N-Acetyl Serotonin Provides Neuroprotective Effects by Inhibiting Ferroptosis in the Neonatal Rat Hippocampus Following Hypoxic Brain Injury

Hypoxic-ischemic encephalopathy is the main cause of infant brain damage, perinatal death, and chronic neonatal disability worldwide. Ferroptosis is a new form of cell death that is closely related to hypoxia-induced brain damage. N-Acetyl serotonin (NAS) exerts neuroprotective effects, but its effects and underlying mechanisms in hypoxia-induced brain damage remain unclear. In the present study, 5-day-old neonatal Sprague–Dawley rats were exposed to hypoxia for 7 days to establish a hypoxia model. Histochemical staining was used to measure the effects of hypoxia on the rat hippocampus. The hippocampal tissue in the hypoxia group showed significant atrophy. Hypoxia significantly increased the levels of prostaglandin-endoperoxide synthase 2 (PTGS2) and the iron metabolism-related protein transferrin receptor 1 (TfR1) and decreased the levels of glutathione peroxidase 4 (GPX4). These changes resulted in mitochondrial damage, causing neuronal ferroptosis in the hippocampus. More importantly, NAS may improve mitochondrial function and alleviate downstream ferroptosis and damage to the hippocampus following hypoxia. In conclusion, we found that NAS could suppress neuronal ferroptosis in the hippocampus following hypoxic brain injury. These discoveries highlight the potential use of NAS as a treatment for neuronal damage through the suppression of ferroptosis, suggesting new treatment strategies for hypoxia-induced brain damage.


Introduction
Neonatal hypoxia can result in severe lifelong sequelae and cognitive impairments and can lead to infant death.This condition has become a critical healthcare problem, and 23% of neonatal deaths occur due to neonatal hypoxia [1].
There are various events that cause hypoxic-ischemic (HI) insult, and brain damage is ultimately caused by impaired oxygen delivery to the brain [2] and cerebral blood flow [3].Severe HI stress can cause mitochondrial dysfunction [4], cellular energy depletion, and brain edema and enhance the release of intracellular calcium [5,6] and neurotransmitters.Hypoxic-ischemic brain damage (HIBD) is thought to involve neuroinflammation, oxidative stress, and mitochondrial dysfunction.Hypoxia in childhood can induce bilateral pathological changes in the hippocampus at an early age and precipitate memory issues [7].There are few effective treatments available to prevent hypoxic injury in fetal brains during and after birth [8], and hypothermia is the only clinical treatment in use of moderate hypothermia, which is efficient for less than 60% of infants [9].Thus, identifying new treatment strategies is important.
Recent research has reported that ferroptosis is associated with the development of a few neurological diseases.As a type of cell death, ferroptosis can be induced by the inhibition of glutathione peroxidase 4 (GPX4) or the disruption of glutathione synthesis; iron accumulation can exacerbate this process [10].Researchers have shown that iron levels were increased in the brain tissue of neonatal HIBD patients [11].In addition, some studies have shown that the prognosis of HIBD may be improved by desferrioxamine and erythropoietin, which modulate iron metabolism [12,13].Accumulating evidence shows that ferroptosis plays a critical role in central nervous system (CNS) disorders, including HIBD and traumatic brain injury, and inhibiting ferroptosis can prevent neuronal death [14,15].However, in brain diseases, the efficacy of ferroptosis inhibitors is restricted because of the blood-brain barrier.Thus, determining the mechanisms of ferroptosis in HIBD and investigating potential antiferroptotic agents to treat HIBD are important.
As a precursor of melatonin, N-acetyl serotonin (NAS) is produced from serotonin by serotonin N-acetyltransferase; its antioxidant activity is stronger than melatonin [16].NAS activates tropomyosin-related kinase-derived neurotrophic factor (TrkB) in a circadian manner [17].Previous studies have shown that NAS can play distinctive roles in the CNS, which was associated with its increasing antioxidant capacity at higher concentrations [18].ANA12 is a novel small-molecule TrkB antagonist.NAS is supposed to alleviate hypoxia induced hippocampus injury associated with brain-derived neurotrophic factor (BDNF)-TrkB pathway.In our research, ANA12 was used to antagonize the neuroprotective effect of NAS.
Thus, we hypothesize that NAS can be used to treat hypoxia/ischemia-induced injury.To validate this hypothesis, the molecular mechanisms by which NAS inhibits ferroptosis were examined, and we showed that NAS has neuroprotective effects against hypoxia-induced injury in the hippocampus of neonatal Sprague-Dawley (SD) rats.

Animals
We purchased the SD rats (5-8 g, 3 days old) from Pengyue Laboratory Animal Breeding Co., Ltd.(located in Jinan, Shandong).Postnatal day 5 (P5) pups were used in our experiments.All rats were bred in a room with a 12/12-h light/dark cycle and a temperature of 25 ± 1 °C.The room was ventilated, and the bedding, water, and feed were replaced regularly to ensure the health of the animals.All animal experimental protocols were approved by the Animal Experimental Ethics Committee at Qilu Hospital of Shandong University and were conducted with the guidance of the Animal Care and Use Committee of Qilu Hospital at Shandong University (Approval number: ECAESDUSM 2012029).

Neonatal Hypoxia Brain Injury Models
To induce hypoxia, we exposed the rats to hypoxia for 7 days in a sealed chamber beginning on P5.The 10% oxygen concentration was maintained for 7 days, during which time the animals were returned to their mothers to suckle for 15 min every day and were monitored daily.Age-matched control rats that were not subjected to hypoxia treatment were placed in the same chamber for the same amount of time.

Drug Administration and Experimental Groups
NAS (purity > 99%, #A1824) was purchased from Sigma-Aldrich (St. Louis, USA) and was dissolved in DMSO and then diluted with saline.The rats were administered NAS (10 mg/kg) by intraperitoneal injection on days 2, 4, and 6 after hypoxia.We purchased ANA-12 from Sigma-Aldrich (St. Louis, USA), which was dissolved in DMSO (35 mg/ml) and then diluted with saline.Before hypoxia was induced, the rats were administered ANA-12 (0.5 mg/kg) by intraperitoneal injection.

Nissl Staining
The sections were dewaxed, washed with distilled water, and then incubated with Nissl staining solution at room temperature for 10 min.Then, 95% and 100% ethanol were used to dehydrate the sections, which were then made transparent using xylene, placed under coverslips, and analyzed by microscopy.

H&E Staining
The rats were anesthetized and perfused with PBS.The brain tissues were rapidly removed and fixed with 4% paraformaldehyde at 4 °C overnight.Then, the tissues were embedded in paraffin and sectioned into 4-μm-thick slices.Next, the sections were deparaffinized in xylene and rehydrated using graded alcohol solutions.Finally, the sections were stained with H&E, and pathological changes in the brain tissue were observed under a light microscope (400-fold magnification).

Western blot analysis
All protein samples were lysed by homogenization in RIPA buffer.The sample was centrifuged at 12,000 × g at 4 °C for 15 min, and the total protein was harvested.The proteins were separated by electrophoresis and transferred onto polyvinylidene difluoride membranes based on the different molecular weights of each protein at low temperature.The membranes were blocked with 5% nonfat dry milk in TBS at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C.Then, the membranes were incubated for 1 h at room temperature with a secondary antibody.The protein signals were detected by enhanced chemiluminescence.The signal bands were analyzed using a ChemiDoc XRS instrument and Image Lab Software.The relative expression was calculated as the ratio between the protein of interest and GAPDH in the same sample and is displayed graphically.

Real-Time Quantitative Polymerase Chain Reaction
The PCR solution containing SYBR Green I was prepared on ice.The finished reaction solution was dispensed into the reaction plate and then sealed and centrifuged at high speed.The reaction plate was placed into the real-time PCR amplification instrument.All samples were analyzed in triplicate.The cycling program was as follows: step 1, 95 °C for 30 s; step 2, 40 cycles of 95 °C for 3 s, followed by 60 °C for 30 s.The amplification curve and the melting curve were confirmed after the reaction.Melting curve analysis was performed to confirm amplification specificity.Finally, the data were exported and copied, and the results were calculated and counted.Transcript levels of each mRNA was normalized to GAPDH and were calculated using the 2 −∆∆CT method.

Immunohistochemistry
Paraffin sections (4 μm) of brain tissue were deparaffinized in xylene solution.Subsequently, rehydration was performed in graded ethanol solutions (100%, 95%, 80%, and 75%) and the antigen was recovered.Sections were then incubated in 3% H 2 O 2 for 15 min at 37 °C and washed with PBS to eliminate the endogenous peroxidase activity.Goat serum was added and incubated for 20 min at room temperature, and the primary antibody of TfR1 (abcam, USA) was added at 4 °C overnight.The following day, after washing in PBS, sections were incubated for 20 min at 37 °C with the secondary antibody.We used the chromogenic agent DAB to colorize and re-stain the sections.The results of immunohistochemistry were visualized by microscopy.

Transmission Electron Microscopy (TEM)
Hippocampal tissue samples were cut into 2 × 2 mm pieces and quickly fixed in electron microscopy fixation solution at room temperature for 2 h.Then, the samples were dehydrated, embedded, and cut into 50-nm thickness.The stained samples were then observed and imaged with a transmission electron microscope (JEOL, Japan).

Cell Cultures and Induction of Cell Death
Primary hippocampal neurons (PHNs) were isolated from E16 rat.Briefly, we euthanized a pregnant rat at approximately 16 days post-fertilization by decapitation and remove pups from the uterus, then decapitated pups with fresh sterile scissors and placed removed head on sterile gauze under a dissecting microscope.After the dissection, gently lift hippocampus with sterile tissue forceps and transfer into a small tissue culture dish with warmed (37 °C) HBSS under a cell culture hood.After grinding and filtering the tissue, cells can be plated with indicated volume of neurobasal plating media (neurobasal media containing B27 supplement, 0.5 mM glutamine solution, 25 uM glutamate, penicillin/streptomycin, 1 mM HEPES, 10% Heat Inactivated Donor Horse Serum).The cells are subsequently weaned from the serum and returned to a Serum-Free environment by serial reduction of the serum at each media replacement.Place neurons in a 37 °C, 5% CO 2 incubator overnight.Neurons should be fed every 4 days by removing half of the old media and replacing it with the same volume of fresh Neurobasal Feeding media.Ten days later, hypoxia model for neurons was induced by exposing the neurons to hypoxia conditions.PHNs were preincubated for 2 h with NAS (10 nM, 100 nM, 1 µM, 10 µM, 100 µM), then they were kept in a hypoxia chamber, with 1% O 2 , 5% CO 2 , and 94% N 2 , for 3 h, 6 h, or 12 h.
Cell death of PHNs was quantitatively evaluated by the lactate dehydrogenase assay according to the manufacturer's instructions (C0016, Beyotime Biotechnology, China).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0, and the data are expressed as the mean ± SD.The statistical significance of multiple comparisons was analyzed by one-way analysis of variance.SPSS 19.0 software was used to perform all statistical analyses.P < 0.05 was considered to be statistically significant.

Hypoxia Induces Hippocampal Injury
The hypoxia rat model was established, and then the next experiments were performed (Fig. 1A).Nissl body staining of neurons showed that many granule cells and pyramidal cells were present in the hippocampus in the sham group, and no neuronal damage was observed.Following hypoxiainduced brain injury, there was a significant decrease in the number of neurons, as shown by Nissl staining, compared with that in the sham group (Fig. 1B).

Hypoxia Induces Hippocampal Ferroptosis and NAS Inhibits Hypoxia-Induced Expression of TfR1
Real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine whether hypoxia was involved in hippocampal ferroptosis, and we measured ferroptosis marker expression (GPX4, transferrin receptor 1 (TfR1) and prostaglandin-endoperoxide synthase 2 (PTGS2).Compared with those in the sham group, GPX4 expression was significantly decreased and TfR1 and PTGS2 expression was increased the hypoxia group (Fig. 2A).The results of immunohistochemistry are shown in the Fig. 2B, TfR1 protein was mainly expressed in the cytoplasm and its positive expression was stained with brownish-yellow.The brownish-yellow positive particles of hippocampus tissues in the hypoxia group were more than those in the sham group.Compared with the hypoxia group, the positive expression rate of TfR1 protein in

NAS Protects the Hippocampus Against Hypoxia-Induced Ferroptosis
We examined whether neuronal ferroptosis could be rescued by NAS.First, neuronal morphology in the hippocampus was observed by H&E staining.We found significantly worsened pathological injury in the hypoxia group compared with the sham group, but NAS attenuated neuronal damage.However, ANA-12 administration partly reversed the decrease in neuronal damage (Fig. 3A).Western blot analysis also revealed that the hypoxia group had significantly increased TfR1 expression and decreased GPX4 expression compared with those in the sham group.However, GPX4 expression was increased and TfR1 expression was decreased in the NAS group, and ANA-12 blocked the effect of NAS on the expression of these factors in the ANA-12 + NAS + hypoxia group (Fig. 3B).The expression of GPX4 and TfR1 was also measured by qRT-PCR, and the hypoxia group had significantly decreased GPX4 and increased TfR1 expression.However, GPX4 expression was increased and TfR1 expression was decreased in the NAS group, and the effect of NAS on the expression of these factors was blocked by ANA-12 (Fig. 3C).Furthermore, TEM showed that compared with that in the sham group and NAS + hypoxia group, mitochondrial atrophy in hippocampal neurons was significantly increased in the hypoxia group and ANA-12 + NAS + hypoxia group (Fig. 3D).In summary, these findings suggested that NAS rescued hypoxia-induced ferroptosis by inhibiting TfR1 expression and that ANA-12 eliminated this effect.

NAS Inhibits Hypoxia-Induced Cell Death in Primary Neurons In Vitro
To provide evidence of NAS protection against neuronal cell death, the hypoxia model was used.Indeed, incubation of PHNs with NAS (0.01 to 100 µM) resulted in statistically significant inhibition of hypoxia-mediated PHN cell death (Fig. 4).

Discussion
In this study, a hypoxia model was established in neonatal rats to examine whether NAS could ameliorate brain damage by inhibiting ferroptosis.First, we confirmed the successful establishment of a hypoxia-induced brain injury model in neonatal rats by Nissl staining.Then, we found that ferroptosis occurred in the hippocampus after hypoxia.Furthermore, NAS treatment decreased hypoxia-induced hippocampal ferroptosis, and ANA-12 blocked the NAS-mediated inhibition of ferroptosis.Our data demonstrated that NAS suppressed hypoxia-induced ferroptosis, thereby alleviating brain injury.
Hypoxic and HI damage in utero or at birth are the principal causes of neonatal morbidity and mortality [19].The hippocampus plays important roles in the development of nerves and is highly sensitive to HI injury [20].HI injury could result in hippocampal atrophy, which is associated with long-term impaired memory and learning, in human term newborns with neonatal encephalopathy [21,22].Additionally, there are decreased expression levels of early neuronal markers and late/mature neuronal markers due to HI injury in patients, suggesting the presence of impairments in neonatal stem cells to mature neurons during the development of normal neurons [23].In recent years, numerous studies have shown that HI injury in neonatal animals is similar to the clinically manifestations in humans [24,25].In our neonatal hypoxia rat model, we observed injury in the hippocampus.
As a newly discovered mode of regulated cell death, ferroptosis is different from apoptosis, programmed necrosis, and autophagy.The accumulation of reactive oxygen species and iron-dependent lipid peroxidation result in ferroptosis [26].Iron is an important trace element, which abnormal distribution and content of iron can affect the normal physiological processes [27].Excess Fe 2+ is oxidized to Fe 3+ by ferroportin.This recycling of internal iron strictly controls iron homeostasis in cells.Silencing the gene encoding transferrin receptor 1 (TfR 1) can inhibit erastininduced ferroptosis [28].Furthermore, lipid metabolism is also closely related to ferroptosis.Polyunsaturated fatty acids are one of the essential elements for ferroptosis and are sensitive to lipid peroxidation.Reducing the expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) reduces the accumulation of lipid peroxide substrates in cells, thus inhibiting ferroptosis [29].The most common features of ferroptosis are shrunken mitochondria, mitochondrial aberrations, rupture of the outer mitochondrial membrane, and reduced cristae [30].The density of the mitochondrial membrane also increases [31].There is a central endogenous inhibitor of ferroptosis known as GPX4, and neurons prevent toxic lipid peroxidation by relying on GPX4 [32].A study showed that selective knockout of GPX4 resulted in rapid death and the loss of hippocampal neurons in adult mice.Cognitive impairment and hippocampal degeneration are caused by targeted knockout of the GPX4 gene in forebrain neurons [33].Although ferroptosis is involved in various pathological conditions, such as neoplastic diseases, glutamate-induced neurotoxicity, neurodegenerative diseases, and ischemia/reperfusion injury, ferroptosis has rarely been examined in an in vivo epilepsy model.In the present study, qRT-PCR showed changes in GPX4, PTGS2, and TfR1 expression in the hippocampus of hypoxia-treated rats, confirming the involvement of ferroptosis in the pathological process of hypoxia-induced brain damage.
Despite many studies and advances in intensive care and neonatology, the approved treatment available for hypoxia-ischemia is hypothermia [34].As a naturally occurring chemical intermediate, NAS is generated from serotonin.Melatonin is converted from NAS and has neuroprotective effects against ischemic stroke and other neurological diseases [35][36][37].NAS has many biological effects that are similar to those of melatonin, such as antioxidant, antiaging, antianxiety, and neuroprotective effects.However, there is evidence indicating that NAS may play unique roles in the CNS, and its robust antioxidant activity [38] and antioxidant capacity [18] may be unrelated to its conversion to melatonin.In addition, NAS acts as a potent TrkB receptor agonist [39].ANA-12 is a low-molecular-weight molecule and it can prevent TrkB activation and inhibit downstream processes with a high potency in a non-competitive manner with BDNF [40].And BDNF specifically binds to the TrkB [41].However, the underlying mechanisms by which NAS can treat hypoxia-induced brain damage are largely unknown.In our study, we found that NAS attenuated hypoxia-induced ferroptosis.Further experiments showed that TrkB activity in the hippocampus was efficiently inhibited and that the protective effect of NAS was blocked by the administration of ANA-12 in rats.A possible neuroprotective mechanism of NAS may be related to inhibiting ferroptosis.In this study, we demonstrated that early brain injury could be decreased In conclusion, ferroptosis is involved in hippocampal impairment associated with hypoxia-induced brain damage.NAS attenuates ferroptosis in the brain following hypoxiainduced brain damage.To the best of our knowledge, this study is the first to report that NAS improves hypoxiainduced brain damage and inhibits ferroptosis in neonatal rats, which could be a potential therapeutic strategy.

Fig. 1
Fig. 1 Experimental design and hypoxia-induced hippocampal injury.A Schematic diagram of the experimental procedure.B Nissl-stained sections of the hippocampus.Values are displayed as the mean ± SD; n = 5; ***p < 0.01

Fig. 3
Fig. 3 NAS treatment decreased the expression of GPX4, but increased the level of TfR1 expression following hypoxia.A Morphologic alteration of hippocampus was measured by HE staining.B GPX4 and TfR1 protein expressions were determined by western blot.Values are displayed as the mean ± SD; n = 5; ***, p < 0.01.C The GPX4 and TfR1 mRNA levels were checked by qRT-PCR.Values are displayed as the mean ± SD; n = 5; *p < 0.05; **p < 0.02; ***p < 0.01.D Representative TEM images of the mitochondrial morphology of hippocampal neurons.The arrow indicates the atrophied mitochondria

Fig. 4
Fig. 4 Neuroprotective effects of NAS on the cell death of PHNs.Cell death was induced by 0-, 3-, 6-, and 12-h exposure to hypoxia with or without a series of concentrations of NAS.Cell death was