, and
star symbols represent the characteristic peaks of Cu[2]O and Cu,
respectively. e XAES spectra of the Cu[5.4]O USNPs. Source data are
provided as a Source Data file.
Transmission electron microscopy (TEM) images showed that these
Cu[5.4]O USNPs were uniform, with an average diameter of 3.5–4.0 nm in
the dry state (Fig. [89]2b). The average hydrodynamic diameter of
Cu[5.4]O USNPs was approximately 4.5 nm (Fig. [90]2c), which meets the
kidney filtration threshold of 5.5 nm^[91]21,[92]22, enabling renal
uptake, accumulation, and clearance. There were slight increases in the
hydrophilic diameters of Cu[5.4]O USNPs in FBS and rat serum
(Supplementary Fig. [93]4a), possibly due to surface protein
adsorption^[94]31. Cu[5.4]O USNPs were uniformly dispersed, with nearly
identical morphologies and particle sizes, indicating that the Cu[5.4]O
USNPs were stable in the media for at least 20 days (Supplementary
Fig. [95]4b). The oxidation state of Cu was investigated through powder
X-ray diffraction (Fig. [96]2d). The dominant peaks at 2θ = 41.6°,
50.7°, and 77.5°, which can be assigned to the (111), (200), and (220)
lattice planes of Cu(0), are consistent with the presence of
face-centered cubic copper. Three additional minor diffraction peaks at
2θ = 29.6°, 36.4°, and 60.1° can be assigned to the (111), (200), and
(220) lattice planes of cuprous oxide (Cu[2]O), respectively. The
results suggest that the reaction did not generate pure copper NPs, but
rather a mixture of Cu^0 and Cu^+ (Cu and Cu[2]O NPs). As shown in
Fig. [97]2e, the mass fractions of Cu and Cu[2]O after normalization
were calculated based on the peak area of Cu 2p, indicating that the
proportions of Cu and Cu[2]O were approximately 3.4. Therefore, the
resulting ultrasmall copper-based NPs are denoted as Cu[5.4]O USNPs.
ROS scavenging activities of Cu[5.4]O USNPs
Three representative ROS, H[2]O[2], O[2]^−, and ·OH, were selected to
investigate the ROS scavenging activities of Cu[5.4]O USNPs. As shown
in Fig. [98]3, the Cu[5.4]O USNPs exhibited a high ROS scavenging
activity in a concentration-dependent manner. Approximately 80% of the
total H[2]O[2] was decomposed by 200 ng mL^−1 Cu[5.4]O USNPs
(Fig. [99]3a). Approximately half of the O[2]·^− was decomposed when
treated with 150 ng mL^−1 Cu[5.4]O USNPs (Fig. [100]3b). More than 80%
of the ·OH was scavenged when the Cu[5.4]O USNP concentration was
150 ng mL^−1 (Fig. [101]3c, Supplementary Fig. [102]5). To further
confirm the antioxidative properties of Cu[5.4]O USNPs, a free radical
scavenging experiment was performed using the classic
2,2′-azino-bis(3-ethylbenzothiazoline 6-sulfonate) (ABTS) radical
assay. As presented in Fig. [103]3d, more than 89% of the free radicals
were eliminated by a very low concentration of Cu[5.4]O USNPs
(150 ng mL^−1).
Fig. 3. ROS scavenging and multienzyme-like antioxidative activity of
Cu[5.4]O USNPs.
[104]Fig. 3
[105]Open in a new tab
a H[2]O[2], b O[2]^−, c OH·, and d free radical scavenging ability of
Cu[5.4]O USNPs. e H[2]O[2]-scavenging capacities of Cu[5.4]O USNPs and
Cu[5.4]O@PEG USNPs. f CAT-like, g SOD-like, and h GPx-like activity of
Cu[5.4]O USNPs. i, j The CAT-like activity of the Cu[5.4]O USNPs after
different treatments. H[2]O[2] (2 mM) elimination efficiencies of
Cu[5.4]O USNPs (250 ng mL^−1) and natural CAT (40 U mL^−1),
respectively, pretreated with different i temperature and j pH
conditions. k Stability test of Cu[5.4]O USNPs (250 ng mL^−1) when used
as CAT-like catalyst at different cycles. The concentration of H[2]O[2]
is 2 mM. l XPS analysis of Cu[5.4]O USNPs before and after treatment
with H[2]O[2]. In a–j, data represent means ± s.d. from five (a–d) or
three (e–j) independent replicates. Source data are provided as a
Source Data file.
Compared with the working concentrations of reported metal-based
nano-antioxidants for ROS scavenging at cellular level, such as
Ce^[106]32, Au^[107]33, TiO[2] (ref. ^[108]34), MnO[2] (ref. ^[109]35),
Co[3]O[4] (ref. ^[110]36), and V[2]O[5] (ref. ^[111]37), which are
typically in the range of 20–50 μg mL^−1 in vitro, the working
concentration of Cu[5.4]O USNPs (25 ng mL^−1) was 2–3 orders of
magnitude lower, indicating higher antioxidant efficacy. The working
concentration of Cu[5.4]O USNPs was 200-fold lower than that of
previously reported Cu[x]O (x = 1−2)^[112]19 for the same
H[2]O[2]-scavenging capacity. Approximately 80–90% of H[2]O[2] (1 mM)
can be scavenged by 0.4 mg mL^−1 of vitamin C^[113]38, whereas
200 ng mL^−1 of Cu[5.4]O USNPs could scavenge approximately 85% of
H[2]O[2] (2 mM). Therefore, Cu[5.4]O USNPs showed much stronger
H[2]O[2]-scavenging efficiency (approximately 2000-fold) than commonly
used small molecular antioxidants, such as vitamin C. To the best of
our knowledge, the working concentration of Cu[5.4]O USNPs was also the
lowest among all ROS scavenging nanomaterials that have been reported
so far (Supplementary Table [114]1).
Mechanism of ROS scavenging activities of Cu[5.4]O USNPs
Since AA has strong antioxidant activity and can eliminate harmful free
radicals both in vitro and in vivo^[115]39, we initially suspected that
the ROS scavenging activities of Cu[5.4]O USNPs originated from the
surface-coated AA. We used chemically inert HS-PEG-OH to replace AA
molecules on the surface of Cu[5.4]O USNPs (Supplementary Fig. [116]6).
Surprisingly, the H[2]O[2]-scavenging abilities of Cu[5.4]O and
Cu[5.4]O@PEG USNPs were very similar (Fig. [117]3e), suggesting that
the presence of AA is not essential for the excellent ROS scavenging
abilities of Cu[5.4]O USNPs. According to the previous
reports^[118]40,[119]41 and our results in Supplementary Figs. [120]7
and [121]8, the AA molecules were possibly converted into
dehydroascorbic acid through oxidation of Cu^2+, and thus lost their
reduction activity.
Multienzyme-like antioxidative activity of Cu[5.4]O USNPs
We further investigated the antioxidant enzyme-mimetic activities of
Cu[5.4]O USNPs. Among biologically relevant ROS, H[2]O[2] is of
greatest importance because of its membrane permeability, longer
half-life than O[2]^− and ·OH, and consequently highest intracellular
concentration^[122]42. Therefore, we focused on the CAT-like property
of Cu[5.4]O USNPs, which was responsible for decomposition of H[2]O[2].
As shown in Fig. [123]3f, the concentration-dependent CAT-like activity
of Cu[5.4]O USNPs was investigated. Terephthalic acid (TPA) was used as
a fluorescence probe, which reacts with ·OH from H[2]O[2], forming
highly fluorescent 2-hydroxyterephthalic acid. Gradual reduction of
fluorescence intensity was observed as the concentration of Cu[5.4]O
USNPs increased (Supplementary Fig. [124]9). Gradual reduction in the
characteristic absorbance of H[2]O[2] at 240 nm (Supplementary
Fig. [125]10a) and gradual enhancement of the O[2] content
(Supplementary Fig. [126]10b) were observed with a prolonged reaction
time in the presence of Cu[5.4]O USNPs.
A steady-state kinetic assay was performed to confirm the enzymatic
activity by varying the concentration of H[2]O[2] in the presence of
Cu[5.4]O USNPs. This reaction showed typical Michaelis–Menten kinetics
(Supplementary Fig. [127]11). The K[m] value of the Cu[5.4]O USNPs with
H[2]O[2] as the substrate was 0.065 mM, and the V[max] value was
3.92 × 10^−6 Ms^−1. The K[m] value of Cu[5.4]O USNPs was smaller than
that of natural CAT (0.134 mM), indicating that our Cu[5.4]O USNPs
possessed greater affinity for substrate H[2]O[2], possibly due to
their ultrasmall size and greater numbers of exposed active sites.
Moreover, the concentration-dependent SOD-like activity of Cu[5.4]O
USNPs was also investigated. As shown in Fig. [128]3g, the percentage
of formed formazan reduced significantly in the presence of Cu[5.4]O
USNPs, indicating the SOD-like activity of Cu[5.4]O USNPs. According to
the previous literature, the EC[50] (an indicator for comparing the
efficiencies of enzymes and enzyme mimics) of native Cu–Zn
SOD^[129]25,[130]43 was 41.6 ng mL^−1. The EC[50] of Cu[5.4]O USNPs was
calculated to be 191.4 ng mL^−1. Thus, the EC[50] of Cu[5.4]O USNPs was
approaching 21.7% of the native SOD activity, which was nearly twice as
much as that of the Cu-TCPP MOF nanodots (12.6% of native SOD activity)
reported by Qu and co-workers^[131]25. Besides, the SOD-like activity
of Cu[5.4]O USNPs was further confirmed by electron paramagnetic
resonance (EPR) spectroscopy (Supplementary Fig. [132]12). In addition,
Cu[5.4]O USNPs also displayed concentration-dependent GPx-like
activity, eliminating H[2]O[2] and catalyzing the oxidation of reduced
glutathione (Fig. [133]3h).
Natural enzymes often exhibit intrinsic shortcomings, such as low
operational stability, temperature and pH sensitivity, and recycling
difficulties. Hence, the thermal and pH stabilities of Cu[5.4]O USNPs
were investigated and compared with those of the natural enzyme CAT.
The results (Fig. [134]3i, j) suggested that the stabilities of
Cu[5.4]O USNPs when exposed to pH and temperature variations were
significantly greater than those of natural CAT. Besides, the recycled
Cu[5.4]O USNPs showed nearly identical catalytic activity as the
original solution (Fig. [135]3k), indicating good stability and
recyclability of Cu[5.4]O USNPs.
The X-ray photoelectron spectroscopy (XPS) spectra of the Cu 2p core
level region for Cu[5.4]O and Cu[5.4]O oxidized by H[2]O[2] showed only
two intense peaks at 932.4 and 952.0 eV for Cu[5.4]O USNPs before and
after oxidation, which were assigned to the binding energies of Cu
2p[3/2] and Cu 2p[1/2]; these corresponded to Cu^+ and/or Cu^0 species,
respectively (Fig. [136]3l). Upon interaction with H[2]O[2], the
positions of the two main peaks did not shift and new peaks rarely
appeared. Hence, we concluded that the ROS scavenging performance of
Cu[5.4]O USNPs can be attributed to its intrinsic multienzyme-mimicking
properties.
Scavenging ROS ability in vitro
Compared with the glomerulus, renal tubules are more susceptible to
oxidative stress in AKI^[137]44. Therefore, protection of renal tubules
against ROS damage and subsequent initiation of a cascade of
pathological processes during the early stage of AKI would
significantly reduce kidney dysfunction^[138]44. For this analysis, the
human embryonic kidney 293 (HEK293) cell line was used to examine the
cytoprotective properties of Cu[5.4]O USNPs against ROS damage in
vitro. As shown in Fig. [139]4a, the intracellular ROS level (green
fluorescent signal) of HEK293 cells increased dramatically after
treatment with 250 μM H[2]O[2], leading to abnormal and shrunken cell
morphology. In comparison, the intracellular ROS level obviously
decreased when the cells were pretreated with Cu[5.4]O USNPs.
Quantitative analysis of intracellular ROS levels via flow cytometry
further confirmed this trend (Fig. [140]4b, c, Supplementary
Fig. [141]13a). The results of CCK-8 analysis (Fig. [142]4d) revealed
that an extremely low concentration (25 ng mL^−1) of Cu[5.4]O USNPs was
able to completely protect the cells against 250 μM H[2]O[2], which is
higher than the concentration in the classical AKI pathological
microenvironment.
Fig. 4. Scavenging ROS with Cu[5.4]O USNPs in vitro.
[143]Fig. 4
[144]Open in a new tab
a Representative ROS staining (green fluorescence) of HEK293 cells
under different treatment conditions. b ROS levels in untreated and
Cu[5.4]O USNPs-treated HEK293 cells incubated with 250 μM H[2]O[2]. c
Statistical analysis of ROS levels in HEK293 cells under different
treatment conditions. d In vitro cell viabilities of HEK293 cells under
different treatment conditions. e FACS results of cell apoptosis and
necrosis distribution in untreated and Cu[5.4]O USNPs-treated HEK293
cells. f Statistical analysis of necrotic and apoptotic cell ratios in
HEK293 cells under different treatment conditions. In c, d and f, data
represent means ± s.d. from four (c, f) or six (d) independent
replicates. ***P < 0.001; n.s., no significance, one-way ANOVA. Source
data are provided as a Source Data file.
We further examined the effect of Cu[5.4]O USNPs against
H[2]O[2]-induced cell apoptosis and necrosis via flow cytometry. In
Fig. [145]4e, f and Supplementary Fig. [146]13b, the addition of
Cu[5.4]O USNPs significantly reduced the ratios of apoptotic and
necrotic cells induced by H[2]O[2] treatment, further confirming the
ROS scavenging and cytoprotective properties of Cu[5.4]O USNPs at the
cellular level.
As shown in Supplementary Fig. [147]14, Cu[5.4]O USNPs could be found
in the mitochondria of cells, which was consistent with previous
reports that ultrasmall NPs could easily enter the mitochondria through
the mitochondrial permeability transition pore^[148]45,[149]46,
indicating that Cu[5.4]O USNPs could alleviate mitochondrial oxidative
stress and maintain mitochondrial function. Besides, Cu[5.4]O USNPs
could also be observed in the phagosome, suggesting that Cu[5.4]O USNPs
might enter into the cells through phagocytosis.
In vitro and in vivo biocompatibility of Cu[5.4]O USNPs
The CCK-8 assay results (Supplementary Fig. [150]15) suggested that
Cu[5.4]O USNPs at test concentrations did not exhibit noticeable
cytotoxicity. HEK293 cells exposed to 200 ng mL^−1 Cu[5.4]O USNPs for
48 h showed normal polygonal cytoskeleton morphology (Supplementary
Fig. [151]16), indicating good biocompatibility. Such results are in
accordance with previously findings that copper NPs are generally
non-toxic at relatively low concentrations (<5 μg mL^−1)^[152]47.
Additionally, hemolysis rate of nanomaterials must be less than 5% to
ensure safety during intravenous administration^[153]48,[154]49. As
shown in Supplementary Fig. [155]17, the hemolysis rate of 500 ng mL^−1
Cu[5.4]O USNPs, which is 10-fold greater than the concentration applied
in vivo to treat AKI (2 μg kg^−1, corresponding to 50 ng mL^−1 for a
25 g mouse with 1 mL of blood), was less than 5%.
Next, we evaluated the impacts of Cu[5.4]O USNPs (4 μg kg^−1, twofold
greater than the concentration used to treat AKI) on blood chemistry,
inflammatory cytokine levels, and major organ histopathology in healthy
mice to reveal their in vivo biocompatibility. As shown in Fig. [156]5a
and Supplementary Fig. [157]18, no necrosis, congestion, or hemorrhage
was observed in the heart, liver, spleen, and lung at 1 and 30 days
after single dose intravenous injection of Cu[5.4]O USNPs. Moreover, no
distinguishable inflammatory lesions or tissue damage were observed in
the glomerulus, tubules, collecting ducts, and urethra at 24 h after
single dose intravenous injection of Cu[5.4]O USNPs (Fig. [158]5b).
Fig. 5. In vivo biocompatibility assessment of Cu[5.4]O USNPs.
[159]Fig. 5
[160]Open in a new tab
a Evaluation of in vivo toxicity of Cu[5.4]O USNPs to major organs
(heart, liver, spleen, and lung) at 24 h after intravenous
administration. b Assessment of in vivo toxicity of Cu[5.4]O USNPs to
the kidney at 24 h after intravenous administration. c Serum levels of
inflammatory factors of interleukin-6 (IL-6) and tumor necrosis factor
alpha (TNF-α). d Serum levels of liver function indicators: aspartate
transaminase (AST) and alanine transaminase (ALT). e Serum levels of
kidney function indicators: blood urea nitrogen (BUN) and creatinine
(CRE). f–j Blood parameters in normal mice (control group), and mice
intravenously injected with Cu[5.4]O USNPs, 24 h after injection. In
c–j, data represent means ± s.d. from five independent replicates.
Source data are provided as a Source Data file.
As shown in Fig. [161]5c, the serum levels of interleukin-6 (IL-6) and
tumor necrosis factor-α (TNF-α) in the Cu[5.4]O USNPs-treated group
were identical to the levels in the control group (P > 0.05),
indicating that Cu[5.4]O USNPs would not trigger obvious immune
responses in vivo at the tested concentration. The serum biochemistry
analysis results (Fig. [162]5d, e) showed that serum concentrations of
liver function indicators (aspartate transaminase (AST) and alanine
transaminase (ALT)) and kidney function indicators (BUN and CRE) in the
Cu[5.4]O USNPs-treated group were similar to those in the control group
(P > 0.05), revealing good biocompatibility in the liver and kidney.
Moreover, the results of complete blood panel analysis (Fig. [163]5f–j)
showed no obvious differences in the hematology of the Cu[5.4]O
USNPs-treated group when compared to that of the control group
(P > 0.05).
We also investigated the accumulation of Cu[5.4]O USNPs in the major
organs after repeated daily administration of Cu[5.4]O USNPs for seven
consecutive days. As shown in Supplementary Fig. [164]19, the liver and
kidneys exhibited higher normalized dosage distribution of Cu[5.4]O
USNPs, reaching 7.92 and 7.97 %ID g^−1 respectively, while the
distributions of NPs in the heart, spleen, lung, and blood reached
2.25, 5.07, 3.02, and 2.84 %ID g^−1, respectively. As shown in
Supplementary Fig. [165]20, Cu[5.4]O USNPs could be found in the
tissues of major organs by TEM, which is consistent with the above
results.
The in vivo toxicity of Cu[5.4]O USNPs accumulated in the major organs
after repeated intravenous administration was also evaluated. As shown
in Supplementary Fig. [166]21, no necrosis, congestion, or hemorrhage
was found in the heart, liver, spleen, lung, and kidney of the mice
after intravenous repeated administration. Besides, the serum
biochemistry analysis and complete blood panel analysis (Supplementary
Fig. [167]22) results further indicated no obvious toxicity of Cu[5.4]O
USNPs. All the results confirmed that the synthesized Cu[5.4]O USNPs
exhibited negligible short-term and long-term in vivo toxicity.
Pharmacokinetics and biodistribution of Cu[5.4]O USNPs in mice
The time-dependent blood circulation profiles of Cu[5.4]O USNPs in
Fig. [168]6a demonstrated a classical two-compartment pharmacokinetic
model. The terminal elimination half-lives of the central component and
peripheral component were 0.77 and 71.2 h, respectively. The
biodistribution of Cu[5.4]O USNPs in the major organs of AKI mice at
24 h post injection was detected by inductively coupled plasma-atomic
emission spectrometry. As shown in Fig. [169]6b, the kidney exhibited
the highest normalized dosage distribution of Cu[5.4]O USNPs
(4.4 %ID g^−1), while the distributions of NPs in the heart, liver,
spleen, and lung reached 0.89, 3.2, 2.1, and 0.94 %ID g^−1,
respectively. The accumulation of Cu[5.4]O USNPs in the kidneys might
be attributed to the high renal uptake resulting from their ultrasmall
hydrodynamic diameter (approximately 4.5 nm) and excellent
hydrophilicity. Previous studies showed that the size threshold of the
glomerular basement membrane (GBM) was approximately 5.5 nm, and
nanomaterials with a diameter less than 5.5 nm could be effectively
cleared from the blood to the renal tubules through the
GBM^[170]50–[171]52. As shown in Supplementary Fig. [172]23, TEM
analysis revealed Cu[5.4]O USNPs in the urine of AKI mice after
injection, further confirming that the Cu[5.4]O USNPs could pass
through the GBM to the tubules and then undergo excretion in the urine.
The time-dependent accumulation of Cu[5.4]O USNPs in the kidneys of
normal and AKI mice indicated that Cu[5.4]O USNPs could effectively
accumulate in the kidneys, regardless of the permeability of the GBM
(Supplementary Fig. [173]24). Moreover, time-dependent accumulation of
Cu[5.4]O USNPs in the urine revealed that renal clearance is the main
excretion pathway for these particles, rather than intestinal excretion
(Fig. [174]6c). Approximately 70% of Cu[5.4]O USNPs could be excreted
within 48 h post injection (60% through the kidneys and 10% through the
intestine), suggesting relatively rapid clearance and potentially low
long-term toxicity of Cu[5.4]O USNPs.
Fig. 6. Pharmacokinetics, biodistribution, and clearance of Cu[5.4]O USNPs in
AKI mice.
[175]Fig. 6
[176]Open in a new tab
a In vivo blood pharmacokinetic curves after intravenous injection of
Cu[5.4]O USNPs. b Biodistribution of Cu[5.4]O USNPs in major organs at
24 h post injection. c Cumulative urine and feces excretion at
different time points. d Biodistribution of Cu[5.4]O USNPs in renal
tissues by TEM observation. Red dashed lines indicate the magnified
area. Dots with red pseudocolor indicate Cu[5.4]O USNPs. In a–c, data
represent means ± s.d. from three independent replicates. Source data
are provided as a Source Data file.
To further analyze the distribution of Cu[5.4]O USNPs in the kidneys,
renal tissues were collected, ultrathin sectioned, and observed under
TEM. As shown in Fig. [177]6d and Supplementary Fig. [178]25, Cu[5.4]O
USNPs (arrowheads) were found in the GBM, urinary space, and epithelial
cell cilia of renal tubules, confirming that the Cu[5.4]O USNPs could
pass through the GBM and reach tubules containing urine.
In vivo therapeutic efficacy of Cu[5.4]O USNPs on AKI mice
The therapeutic effect of Cu[5.4]O USNPs on AKI mice was tested
(Fig. [179]7a). As shown in Fig. [180]7b, compared with the control
group (90% of mice died within 5 days following establishment of the
AKI model), all AKI mice treated with Cu[5.4]O USNPs survived for more
than 14 days. In addition, AKI mice treated with Cu[5.4]O USNPs
exhibited a weight increase similar to that of healthy mice
(Fig. [181]7c), while control AKI mice underwent dramatic body weight
loss within 24 h. Furthermore, the serum levels of two important kidney
function indicators, BUN and CRE levels, of AKI mice treated with
Cu[5.4]O USNPs were significantly lower than those of AKI mice in the
control group (P < 0.001, Fig. [182]7d, e). There were no detectable
differences in BUN and CRE levels between healthy mice and Cu[5.4]O
USNPs-treated AKI mice (P > 0.05), confirming the excellent therapeutic
efficacy of Cu[5.4]O USNPs in the treatment of glycerol-induced AKI.
Fig. 7. Ttherapeutic efficiency of Cu[5.4]O USNPs on AKI mice.
[183]Fig. 7
[184]Open in a new tab
a Schematic illustration of the establishment and treatment schedule of
AKI mice. b Survival curves of AKI mice with different treatment. c
Weight variation of AKI mice at 24 h after treatment with Cu[5.4]O
USNPs. Serum levels of d CRE and e BUN in AKI mice at 24 h after
different treatment. f H&E staining of kidney tissues from each group.
Triangles indicate the formation of casts. g Dihydroethidium (red
fluorescence) and DAPI (blue fluorescence) staining of kidney tissues
from each group. h SOD, i KIM-1, and j HO-1 levels measured in renal
tissue homogenates from each group. In c–e and h–j, data represent
means ± s.d. from four independent replicates (***P < 0.001; n.s., no
significance, One-way ANOVA). Source data are provided as a Source Data
file.
In kidney diseases, the precipitation of denatured proteins in tubules
forms a cast structure, which is regarded as an important diagnostic
marker of pathological changes. As shown in Fig. [185]7f, many casts
(marked as triangles) could be found in the renal tissues of AKI mice,
whereas only a few casts could be observed in AKI mice that were
treated with Cu[5.4]O USNPs, suggesting that renal tissue integrity was
maintained via adoption of the NPs.
To further demonstrate the therapeutic activity of Cu[5.4]O USNPs
acting as a ROS scavenger in vivo, NAC molecules with different
concentrations (8, 40, and 160 mg kg^−1) were intravenously injected
into AKI mice as controls. As shown in Supplementary Fig. [186]26, the
CRE, BUN levels and survival percentage of AKI mice treated with
Cu[5.4]O USNPs (at 2 μg kg^−1 concentration) were similar to those of
AKI mice treated with 160 mg kg^−1 of NAC, which was also in accordance
with the histological analysis of renal tissues. The overall results
indicated the in vivo ROS scavenging activity of Cu[5.4]O USNPs.
The ROS levels and SOD activity in the kidneys were further
investigated to understand the molecular mechanism underlying the
protection process. Compared with PBS-treated AKI mice, the levels of
superoxide in Cu[5.4]O USNPs-treated AKI mice were significantly
reduced, reaching the levels exhibited in normal mice (Fig. [187]7g,
Supplementary Fig. [188]27). As shown in Fig. [189]7h, the renal SOD
activity in Cu[5.4]O USNPs-treated AKI mice was similar to that of
healthy mice, whereas a significant reduction in SOD activity was
observed in AKI mice in the control group (P < 0.001). This result
suggested that Cu[5.4]O USNPs could protect renal cells by functioning
as antioxidants to scavenge ROS and maintain SOD activity in vivo.
Furthermore, the renal expression levels of two important kidney injury
biomarkers^[190]53,[191]54, heme oxygenase-1 (HO-1) and kidney injury
molecule-1 (KIM-1), were detected. As shown in Fig. [192]7i, j,
compared with AKI mice in the control group, Cu[5.4]O USNPs-treated AKI
mice exhibited significantly reduced levels of KIM-1 and HO-1
(P < 0.001), consistent with the results we found for CRE and BUN.
The cisplatin-induced AKI (Cis-AKI) mouse model was also used to
demonstrate the broad application of Cu[5.4]O USNPs for ROS-mediated
AKI diseases. As shown in Supplementary Fig. [193]28, kidney function
examination and histological analysis revealed that kidney damage in
Cis-AKI mice treated with Cu[5.4]O USNPs was significantly lower than
that in Cis-AKI mice treated with PBS (P < 0.001; P < 0.05), confirming
the therapeutic effect of Cu[5.4]O USNPs against Cis-AKI.
Therapeutic effect on acute liver injury and wound healing
It is intriguing for us to discover whether the Cu[5.4]O USNPs could be
applicable to ROS-related pathological conditions other than AKI.
Therefore, acetaminophen (APAP)-induced acute liver injury (AILI) and
full-thickness skin defect were chosen as the representative
ROS-related disease models as well. As shown in Fig. [194]8a, b, the
ALT and AST levels of Cu[5.4]O USNPs-treated AILI mice were
significantly lower than those of AILI mice without treatment
(P < 0.001), indicating the excellent therapeutic effect of Cu[5.4]O
USNPs on AILI. Histological observation of liver tissues further
confirmed the therapeutic effect of Cu[5.4]O USNPs on AILI
(Fig. [195]8c).
Fig. 8. Therapeutic efficiency of Cu[5.4]O USNPs on AILI and wound healing.
[196]Fig. 8
[197]Open in a new tab
Serum levels of a AST and b ALT in AILI mice at 24 h after different
treatment. c H&E staining of liver tissues from each group. Blue dashed
lines indicate the range of hepatic necrosis. The percentage of d
closed diabetic wound area and representative macroscopic appearance of
e diabetic wound at different time points. A 6-mm-diameter standard
green disc was used as the reference when taking photos. f
Representative histological images and g quantitative determination of
the length of regenerated epidermis on day 15 post-surgery. The yellow
double-headed arrows indicate the regenerated epidermis. h
Representative histological images and i quantitative measurement of
granulation tissue thickness on day 15 post-surgery. The yellow
double-headed arrows indicate the granulation tissue. In a, b, data
represent means ± s.d. from five independent replicates (one-way
ANOVA). In d, g and i, data represent means ± s.d. from three
independent replicates (Student’s t-test, **P < 0.01; ***P < 0.001).
Source data are provided as a Source Data file.
Elevated ROS production in the wound site has also been implicated in
delaying wound healing^[198]32,[199]55, especially in chronic diabetic
wounds where high glucose and proinflammatory environment caused the
large production of ROS in the wound bed^[200]56,[201]57. As shown in
Fig. [202]8d, e, the diabetic wound healing rate of Cu[5.4]O USNPs
group was always significantly faster than that of the corresponding
control group on days 4, 7, 9, and 15 post-surgery (P < 0.01). The
results clearly indicated that Cu[5.4]O USNPs could accelerate the
diabetic wound healing process. As shown in Fig. [203]8f–i, the length
of newly regenerated epidermis and the thickness of granulation tissue
in the Cu[5.4]O USNPs group were significantly greater than those in
the control group, further confirming that Cu[5.4]O USNPs could promote
diabetic wound healing.
Therapeutic mechanisms of Cu[5.4]O USNPs on AKI
To further elucidate the underlining therapeutic mechanisms, AKI was
chosen as the representative disease model for further transcriptomics
analysis. An unguided principal component analysis (PCA) of the data
revealed substantially different transcriptomic profiles between
Cu[5.4]O- and PBS-treated AKI mice kidneys (Fig. [204]9a). The Venn
diagram in Fig. [205]9b showed that 13025 genes were co-expressed by
two groups, while 584 genes were exclusively expressed by
Cu[5.4]O-treated group. Volcano plots (Fig. [206]9c) showed 5819
significantly differentially expressed genes (DEGs), of which 2813 and
3006 genes were upregulated and downregulated, respectively.
Fig. 9. Therapeutic mechanisms of Cu[5.4]O USNPs on AKI.
[207]Fig. 9
[208]Open in a new tab
a Principal component analysis (PCA) was performed based on
differentially expressed genes from the kidneys of two groups. Each
data point corresponds to the PCA analysis of each sample. b Venn
diagram of the transcriptomic profiles between Cu[5.4]O USNPs and PBS
groups. c Volcano plots showing the identified upregulated and
downregulated genes by Cu[5.4]O USNPs. d KEGG pathway enrichment
analysis of the identified differentially expressed genes. The 20 most
significantly enriched pathways are shown. Heat maps of significantly e
upregulated and f downregulated genes involved in the oxidative stress
after Cu[5.4]O USNPs treatment (fold change ≥2 and P < 0.05). g
Protein–protein interaction network of differentially expressed genes
involved in the oxidative stress. h qRT-PCR analysis of the mRNA
expression levels of antioxidant genes. i Western blot analysis of the
expression of phospho-NF-κB p65, total NF-κB p65, phospho-IκB-α, and
total IκB-α in kidney tissues of AKI mice. Serum levels of j TNF-α and
k IL-1β. Renal levels of l TNF-α and m IL-1β. In h, j–m, data represent
means ± s.d. from three independent replicates, and P values were
calculated by Student’s t-test (h) and one-way ANOVA (j–m). *P < 0.05;
**P < 0.01; ***P < 0.001. Source data are provided as a Source Data
file.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis (Fig. [209]9d) indicated that the glutathione (GSH)
metabolism, MAPK signaling pathway, and TNF signaling pathways were
highly associated with the therapeutic mechanisms of Cu[5.4]O USNPs.
Literature has reported that ROS could activate the MAPK signaling
pathway to induce renal cell apoptosis and the release of local or
systemic inflammatory mediators to aggravate renal
injury^[210]58–[211]60. Notably, the MAPK signaling pathway
(Supplementary Fig. [212]29) was significantly inhibited after Cu[5.4]O
USNPs treatment, indicating that Cu[5.4]O USNPs could alleviate renal
injury via inhibiting the MAPK signaling pathway through decreasing the
ROS level. In addition, it has been reported that ROS could promote the
production of proinflammatory cytokines (e.g. TNF-α, IL-1β,
IL-6)^[213]58,[214]61 and that TNF-α could further trigger a strong
cascade inflammatory response throughout the TNF-α/MAPK and TNF-α/NF-κB
signaling pathways, resulting in an excessive inflammation response and
more pronounced renal damage^[215]62. Notably, the TNF signaling
pathway (Supplementary Fig. [216]30) was also significantly inhibited
after Cu[5.4]O USNP treatment, suggesting that other therapeutic
mechanisms of renal protection occur through inhibiting the TNF
signaling pathway.
We also investigated the impact of Cu[5.4]O USNPs on the expression of
genes related to oxidative stress. As shown in Fig. [217]9e, f, several
important antioxidant genes, including SOD1, SOD2, SOD3, GPX1, GPX3,
GPX4, GPX6, and CAT, were significantly upregulated after the Cu[5.4]O
USNPs treatment. The expression trend of the SOD and HMOX1 genes was
consistent with the aforementioned result regarding the SOD
(Fig. [218]7h) and HO-1 proteins level (Fig. [219]7j) in AKI mice,
respectively. Moreover, the genes associated with oxidative stress that
significantly changed after Cu[5.4]O USNP treatment were used in the
protein–protein interactions network analysis (Fig. [220]9g). We
discovered that the neighboring proteins connected to the leading
proteins contained SOD1, SOD3, CAT, etc., indicating that these genes
play an important role in ROS scavenging after Cu[5.4]O treatment. As
shown in Fig. [221]9h, the mRNA expression levels of antioxidant genes
in Cu[5.4]O-treated mice kidneys were significantly higher than those
of the corresponding control group, confirming that Cu[5.4]O USNPs
could maintain a high expression of antioxidant genes in AKI by
protecting renal cells from ROS damage.
The phosphorylation of NF-κB and IκB were significantly enhanced in the
AKI mice (Fig. [222]9i, Supplementary Fig. [223]31), indicating the
activation of NF-κB signaling pathway in AKI. Besides, the
phosphorylation of NF-κB and IκB were significantly decreased in the
Cu[5.4]O USNPs group, indicating that the NF-κB signaling pathway was
inhibited after Cu[5.4]O USNPs treatment, which was also in accordance
with the aforementioned transcriptomics analysis result (Supplementary
Fig. [224]30). We also detected the downstream inflammatory factors of
the NF-κB signaling pathway. As shown in Fig. [225]9j–m, Cu[5.4]O USNPs
could significantly reduce the serum and tissue levels of TNF-α and
IL-1β, indicating that Cu[5.4]O USNPs could protect kidney tissues from
oxidative stress by inhibiting the production of excessive
proinflammatory factors.
Additionally, we found that several important genes related to tissue
repair, including fibroblast growth factor 10 (FGF10), hepatocyte
growth factor (HGF), NOTCH1, and wingless-type MMTV integration site
family member 7A (WNT7A), were significantly upregulated after the
Cu[5.4]O USNPs treatment (Supplementary Fig. [226]32a, b). The
protein–protein interaction network further confirmed the importance of
these genes in tissue repair and regeneration, as shown in
Supplementary Fig. [227]32c. The overall results indicated in addition
to the ROS scavenging ability, Cu[5.4]O USNPs may also promote the
expression of genes related to renal repair and regeneration.
In summary, we have presented uniform and stable Cu[5.4]O USNPs that
mimic an intracellular antioxidant enzyme-based defense system. The
distinctive advantages of Cu[5.4]O USNPs are the ultrasmall particle
size, rapid renal clearance, high biocompatibility, and broad ROS
scavenging abilities. Both in vitro and in vivo experiments
demonstrated excellent biocompatibility and cytoprotective effects of
Cu[5.4]O USNPs against ROS-mediated damage. Cu[5.4]O USNPs could be
applicable to various ROS-related pathological conditions such as AKI,
ALI, and diabetic wound. Taken together, the synthesized ultrasmall
Cu[5.4]O USNPs with robust ROS scavenging abilities and excellent
biocompatibility could represent a promising antioxidant for the
treatment of AKI and other oxidative stress-related diseases. We expect
that our findings will promote the development of nanomaterials with
multiple enzyme-mimicking properties and enable further clinical
applications of copper-based ROS scavengers in biomedical treatment and
research.
Methods
Materials
Cupric chloride (CuCl[2]), l-ascorbic acid (AA), and sodium hydroxide
(NaOH) were purchased from J&K Scientific (Beijing, China).
3,3′,5,5′-Tetramethylbenzidine (TMB) and
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was bought
from Macklin (Shanghai, China). Acetic acid (HAc), sodium acetate
(NaAc), and potassium persulfate (K[2]S[2]O[8]) was obtained from
Sigma-Aldrich (Shanghai, China). Thiol-polyethylene glycol-OH
(SH-PEG-OH) (Mw: 1000 Da) was purchased from Yare Biotech. Inc.
(Shanghai, China). Catalase (purified powder from bovine liver),
superoxide dismutase (purified powder from bovine erythrocytes), and
peroxidase (purified powder from horseradish roots) were from Sangon
Biotech (Shanghai, China). Phosphate-buffered saline (PBS, pH 7.4,
Na[2]HPO[4]–NaH[2]PO[4], 10 mM) solution was prepared in the
laboratory. All chemicals and reagents were of analytical grade and
used as received without further purification. Ultrapure water
(18.2 MΩ cm^−1 at 25 °C) purified by a Milli-Q system was used
throughout the experiment.
Instrumentation
TEM imaging was conducted on a Tecnai G220 (Shimadzu, Japan) at
200 keV. The sample was prepared by dispersing a small amount of
freeze-dried powder in the PBS. Then, the suspension was dropped on 230
mesh copper TEM grids covered with thin amorphous carbon films. Fourier
Transform infrared spectroscopy (FTIR) spectra were measured by a
FTIR-8300 series spectrometer (Shimadzu, Japan) in the range of
4000–400 cm^−1. The fluorescence spectra were determined by using an
F-2500 spectrofluorometer (Hitachi, Japan). The UV-vis spectra were
obtained using a Hitachi U-3010 spectrometer (Hitachi, Japan). A
dynamic light scattering (DLS) particle size analyzer (Malvern 2000,
USA) was used to determine the hydrophilic diameters of the particles.
XPS and X-ray Auger electron spectroscopy measurement were performed by
an ESCALAB 250 Xi Mg (Thermo Scientific, Japan) X-ray resource. Crystal
structure and oxidation state of Cu[5.4]O USNPs were analyzed using
X-ray diffractometer (XRD, Bruker AXS D8) with a scan rate of 1 s per
step with a step size of 0.02. The concentration of Cu was detected by
inductively coupled plasma-atomic emission spectrometry (ICP-AES)
(Thermo Scientific, iCAP 7400, USA). The concentration of Cu[5.4]O
USNPs used in following studies is calculated based on Cu element. The
EPR spectroscopy signal was obtained on a Bruker A300 (X-band)
spectrometer (Bruker, Germany). All the measurements were performed at
room temperature if not specially mentioned.
Synthesis of fluorescent Cu[5.4]O USNPs
The Cu[5.4]O USNPs was synthesized according to previous report with
some modifications^[228]40. In a typical preparation process, 10 mM
CuCl[2] powders were dissolved in 50 mL deionized water and stirred for
10 min at 80 °C in an oil bath with magnetic stirring. Then, l-ascorbic
acid aqueous solution (100 mM, 50 mL) was added slowly to the above
CuCl[2] solution. Afterwards, the pH of the solution was adjusted to
8.0–9.0 using NaOH solution (1 M). The mixture was kept at 80 °C for
12 h with constantly stirring. After reaction, the larger aggregates
were removed by centrifugation (6577 × g, 15 min), and then the
supernatant was dialyzed against water (Mw cutoff: 10,000 Da) for 2
days to remove small molecules. Purified Cu[5.4]O USNPs were
concentrated with centrifugation.
Surface functionalization of Cu[5.4]O USNPs by SH-PEG-OH
In total, 0.1 g Cu[5.4]O USNPs were dispersed into 5 mL water under
ultrasonication for 1 min and then stirred for 1 h. In all, 0.1 wt% of
SH-PEG-OH was added. The reaction mixture was stirred for another 24 h,
dialyzed to remove unreacted SH-PEG-OH molecules, and then concentrated
by centrifugal ultrafiltration.
H[2]O[2] scavenging activity of Cu[5.4]O USNPs
H[2]O[2] scavenging capacity of Cu[5.4]O USNPs was tested by the
Hydrogen Peroxide Detection Kit (Nanjing Jiancheng Bioengineering
Institute, China). H[2]O[2] reacts with ammonium molybdate to form a
stable yellow complex, which displays an absorbance peak at 405 nm.
Various concentrations of Cu[5.4]O USNPs (25–200 ng mL^−1) were
incubated with 2 mM H[2]O[2] at 37 °C for 2 h, respectively. After
reaction, the concentration of remaining H[2]O[2] was determined
according to the manufacturer’s instructions, and the
H[2]O[2]-eliminating capacity was calculated.
·OH scavenging activity of Cu[5.4]O USNPs
The TMB chromogenic method was performed for ·OH scavenging activity
test. The ·OH was generated by the classical Fenton reaction between
H[2]O[2] and Fe^2+, which can convert the TMB to a oxidized TMB (oxTMB)
with a characteristic absorption at 652 nm. Therefore, the
concentration of remaining ·OH can be determined via monitoring the
absorption at 652 nm of oxTMB. In detail, The working test solutions
containing 250 μM TMB, 2 mM H[2]O[2], 1 mM FeSO[4] and different
concentrations of Cu[5.4]O USNPs (25–175 ng mL^−1) in HAc/NaAc buffer
(0.5 M, pH 4.5) were prepared in the dark and rest for 5 min.
Afterwards, the absorbance peak in 652 nm of the solution was monitored
with a UV-vis spectroscopy.
The EPR spectroscopy signal was measured by a Bruker A300 spectrometer
(Bruker, Germany). Typically, 2 mM H[2]O[2], 20 μM FeSO[4], 100 mM
DMPO, and different concentration of Cu[5.4]O USNPs (0, 50,
150 ng mL^−1, respectively) were added into the HAc/NaAc buffer (0.5 M,
pH 4.5); EPR signal was detected immediately.
O[2]^− scavenging activity of Cu[5.4]O USNPs
The superoxide anion (O[2]^−) scavenging activity was assessed using a
superoxide anion assay kit (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China) according to the manufacturer’s instructions. Different
concentrations of Cu[5.4]O USNPs (0–150 ng mL^−1) were added to the
working solution. The absorbance at 550 nm was measured using a
multiple plate reader after standing for 10 min.
ABTS radical scavenging activity of Cu[5.4]O USNPs
The evaluation of ABTS radical scavenging activity was based on the
method reported by Wang et al.^[229]63. Briefly, the ABTS radicals were
generated by reacting 7 mM ABTS stock solution with 2.45 mM potassium
persulfate in the dark for 16 h. Then, the ABTS radical solution was
diluted by PBS to reach a proper absorbance at 734 nm. Two milliliters
Cu[5.4]O USNPs solutions (0, 50, 75, 100, 125, 150 ng mL^−1,
respectively) were mixed with 2 mL ABTS solution and placed in dark for
10 min. Then the absorbance peak at 734 nm was monitored with a UV–vis
spectroscopy. The ABTS radical scavenging abilities were calculated as
follows:
[MATH: ABTSscavengingratio%=Acontrol−Asample/Acontrol×100, :MATH]
1
where A[control] is the absorbance of a standard without any radical
scavengers, and A[sample] is the absorbance after the reaction with the
radical scavengers, respectively.
CAT-like activity of Cu[5.4]O USNPs
The CAT-like activity of the Cu[5.4]O USNPs and the steady-state
kinetic were assayed using a fluorescence spectroscopy^[230]19,[231]36.
Typically, TPA is a non-fluorescent compound, which can react with ·OH,
decomposed from H[2]O[2], to produce a fluorescent aromatic
hydroxylated product (2-hydroxyterephthalic acid) with
excitation/emission peaks at 315/425 nm respectively. In the presence
of CAT or CAT analogs, the H[2]O[2] decomposed into H[2]O and O[2]
could not produce the fluorescent 2-hydroxyterephthalic acid. Hence,
the CAT-like activity of the Cu[5.4]O USNPs was investigated by
monitoring the fluorescence signal of the system. The steady-state
kinetic assay of Cu[5.4]O USNPs and CAT were performed to confirm the
enzymatic catalysis mechanism, by varying the concentration of H[2]O[2]
(0–6 mM) in the presence of Cu[5.4]O USNPs (1000 ng mL^−1) and H[2]O[2]
(0–1 mM) in the presence of CAT. Firstly, 200 μL 1000 ng mL^−1 of
Cu[5.4]O USNPs solution or 20 U mL^−1 CAT was added into the PBS
(10 mM, pH 7.4), different concentrations of H[2]O[2] were added and
incubated at 40 °C for 5 h. Finally, the TPA in 2 mM NaOH was added and
the fluorescence spectra of the resultant solution were recorded under
an excitation of 315 nm, and the variation of fluorescence emission at
425 nm was adopted to quantify remaining H[2]O[2]. Apparent kinetic
parameters were calculated on the flowing basis of the Michaelis–Menten
equation^[232]64.
[MATH:
V0=VmaxSS+Km,
:MATH]
2
where V[0] is the initial catalytic rate, V[max] is the maximum rate
conversion, which is obtained when the catalytic sites on the enzyme
are saturated with substrate concentration, and K[m] is the apparent
Michaelis–Menten equation. Maximum initial velocity (V[max]) and
Michaelis–Menten constant (K[m]) were obtained using Lineweaver–Burk
plots^[233]65.
SOD-like activity of Cu[5.4]O USNPs
The SOD-like activity of Cu[5.4]O USNPs was determined by formazan
formation using a SOD assay kit (WST-1 method) (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China)^[234]19. Briefly, O[2]^− was
generated through the oxidation of xanthine by xanthine oxidase (XO),
which can convert the WST-1 into WST-1 formazan with a characteristic
absorption at 450 nm. The formazan concentration was determined at
450 nm using a multiple plate reader. The SOD-like activity of Cu[5.4]O
USNPs was further confirmed with EPR spectroscopy. Briefly, a series of
samples containing xanthine (5 mM) and xanthine oxidase (0.5 U mL^−1)
in 10 mM PBS and incubated for 10 min at 37 °C. Different Cu[5.4]O
USNPs solutions (0, 20, 100, 1000 ng mL^−1) were added and then the EPR
signals were recorded immediately.
GPx-like activity of Cu[5.4]O USNPs
The GPx-like activity of Cu[5.4]O USNPs was estimated using a GPx assay
kit (Solarbio, Shanghai, China) according to the manufacturer’s
instructions. Glutathione (GSH) can react with
5,5′-Dithiobis-2-nitrobenzoic acid (DTNB) to form a compound with
characteristic absorption at 412 nm, which could be monitored using a
UV-Vis spectroscopy. During the test, GSH was oxidized to oxidized
glutathione (GSSG). The decrease of GSH concentration is proportional
to the catalytic activity of Cu[5.4]O USNPs.
O[2] generation from H[2]O[2] catalyzed by Cu[5.4]O USNPs and CAT
A commercial O[2] probe (Oxford-optronix, Oxford, UK) was utilized to
measure the real-time O[2] level according to the manual instructions.
Ten milliliters of PBS was poured into a small three-necked flask and
stirred at 37 °C. The flask was then sealed with parafilm and rubber
stoppers, and inserted with two needles for gassing. 95% N[2]/5% CO[2]
gas mixture was bubbled into the flask for 20 min to create hypoxic
condition. Cu[5.4]O USNPs (500 μL, 200 ng mL^−1) and CAT (500 μL,
20 U mL^−1) solutions were injected to the mixture, while PBS was used
in the control group. Then H[2]O[2] (500 μL, 2 mM) was injected and the
amount of generated O[2] was monitored at predetermined time intervals.
Cell culture
The human embryonic kidney 293 (HEK293) cell line was purchased from
the American Type Culture Collection (ATCC). The HEK293 cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 100 μg mL^−1 streptomycin, and
100 U mL^−1 penicillin at 37 °C in an incubator supplied with a
humidified atmosphere of 5% CO[2].
In vitro ROS scavenging using Cu[5.4]O USNPs
To investigate the ROS scavenging ability of Cu[5.4]O USNPs in cells,
HEK293 cells were seeded into 96-, 48-, and 24-well plates at the
density of 1 × 10^4 cells per well, 5 × 10^4 cells per well, and
10 × 10^4 cells per well, respectively. After 24 h incubation, Cu[5.4]O
USNPs with different concentrations (0–50 ng mL^−1) were added to each
group of wells and incubated for 30 min. Then, cells were treated with
250 μM H[2]O[2] and further incubated at 37 °C for 24 h. Cells seeded
in 96-well plates were incubated with cell counting kit-8 (CCK-8,
Dojindo, Japan) to detect the cell viability. Wells without the
addition of H[2]O[2] were regarded as the negative control.
Furthermore, cells seeded in 48-well plates were stained with Annexin
V-FITC apoptosis detection kit (C1062, Beyotime, China) to detect the
ratio of apoptotic and necrotic cells^[235]66. In brief, HEK293 cells
in a well were collected, washed with cold PBS, and re-suspended in
195 μL binding buffer after the aforementioned incubation with H[2]O[2]
for 24 h. Then, 5 μL Annexin V-FITC and 10 μL PI were sequentially
added to the cell suspension and incubated at room temperature in dark
for 15 min. After that, cells were analyzed by the Attune Acoustic
Focusing Cytometer (Life Technologies, USA). At least 50,000 cells were
analyzed in each sample.
2′,7′-Dichlorofluorescin diacetate (DCFH-DA, D6883, Sigma-Aldrich,
USA), an oxidation sensitive fluorescent dye, was used to detect the
intracellular ROS level according to the literature^[236]25. Briefly,
DCFH-DA is a non-fluorescent chemical compound which could diffuse
through cell membrane freely and could be hydrolyzed by intracellular
esterase to DCFH. The non-fluorescent DCFH could be oxidized by the
intracellular ROS to fluorescent DCF. Therefore, the quantity of
intracellular ROS is correlated with the fluorescent intensity of DCF.
After the aforementioned incubation with H[2]O[2] for 24 h, cells were
gently rinsed thrice with serum-free medium to remove the free Cu[5.4]O
USNPs. Then, a final concentration of 10 μM of DCFH-DA in serum-free
medium was added to the cells and incubated in dark at 37 °C for
30 min. Afterwards, the cells were washed with serum-free medium thrice
to remove unloaded DCFH-DA probe, then were imaged using a laser
confocal microscope (Zeiss LSM780, Germany), and were subjected to a
flow cytometry analysis to quantify the intracellular ROS levels
respectively.
To determine the location of Cu[5.4]O USNPs in the cells, HEK293 cells
(1 × 10^6 cells) were collected after incubation with 50 ng mL^−1 of
Cu[5.4]O USNPs at 37 °C for 24 h. The collected cells were fixed with
2.5% glutaraldehyde at 4 °C for 2 h, then post-fixed with 1% osmium
tetroxide and prepared for TEM observation.
In vitro biocompatibility evaluation of Cu[5.4]O USNPs
The cytotoxicity of Cu[5.4]O USNPs was determined by the CCK-8 assay in
vitro. Briefly, HEK293 cells were seeded into 96-well culture plates at
the density of 1 × 10^4 cells per well and incubated at 37 °C in an
incubator with 5% CO[2] for 24 h. Afterwards, the cell culture medium
was aspirated and fresh culture media containing various concentrations
of Cu[5.4]O USNPs (0–200 ng mL^−1) were added. After 24 or 48 h
incubation, cells were gently washed once with sterile PBS and then
treated with 100 μL fresh culture medium and 10 μL CCK-8 solution, and
further incubated at 37 °C for 2 h. The cell viability was then
quantified by measuring the absorbance value at 450 nm by a microplate
reader (Thermo Varioskan Flash, USA).
The hemolysis assay was performed on the basis of previously reported
methods with some modifications^[237]67. All the animal experiments
were carried out under the approval of the Institutional Animal Care
and Use Committee of the Third Military Medical University (Army
Medical University). Fresh whole-blood samples were collected from the
orbital venous of healthy Sprague-Dawley rats. The collected blood
samples were centrifugated for 15 min at 231 × g to collect
erythrocytes and gently washed thrice with saline solution. Then,
3.67 mL of saline solution was added to erythrocytes collected from
1 mL blood. Afterwards, 100 μL of diluted erythrocytes suspension was
mixed with 1 mL Cu[5.4]O USNP dispersion at various concentrations
(50–5000 ng mL^−1). The mixed dispersions were incubated for 3 h at
37 °C and then centrifugated for 15 min at 13800 × g before observing
and recording the hemolysis phenomenon. The hemolysis ratio was
quantified by measuring the absorbance value of supernatant at 540 nm
with a microplate reader. Deionized water and saline solution were used
as the positive and negative control, respectively.
In vivo biocompatibility evaluation of Cu[5.4]O USNPs
To evaluate the biocompatibility of Cu[5.4]O USNPs in vivo, BALB/c mice
(aged 8–10 weeks, 20–25 g) were intravenously administrated with
Cu[5.4]O USNPs at a single dose of 4 μg kg^−1. The mice injected with
PBS were used as the control group. One day post injection, the blood
samples were collected for complete blood panel analysis and serum
biochemistry test. The serum biochemistry test included two important
indicators of hepatic function as aspartate aminotransferase (AST) and
alanine aminotransferase (ALT), and two indicators of kidney function
as blood urea nitrogen (BUN) and creatinine (CRE). Serum IL-6 and TNF-α
levels were quantified by the ELISA assay. One day and thirty days post
injection, the mice were sacrificed to harvest major organs (including
heart, liver, spleen, lung, and kidney) for hematoxylin and eosin (H&E)
staining and histological analysis.
To evaluate the in vivo toxicity of Cu[5.4]O USNPs after repeated
administration, BALB/c mice (aged 8–10 weeks, 20–25 g) were
intravenously administrated with Cu[5.4]O USNPs at a dose of 4 μg kg^−1
every day for seven consecutive days. On eighth day, mice were
sacrificed to harvest major organs for H&E staining and collect blood
samples for complete blood panel analysis and serum biochemistry test.
To detect the accumulation of Cu[5.4]O USNPs in the major organs after
repeated administrations, BALB/c mice (aged 8–10 weeks, 20–25 g, n = 8)
were intravenously administrated with Cu[5.4]O USNPs at a dose of
4 μg kg^−1 every day for seven consecutive days. On the eighth day,
mice were sacrificed to harvest major organs. The organs of four mice
were weighed, homogenized, and then dissolved in aqua regia to
calculate the percentage of injected dose per gram of tissue (%ID g^−1)
by ICP-AES. The organs of the other four mice were fixed with the
mixture of 2.5% glutaraldehyde and 4% formaldehyde for 24 h, and then
post-fixed with 1% osmium tetroxide and prepared for TEM observation.
AKI models in mice
The glycerol-induced AKI model was established according to the
previously reported protocol^[238]10. Briefly, female BALB/c mice (aged
8–10 weeks, 20–25 g) were deprived of water but given free access to
food for 15 h. After water deprivation, the two hindlimbs of mice were
equally intramuscularly injected with 50% glycerol at a dose of
8 mL kg^−1. After that, all the mice had free access to water and food.
Symptoms of AKI, such as a lack of activities and decreased urine
output, could be observed in a few hours after glycerol injection.
The cisplatin-induced AKI model was also established according to
previously reported method^[239]68. In brief, female BALB/c mice (aged
8–10 weeks, 20–25 g) were given one time intraperitoneal injection of
cisplatin (BP809, Sigma-Aldrich, USA) at a dose of 20 mg kg^−1. Mice in
the treatment group were intravenously injected with Cu[5.4]O USNPs at
2 μg kg^−1 dose 2 h after intraperitoneal cisplatin injection. Mice
received saline injection was used as control. Three days post
injection, mice were sacrificed to collect blood and renal tissues for
kidney function analysis and histological analysis, respectively.
Pharmacokinetics and biodistribution of Cu[5.4]O USNPs
To evaluate the blood circulation half-life of Cu[5.4]O USNPs, BALB/c
mice (n = 3) with established glycerol-induced AKI model were
intravenously injected with Cu[5.4]O USNPs at a dose of 2 μg kg^−1. At
different time points post injection (10 min, 1, 2, 4, 8, 24, 48, and
72 h), 10 μL of whole-blood samples were collected from the mouse tail
vein. The collected blood samples were dissolved in aqua regia and the
concentrations of NPs were quantified by ICP-AES. A two-compartment
pharmacokinetic model was utilized to calculate the pharmacokinetics
parameters of Cu[5.4]O USNPs. Simultaneously, the urine and feces of
mice were collected at different time points and dissolved in aqua
regia to quantify the content of Cu by ICP-AES. Furthermore, the
collected urine was also diluted with PBS and dropped on a
carbon-coated copper grid to detect the presence of Cu[5.4]O USNPs in
the urine under TEM (JEOL JEM-1400, Japan).
To detect the biodistribution of Cu[5.4]O USNPs in the major organs,
BALB/c mice (n = 3) with established glycerol-induced AKI model were
intravenously injected with Cu[5.4]O USNPs at a dose of 2 μg kg^−1. One
day after injection, mice were sacrificed to harvest major organs
including heart, liver, spleen, lung, and kidney. The tissues were
weighed, homogenized, and then dissolved in aqua regia to calculate the
percentage of injected dose per gram of tissue (%ID g^−1) by ICP-AES.
To study the accumulation of Cu[5.4]O USNPs in the kidney at different
time periods, healthy female BALB/c mice or the ones with established
AKI model (n = 3) were intravenously injected with Cu[5.4]O USNPs at a
dose of 2 μg kg^−1. At desired time points (6, 12, 24 h), three mice in
each group were sacrificed. Kidney tissues were harvested, weighed,
homogenized, and dissolved in aqua regia to calculate the %ID g^−1 of
Cu by ICP-AES. The same experiment setup was applied for another batch
of mice to investigate the biodistribution of Cu[5.4]O USNPs in the
GBM. At different time points (2, 24, 72 h), three mice in each group
were sacrificed and a small piece of kidney tissues was fixed with the
mixture of 2.5% glutaraldehyde and 4% formaldehyde for 24 h, then
post-fixed with 1% osmium tetroxide and prepared for TEM observation.
In vivo therapeutic outcome of Cu[5.4]O USNPs on AKI mice
BALB/c mice with established AKI model were randomly divided into five
groups (n = 4): received PBS injection (control group), NAC
(8 mg kg^−1), NAC (40 mg kg^−1), NAC (160 mg kg^−1), and received with
Cu[5.4]O USNPs at a dosage of 2 μg kg^−1, respectively. The drug or NPs
were administrated once. The body weight variations in each group after
treatment were monitored for 24 h.
After 24 h post injection, mice were sacrificed to collect blood
samples for detecting the BUN and CRE levels. The left kidneys were
harvested and homogenized for the detection of renal biomarkers. The
level of SOD in the kidney was detected with a SOD assay kit (19160;
Sigma-Aldrich, USA). The expression levels of two important kidney
injury biomarkers, heme oxygenase-1 (HO-1) and kidney injury molecule-1
(KIM-1), were detected with HO-1 (ab204524, Abcam, USA) and KIM-1
(ab213477, Abcam, USA) ELISA kits, respectively.
The right kidneys were cut into two equal sections. One section was
fixed with 4% paraformaldehyde and embedded in paraffin for H&E
staining. The other section was frozen and embedded in optimum cutting
temperature (O.C.T.) specimen matrix (Sakura, Leiden, The Netherlands)
for cryostat sectioning at −20 °C. The renal tissue was sectioned into
approximately 5 μm thickness. Frozen renal tissue sections were stained
with DAPI (C1005, Beyotime, China) and dihydroethidium (DHE, D7008,
Sigma-Aldrich, USA) at 37 °C for 30 min, gently washed thrice with PBS
to remove excessive dyes and then imaged under a fluorescence
microscope to qualitatively detect the ROS levels in the renal tissues.
The same experiment setup was applied for another batch of mice
(n = 10) to draw the survival curves of AKI mice within 2 weeks. For
therapeutic mechanism investigation of Cu[5.4]O USNPs, after 24 h post
treatment, another batch of mice (n = 3) were sacrificed to collect
serum and kidney tissues for measurement of inflammatory factors by
ELISA kit.
In vivo therapeutic effect of Cu[5.4]O USNPs on AILI mice
The acetaminophen (APAP)-induced acute liver injury model was
established according to the previously reported protocol^[240]69.
Briefly, female C57BL/6 mice (aged 8–10 weeks, 18–20 g) were deprived
of food but given free access to water for 15 h. Then, 10 mice were
intraperitoneally injected with APAP (dissolved in warm saline,
15 mg mL^−1) at a dose of 300 mg kg^−1 and randomly divided into two
groups (n = 5). Mice (n = 5) without APAP injection served as control.
Afterwards, all the mice had free access to water and food. Mice in the
treatment group were intravenously injected with Cu[5.4]O USNPs at
6 μg kg^−1 dose 2 h after APAP injection. At 24 h post injection, mice
were sacrificed to collect blood and liver tissues for liver function
test and histological analysis, respectively.
In vivo therapeutic effect of Cu[5.4]O USNPs on wound healing
To investigate the effect of Cu[5.4]O USNPs on diabetic wound healing,
the STZ-induced diabetic mice model was established according to the
previous literature^[241]70. Briefly, male BALB/c mouse (aged 8–10
weeks, 20–25 g, n = 3) were intraperitoneal injected with 100 mg kg^−1
streptozotocin (STZ, Sigma-Aldrich, USA) for six consecutive days. Then
all mouse were provided with normal food and water. The glucose level
of mice was then monitored everyday from the tail venous blood using a
blood glucose meter (Roche Diagnostics, Shanghai, China). Mice with
sustained blood glucose levels exceeding 250 mg dL^−1 were considered
diabetic mice. These diabetic mice were used for further wounding model
at 2 weeks after the initiation of STZ treatment. Full-thickness wounds
were simultaneously created in the dorsal skin using a sterile 6-mm
diameter punch. A green round marker (6-mm diameter) was placed beside
each wound to represent the initial wound area and the wounds were
photographed immediately using a digital camera. Afterwards, each wound
in the treatment group was topically administrated with 20 μL of
Cu[5.4]O USNPs at 400 ng mL^−1. Wounds in the control group were
treated with 20 μL of PBS instead. Then, the wounds were covered with a
piece of biological membrane (NPWT-1, Negative Pressure Wound Therapy
Kit, China). The wounds were photographed and Cu[5.4]O was topically
administrated at days 1, 4, 7, 9, 15 post-surgery, respectively. Wound
areas were measured using ImageJ software. The wound healing rate was
calculated based on the following formula:
[MATH: Woundhealingrate%=I−R/I×100%, :MATH]
3
where I represented the initial wound area and R represented the
remaining wound area on the determined day post-surgery.
At day 15 post-surgery, mice were sacrificed to harvest wound tissues
for histological analysis. The wound tissues were fixed with 4%
paraformaldehyde and embedded in paraffin for H&E staining. The length
of regenerated epidermis and the thickness of granulation tissues were
quantified using ImageJ software.
Transcriptome analysis of AKI mice
BALB/c mice with established AKI model were randomly divided into two
groups: received PBS injection (control group, n = 5) and received with
Cu[5.4]O USNPs at a dosage of 2 μg kg^−1 (experimental group, n = 5),
respectively. After 24 h post injection, mice were sacrificed to
collect the kidneys. Total RNA of kidney tissues were prepared using
Trizol Reagent (Invitrogen, USA) according to the manufacturer’s
instructions and genomic DNA was removed using DNase I (TaKara, Japan).
Then RNA quality was determined by 2100 Bioanalyser (Agilent) and
quantified using the ND-2000 (NanoDrop Technologies). Only high-quality
RNA sample (OD[260/280] = 1.8–2.2, OD[260/230] ≥ 2.0, RIN ≥ 6.5,
28S:18S ≥ 1.0, >2 μg) was used to construct sequencing library. The RNA
purification, reverse transcription, library construction, and
sequencing were performed at Majorbio Bio-pharm Biotechnology Co., Ltd
(Shanghai, China) using Illumina HiSeq X10 (Illumina, San Diego, CA)
according to the manufacturer’s instructions. The raw paired end reads
were trimmed and quality controlled by SeqPrep
([242]https://github.com/jstjohn/SeqPrep) and Sickle
([243]https://github.com/najoshi/sickle) with default parameters. Then
clean reads were separately aligned to reference genome with
orientation mode using TopHat ([244]http://tophat.cbcb.umd.edu/,
version2.1.1) software.
For bioinformatics analysis, the expression level of each transcript
was calculated according to the fragments per kilobase of exon per
million mapped reads (FPKM) method. RSEM
([245]http://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene
abundances. DEGs were identified using R statistical package software
DESeq2 ([246]http://bioconductor.org/packages/stats/bioc/DESeq2.html)
(fold change ≥2 and P value <0.05) with a false discovery rate (FDR)
cutoff <0.05. KEGG functional enrichment analysis was performed to
identify which DEGs were significantly enriched in KEGG signaling
pathways at Bonferroni-corrected P value <0.05 compared with the
whole-transcriptome background. KEGG enrichment analysis was performed
by KOBAS 2.1.1 ([247]http://kobas.cbi.pku.edu.cn/download.php).
Besides, protein−protein interactions of genes were analyzed by Search
Tool for the Retrieval of Interacting Genes/Proteins (STRING) algorithm
([248]http://www.string-db.org/).
Quantitative real-time PCR and Western blot analysis
The total RNA from kidney tissue was extracted using Trizol Reagent.
cDNA was synthesized using a reverse transcription system kit according
to the manufacturer’s instructions (PrimeScript™ RT reagent Kit with
gDNA Eraser, RR047A, TaKaRa, Japan). Real-time PCR was performed using
the TB Green Premix Ex Taq™ II kit (RR820A, Takara, Japan) following
the manufacturer’s protocol. Gene expression levels were normalized to
GAPDH and analyzed using the comparative cycle threshold (F = 2^−ΔΔCt)
method. Primer sequences for qRT-PCR are listed in Supplementary
Table [249]2.
The total proteins from kidney tissue were extracted using ice-cold
RIPA lysis buffer containing phosphatase and protease inhibitor
cocktail (KeyGEN, China). The concentrations of extracted proteins were
determined using a BCA protein assay kit (Thermo Scientific, USA). An
equal amount of protein from each sample was run in 8% SDS-PAGE gel,
then transferred to polyvinylidenedifluoride (PVDF) membranes
(Millipore, USA). PVDF membranes were blocked with 5% skim milk at room
temperature for 2 h and then incubated with primary antibodies at 4 °C
overnight, followed by incubation with secondary horseradish
peroxidase-conjugated antibodies (1:1000, Sungene Biotech, China) for
1 h at room temperature. The intensity of bands was visualized and
determined using a ChemiDoc™ XRS detection system (Bio-Rad, USA).
Primary antibodies used were: NF-κB p65 (L8F6), phospho-NF-κB p65
(Ser536, E1Z1T), IκB-α (L35A5), phospho-IκB-α (Ser32/36, 5A5) (1:1000,
Cell Signaling Technology, USA) and β-actin (1:1000, Sungene Biotech,
China). The primary images (Fig. [250]S31) were cropped for
presentation.
Reporting summary
Further information on research design is available in the [251]Nature
Research Reporting Summary linked to this Article.
Supplementary information
[252]Supplementary Information^ (9.7MB, pdf)
[253]Peer Review File^ (261.1KB, pdf)
[254]Reporting Summary^ (207.4KB, pdf)
Source data
[255]Source Data^ (343.4KB, xlsx)
Acknowledgements