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As annd, exposure anf oxidative stress by addition of hydrogen peroxide in cultured endothelial cells markedly upregulated expression of antioxidant genes Supplementary Fig.
However, mRNA expression levels of ARE-containing genes Heme Oxygenase 1 HO-1Thioredoxin Reductase 1 TXNRD-1 and NAD P H Quinone Oxidoreductase 1 NQO-1 as well as SuperOxide Dismutase 1 SOD1 and catalasemarkers of antioxidant enzymatic activity, were similar in ECs harvested from participants after adequate sleep and sleep restriction Fig.
Studies in Drosophila and rodent models have shown that sleep restriction upregulates antioxidant response via induction of the antioxidant regulator Nrf2, a redox sensitive transcription factor that translocates into the nucleus and initiates the transcription of antioxidant genes 121314 To probe potential mechanisms that might account for lack of antioxidant responses, we first investigated whether expression and cellular localization of Nrf2 are altered in ECs after sleep restriction.
We confirmed that exposure to oxidative stress leads to increased nuclear localization of Nrf2 in cultured ECs Supplementary Fig. In contrast, in ECs harvested from participants, expression of Nrf2 mRNA and total protein was similar in adequate sleep and sleep restriction Supplementary Fig.
Whereas readily detected in cytoplasm, nuclear fluorescence of Nrf2 was almost undetectable in harvested ECs after both adequate sleep and sleep restriction Supplementary Fig.
S4 Dindicating that Nrf2 does not translocate into the nucleus of ECs after sleep restriction despite increased endothelial oxidative stress. We next investigated why Nrf2 fails to transfer into the nucleus of harvested ECs after sleep restriction. Nrf2 function is regulated by the Cul3-Keap1-E3 ligase, a part of the Cullin-Ring-Ligase complexes Under basal conditions, Cul3 neddylation activates the E3-Cul3-Keap1 complex, which ubiquitinates Nrf2 and targets it for proteasomal degradation thereby maintaining the low basal levels of Nrf2 1428 Under conditions of increased oxidative stress, ubiquitination of Nrf2 is suppressed, resulting in increased availability of Nrf2 and its translocation into the nucleus, binding to ARE and consequent activation of the antioxidant genes 1428 Both protein and mRNA expression of Cul3 were similar in adequate sleep and sleep restriction Supplementary Fig.
S5 A—B. In contrast, cytoplasmic co-localization of Cul3 and Nrf2 was significantly increased after sleep restriction compared with adequate sleep Fig. Identification of mediators of impaired antioxidant response in sleep restriction.
To identify potential mediators of the altered Nrf2 and Cul3-Keap1-E3 complex interaction, we performed bulk RNA-seq in ECs harvested at the end of adequate sleep phase and sleep restriction phase in 5 participants Supplementary Table S1.
Sleep restriction altered expression of 13 genes Fig. Using the predicted protein—protein interaction database BioGRID 30we interrogated protein products of those 13 genes for binding probability specifically to Nrf2, Keap1 and Cul3.
While none were predicted to bind to Nrf2 or Keap1, we identified Defective in Cullin Neddylation-1 Domain Containing 3 DCUN1D3 as the binding partner of Cul3 among genes altered by sleep restriction.
Cul3 was the top predicted binding partner of DCUN1D3 In non-endothelial cells, DCUN1D3 sequesters Cul3 to the plasma membrane thereby preventing its neddylation, reducing Nrf2 degradation and facilitating Nrf2 nuclear translocation and activation of antioxidant response 31 We confirmed that DCUN1D3 interacts with Cul3 in human umbilical vein endothelial cells HUVECs indicating a function similar to that described in other cell types Supplementary Fig.
Reduced expression of endothelial DCUN1D3 coupled with increased interaction between Cul3 and Nrf2 suggest reduced sequestration of Cul3 to the plasma membrane and its greater availability in the Cul3-Keap1-E3 ubiquitin ligase complex, which traps Nrf2 thereby precluding Nrf2 nuclear translocation and activation of endothelial antioxidant response in sleep restriction.
We next investigated potential mechanisms linking sleep restriction to reduced expression of DCUN1D3. DCUN1D3 gene was first identified during high-throughput screening of novel human genes that contain serum response element SRE 33 SRE binds serum response factor SRFa transcription factor that regulates a variety of cellular processes Interestingly, SRF has recently emerged as a leading candidate transcription factor for priming the cerebral cortex response to short-term sleep restriction in mice SRF plays a key role in activity-dependent modulation of synaptic strength and its ortholog blistered is required to increase sleep duration after social enrichment in Drosophila 3537 Expression of SRF follows circadian pattern and its abundance is reduced during the wake period following a short-term sleep restriction in mice, which corresponds with the timing of EC harvesting in our participants Indeed, expression of SRF mRNA in harvested ECs was reduced after sleep restriction compared with adequate sleep Fig.
Interestingly, binding of SRF to SRE, in coordination with other transcription factors, is required for activation of growth hormone GH -responsive genes that contain SRE Since DCUN1D3 contains SRE, we investigated whether its regulation by SRF is mediated by GH.
This is of interest because a major pulsatile release of GH occurs during the slow wave sleep immediately after the sleep onset and sleep restriction blunts GH release The number of GH pulses and GH release are greatest for both sexes between and h Therefore, delaying bedtime by 1.
Because GH assessment requires frequent blood sampling over 24 h, which is not feasible in a prolonged, outpatient study, we incubated HUVECs with GH to assess its effects on SRF and DCUN1D3 expression.
As expected, addition of GH did not alter mRNA expression of SRF ; however, it upregulated mRNA expression of DCUN1D3 Supplementary Fig. Silencing of SRF in HUVECs using siRNA Supplementary Fig. S6 Dsuggesting that SRF indeed regulates its expression.
Interestingly, mRNA expression of DCUN1D3 both at baseline and after a 4 h exposure to oxidative stress remained suppressed even after addition of GH in HUVECs with SRF knockdown compared with control Supplementary Fig.
S6 Dindicating that SRF mediates effects of GH on DCUN1D3 expression. To investigate whether DCUN1D3 independently regulates antioxidant response in ECs, we silenced DCUN1D3 in HUVECs using siRNA Supplementary Fig.
Remarkably, the expression of HO-1 mRNA, a major ARE-containing Nrf2 target gene, in response to oxidative stress was almost completely abrogated in HUVECs with DCUN1D3 knockdown Fig. These finding suggest that DCUN1D3 is required for activation of Nrf2-mediated antioxidant response in ECs.
In addition, NRF2 mRNA expression was similar in HUVECs with silenced DCUN1D3 and controls both at baseline and after exposure to oxidative stress Fig. NRF2 protein expression was similar in HUVECs with DCUN1D3 knockdown and controls at baseline.
However, after exposure to oxidative stress, NRF2 protein expression was significantly reduced in HUVECs with DCUN1D3 knockdown compared with controls Fig. DCUN1D3 Regulates endothelial antioxidant response. Abbreviations as in Fig. We used a rigorous, randomized crossover design and freshly harvested ECs to show directly that insufficient sleep increases endothelial oxidative stress in healthy female persons.
Remarkably, endothelial antioxidant responses were completely lacking after sleep restriction despite markedly increased endothelial oxidative stress.
We identified reduced expression of endothelial DCUN1D3, a protein that facilitates Nrf2-mediated antioxidant response in ECs, as a novel mechanism mediating the lack of endothelial antioxidant response to sleep restriction-induced oxidative stress.
Curtailing sleep by delaying bedtime reduced expression of endothelial DCUN1D3 regulator SRF, a transcription factor that primes cortical response to sleep restriction These findings provide direct evidence that curtailing sleep, a highly prevalent behavioral pattern among adults, has detrimental effects on vascular health Fig.
Endothelial cell function during wakefulness is impaired after sleep restriction compared with adequate sleep. After adequate sleep, endothelial oxidative stress that accumulates during wakefulness is cleared by an appropriate antioxidant response.
SRF mRNA expression increases after sleep pressure build-up during wakefulness, which upregulates DCUN1D3 and sequesters Cul3 toward plasma membrane.
Reduced Cul3 availability in the Nrf2 ubiquitination complex releases Nrf2 and allows for its nuclear translocation and activation of antioxidant genes.
After sleep restriction, endothelial antioxidant responses are not appropriately upregulated leading to increased endothelial oxidative stress. Reduced SRF mRNA expression during wakefulness after sleep restriction leads to reduction in DCUN1D3 expression and consequent increase in Cul3 availability in Nrf2 ubiquitination complex, which traps Nrf2 and precludes its nuclear translocation and activation of antioxidant genes.
Impaired endothelial antioxidant response after insufficient sleep results in increased oxidative stress during wakefulness, which impairs endothelial function and may increase cardiovascular risk. Insufficient sleep has been long-linked to increased intracellular oxidative stress in model organisms.
Short-term sleep restriction impairs the mitochondrial electron transport chain and increases reactive oxygen species production in Drosophila and mouse models thereby increasing oxidative stress 1142 A pro-inflammatory transcription factor NF-κB is in part regulated by the redox status of the cell and reactive oxygen species activates NF-κB in venous ECs We reported recently that mild, prolonged sleep restriction activates NF-κB in ECs, suggesting a mechanistic link between increased oxidative stress and inflammation in ECs after sleep restriction in healthy female persons 7.
Female persons have a greater pro-inflammatory response than males both during adequate and restricted sleep, suggesting that this may be an important underlying mechanism responsible for the sex difference in cardiovascular risk associated with insufficient sleep 45 We have identified DCUN1D3, a protein that sequesters Cul3 to the plasma membrane, as a novel mediator of impaired antioxidant response in insufficient sleep.
Cul3-containing ubiquitin ligase complex targets Nrf2, a major activator of antioxidant response, for proteasomal degradation.
: Antioxidants and sleep quality| Antioxidant benefits of sleep | ScienceDaily | Chronobiology in Medicine, 1 3 , — We used actigraphy to objectively assess sleep duration during this study. A specific formula proposed by Juda et al. RELATED TERMS Polyphenol antioxidant Drosophila melanogaster Fly Obstructive sleep apnea Sleep Housefly Myopia Seedless Fruit. Last Name. |
| Foods You Should Eat to Get Better Sleep | Silent Night Therapy | Evaluation of the measurement properties of the Epworth sleepiness scale: a systematic review. Ramanathan, L. Why do we sleep? FULL STORY. In particular, compared with the SD group mice, Iba-1 positive cells decreased significantly in the FNAO treated group, suggesting that microglia returned to a relatively normal state. Deczkowska A , Amit I , Schwartz M. |
| Antioxidant Benefits of Sleep | Neuron 72 , — Stem-leaf saponins from Panax notoginseng counteract aberrant autophagy and apoptosis in hippocampal neurons of mice with cognitive impairment induced by sleep deprivation. Public Health Dedicated to Prof. Immunofluorescence Nuclear fluorescence intensity of the fluorogenic probe activated by reactive oxygen species and subsequent binding to DNA, a marker of oxidative stress was assessed using fluorescence microscopy. Get training in Lab Safety and earn CEUs. |
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Health experts warn of risks with taking melatoninAntioxidants and sleep quality -
The protein in turkey may also contribute to its ability to promote tiredness. Flavones are a class of antioxidants that reduce the inflammation that often leads to chronic diseases, such as cancer and heart disease. In addition, chamomile tea has some unique properties that may help improve sleep quality.
Specifically, chamomile tea contains apigenin. This antioxidant binds to certain receptors in your brain that may promote sleepiness and reduce insomnia. Kiwis are a low-calorie and very nutritious fruit, and eating them may benefit your digestive health, reduce inflammation, and lower your cholesterol.
These effects are due to the high amounts of fiber and carotenoid antioxidants that they provide. Kiwis may also be one of the best foods to eat before bed.
The sleep-promoting effects of kiwis are sometimes attributed to serotonin. Serotonin is a brain chemical that helps regulate your sleep cycle.
Eating a diet rich in fruit like kiwis may help promote better sleep. However, more scientific evidence is needed to determine the effects that kiwis may have in improving sleep. Tart cherry juice provides modest amounts of a few important nutrients, such as magnesium phosphorus, and potassium.
For these reasons, drinking tart cherry juice before bed may improve your sleep quality. That said, more extensive research is necessary to confirm the role of tart cherry juice in improving sleep and preventing insomnia.
Fatty fish , such as salmon, tuna, trout, and mackerel, are incredibly healthy. What makes them unique is their exceptional amounts of vitamin D. For example, a 3-ounce gram serving of sockeye salmon contains international units IU of vitamin D. Additionally, fatty fish are high in healthy omega-3 fatty acids, specifically eicosapentaenoic acid EPA and docosahexaenoic acid DHA , which are known for reducing inflammation.
In combination with the vitamin D in fatty fish, Omega-3 fatty acids may help protect against heart disease and boost brain health. Walnuts are a popular type of tree nut abundant in nutrients and a great source of healthy fats, including omega-3 fatty acids and linoleic acid. Walnuts have been studied for their ability to reduce high cholesterol levels, which are a major risk factor for heart disease.
The fatty acid makeup of walnuts may also contribute to better sleep, according to a study on mice. More human studies are needed to support the claims about walnuts improving sleep.
Additionally, passionflower tea has been studied for its potential to reduce the symptons of anxiety, depression, and other psychiatric disorers.
Specifically, the results of a small study suggest that passionflower increases the production of the brain chemical gamma aminobutyric acid GABA.
GABA works to inhibit other brain chemicals that induce stress, such as glutamate. The calming properties of passionflower tea may promote sleepiness, so it may be beneficial to drink it before going to bed.
The major difference between white and brown rice is that white rice has had its bran and germ removed. This makes it lower in fiber, nutrients, and antioxidants. White rice is high in carbs. Its carb content and lack of fiber contribute to its high glycemic index GI. That said, this research was based on professional athletes who may need to consume more cabrs than the average person.
A review suggests, however, that the evidence that high GI foods can help with sleep is mixed and more study is necessary.
Several other foods and drinks have sleep-promoting properties. In particular, the intestinal epithelial barrier hinders the damage of external risk factors to the body, playing a critical role in regulating systemic immunity homeostasis [ 14 ]. Additionally, the studies on the brain-gut axis further elucidate the bidirectional communications between the gut and brain [ 15—17 ].
Once sleep deprived, it could be a risk factor for more frequent episodes of inflammatory bowel disease, which in turn worsens sleep abnormalities [ 18 ]. The latest studies have revealed that SD leads to the accumulation of excessive reactive oxygen species ROS , specifically in the intestine [ 19 ].
These could result in high expressions of pro-inflammatory factors and continuous releases of danger signals into the blood, which leads to systemic inflammation and injury to nerves [ 20 , 21 ].
Therefore, in the intestine, researchers have used oral antioxidants, or modified antioxidant enzyme genes, to targeted peroxides to reduce the intestinal damage caused by SD in animal models [ 19 ].
However, there are few studies investigating the effects of antioxidants on sleep quality or sleep duration. Fullerene, as a representative of carbon nanomaterials, has a wide range of applications in the biomedical field.
Previous studies have reported that fullerene nanoparticles have superior antioxidant activity and anti-inflammatory properties [ 22—26 ].
Due to the large conjugated π bonds with high electron affinity on its carbon cage, it can efficiently capture the unpaired electrons of excessive free radicals at the site of lesions and regulate the body's redox balance [ 27 ].
Emerging studies showed that fullerene nanoparticles can restore damaged intestinal barrier function and further hinder entry of harmful lipopolysaccharides LPSs into the circulation of atherosclerotic mice [ 28 ]. Importantly, SD is closely related to the occurrence and development of cardiovascular diseases such as atherosclerosis [ 29 ].
This encourages us to study the effects of fullerene nanomaterials on both SD and sleep quality. Additionally, oral administration is a more commonly used method for administering small-molecule drugs and traditional medicines, as it offers better patient compliance compared to injection [ 30 ].
Given the central role of intestinal ROS in severe SD [ 13 ] and the efficient ability of fullerenes to eliminate ROS [ 22 , 24 ], we attempted to explore the role of oral administration of fullerene nanoparticles as an antioxidant in SD research.
In this work, we report for the first time that orally delivered fullerene nano-antioxidants FNAO are adopted to restore intestinal barrier integrity and systematically regulate the inflammatory microenvironment by acting on the brain-gut axis to achieve superior improvement in SD Fig.
FNAO could reduce excessive intestinal ROS and improve intestinal barrier function both in zebrafish and mouse models, thereby restraining intestinal and systemic inflammatory responses. More importantly, FNAO remarkably avoids SD-induced neuroinflammation damage, conserving circadian regulation and sleep homeostasis.
Of note, following oral administration, FNAO is merely distributed in the intestine without diffusion into the whole body and could be excreted without causing apparent toxicity to the major organs. Our work provides a powerful candidate for improving SD and broadens the mind for therapy of SD-related nerve injury by maintaining intestinal homeostasis.
Schematic depiction and characterizations of FNAO. a SD improvement and anti-nerve injury mechanism diagram of FNAO. b Schematic diagram of the synthesis of FNAO. c Representative pictures of FNAO dispersion liquid the left picture shows the dispersion state of pharmaceutical excipients without C 60 , and the right picture shows the dispersion state of FNAO.
d TEM image of FNAO. Scale bar: nm. e The MALDI-TOF-MS of FNAO. f The ESR spectrum of the hydroxyl radicals captured by DMPO after treatment with FNAO blue line and pharmaceutical excipients without C 60 red line , using water as a blank control blank line.
g The quantitative statistics intensity of ESR. First, we prepared FNAO with an effective free radical scavenging capacity through a simple method. To be orally delivered, they were compressed into tablets and dissolved in ultrapure water to form a dispersion system for further use Fig.
Subsequently, transmission electron microscopy TEM was adopted to determine the morphology and size of FNAO, which indicated that the particle size was around 1 μ m Fig. Zebrafish, as a new model organism, show great advantages in SD-related research.
Compared with rats and mice, zebrafish could better simulate mammalian sleep structure as it moves during the day and sleeps at night [ 31 ]. Before the formal SD experiment, an in-situ matrix-assisted laser desorption ionization mass spectrometry imaging MALDI-MSI detection method was applied to investigate the visualization of tissue distributions of FNAO in zebrafish.
Compared with the control group without FNAO, the C 60 of the FNAO group was mainly distributed in the intestine especially in the intestinal contents , with very little in the gills inevitably brought in by respiration.
It indicated that fullerene C 60 as the active ingredient of FNAO suspension could directly act on the intestine of zebrafish rather than other tissues, and that the properties of C 60 are stable in the gastrointestinal tract, which is highly consistent with our previous biodistribution study of FNAO [ 28 ].
Assessments of FNAO on the levels of ROS and inflammatory-associated immune cells in the gut of zebrafish. b Schematic diagram of SD improvement study by FNAO in zebrafish. c The DCF fluorescence images left and quantitative statistics intensity right in gut section of 5 days sleep-deprived zebrafish treated by FNAO.
Scale bar, μ m. d Measurements of the level of CAT activities, SOD activities, and MDA content of the zebrafish after 5 days of SD. Scale bar, 25 μ m. f The fluorescence images of neutrophils and macrophages in the gut of 5 days sleep-deprived zebrafish.
Scale bars, μ m left , μ m right. Since persistent SD can lead to ROS accumulation and damage caused by systemic oxidative stress [ 19 ] especially in the gut [ 13 ], we first examined the effects of FNAO on the gut of SD zebrafish.
We first established a SD model in zebrafish larvae by continuous illumination. Briefly, 6-day-old zebrafish larvae were exposed to lux unit of light illuminance white light for 5 days, thus the zebrafish larvae were deprived of sleep Fig. Due to the organism's powerful balance mechanism, there may be varying degrees of repair of SD damage after the cessation of SD [ 32 ].
Therefore, we chose to administer FNAO during continuous SD to reflect the role of FNAO in SD as much as possible, eliminating interference from self-repair mechanisms in the organism. It showed that excessive ROS were particularly generated in the gut by SD Fig. Notably, FNAO suspensions reduced the DCF fluorescence intensity in the SDF group.
Importantly, the DCF fluorescence intensity was much lower in the SDF group than that in the SDM group. Furthermore, we examined the effects of FNAO on overall oxidative stress in sleep-deprived zebrafish. Thirty AB zebrafish were taken from each group, ground on ice, and total protein was extracted and measured for concentration.
The expression of the target molecules was detected according to the manufacturer's instructions. After 1 day of SD, the overall levels of catalase CAT , superoxide dismutase SOD , and malonaldehyde MDA in zebrafish showed little variation among the groups Supplementary Fig.
After 5 days of SD, the levels of CAT, SOD, and MDA tended to increase. Of note, they were decreased almost back to the control group level after FNAO and melatonin treatment Fig. This indicated that the oxidative stress levels in zebrafish increased with prolonged SD, and FNAO could actively regulate redox balance during the process of SD.
The overproduction of ROS and oxidative stress would further lead to the occurrence of inflammation, and the cell structure would be vulnerable to damage. Our results revealed that the intestinal cavities had regular shape, the intestinal villus owned complete structure and the outline of intestinal epithelial cells were clear in the non-SD group control group.
On the contrary, the gut was severely damaged in the SD group. Specifically, the intestinal villus was exfoliated and intestinal epithelial cells were severely disrupted after SD. Interestingly, the villus and epithelial structures of the intestine were observably repaired after FNAO treatment.
It indicated that FNAO could inhibit inflammation and improve the intestinal environment. To clarify the mechanism by which FNAO eliminates intestinal inflammation, we selected Tg lyz: DsRed2; coro1a: EGFP transgenic larvae as the optimal tool, which selectively expressed red fluorescent protein to track the neutrophils and green fluorescent protein to track the macrophages [ 33—35 ].
We established the SD model in transgenic larvae and treated them with FNAO using the same method. The results showed that both the macrophages and neutrophils were remarkably aggregated in the gut of SD larvae, especially in the intestinal bulb labeled with ellipsoid and hind-intestine Fig. Of note, the aggregations of macrophages and neutrophils had dramatically vanished after FNAO suspension treatment.
This indicated that FNAO improved intestinal damage by regulating the aggregation and migration of zebrafish macrophages and neutrophils. Taken together, FNAO significantly reduced oxidative stress and inflammation caused by SD in the zebrafish gut. Few studies have directly shown that improving the intestinal environment can improve sleep or reduce neuroinflammation [ 36 , 37 ], which greatly stimulated our exploration of the effects of FNAO on sleep and neurological function, in order to further confirm whether FNAO acts in the afferent direction gut-to-brain of the brain.
Having shown the favorable effects of FNAO on the gut of sleep-deprived zebrafish, we sought to assess the impact of FNAO on sleep improvement in this model.
First, zebrafish larvae were deprived of sleep for either 1 day or 5 days, and sleep-deprived larvae were treated with FNAO and melatonin, respectively. At zeitgeber time ZT 0 on days 2 and 6, we consecutively monitored the h behavior changes of larvae under normal day-night environment light for 14 h and dark for 10 h by an automatic video tracking system Fig.
Total sleep time, ZT0—ZT14 sleep time and ZT14—ZT24 sleep time were analyzed, respectively. As shown in Fig. However, the cumulative activity time curve of the FNAO treatment group approached that of the control group and was superior to the melatonin treatment group especially during the ZT0—ZT14 period.
As SD extended to 5 days, the cumulative activity time of zebrafish in the SD group also significantly decreased during the ZT0—ZT14 period.
Surprisingly, both FNAO and melatonin were able to improve this detrimental impact Fig. After further analysis of sleep time, we found that the sleep-deprived zebrafish showed shortened duration of movement and lengthened sleep time during ZT0—ZT14, while showing longer duration of movement and shorter sleep time during ZT14—ZT24 compared with the control group.
Interestingly, the prolonged total sleep time in SD zebrafish was almost back to a normal level after FNAO and melatonin treatments Fig.
Subsequently, we evaluated the potential effects of FNAO on brain nerves in sleep-deprived zebrafish using Tg elavl3: YC2 , which labeled the neonatal neurons with YC2 that could be detected under fluorescence microscopy [ 39 ].
It showed a marked decline in fluorescence intensity after sleep loss in the SD group compared to the control group Fig. Notably, the fluorescence intensity in neurons had increased back to almost normal levels after FNAO treatment Fig.
The results indicated that FNAO restored neuronal damage from SD-induction by increasing the transcription or translation of elavl3. We also recorded the survival rate of zebrafish larvae under SD. Each group consisted of 40 AB type zebrafish larvae, and the number of dead larvae in each group was recorded from the first day of SD until the day The result indicated that continuous SD caused high mortality in zebrafish larvae, and after 8 days of SD, all zebrafish had died Fig.
Notably, the FNAO-treated group had a higher survival rate, suggesting that FNAO provided efficient protection to sleep-deprived zebrafish and prolonged their lifespan. Sleep improvement effects of FNAO on SD in zebrafish.
a Schematic diagram of SD improvement study in zebrafish. d The increased green fluorescence intensity of neurons in the sleep-deprived zebrafish brain after FNAO treatment. Scale bars, μ m. e Quantification. f Overall Kaplan—Meier survival curves of sleep-deprived red , FNAO therapy blue and non-deprived black zebrafish.
Differences were assessed by ANOVA and symbolized as follows: ns is no significant difference. Taken together, for zebrafish larvae, FNAO treatment during SD could prevent pathological damage to the gut by regulating the body's redox balance and the migration of macrophages and neutrophils.
In addition, SD was the cause of oxidative stress in organisms. As SD was prolonged from 1 day to 5 days, zebrafish behavior continued to change Fig. Importantly, FNAO can efficiently regulate the body's redox balance to improve sleep in zebrafish.
Furthermore, FNAO therapy could alleviate brain and nerve damage Fig. This indicated that even under conditions of continuous SD where the organism did not have enough time for self-repair, FNAO could effectively reduce the damage caused by prolonged SD and improve the organism's sleep condition.
However, we still do not know how FNAO sent signals to the brain to improve sleep and reshape neural function. Due to the fact that C 60 was exclusively distributed in the gut Fig. Sleep regulation is conserved across species [ 40 ]. First, a mouse model of SD was constructed. It showed that the sleep structures of mice after SD were obviously disrupted.
Specifically, the sleep latency of the mice in the SD group became longer compared with those in the control group during ZT2—ZT Moreover, the sleep cycle was significantly fragmented in SD mice in the total h record [the frequency of cycle replacement of awakening time blue strip and sleep time yellow strip and green strip was relatively quick].
Interestingly, these abnormalities in SD mice were observably improved after FNAO treatment. It could be found that the sleep latency of FNAO-treated mice was distinctly shortened, the phenomenon of sleep fragmentation was markedly reversed, and the sleep cycle mostly returned to normal.
Together, FNAO notably improved the sleep status of SD mice. Effects of FNAO on SD in mice. a Schematic diagram of the mouse SD study. d The Nissl staining in the hippocampus CA1 subfield scale bar is 2. e The Iba-1 immunohistochemistry images in the hippocampus field scale bar is μ m and the corresponding magnification scale bars: μ m; 25 μ m.
f Measurements of the levels of 5-HT, Melatonin MLT and the normalized levels of IL-1 β , IL-6, and TNF- α g in serum of the mice in the non-SD Control , SD and FNAO treatment SDF groups via ELISA tests.
After confirming the rescue of sleep fragmentation by FNAO in SD mice, we further studied the effect of FNAO treatment on brain nerve protection. First, neuron loss was one of the hallmarks in SD [ 42 ]. The Nissl body is one of the characteristic structures of neurons. To observe the effects on neuron loss by FNAO treatment, mouse brain tissues were fixed and paraffin-sealed.
Then, we used the Nissl staining of the paraffin sections to evaluate the morphological changes in mice hippocampal neurons Fig. We found that the Nissl bodies boundary of mice in the SD group had an unclear cell-boundary compared with the control group, and the nucleus of neurons were mostly karyolitic.
Notably, the neurons had a clear nucleus, abundant Nissl bodies and regular sequence after FNAO treatment. These results suggested that FNAO maintained the normal morphological structure of Nissl bodies in SD mice.
In addition, as important immune cells in the central nervous system CNS , microglia actively regulate the immune function of the brain and reduce inflammation [ 43 , 44 ].
We assessed the contents of ionized calcium-binding adapter molecule 1 Iba-1 by immunohistochemical staining, which is a marker of reactive microglia in the brain. It showed that the number of Iba-1 positive cells in the hippocampal CA1 region of the control group mice was less Fig.
However, the number of Iba-1 positive cells in SD group mice was significantly increased, and the cell body became larger, accompanied by the deepened staining. In particular, compared with the SD group mice, Iba-1 positive cells decreased significantly in the FNAO treated group, suggesting that microglia returned to a relatively normal state.
Collectively, these observations suggested that FNAO could protect mouse nerves from damage by inhibiting the excessive activation of CNS microglial cells.
We next assessed the effect of FNAO on neurotransmitters and systemic inflammation. The levels of representative neurotransmitters and inflammatory cytokines were assessed using an enzyme linked immunosorbent assay ELISA. Serotonin, also referred to as 5-hydroxytryptamine 5-HT , is an important neurotransmitter that plays an important role in controlling sleep and arousal [ 45 ].
Besides, 5-HT could convert into melatonin MLT , which is the dominant regulator of the sleep cycle and circadian rhythm [ 46 ]. After treatment with FNAO, the level of 5-HT returned to normal, but the melatonin level did not, which reminded us that after FNAO treatment, the upregulation rate of 5-HT seemed to be much higher than the conversion rate of 5-HT to melatonin.
This suggested that FNAO may selectively regulate neurotransmitters through indirect means. In addition, FNAO improved sleep most likely through the 5-HT pathway rather than the melatonin pathway. Continuous SD can result in an increase in pro-inflammatory cytokines [ 47 ].
ELISA results showed that the levels of proinflammatory cytokine in serum, such as IL-1 β , IL-6 and TNF- α , were increased after SD which were then significantly reduced after FNAO treatment Fig. The above results demonstrated that FNAO had a positive regulatory effect on sleep structure disorder in SD mice, which may be due to the significant regulatory effect of FNAO treatment on Nissl body dissolution and excessive activation of microglia in the CNS of SD mice.
Furthermore, FNAO could capture the signal in the efferent direction of the brain, effectively reducing systemic inflammation and regulating the release of neurotransmitters in order to inhibit neural damage caused by SD stress. After receiving signals of SD, the brain induced corresponding changes in the efferent direction brain-to-gut which can affect peripheral organs or tissues.
In order to further explore whether the mechanism of FNAO improving sleep in mice was related to the intestine, we next detected the contents of ROS in both the ileum and colon of sleep-deprived mice using cryo-sectioning and flow cytometry.
The superoxide anion level was detected by superoxide anion fluorescence probe dihydroethidium DHE staining based on the intestinal cryosection technique. The total intracellular ROS level was detected by DCFH-DA based on flow cytometry.
For the ileum, as shown in Fig. In addition, we tested the levels of IL-1 β , IL-6 and TNF- α in the ileum by ELISA kits.
It revealed that the excessive ROS in the ileum mainly induced the high levels of these pro-inflammatory cytokines in SD mice, which were decreased after FNAO treatment Fig. Control flies that had normal sleep lived up to approximately 40 days in the same environmental conditions.
Because mortality increased around day 10, the researchers looked for markers of cell damage on that and preceding days. Most tissues, including in the brain, were indistinguishable between sleep-deprived and non-deprived flies, with one notable exception.
The guts of sleep-deprived flies had a dramatic buildup of ROS—highly reactive, oxygen-containing molecules that in large amounts can damage DNA and other components within cells, leading to cell death. The accumulation of ROS peaked around day 10 of sleep deprivation, and when deprivation was stopped, ROS levels decreased.
Additional experiments confirmed that ROS builds up in the gut of only those animals that experienced sustained sleep loss, and that the gut is indeed the main source of this apparently lethal ROS. That almost never happens in lab research.
The team also examined whether ROS accumulation occurs in other species by using gentle, continuous mechanical stimulation to keep mice awake for up to five days. Compared to control animals, sleep-deprived mice had elevated ROS levels in the small and large intestines but not in other organs, a finding consistent with the observations in flies.
To find out if ROS in the gut play a causal role in sleep deprivation-induced death, the researchers set out to determine whether preventing ROS accumulation could prolong survival. They tested dozens of compounds with antioxidant properties known to neutralize ROS and identified 11 that, when given as a food supplement, allowed sleep-deprived flies to have a normal or near-normal lifespan.
These compounds, such as melatonin, lipoic acid and NAD, were particularly effective at clearing ROS from the gut. Notably, supplementation did not extend the lifespan of non-deprived flies. The role of ROS removal in preventing death was further confirmed by experiments in which flies were genetically manipulated to overproduce antioxidant enzymes in their guts.
These flies had normal to near-normal lifespans when sleep-deprived, which was not the case for control flies that overproduced antioxidant enzymes in the nervous system.
The results demonstrate that ROS buildup in the gut plays a central role in causing premature death from sleep deprivation, the researchers said, but cautioned that many questions remain unanswered.
Similarly, death could be due to damage in the gut or because high levels of ROS have systemic effects, or some combination of these.
They found that some sleep-deprived flies ate more throughout the day compared with non-deprived controls. However, restricting access to food had no effect on survival, suggesting that factors beyond food intake are involved.
The researchers are now working to identify the biological pathways that lead to ROS accumulation in the gut and subsequent physiological disruptions.
The team hopes that their work will inform the development of approaches or therapies to offset some of the negative consequences of sleep deprivation. One in three American adults gets less than the recommended seven hours of sleep per night, according to the U.
Centers for Disease Control and Prevention, and insufficient sleep is a normal part of life for many around the world. Additional authors on the study include Keishi Nambara, Elizabeth Pollina, Cindy Lin and Michael Greenberg.
The study was supported by the New York Stem Cell Foundation, the Pew Charitable Trusts and the National Institutes of Health R73 NSO Additional support includes an EMBO long-term fellowship, a Fondation Bettencourt Schueller fellowship, an Edward R.
and Anne G. Lefler Center postdoctoral fellowship, an Alice and Joseph E.
Qualit oxygen species Dark chocolate exploration Antioxidants and sleep quality in the gut of sleep-deprived fruit flies, one leftseven sldep and ten right days without sleep. Image: Vaccaro et al, The first signs of insufficient sleep are universally familiar. Far fewer people have experienced the effects of prolonged sleep deprivation, including disorientation, paranoia and hallucinations. Total, prolonged sleep deprivation, however, can be fatal.
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