Treatment of preterm brain injury via gut‐microbiota–metabolite–brain axis

Abstract Background Brain injury in preterm infants potentially disrupts critical structural and functional connective networks in the brain. It is a major cause of neurological sequelae and developmental deficits in preterm infants. Interesting findings suggest that the gut microbiota (GM) and their metabolites contribute to the programming of the central nervous system (CNS) during developmental stages and may exert structural and functional effects throughout the lifespan. Aim To summarize the existing knowledge of the potential mechanisms related to immune, endocrine, neural, and blood–brain barrier (BBB) mediated by GM and its metabolites in neural development and function. Methods We review the recent literature and included 150 articles to summarize the mechanisms through which GM and their metabolites work on the nervous system. Potential health benefits and challenges of relevant treatments are also discussed. Results This review discusses the direct and indirect ways through which the GM may act on the nervous system. Treatment of preterm brain injury with GM or related derivatives, including probiotics, prebiotics, synbiotics, dietary interventions, and fecal transplants are also included. Conclusion This review summarizes mechanisms underlying microbiota‐gut‐brain axis and novel therapeutic opportunities for neurological sequelae in preterm infants. Optimizing the initial colonization and microbiota development in preterm infants may represent a novel therapy to promote brain development and reduce long‐term sequelae.


| OVERVIE W OF PRE TERM B R AIN INJ U RY
Brain injury is a common complication of preterm infants, including Intraventricular hemorrhage, periventricular leukomalacia, and diffuse white matter injury (WMI). 10In very low birth weight preterm infants, the germinal stroma blood vessels are immature and the tissue blood vessels are poorly supported, making the lateral ventricle particularly susceptible to intraventricular hemorrhage, white matter coagulation, and necrosis. 11,12As the most common form of preterm brain injury, periventricular WMI or periventricular leukomalacia often occurs in infants between 24 and 32 weeks of gestational age, which may be accompanied by reduced cortical volume, thalamus, and basal ganglia volume. 13Necrotic lesions can develop into multiple cystic lesions within a few weeks, which is the most severe injury leading to cerebral palsy. 14Diffuse WMI is associated with impaired cognitive, sensory, and psychological functions, and is increasingly recognized as a risk factor for autism spectrum disorders, attention deficit hyperactivity disorder, social difficulties, anxiety, behavioral disorders, and other psychological disorders. 15,16enatal or postnatal factors (such as intrauterine infection and/ or inflammation, fetal immune response syndrome, and hypoxiaischemia) may affect the pathophysiology of preterm brain injury and is related to adverse neurological outcomes. 17These factors may trigger oxidative stress reaction to produce a large number of oxygen free radicals (FRs).Because the brain of preterm infants cannot activate antioxidant defense, it is particularly vulnerable to OS damage and sensitive to FRs, which causes neuronal degeneration, edema, death, and can lead to cerebral palsy, cognitive, behavioral, and attention deficits. 18,19FRs have also been shown to disrupt the maturation and differentiation of pre-oligodendrocytes into mature oligodendrocytes, thus leading to WMI, which is the dominant mode of brain injury in preschoolers. 20terestingly, it has been demonstrated that the microbiome and its metabolites can regulate oxidative stress-induced damage, affecting the differentiation and myelination of precursor oligodendrocytes. 21Brittany Needham and his colleagues observed that 4-ethylphenol sulfate, an intestinal-derived metabolite, can enter the brain of mice with neurodevelopmental disorders, and promote anxiety-like behavior by affecting oligodendrocyte function and neuronal myelin sheath pattern in the brain. 22Ciara E Keogh and colleagues studied ABX-induced intestinal disorders in mice and observed that antibiotic treatment can lead to up-regulation of myelin-related genes in the prefrontal cortex, mature oligodendrocyte precursor cells, increase in myelin production, and impairment to hippocampal neurogenesis.Supplementation of tributyl glyceride, a metabolite of intestinal bacteria, can effectively alleviate behavioral defects, intestinal pathology, and myelin disorder in mice. 23Another mouse study found that trimethylamine N-Oxide, a metabolite of GM, can cross the BBB through NLR family pyrin domain-containing 3 (NLRP3) inflammasome signaling and mitochondrial dysfunction, promoting oligodendrocyte pyroptosis and demyelination. 24In the future, the specific role of GM in myelination may be further investigated by using myelin-specific knockout mouse models.

| MA JOR PRO CE SS E S IN NEURO DE V ELO PMENT COIN CIDE WITH CHANG E S IN THE NEONATAL G UT MICROB I OME
Neurodevelopment can be roughly divided into neural induction, neural pattern formation, neurogenesis, and axon growth. 25Recent studies have found that basic neurodevelopmental processes are synchronized with the maturation of GM in newborns.Oliphant et al. investigated the relationship between head circumference growth and the establishment of GM from the first week of life by collecting fecal samples from 58 preterm infants, finding that poor head circumference growth was associated with low abundance of Bacteroidetes and Uncaria. 26Another prospective observational study explored the relationship between the dynamic sequence of Bifidobacterium in extremely low birth weight infants during the first month of life and their neurodevelopment at 24 months of corrective age.Bifidobacteria in preterm infants with normal and damaged neurodevelopment followed different time trajectories and had different component rearrangements. 27This possibly indicates that the developmental trajectory of the nervous system might somehow be shaped by the maturation of GM.9][30][31] In the future, it is possible to interfere with neurodevelopmental trajectories by early regulation of GM and their metabolites.

| P OTENTIAL MECHANIS MS BY WHICH G UT MI CROB I OTA AFFEC TS NEURO DE V ELO PMENT
Early in life, the GM maturation coincides with crucial neurodevelopmental period, during which time the neural circuits are highly plastic and vulnerable. 32Experiments in germ-free mice have confirmed the importance of the GM in many fundamental processes in the brain, including BBB permeability, brain volume, neural circuits, myelination, and microglial alterations [33][34][35] (Figure 1).These processes are critical in shaping animal behavior and neurodevelopment and may involve the role of GM and their metabolites. 36

| Immune system
Innate immune cells can recognize pathogen-associated molecular patterns through pattern recognition receptors and rapidly induce non-specific inflammatory responses. 37GM have an important influence on brain development by regulating the production of components of innate immune response, macrophages, microglia, and cytokines. 38Toll-like receptor 4 (TLR4) has been shown to mediate microbiome-brain communication. 39Huiling Wei and colleagues observed in mice that the dietary microbial metabolite butyrate inhibits microglia-mediated neuroinflammation and regulates the microbiota-gut-brain axis (MGBA) by binding to GPR109A, upregulating PPARγ expression, and downregulating TLR4 and Nuclear factor Κb (NF-κB) activation, improving memory and cognitive function. 40In addition, ProBiotic-4 inhibits bacterial-related TLR4 and NF-κB signaling pathway shows improvement in memory impairment, synaptic and brain neuron damage, and microglial activation. 41croglia are innate immune cells of the brain that fine-tune neurogenesis and synaptogenesis by secreting pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), IFNα, and interleukin-1β (IL-1β).Meanwhile, microglia are important neuroimmune components of the MGBA. 42,43Studies using germ-free mice have shown changes in cell size and proportion in microglia, as well as their abnormal development and function. 44Dysbiosis can significantly affect the cell-specific transcriptome, especially in microglia and their subtypes. 45Moreover, changes in GM caused by diet, F I G U R E 1 Neurophysiological and behavioral changes were induced by intestinal microbial defects in germ-free mice.Germ-free mice have intestinal microbial defects and are prone to abnormal intestinal microbial colonization after birth.Intestinal microbiota may regulate brain function and development, such as myelination, microglia maturation, HPA axis development, HPA axis stress response, and the development and maintenance of BBB integrity through immune signal transduction, endocrine, and neural pathways.Therefore, germ-free mice were prone to stress, reduced social interaction, and cognitive deficits.HPA hypothalamic-pituitary-adrenal; BBB blood-brain barrier (Figure created with Biore nder.com).
exercise, and antibiotics can act on microglia and then affect the recombination of neural networks, 46 and alter the neurophysiology of adolescence, including the expression of myelin-related genes in the prefrontal cortex and the morphology of microglia in the basolateral amygdala. 47In another CLP-induced septicemia study in rats, the diversity and abundance of Bacteroides distasonis, Lactobacillus salivarius, and Clostridium cluster decreased, the activation of microglia increased, and the inflammatory factors in hippocampus and cortex increased. 48Neuroinflammatory responses and microglia synaptic damage may induce neuropsychiatric disorders related to rodent behavioral phenotype. 49t microbiota is widely and comprehensively involved in the shaping and function of the adaptive immune system. 50GM not only induces the differentiation and maturation of antigen-specific T-and B cells by producing specific antigens and metabolites, but also interacts with lymphocytes in the intestinal mucosal immune system to play a part in CNS diseases.By analyzing the microbiome characteristics of stress-sensitive mice, it was found that Lactobacillus (involved in T-cell differentiation to protect host immune system) decreased, and the differentiation of IL-17-producing colon γ δ T cells (γ δ 17 T cells) increased.2][53] In addition, peripheral-induced autoreactive myelin-specific T cells can be activated by Lactobacillus reuteri through molecular simulation after migrating to the gut, and then, highly pathogenic Th17 cells may enter the CNS to induce demyelination response. 54

| Neuroendocrine system
The GM can produce various metabolites, including short-chain fatty acids (SCFAs), bile acid metabolites, neurotransmitters (such as GABA, 5-hydroxytryptamine (5-HT)), tryptophan, catecholamines, and its derivatives, which can affect the neural and endocrine pathways in neurodevelopment.Bile acid receptors, including G protein-coupled bile acid receptor 5 and farnesol X receptor, are expressed in the brain, mainly in the cerebral cortex, hippocampus, and hypothalamus.Bile acids play a role through the activation of nuclear hormone farnesol X receptor) and G protein-coupled bile acid receptor 5. 55 GM is an important regulator of bile acid metabolism.The diversity of bile acid is decreased in germ-free and antibiotic-treated rats, and bile acid metabolites can activate receptors and act as signal molecules in the host, which can affect HPA axis and play a role in the brain. 56Intestinal Lactobacillus improved synaptogenesis and maintained glial homeostasis by balancing the metabolism of tryptophan-derived neurotransmitters, increasing the levels of 5-HT in brain and colon, and increasing the levels of BDNF and gamma-aminobutyric acid receptors in the hippocampus and amygdala. 57,58In addition, SCFAs can stimulate the synthesis and secretion of 5-HT in intestine, which plays a role in regulating motility, neurodevelopment and differentiation, and emotion. 59reover, colonization of Lactobacillus or supplementation of SCFAs can increase the level of GABA in the hippocampus of mice, regulate the level of specific neurotransmitters in hippocampus of brain, and play a role in enhancing memory. 60e ecological perturbations to the Bifidobacteria, Proteobacteria, Lactobacillus, and Staphylococcus in early life may alter the signaling associated with developmental programs during the critical developmental trajectory of the stress axis. 61In infants and preterm infants, who are vulnerable to gut microenvironment interventions, dysbiosis may interfere with hypothalamic-pituitary-adrenal (HPA) axisrelated signaling, inducing cognitive or behavioral changes. 62The experiments in germ-free mice showed elevated corticosteroid levels and stress-anxious behaviors under mild stress.These behaviors were normalized when the mice were colonized with Bifidobacterium longum subsp. 63 response to stress, the HPA axis regulates cortisol and catecholamine secretion, directly affecting the intestinal permeability and barrier function, which interferes with the intestinal microbiota composition. 64The experiments in ERβ-deficient mice have shown that dextran sodium sulfate-induced colitis causes over-activation of the HPA axis, which might be associated with anxiety-like behaviors. 65In addition, chronic unpredictable mild stress can reduce the catecholamine synthesis ability of the adrenal gland, disrupt the tryptophan and serotonin metabolic pathways in the hippocampus, prefrontal cortex, and colon, and alter the composition of the gut microbiota, inducing anxiety-like behavior. 66Some specific probiotics may inhibit excessive stress responses in social interactions and improve emotional and anxiety symptoms in response to chronic stress by restraining corticosterone and catecholamine production. 67Probiotics not only improves depressive behavior by increasing glucocorticoid receptor expression and decreasing adrenocorticotropin-releasing factor expression, adrenocorticotropic hormone, and corticosterone levels in the blood, 68 but also attenuates the homeostasis disruption caused by stress, regulates cortical brain-derived neurotrophic factor (BDNF) expression, and inhibits over-response to stress in the hippocampus and HPA axis. 69

| Vagus nervous system
The vagus nerve is considered the most direct pathway for microbiota signals to reach the brain.It receives signals from gut microbial metabolites and neurotoxic agents as well as directly synapses with enteroendocrine cells to transmit sensory signals to the nucleus accumbens, projecting many behavior-related areas of the brain. 70The neuronal recordings and imaging in mice revealed that compounds from intestinal bacteria emit signals through the vagus nerve that transmit intestinal osmotic pressure fluctuations to the brain to modulate thirst and drinking behavior. 71Besides, the impairment on the gut and neurodevelopment may stem from abnormal or pathological vagal connections. 72Therefore, normalizing the function of the vagus nerve may restore neuronal cell homeostasis and optimize brain development, providing a promising strategy for preventing prematurity-associated neurological disorders.

| The blood-brain barrier
Two main barriers exist between the microbiota and brain: the gastrointestinal barrier and BBB.The permeability of these barriers is closely related to the transport of microbiota-derived metabolites and neurotransmitters in the MGBA. 73Animal experiments revealed that increases ratio of the Firmicutes to Bacteroidetes and associated metabolite changes activated NLRP3 inflammatory vesicles in the colon and brain, disrupting the functional integrity of the BBB, stimulating neuroinflammation, and leading to brain cell apoptosis. 74,75The expression of occludin and claudin-5 decreased in the hippocampus and prefrontal cortex after long-term high-fat diet intake, leading to impaired brain function and neurobehavioral changes involving mood, social skills, learning, and memory. 76Correspondingly, maternal GM is involved in regulating the BBB of the fetus in utero by upregulating the expression of tight proteins. 77e deficits in BBB development in early life impair subsequent spatial learning.Maternal supplementation with the Lactobacillus reuteri and their metabolite short-chain fatty acids (SCFAs) may rescue the cognitive impairment associated with fetal BBB dysplasia and dysfunction. 78Studies in germ-free mice have shown that gutderived SCFAs may be a potential epigenetic regulator of learning, memory formation, and storage. 79,80Researchers found that BBB permeability in germ-free mice increased in utero and remained unchanged after birth till adulthood.Supplementation with SCFAproducing Clostridium tyrobutyricum restored the homeostasis in the gut and brain. 81It is because SCFAs may maintain intestinal barrier and BBB integrity by regulating tight junction proteins. 82SCFAs also work with microglia and mitochondria to regulate BBB function and neuroinflammation. 83Although microbial-derived SCFAs are beneficial to the host by promoting intestinal health and BBB integrity, excessive SCFAs have been demonstrated to be harmful or even toxic. 84 seems a promising treatment for preterm infants with brain injury by targeting at factors working in the MGBA.However, as far as we know, the potential mechanistic pathways by which GM affects neurodevelopment are not fully verified in preterm brain injury, and more clinical studies are needed to elucidate its mechanism.

| TRE ATMENT OF B R AIN INJ URY IN PRE TERM INFANTS MED IATED THROUG H THE G UT-B R AIN A XIS
The early postnatal period is critical for delicate neurodevelopment and an important opportunity for therapeutic interventions for gut microbial-associated psychiatric disorders.Previous understanding about the role of GM allows for new nutritional treatment strategies for preterm infants with brain injury.Various gut-related interventions have already been applied to alleviate brain injury in preterm infants, including microecological agents (probiotics, prebiotics, and synbiotics), dietary interventions, FMT, and natural drugs (Figure 2).Moreover, human studies have found that adding probiotics, prebiotics, and synbiotics to dietary nutrition supports the balance of GM and improves cognition, mood, behavior, and psychology in children. 85

| Microecological agents
With the progressive research on MGBA, the application of microecological agents in neurological disorders has gained notable interest in clinical practice (Table 1).The following sections focus on the progress and challenges of using microecological agents in infants and premature infants with neurocognitive disorders (Figure 3).

| Probiotics
Probiotic therapy administers to the subject active microorganisms that benefit the host's well-being, and its effects are highly strain-specific. 86Prophylactic probiotics usage is recommended to support the development of healthy GM, suppress the colonization of potentially pathogenic bacteria, and inhibit opportunistic pathogen overgrowth without altering the overall longitudinal bacterial profile in the neonatal period. 87,88Recent studies suggest that the early microbial colonization affects brain development in preterm infants and microbiome optimization improves neurodevelopmental outcomes. 89,90This anticipates probiotic interventions for improving neurodevelopment, although their safety remains controversial.
Probiotics is beneficial to neurodevelopment, emotion, and cognition in mice.Maternal dysbiosis in mice that are exposed to commonly used broad-spectrum oral antibiotics (ampicillin) can affect the neurobehavioral outcomes of their offsprings, resulting in reduced social motivation and social interaction. 91And in maternal mice, probiotics may stimulate the expression of intestinal butyric acid, brain lactate, and BDNF in the offspring, benefiting their neurological development. 92Although the evidence suggests that probiotics significantly affect gut-brain axis-mediated signaling pathways, no definitive key molecules of action have been identified up to now.
A study on structural alterations in the GM induced by antibiotic exposure in preterm infants found that early microbiome alterations would likely affect the neurological outcomes associated with premature birth. 93Administering complex probiotics during hospitalization reduced the diversity of antibiotic resistance genes in the GM of preterm infants and effectively prevented chronic retention of these resistance genes. 94However, due to the weak intestinal immunity defense of preterm infants, some probiotic strains may convert into opportunistic pathogens, increasing the risk of antibiotic resistance and leading to infections. 95ctobacillus and Bifidobacterium can accelerate the maturation of gut microbial composition and immune system in extremely preterm infants.Early colonization by Bifidobacterium and L. plantarum rebalances GM, increases glutamine, γ-aminobutyric acid, restores anxiety-related BDNF and 5-HT levels, 58,96 and significantly inhibits microglial activation and neuronal apoptosis, contributing to anxiolytic homeostasis. 97Mice experiments showed that supplementation with probiotics such as B. shortum strains and L. plantarum restored cognitive deficits, anxiety, and other abnormal social behaviors early in life. 98In a randomized doubleanonymized controlled trial including 40 healthy human participants, intervention with B. longum altered resting neural activity, which is characterized by enhanced vigor, reduced mental fatigue following social stress, providing a neural mechanism for alleviating stress responses. 99e premature infants are at the risk of dysbiosis and neurodevelopmental defects, for which probiotic supplementation represents a promising therapeutic strategy. 100A randomized intervention trial including 57 extremely preterm infants found that probiotics restored their intestinal microecology, promoted gut microbiome maturation to the level of full-term infants, and suppressed inflammatory responses. 101

| Prebiotics
Prebiotics are substrates that can stimulate certain bacterial taxa or bacterial activities in the GM and can be selectively utilized and converted into beneficial substances for the host's health.They can also modulate inflammatory responses by altering the GM composition and activity, affecting mood and some psychiatric disorders.
Therefore, prebiotics use is considered a practical strategy for treating neurological disorders. 103terventions using prebiotics have potential advantages over using single or multiple strains of probiotics.The ingredients of prebiotics are often very stable, and they can withstand high temperature and acidic environment, which are common in the production of nutritious food and functional food. 104,105Prebiotic ingredients typically have a longer shelf life than probiotic ingredients, and this stability also provides convenience for storage and processing. 106erefore, prebiotics are gaining popularity in the field of digestive health or parenteral health.
In mice, early prebiotic intervention reduced brain lesions by upregulating the neuroprotective phenotype of microglia via the inhibition of relevant pro-inflammatory and neurotoxic signaling pathways. 107A RCT of 161 infants revealed that the prebiotic intervention group cried for significantly shorter durations while awake and the 24-h sleep pattern monitoring showed longer latency to first and second naps than in control group, respectively.Moreover, GM composition was significantly different between the two groups.
These results suggest that prebiotics may influence neurodevelopment through the gut-brain axis, affecting sleep performance. 108 far, prebiotic supplementation mainly refers to the addition of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides, which are metabolized by Bifidobacteria.
Animal experiments revealed that prebiotics (galactooligosaccharide) can influence Lactobacillus and Bifidobacterium composition and induce metabolic, neurochemical, and behavioral changes in adult rodents.Continuous prebiotic supplementation may benefit the ecology and genetic stability of the GM. 109Galactooligosaccharide supplementation in maternal mice increased exploratory behaviors and decreased hippocampal glutamate receptor gene expression in their weaning offsprings, suggesting beneficial effects on brain gene expression and behaviors in the offsprings. 110Another mouse model experiment showed that prebiotic (oligofructose) intake increased Bifidobacteria levels and decreased corticosterone release, thereby increasing the IL-10 level, relieving brain inflammation, and maintaining BBB integrity in mice hypothalamus. 111ebiotics also include fibers such as inulin and resistant starch, which are not absorbed in the small intestine but are selectively fermented by beneficial microorganisms.In the study of chronic unpredictable mild stress mice, inulin intake can protect the integrity of intestinal barrier, increase the formation of SCFAs, inhibit the decrease of BBB permeability, and regulate TLR4/MyD88/NF-κB signal transduction to alleviate neuroinflammatory response. 112erefore, prebiotic interventions might ameliorate neurological disorders in infants and preterm infants.However, with the limited data, further research is required to elucidate their exact mechanisms in neurodevelopment.

| Synbiotics
Synbiotics are food ingredients or dietary supplements that comprise probiotics and prebiotics.Synbiotics are more effective than probiotics or prebiotics alone in improving CNS-related symptoms. 113,114A study reported that the combination of prebiotic inflammation, restoring hippocampal synaptic plasticity, attenuating brain mitochondrial dysfunction, and alleviating hippocampal oxidative stress and apoptosis. 115Given the limited clinical data and relevant clinical studies available, more preclinical and clinical evidence is needed to clarify the effects of synbiotics.

| Dietary interventions
A symbiotic relationship between the GM and the host starts early in life, with initial perturbations affecting neurodevelopment.
Dietary interventions are involved in brain development and function through microbial-dependent and independent mechanisms (Table 2).The emerging role of dietary interventions as a noninvasive and practical adjunct treatment for neurocognitive disorders has been highlighted in early psychiatric disorders.They are usually administered as nutritional supplements, including human milk oligosaccharides (HMOs), dietary fiber, and dietary polyphenols 116 (Figure 4).

| Human milk oligosaccharides
HMOs are ideal supplements because of their good stability and ability to withstand gastric acid and high temperatures in pasteurization.HMOs have been reported to optimize Bifidobacteriaceae and Bacteroidaceae colonization, suppress pathogen invasion, promote immune development, and regulate microbiota-gut-brain communication. 117,118In a pig model fed with HMOs, HMO-diet-induced intestinal alterations were associated with changes in gene expression encoding the BBB, endocrine function, and SCFA receptors in brain tissue. 119Another pig study showed that gut microbes could leverage metabolites produced by HMOs to induce structural changes in the brain and enhance cognitive function. 120Additionally, human studies have demonstrated multiple roles for HMOs as modulators of the GM in children, influencers of colonization of Bifidobacteria in the infant gut, and facilitators of neonatal neurological development. 121,122

| Dietary fiber
Dietary fiber can be used as an additive in formula which can be fermented by GM into host-absorbable SCFAs in the colon. 123As key signaling molecules for gut-brain communication, SCFAs readily cross the BBB to stimulate BDNF expression, thereby regulating the development, and maturation of neurons and microglia and modulating major neurodevelopmental processes during gestation. 124Mice experiments found that high dietary fiber intake improved cognitive and social behavioral deficits in the offspring of obese maternal mice by modulating the composition of the offspring's Clostridiales and Bacteroidales and SCFAs. 125Moreover, sodium butyrate promoted the recovery of SCFA-producing bacteria in neonatal rats with hypoxic-ischemic brain damage, facilitating the recovery of cerebral cortex and hippocampus. 126However, not all fibers are beneficial; unfermented fibers may elicit the production of pro-inflammatory factors by immune cells, activating the NLRP3 and TLR2 pathways and exacerbating inflammation in Inflammatory bowel disease (IBD). 127Therefore, although modulating GM through dietary fiber supplementation is possible, the diverse findings of relevant studies are insufficient to back up individualized regimen.

| Dietary polyphenols
Dietary polyphenols, primarily from plant sources, can inhibit proinflammatory cell signaling pathways and regulate GM.They are also recognized as exogenous molecules capable of modulating neurogenesis.Active metabolites in the brain that come from polyphenol biotransformation are processed by GM.Polyphenol metabolites can either directly cross the BBB or indirectly modulate the cerebrovascular system, thereby improving cognitive function and relieving neuropsychiatric disorders. 128,129review of animal and clinical studies demonstrated that dietary polyphenols can act as drugs for the treatment of intestinal and neurodevelopment-related diseases. 130Resveratrol, a natural polyphenol, is involved in downregulation of Bacteroidetes and TA B L E 2 Effects of dietary intervention and FMT on mental status of preterm infants.

Dietary intervention
HMOs Promote the colonization of beneficial bacteria, and improve the maturation of the intestinal epithelial barrier Improve cognitive development [118-121]   Dietary fibers Regulate the GM diversity, facilitate recovery of SCFAsproducing bacteria, and promote BBB integrity Improve the behavioral deficits, synaptic damage, and cognitive performance [125,126]   Dietary polyphenols Enrich beneficial bacteria, upregulate the expression of SCFAs Restore the integrity of the BBB, and boost mental and brain function [131-133]   FMT Reshape GM, enhance intestinal barrier integrity, upregulate expression of fecal SCFAs Improve neurological functions and neuronal axonal regeneration [138]   Abbreviations: BBB, blood-brain barrier; FMT, fecal microbiota transplantation; GM, gut microbiota; SCFAs, short-chain fatty acids.
Lachnospiraceae and the regulation of Glucagon-like peptide-1, and 5-HT levels, thus restoring the integrity of the BBB, inhibits the activation of microglia, and suppresses CNS inflammation. 131,132lyphenols are also crucial for neurodevelopment in uterus and during lactation. 133They can induce adult hippocampal neurogenesis and enhance learning and memory by increasing synaptic plasticity and promoting long-term hippocampal enhancement, while hippocampal neurogenesis in the offsprings is affected by perinatal maternal nutrition. 134In conclusion, the supplementation of plant-active substance polyphenols provides a novel perspective for the treatment of early-life brain injury.

| Fecal microbiota transplantation
FMT is the delivery of various intestinal microorganisms, metabolites, and natural antimicrobial agents isolated from the feces of healthy individuals (donors) into the intestine of patients (recipients) by oral administration, enema, and colonoscopy.FMT helps re-establish the balance of GM, repair the intestinal mucosal barrier, control inflammatory responses, and regulate host immunity 135 (Figure 5).
Numerous studies have confirmed the effectiveness and safety of FMT for treating recurrent Clostridium difficile infections, 136 IBDs, 137 and other gastrointestinal diseases.FMT can modulate neurological disorders driven by dysbiosis, and it may enhance neural regeneration in mice with spinal cord injury by restraining blood-spinal cord barrier damage, upregulating neurotrophic factors, alleviating spinal cord ischemia, and promoting neuronal survival 138,139 (Table 2).
FMT is a treatment option for neonatal necrotizing enterocolitis (NEC) and related mental disorders in preterm infants based on its regulatory role on GM.NEC is characterized by dysbiosis (i.e., increased TLR4 activity) and is associated with a significant risk of neurodevelopmental disorders. 140In a rat model of NEC, FMT before or after induction of NEC significantly alleviated inflammation, oxidative stress, and intestinal mucosal damage, suggesting that FMT may be a potential therapy for NEC. 141Experimental FMT in neonatal preterm piglets reduced NEC risk, but the effect was donor-specific. 142This process may be related to the promotive effect of early FMT intervention on the development of the innate and adaptive immune systems and the maturation of the GM in piglets. 143Although FMT has been widely used in clinical practice, transplanting live bacteria increases infection risk, making it imperative to develop donor screening protocols to ensure the safety of this therapy. 144Although there is no direct evidence supporting FMT in NEC-related brain injury, future studies are expected to advance and optimize the application of FMT in preterm brain injury, aiming at reducing long-term neurological complications.

| Natural drugs
There are intricate interactions between drugs and GM, with the gutbrain axis being a possible route for conveying the benefits of drugs for brain health (Table 3).Plant-derived anthocyanins may prevent cogni-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no competing interests.
TA B L E 3 Effects of drugs on mental status of preterm infants.

Drugs Impact on microbiota Effects References
Oxymatrine Remodel GM homeostasis Decrease BBB permeability, and ameliorate experimental autoimmune encephalomyelitis [146]   Pterostilbene Increase the levels of SCFAs and maintain normal intestinal permeability Ameliorate cognitive dysfunction, and improve both memory and learning ability [147]   Dimethyl itaconate Reshape the GM profile and maintain gut immune homeostasis Optimize synapse and cognition-associated genes, and improve cognitive impairment [148]   Dectin-1 ligand pachyman Regulate gut immunity homeostasis and control Treg cell differentiation in the intestine Prevent stress-induced behavioral abnormalities [149]   Diosgenin Upregulate the relative abundance of Lactobacillus and the expression of SCFAs, and restore the activity of the HPA axis Alleviate inflammation-related brain injury behavior [150]   Abbreviations: BBB, blood-brain barrier; GM, gut microbiota; HPA, hypothalamic-pituitary-adrenal; SCFAs, short-chain fatty acids.

102 F
However, limited publications in this area make it difficult to determine whether probiotics can directly improve neurodevelopmental outcomes in preterm infants, necessitating further research.Moreover, screening and isolating novel probiotics through next-generation sequencing and bioinformatics platforms are needed for the targeted treatment of specific diseases.I G U R E 2 Early intervention of gut microbiota is a potential therapeutic strategy for preterm brain injury.Early microbial interventions, including probiotics, dietary supplements, FMT, and drug application, can alleviate preterm brain injury by regulating the composition of GM and intestinal environment, increasing beneficial bacterial metabolites such as SCFAs and intestinal barrier function, and regulating nerve, immune, endocrine, and other gut-brain axis pathways.HMOs human milk oligosaccharides; FMT fecal microbiota transplantation; SCFAs short-chain fatty acids; GLP-1 glucagon-like peptide-1; 5-HT 5-hydroxytryptamine; GM gut microbiota (Figure created with Biore nder.com).
Lactobacillus paracasei HII01 as a synbiotic attenuated dysbiosis and improved cognitive function by reducing intestinal F I G U R E 3 Potential mechanism of Microecological agents in preterm brain injury.Microecological agents such as probiotics, prebiotics, and synbiotics can inhibit pathogens, reduce the levels of pro-inflammatory factors and cortisol, promote the colonization of beneficial bacteria, and increase the levels of anti-inflammatory factors and SCFAs.Generally speaking, they play a role in anti-inflammatory, enhancing epithelial barrier function, and diminishing gastrointestinal dysfunction.They can also intervene in the CNS development process through the gut-microbiota-metabolite-brain axis pathways, including increasing BDNF levels and blood-brain barrier function, reducing neuroinflammation and exerting neuroprotective effects, thereby alleviating preterm brain injury.BDNF brain-derived neurotrophic factor; SCFAs short-chain fatty acids; TNFα tumor necrosis factor-alpha; IL-6 interleukin-6; IL-10 interleukin-10; CNS central nervous system (Figure created with Biore nder.com).

F I G U R E 4 | 11 of 16 LI
Potential mechanism of dietary intervention in preterm brain injury.The potential mechanism of dietary intervention in brain injury in premature infants.Dietary fiber, polyphenols, and HMOs can affect the composition and diversity of gut microbiota, and increase the levels of beneficial bacteria and their metabolites (SCFAs).These metabolites can affect the vagus nerve pathway, immune signaling pathway, HPA axis, and blood-brain barrier function, thereby alleviating neuroinflammation, enhancing synaptic plasticity and neurodevelopment.HMOs human milk oligosaccharides; HPA hypothalamic-pituitary-adrenal; SCFAs short-chain fatty acids; 5-HT 5-hydroxytryptamine (Figure created with Biore nder.com).et al.
tive deficit-related behaviors by correcting dysbiosis, restoring the intestinal ecological and functional microenvironment, and inhibiting inflammation mediated by immune cells.145Apart from alleviating cognitive impairment, plant root-derived oxymatrine may alleviate clinical F I G U R E 5 Potential mechanism of FMT in preterm brain injury.FMT involves the transfer of fecal bacteria from healthy individuals to individuals with pathological conditions, which can reduce intestinal inflammation and permeability, increase intestinal beneficial metabolites and barrier function to relieve neurological diseases.It has been found to be an effective method to reduce the pathophysiology of preterm brain injury.FMT fecal microbiota transplantation; SCFAs short-chain fatty acids (Figure created with Biore nder.com).symptoms and pathological manifestations of multiple sclerosis by correcting microbiota dysbiosis, suppressing immune cell-mediated inflammation, and remodeling the gut-brain axis.146Another natural plant antitoxin, pterostilbene, could reduce the release of inflammatory factors by decreasing lipopolysaccharide levels and modulating TLR4/NF-κB signaling pathways in the colon and brain, thereby inhibiting oxidative stress damage, astrocyte and microglia activation, and dopaminergic neuron loss.147Natural plant-derived active agents are promising in treating neurological disorders through the modulation of GM, providing theoretical basis for its future therapeutic value.Microbiota-gut-brain axis links multiple biofeedback systemsthrough which many drugs work on CNS disorders.For example, dimethyl itaconic acid can significantly reduce macrophage infiltration and expression of pro-inflammatory cytokines (TNFα, IL-1β, and IL-6) in high-fat-diet mice, thereby increasing propionate and butyrate levels to restore the ecological and functional microenvironment of the intestine and improve cognition.148However, currently no neuroprotective strategies have been validated to prevent inflammation-related brain damage in preterm infants.Natural drugs that support brain health may provide essential benefits.A decrease in certain Lactobacilli associated with increased differentiation and accumulation of IL-17-producing γδ17 T cells in the meninges was observed in a study analyzing the microbiome characteristics of stress-sensitive mice and patients with major depression.This study demonstrates that this alteration promoted stress-induced social avoidance behaviors in mice and oral administration of the dectin-1 ligand porin glucan modulated the stress response to chronic social stress.149Another study reported that plant-derived diosgenin might reduce inflammation-related brain injury by regulating the Firmicutes and Lactobacillus, enhancing neurotrophic functions, and suppressing inflammatory and neuroendocrine activities.1506 | CON CLUD ING REMARK S AND FUTURE PER S PEC TIVEIn conclusion, the GM plays a crucial role in the pathogenesis of central nervous system diseases and might be considered as a new organ, namely the second brain.The related changes in the composition of gut microbiota and their metabolites may affect neurodevelopment by activating the vagus nerve, stimulating intestinal endocrine cells and immune signals, as well as the function of the blood-brain barrier.However, due to the complexity of microbiota composition and their activities, the mechanisms are far from clearly explored.As different neurodevelopmental disorders may reflect different microbial patterns associated with specific neurological conditions, targeted microbial intervention approaches based on the hypothesis of appropriate recipient-donor matching of gut microbial profiles could be explored concerning the neurology of preterm infants.New analyzing and sequencing methods may be of some help.Future neurotherapeutic research will provide more important information about the GM and neurological diseases in preterm infants.The application of microbial interventions, such as FMT, probiotics, and prebiotics as a promising treatment strategy, also presents some concerns and challenges.Therefore, the safety and tolerance of microbial interventions should be carefully evaluated to avoid adverse effects in the future and to promote the success of targeted microbial therapy clinical trials in preterm brain injury.AUTH O R CO NTR I B UTI O N SLL collected the literature, conceptualized the title, prepared the initial draft, designed the figures, and revised the manuscript according to YYS's comments, modifications, and suggestions.TJL proofread the language problems.All authors read and approved the final manuscript.FU N D I N G I N FO R M ATI O NThis work was supported by the National Natural Science Foundation of China (82171709 and 81801500), the 345 Talent Project of Shengjing Hospital (M1392 and M1415), Key R&D Guidance Plan Project in Liaoning Province (2020JH1/10300001), and Natural Science Foundation of Liaoning Province (No. 2022-MS-207).