Does your doctor and hospital have enough competence to get this research done in humans? NO? So, you DON'T have a functioning stroke doctor or hospital, do you?
Dietary Natural Melanin Nanozymes Delay Aging and Ameliorate Neurodegeneration via Improving Gut Microbiota and Redox Homeostasis
- Yao Xiao
- Shikun Zhang
- Hailong Zhuo
- Xiaoyong Zhang
- Kai Zhu
- Wanyi Chen
- Guoxing You
- Hongwei Chen*
- Qun Luo*
- Hong Zhou*
- Gan Chen*
Abstract
Aging is an inevitable multifactor process that causes a decline in organ function and increases the risk of age-related diseases and death. Thus, the development of highly effective and safe therapeutic strategies to delay aging and age-related diseases is urgently required. In this study, we isolated natural melanin nanozymes (NMNs) from the ink sacs of live octopuses. The NMNs exhibited excellent superoxide-dismutase-mimicking and radical scavenging activities. In SAMP8 mice, treatment with NMNs improved their cognition and memory functions while restoring their aging-impaired liver function and lipid metabolism, thereby prolonging their lifespan. Moreover, the NMNs reversed metabolic changes in their aged brains and reconstructed their gut microbiota composition by enhancing microbial community diversity. Our findings indicate that NMNs treatment could be a promising approach for delaying aging and preventing age-associated physiological decline in humans.
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Copyright © 2025 The Authors. Published by American Chemical Society
Introduction
Aging
is a complex process that involves the progressive deterioration of
physiological homeostasis, thus resulting in impaired functional ability
and increased vulnerability to age-related diseases and death. (1) Globally, the number of individuals over the age of 65 is 617 million and may reach 1.6 billion by 2050. (2)
The acceleration of aging is accompanied by an increased incidence of
undesirable age-related diseases, such as cognitive
decline/neurodegeneration and progressive organ dysfunction, which are
common health threats to the elderly population and pose major societal
and economic burdens. (3)
Although certain drugs or health products, such as metformin,
resveratrol, nicotinamide mononucleotide, and rapamycin, can hinder the
process of aging and age-related diseases, they have not been widely
promoted owing to their extraction difficulty, high cost, and serious
side effects. (4)
Thus, developing accessible, safe, and effective therapeutic strategies
to delay aging and age-related diseases in response to the aging
population is urgently needed.
Increasing
evidence suggests that oxidative stress and gut microbiome dysbiosis are
fundamental triggers of aging and age-related diseases. (5−7)
For example, oxidative stress induced by the excessive generation of
reactive oxygen species (ROS) can cause oxidative damage and result in
impaired organ function and aging. (8)
Furthermore, increasing evidence suggests that the aging process is
often accompanied by gut microbiota dysbiosis and that the gut
microbiota could regulate the process of aging and age-related diseases,
particularly age-related cognitive decline. (6)
Thus, reducing the ROS content and modifying the microbiota are
promising strategies for delaying aging and age-related diseases.
Natural
melanin nanozymes (NMNs), which are widely distributed and naturally
occurring components of living organisms, exhibit excellent
biocompatibility and biodegradability for various biomedical
applications, with negligible side effects. (9) NMNs exhibit various abilities, including radical scavenging, (10) ultraviolet protection, (11) photothermal conversion, (12) and metal-chelating capabilities. (13)
They have been explored as therapeutic agents for various diseases,
including myocardial infarction, cancer, acute kidney injury, and wound
healing. (9,12,14,15) However, the mechanisms by which NMNs delay aging and age-related cognitive decline have been poorly explored.
As
a spontaneous and commonly used model of accelerated aging, the
senescence-accelerated mouse prone 8 (SAMP8) mouse strain exhibits
progressive cognitive decline and pathological alterations in the liver
and kidney. (16)
In this study, NMNs were extracted from living octopuses and explored
as effective therapeutic agents against aging and age-related cognitive
decline. We demonstrated that NMNs exhibit robust superoxide dismutase
(SOD)-mimicking and radical scavenging activities and that dietary NMNs
improve cognition and memory functions, inhibit related pathological
changes in the liver, and prolong the lifespan of SAMP8 mice,
accompanied by intestinal microbiota reshaping. Our study suggests that
NMNs may be a promising and accessible countermeasure to delay aging and
age-related diseases.
Results and Discussion
Characterization of NMNs
NMNs were extracted from the ink sacs of living octopuses via multiple centrifugal washing steps with deionized water. (9)
Typical images from transmission and scanning electron microscopy (TEM
and SEM, respectively) indicated that the NMNs had an average diameter
of 130 ± 50 nm and exhibited a spherical morphology with a rough surface
(Figure 1A,B). The average zeta potential of the NMNs was –27.4 ± 1.1 mV (Figure 1C),
which supported the good dispersibility of the NMNs in water. Moreover,
the average hydrodynamic diameter of the NMNs, measured by using
dynamic light scattering, was approximately 255 nm (Figure 1D). The NMNs were also characterized using UV–vis absorption spectroscopy (Figure 1E), which demonstrated their broad-spectrum absorption from 200 to 1000 nm, consistent with that of previous studies. (17,18) The Fourier-transform infrared (FTIR) spectra of the NMNs are shown in Figure 1F. A peak signal in the range of 3500–3000 cm–1
was attributed to the O–H and N–H stretching vibrations of the
carboxyl, phenolic, and amino functional groups. The bending vibration
of C═O and the in-plane bending of C═C and N–H of the aromatic system
occurred in the spectral range of 1750–1550 cm–1. The hydroxyl group bending of phenol and carboxyl was observed in the spectral range of 1450–1300 cm–1. These characteristic peaks were highly consistent with the natural squid melanin reported in previous studies, (19,20)
suggesting that melanin molecules were well preserved. In addition, a
previous study indicated that NMNs not only contain the primary
component of melanin but also include amino acids, metals, and
polysaccharides; (12)
however, we found the FTIR spectra of the NMNs to be fundamentally
similar to those of pure melanin synthesized using the Stöber-like
method. (21) This suggests that the impact of these other components on the FTIR results is minimal.
Figure 1
Figure 1. Characterization of the NMNs. (A) Representative TEM and (B) SEM images of the NMNs; scale bars, 100 nm. (C) Zeta potential of the NMNs (n = 3). (D) Hydrodynamic diameter of the NMNs measured using dynamic light scattering. (E) UV–vis–NIR absorbance and (F) FTIR curves of the NMNs. (G) SOD-mimicking activity of the NMNs measured using ESR spectroscopy and the (H) hydroxylamine method. (I) ESR spectra of radical adducts trapped by the NMNs on 1O2 and (J) •OH.
NMNs Exhibit Robust SOD-Mimicking and Radical Scavenging Activities
SOD plays a pivotal role in regulating intracellular ROS levels by modulating superoxide (•O2–) degradation. (22)
The SOD-mimicking activity of the NMNs was first confirmed using
electron spin resonance (ESR) spectroscopy by investigating the •O2–
scavenging effect after adding NMNs to a xanthine oxidase + xanthine
(XO + X) reaction system. The ESR spectra indicated that the
characteristic signal of •O2– became significantly weakened following the addition of NMNs (Figure 1G). Furthermore, the SOD-mimicking catalytic activity was evaluated by using the hydroxylamine method. As shown in Figure 1H, the NMNs exhibited robust SOD-mimicking catalytic activity at a concentration of 6.25 μg/mL.
Next, we conducted an ESR spectral assay to confirm the scavenging activity of ROS, including singlet oxygen (1O2) and the hydroxyl radical (•OH). The 1O2 and •OH exhibited robust signals, which gradually decreased upon adding NMNs at concentrations of 1, 10, and 100 μg/mL (Figure 1I,J),
indicating the scavenging activity of NMNs for ROS. Particularly, we
observed that as the concentration of NMNs increased, the rate of
decline in ESR signals slowed, consistent with a previous report
investigating the MnO2 nanozyme. (23) We speculate that this may be associated with reaction saturation in the solution system.
NMNs Improve Cognition and Memory Functions and Prolong Lifespan
Currently,
SAMP8 mice are the ideal model of aging, with age-related impairment in
learning and memory occurring as early as two months. (24)
In this study, 4 month-old SAMP8 mice were administered NMNs in their
daily drinking water, and age-matched SAMP8 and SAMR1 mice with normal
drinking water were used as the model and control groups, respectively.
Behavioral testing, including the Morris water maze and open field
tests, was conducted after 4 months of treatment, and survival was
continuously observed for 12 months (Figure 2A).
Figure 2
Figure 2. NMNs prolong lifespan and improve cognition and memory functions. (A) Experimental timeline for NMN administration (starting at the age of 4 months). Gut microbiota, metabolic profiling of the brain, hepatic parameters, and survival analysis were performed at the indicated time. (B) Kaplan–Meier survival curves of SAMP8 mice treated with and without NMNs. (C) Representative swimming tracks of mice in the Morris water maze test. (D) Escape latency. (E) Crossing the platform numbers. (F) Time of platform crossing. (G) Representative track images of mice in the open-field test. (H) Quantification of rearing. (I) Time spent in the center. (J) Crossing grid. (K) Defecation. *p < 0.05, **p < 0.01.
The effect of the NMN treatment on the lifespan of SAMP8 mice was also determined. As shown in Figure 2B,
SAMP8 mice died quicker than SAMR1 mice─a phenomenon that was
completely reversed by the NMNs during the 8 months of treatment (p < 0.05).
The
Morris water maze test was used to evaluate spatial learning and
memory. No significant differences were detected in the escape latency,
defined as the time taken to find the hidden platform, among the three
groups on the first day of training. NMNs-administered mice exhibited a
significantly decreased escape latency during training, which was
similar to that of SAMR1 mice in the control group. Conversely, no
obvious change in escape latency was observed for SAMP8 mice in the
model group during training (Figure 2C,D).
Compared with the SAMR1 mice in the control group, the SAMP8 mice in
the model group displayed a significantly increased crossing platform
number and time of platform crossing during the probe trial, which was
reversed by NMN administration (Figure 2E,F).
Given the better memory retention in NMNs-treated mice compared with
model mice, we speculated that the NMNs might also affect behavior.
Therefore, NMNs improved aging-related learning and memory impairments
and responded to different stimuli.
The
open-field test was also used to assess exploratory behaviors and
emotional responses in an unfamiliar environment. Time spent in the
center, number of grids crossed, rearing frequency, and defecation
frequency were regarded as indirect indices of activity and anxiety
levels. (25)
A significant reduction in the time spent in the center and the number
of grids crossed was observed in SAMP8 mice compared with those in SAMR1
mice. Particularly, NMN treatment significantly increased the time
spent in the center, number of grids crossed, and rearing frequency (Figure 2G,J) while reducing the defecation frequency of SAMP8 mice (Figure 2K).
These results demonstrated that NMNs promote exploratory behavior and
alleviate anxiety in SAMP8 mice in an unusual environment.
NMNs Alleviate Oxidative Stress and Inflammation in the Brain
Increasing
evidence demonstrates that sustained perturbations induced by oxidative
stress result in inflammation and apoptosis in the brain during the
aging process, ultimately intensifying aging and age-related cognitive
decline. (26)
Malondialdehyde (MDA) is the primary product of ROS-induced lipid
peroxidation and is regarded as a biomarker for evaluating the level of
oxidative stress. (27,28) As illustrated in Figure 3A,
SAMP8 mice exhibited a significant increase in brain MDA compared to
age-matched SAMR1 mice, while SAMP8 mice treated with NMNs exhibited a
reduced MDA content compared to SAMP8 mice.
Figure 3
Figure 3. NMNs alleviate oxidative stress and inflammation in the brain. (A) MDA content in the brain. (B) Interleukin (IL)-1β, (C) IL-6, and (D) tumor necrosis factor (TNF)-α contents in the brain. (E) Immunofluorescence staining and corresponding quantitative results of the Iba+ cells in the brain. Scale bars, 20 μm. *p < 0.05 and **p < 0.01.
Neuroinflammation, represented by proinflammatory cytokine secretion and microglial activation, (29,30)
was evaluated. Significantly increased levels of interleukin (IL)-6 and
tumor necrosis factor (TNF)-α in the brains of SAMP8 mice were observed
compared with those in age-matched SAMR1 mice. Moreover, NMNs treatment
significantly suppressed the production of IL-6, TNF-α, and IL-1β
compared with that in the SAMP8 model mice (Figure 3B–D). Microglia are a primary source of proinflammatory cytokines in the central nervous system. (31)
Thus, immunofluorescence staining of Iba1 is the most commonly used
marker of microglia. Compared with SAMR1 mice, SAMP8 mice displayed a
higher number of Iba1+ microglia in the brain. In contrast, NMN treatment significantly decreased the number of Iba1+ microglia in the brain of SAMP8 mice (Figure 3E).
These results indicated that NMN treatment may suppress proinflammatory
cytokine secretion by inhibiting abnormal microglial activation.
Therefore, dietary supplementation with NMNs may improve age-related
cognitive decline by reducing ROS levels and inhibiting
neuroinflammation.
NMNs Remodel Metabolic Profiling in the Brain of SAMP8 Mice
Brain metabolic disturbances play a key role in the development of age-related cognitive decline. (32,33)
Thus, we analyzed the differences in the metabolomic profiles of the
brain among the study groups. Compound identification was based on
precise mass-to-charge ratio (M/z), secondary fragments,
and isotopic distribution using the human metabolome database, Lipidmaps
(V2.3), Metlin, and self-built databases. Using a partial
least-squares-discriminant analysis (PLS-DA), we identified metabolites
with the strongest influence on defining the separation between MO and
NMNs groups (Figure S1).
Next, we found that NMNs can exert a significant influence on mineral
absorption, nonalcoholic fatty liver disease, and the mTOR signaling
pathway in mice (Figure 4A,B).
Furthermore, we found a total of 91 and 215 differential metabolites in
the CK and NMNs groups, respectively, compared to the MO group (p < 0.05, VIP >1; Figures S2 and S3 and Tables S1–S3).
Certain classes of lipids and lipid-like molecules, such as
glycerophospholipids [lysoPE(0:0/20:1(11Z))], fatty acyls (nonadecanoic
acid, japanic acid, heptadecanoic acid, and 3-hydroxynonyl acetate), and
prenol lipids (pristanic acid), were significantly elevated in SAMP8
mice compared to those in SAMR1 mice; this was reversed via NMN
intervention (Figure 4C–H).
Furthermore, brain levels of organic acids and derivatives (camphoric
acid and triethyl citrate) and organic oxygen compounds (d-glucose)
were increased, and the levels of Leu-Gly-Pro, OSU03012, and dihydroxy
melphalan were significantly decreased in SAMP8 mice compared to those
in SAMR1 mice (Figure 4I–N).
These differences were reversed following NMN administration. These
results imply that NMNs can reverse metabolic changes in the aged brain.
Figure 4
Figure 4. NMNs remodel metabolic profiling in the brain. (A) Enriched pathway of all metabolites in the brain associated with the CK and MO groups. Colors indicate the significance of enrichment, and circle sizes indicate the number of genes falling into respective categories. (B) Enriched pathway of all metabolites in the brain associated with the MO and NMNs groups. (C–N) Changes in the metabolite levels of the brain among the three groups. *p < 0.05 and **p < 0.01.
NMNs Restore Aging-Impaired Liver Function and Lipid Metabolism
Aging
is accompanied by abnormal lipid metabolism and widespread and
coordinated functional decline in numerous tissues, including the liver.
(34) As shown in Figure 5A–C,
a significant increase was observed in plasma cholesterol (CHO) and
low-density lipoprotein (LDL) levels in SAMP8 mice compared to those in
SAMR1 mice. NMN treatment reduced the plasma levels of triglycerides
(TGs), CHO, and LDLs in SAMP8 mice. In addition, NMN treatment
significantly elevated plasma HDL and diminished blood glucose levels
compared to those of SAMP8 mice in the model group (Figure 5D,E).
Figure 5
Figure 5. NMNs restore aging-impaired liver function and lipid metabolism. Levels of (A) TG, (B) CHO, (C) LDL, (D) HDL, (E) GLU, and (F) ALT in plasma. (G) Representative images show the effect of NMN administration on hepatic lipid accumulation and histological injury. Scale bar, 50 μm. *p < 0.05 and **p < 0.01.
Regarding
liver function, the plasma alanine transaminase (ALT) level in SAMP8
mice was significantly higher than that in the normal control group,
whereas SAMP8 mice in the NMN-treated group exhibited a decrease (Figure 5F). Oil Red O staining revealed that hepatic lipid accumulation in SAMP8 mice was inhibited by NMN treatment (Figure 5G).
NMN treatment significantly and consistently ameliorated age-associated
liver damage, as evidenced by hematoxylin and eosin (H&E) staining
results (Figure 5G).
NMNs Restored Gut Microbiota Balances in SAMP8 Mice
The gut microbiota plays a pivotal role in the onset and development of aging and age-related cognitive decline. (35,36)
After demonstrating the beneficial effects of NMNs on aging and
age-related organ dysfunction, we investigated whether the gut
microbiota was also involved in these protective effects. As shown in Figure 6A,B,
the Simpson index was lower in SAMP8 mice than in SAMR1 mice. SAMP8
mice treated with NMNs exhibited significantly increased Simpson and
Shannon indices, indicating that dietary supplementation with NMNs
effectively enhanced the diversity of microbial communities in SAMP8
mice. The characteristic phylum-level microbiome was further compared
among all of the groups (Figure 6C,D).
SAMP8 mice demonstrated decreased abundances of Proteobacteria,
Actinobacteria, Fusobacteria, Epsilonbacteraeota, and Acidobacteria
compared with SAMR1 mice. NMN intervention increased the abundance of
Proteobacteria, Actinobacteria, Epsilonbacteraeota, Fusobacteria,
Bacteroidetes, Gemmatimonadetes, Patescibacteria, and Cyanobacteria and
decreased the abundance of Firmicutes, Deferribacteres, and Nitrospirae
in SAMP8 mice.
Figure 6
Figure 6. NMNs enhance microbial community diversity. (A) Shannon index, (B) Simpson index, and (C) relative abundance and heatmap (D) of gut microbiota at the phylum level. *p < 0.05 and **p < 0.01.
Cognitive
decline is a multifaceted disorder involving various biological
mechanisms, such as oxidative stress, neuroinflammation, and gut
microbiome dysbiosis. (37,38)
Previous studies have indicated that inhibiting excessive oxidative
stress and inflammation, along with modulating gut microbiota, can
effectively prevent cognitive decline. (39,40)
In our study, we demonstrated that NMNs have potent SOD-mimicking and
radical scavenging activities, significantly reducing oxidative stress
and inflammation in a senescence-accelerated mouse model. Additionally,
NMNs were found to restore the gut microbiota balance by enhancing
microbial diversity. Thus, we believe that NMNs may delay aging and
age-related cognitive decline by inhibiting oxidative stress and
inflammation and modulating the gut microbiota.
Furthermore,
a previous study has indicated that natural melanin not only contains
the primary component of melanin but also includes amino acids, metals,
and polysaccharides, (12)
which may have an impact on the therapeutic efficacy of the natural
melanin. Previous studies have revealed that some polysaccharides from
different natural sources possess antioxidant activity, gut microbiota
modulation, and immunostimulatory function. (41,42)
Therefore, we believe that although melanin is the main component
responsible for the antiaging effects within natural melanin, other
components might also contribute in a complementary fashion.
Conclusions
Dietary
NMNs can efficiently delay aging and improve age-related
neurodegeneration and liver dysfunction. These beneficial effects are
associated with brain inflammation and oxidative stress inhibition,
metabolic imbalance restoration, and enhanced microbial community
diversity. Given the desirable functions, easy accessibility, and safe
consumption history of NMNs, translating this research into a promising
intervention to delay aging and prevent age-associated physiological
decline in humans is of significant interest.
Materials and Methods
Materials
Fresh
octopuses were purchased from the Yuegezhuang market (Beijing, China).
The enzyme-linked immunosorbent assay (ELISA) test kits for TNF-α, IL-6,
and IL-1β were purchased from PeproTech Co., Ltd. (PeproTech, NJ, USA).
All reagents, unless otherwise stated, were obtained from commercial
sources and directly used without further purification.
NMN Extraction and Characterization
NMNs
were extracted from the dissected ink sacs of fresh octopuses by using a
simple centrifugation method. Briefly, after removing large
precipitates, the NMNs solution was centrifugated 14,000g at 4 °C for 15
min to obtain clean NMNs. Subsequently, the NMNs were washed with
ultrapure water five times, followed by drying at 37 °C for 24 h, to
obtain dried NMNs, which were stored at 4 °C for further use.
The
morphology of the NMNs was characterized using a JEM-2100Plus TEM
(JEOL, Tokyo, Japan) system and a JEOL JSM7500F SEM (JEOL, Tokyo, Japan)
system. The size distribution and zeta potential of the NMNs were
evaluated by using a Zetasizer Nano-ZS system (Malvern Nano Series,
Malvern, UK). FTIR spectra of the NMNs were recorded using an FTIR
spectrophotometer (Nicolet 6700, Thermo Scientific, USA) in the range of
400–4000 cm–1. The absorption spectra were measured by using a Spectra Max M5 microtiter plate reader (Molecular Devices, USA).
Measurement of SOD-Mimicking and Radical Scavenging Activities
The
SOD-mimicking activity of the NMNs was determined using an SOD assay
kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)
according to the manufacturer’s instructions and measured using an ESR
spectrometer (Bruker ELEXSYS-II E500, Germany). Briefly, the XOD system
and BMPO were selected as the generator and detector of •O2–,
respectively. The sample contained 25 mM BMPO, 5 mM hypoxanthine, and
0.25 mM DTPA in 15 μL of PBS, to which the NMNs were introduced to
scavenge the radicals. The reaction was initiated by the addition of
XOD. ESR measurements for the detection of BMPO/•OOH spin-adducts were as follows: microwave power, 2 mW; scan range, 100 G; and field modulation, 1 G.
The generation of •OH and 1O2 was qualitatively measured by using ESR spectroscopy, with DMPO and TEMP as the •OH and 1O2 spin-trapping agents, respectively. Various concentrations of H2O2,
magnetic nanoparticles (1, 10, and 100 μg/mL), and DMPO/TEMP were mixed
and placed into quartz capillary tubes for ESR measurements under the
following conditions: microwave power, 2 mW; field modulation, 1 G;
sweep width, 100 G.
Animals and Drug Administration
Four-month-old
male SAMP8 and SAMR1 mice were purchased from the Department of
Experimental Animal Science, Peking University Health Science Center
(Certificate No: SCXK 2016-0010, Beijing, China) and housed in specific
pathogen-free and controlled environmental conditions (21 ± 2 °C, 12 h
light/dark cycle) with free access to food and water. All animal
experimental procedures were approved by the Institutional Animal Care
and Use Committee of the Academy of Military Medical Sciences (Beijing,
China). This research was performed in accordance with the guidelines
and ethical standards of the Chinese Council on Animal Care to minimize
animal suffering throughout the study.
To evaluate the therapeutic effect of NMNs, SAMP8 mice were randomly divided into two groups: SAMP8 model (MO, n = 9) and NMN-treated groups (NMNs, n = 12). Moreover, age-matched SAMR1 mice were allocated to the control group (CK, n
= 9). The mice in the NMN group were continuously administered NMNs
dispersed in drinking water at doses of 10 μg/mL, and mice in the CK and
MO groups were provided with normal drinking water. Following 16 weeks
of treatment, the mice were sacrificed, and their plasma, major organs
(brain, liver, and kidney), and intestinal contents were immediately
collected and stored for further analysis. We observed that NMN doses
over 10 μg/mL, especially at higher concentrations, would cause the
solution to exhibit a pronounced color and odor, resulting in decreased
water intake by the mice. Therefore, we chose 10 μg/mL NMNs as the
treatment concentration in drinking water.
For the survival study, 30 four-month-old SAMP8 and 15 age-matched SAMR1 mice were grouped as follows: SAMR1 control (CK, n = 15), SAMP8 model (MO, n = 15), and NMN-treated groups (NMNs, n
= 15). NMNs were supplied daily in drinking water until the mice
reached their natural death. The number of live and dead mice in each
group was recorded.
Behavior Study
The Morris water maze experiment was performed as previously described, (43)
with minor modifications. Briefly, a pool (120 cm in diameter and 50 cm
in height) filled with opacified water (22 ± 0.5 °C) was divided into
four virtual quadrants, and an escape platform was randomly placed 1 cm
below the surface of the water in the center of one of the quadrants.
The participating mice were trained to find the platform once a day and
then assessed for their ability to find the platform for five
consecutive days. Mice that found the platform within 60 s were allowed
to remain there for 20 s. Conversely, if the mouse failed to locate the
platform within 60 s, then it was guided to the platform and allowed to
stay there for 20 s, and its escape latency was recorded as 60 s. The
paths, distances, and latencies required to swim on the platform were
recorded. During the probe trial, the platform was removed, and the mice
could explore the pool for 60 s. The number of crossings in the
previous position of the platform and the number of platform crossings
were evaluated.
The open field test was performed according to a previous report (44)
in an open field box (50 cm × 50 cm × 25 cm) with a bottom. Mice were
placed in the center of the box and allowed to explore freely for 5 min.
Four parameters were collected: time spent in the center, number of
crossing grids, rearing frequency, and defecation frequency.
Measurement of Biochemical Indicators
Plasma biochemistry was measured using a Chemray 800 biochemistry autoanalyzer (Shenzhen Redu Life Sciences, China).
Antioxidant Capacity and Immune Response Detection
Mice
brains were homogenized in ice-cold normal saline and centrifuged, and
the level of MDA in the brain supernatant was determined using a
commercial kit (Nanjing Jiancheng Biological Institute, Nanjing, China)
according to the manufacturer’s instructions, as previously described. (45,46) The levels of IL-1β, IL-6, and TNF-α in the brain supernatants were measured using ELISA kits, as previously described. (47,48)
Histological Examination
Fresh
mice livers were isolated and fixed in 4% paraformaldehyde for at least
24 h. Subsequently, they were dehydrated using an ethanol gradient, and
the paraffin-embedded tissues were cut into 4 μm-thick slices and
stained with H&E, as previously described. (49,50)
For
Oil Red O staining, the slides were washed with 60% isopropyl alcohol,
stained with Oil Red O solution at room temperature for 10 min, rinsed
with distilled water, and covered with an aqueous mounting medium.
Metabolomic Analysis
First, 30 mg of brain samples was obtained, and 20 μL of internal standard and a 400 μL mixture of methanol and water (v/v
= 4:1) were added to the samples. Following storage at –80 °C for 2
min, the obtained samples were ground at 60 Hz for 2 min, ultrasonicated
for 10 min, and stored at –20 °C for 30 min. Following centrifugation,
300 μL of the supernatant was placed in a liquid chromatography (LC)
bottle. After evaporation, a 200 μL mixture of methanol and water (v/v
= 1:4) was added to each sample, vortexed for 2 min, and stored at 4 °C
for 2 min. Following centrifugation, the supernatants were filtered
into LC vials using a 0.22 μm microfilter and stored at –80 °C for
liquid chromatography–mass spectrometry (LC–MS) analysis.
LC–MS
was performed by using a Waters ACQUITY UPLC I-Class system (Waters
Corporation, Milford, MA, USA) coupled with a VION IMS QTOF mass
spectrometer (Waters Corporation, Milford, MA, USA) in both positive and
negative electrospray ionization modes. Measurements were performed
using an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) with a
mobile phase comprising A-water (containing 0.1% formic acid) and
B-acetonitrile/methanol (containing 0.1% formic acid). The flow rate,
column temperature, and injection volume were 0.4 mL/min, 45 °C, and 1
μL, respectively. Data acquisition was performed in the full-scan mode (m/z
range from 50 to 1000) combined with the MSE mode. Finally, the
acquired LC–MS raw data were resolved using Progenesis QI software
(Waters Corporation, Milford, USA).
Microbiome Analysis
The
16S rRNA sequencing of mouse cecal contents was conducted at OEbiotech
Co., Ltd. (Shanghai, China), following a previously reported
methodology. (51)
The total genomic DNA of mouse cecal contents was isolated using a
DNeasy PowerSoil kit (Qiagen, Hilden, Germany) and quantitatively
measured using a NanoDrop2000 instrument (Thermo Fisher Scientific,
Waltham, MA, USA). Raw data in the FASTQ format were obtained by
performing DNA amplification, library construction, and sequencing using
an Illumina HiSeq platform (San Diego, CA, USA). The software and
platform used for additional bioinformatics analysis were provided by
OEbiotech Co., Ltd.
Immunofluorescent Staining
The
paraformaldehyde-fixed brains were dehydrated, embedded in paraffin,
and cut into 5 μm-thick sections before immunofluorescence analysis. The
brain sections were incubated with anti-Iba1 (1:200, Cell Signaling
Technology, MA, USA) primary antibodies, washed, incubated with a
fluorophore-labeled secondary antibody, and visualized using a confocal
microscope (Nikon, Tokyo, Japan).
Statistical Analysis
All
data were expressed as the mean ± standard error of the mean.
Statistical differences between the groups were analyzed using one-way
analysis of variance (ANOVA), followed by Dunnett’s multiple comparison
test. Survival data were analyzed by using the log-rank test.
Statistical significance was set at p-value < 0.05.
Data Availability
The data underlying this study are not publicly available due to a patent being filed for that data.
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c08419.
Dietary Natural Melanin Nanozymes Delay Aging and Ameliorate
Neurodegeneration via Improving Gut Microbiota and Redox Homeostasis
Yao Xiao
1#
, Shikun Zhang
1#
, Hailong Zhuo
2#
, Xiaoyong Zhang
1
, Kai Zhu
1
, Wanyi
Chen
1
, Guoxing You
1
, Hongwei Chen
3*
, Qun Luo
2*
, Hong Zhou
1*
, Gan Chen
1*
1
Academy of Military Medical Sciences, Beijing 100850, China
2
Department of Transfusion, The Fifth Medical Center of Chinese PLA General
Hospital, Beijing 100071, China
3
Fuyang Normal University, Fuyang 236037, China
*
Corresponding authors:
Hongwei Chen,
chen790514@126.com;
Qun Luo, luoq66@aliyun.com;
Hong Zhou,
zhouhtt1966@163.com;
Gan Chen,
chenlzu2005@163.com.
#
These authors contributed equally to this work.
Figure S1. PLS-DA scores of MO and NMNs groups
Figure S2. Volcano plot of differentially metabolites between MO and CK groups.
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- Hong Zhou - Academy of Military Medical Sciences, Beijing 100850, China;
https://orcid.org/0000-0003-2987-7781;
- Gan Chen - Academy of Military Medical Sciences, Beijing 100850, China;https://orcid.org/0000-0003-4586-4526;
Y.X., S.Z., and H.Z. have contributed equally to this work. Designed the study: GC, HZ, HC, and QL. Performed the experiments and collected the data: YX, SZ, HZ, XZ, and KZ. Analyzed the data: GC, YX, SZ, HZ, WC, and GY. Prepared the manuscript: CG, YX, HZ, HC, and QL.
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Abstract
Figure 1
Figure 1. Characterization of the NMNs. (A) Representative TEM and (B) SEM images of the NMNs; scale bars, 100 nm. (C) Zeta potential of the NMNs (n = 3). (D) Hydrodynamic diameter of the NMNs measured using dynamic light scattering. (E) UV–vis–NIR absorbance and (F) FTIR curves of the NMNs. (G) SOD-mimicking activity of the NMNs measured using ESR spectroscopy and the (H) hydroxylamine method. (I) ESR spectra of radical adducts trapped by the NMNs on 1O2 and (J) •OH.
Figure 2
Figure 2. NMNs prolong lifespan and improve cognition and memory functions. (A) Experimental timeline for NMN administration (starting at the age of 4 months). Gut microbiota, metabolic profiling of the brain, hepatic parameters, and survival analysis were performed at the indicated time. (B) Kaplan–Meier survival curves of SAMP8 mice treated with and without NMNs. (C) Representative swimming tracks of mice in the Morris water maze test. (D) Escape latency. (E) Crossing the platform numbers. (F) Time of platform crossing. (G) Representative track images of mice in the open-field test. (H) Quantification of rearing. (I) Time spent in the center. (J) Crossing grid. (K) Defecation. *p < 0.05, **p < 0.01.
Figure 3
Figure 3. NMNs alleviate oxidative stress and inflammation in the brain. (A) MDA content in the brain. (B) Interleukin (IL)-1β, (C) IL-6, and (D) tumor necrosis factor (TNF)-α contents in the brain. (E) Immunofluorescence staining and corresponding quantitative results of the Iba+ cells in the brain. Scale bars, 20 μm. *p < 0.05 and **p < 0.01.
Figure 4
Figure 4. NMNs remodel metabolic profiling in the brain. (A) Enriched pathway of all metabolites in the brain associated with the CK and MO groups. Colors indicate the significance of enrichment, and circle sizes indicate the number of genes falling into respective categories. (B) Enriched pathway of all metabolites in the brain associated with the MO and NMNs groups. (C–N) Changes in the metabolite levels of the brain among the three groups. *p < 0.05 and **p < 0.01.
Figure 5
Figure 5. NMNs restore aging-impaired liver function and lipid metabolism. Levels of (A) TG, (B) CHO, (C) LDL, (D) HDL, (E) GLU, and (F) ALT in plasma. (G) Representative images show the effect of NMN administration on hepatic lipid accumulation and histological injury. Scale bar, 50 μm. *p < 0.05 and **p < 0.01.
Figure 6
Figure 6. NMNs enhance microbial community diversity. (A) Shannon index, (B) Simpson index, and (C) relative abundance and heatmap (D) of gut microbiota at the phylum level. *p < 0.05 and **p < 0.01.
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