Intercellular communication in the central nervous system (CNS) is essential for brain growth, development, and homeostasis maintenance and, when dysfunctional, is involved in the occurrence and development of neurodegenerative diseases. Increasing evidence indicates that extracellular vesicles, especially exosomes, are critical mediators of intercellular signal transduction. Under physiological and pathological conditions, neural cells secret exosomes with the influence of many factors. These exosomes can carry specific proteins, lipids, nucleic acids, and other bioactive substances to the recipient cells to regulate their function. Depending on the CNS environment, as well as the origin and physiological or pathological status of parental cells, exosomes can mediate a variety of different effects, including synaptic plasticity, nutritional metabolic support, nerve regeneration, inflammatory response, anti-stress effect, cellular waste disposal, and the propagation of toxic components, playing an important role in health and neurodegenerative diseases. This review will discuss the possible roles of exosomes in CNS intercellular communication in both physiologic and neurodegenerative conditions. Normal functions of the central nervous system (CNS) rely critically on the exchange and integration of information among neural cells, including neurons, microglia, astrocytes, and oligodendrocytes. Communication of neural cells is widely involved in brain development and homeostasis, as well as the occurrence of nervous system diseases such as neurodegenerative diseases . In geneExtracellular vesicles are membrane vesicles secreted by cells into the extracellular matrix and have been observed in most types of cells . AccordiIncreasing evidence showed that neural cell-derived exosomes are vital signal carriers of the CNS. By carrying bioactive molecules and mediating signal transmission of adjacent or distant cells, exosomes may play a crucial role in intercellular communication and neurodegenerative diseases. Besides, neural exosomes can cross the blood-brain barrier (BBB), implicating that exosomes may be a potential window into brain physiological and pathological activities. Exosomes can be used as molecular markers as well as carriers of drugs and bioactive molecules, and have good potential in the diagnosis and therapy of neurodegenerative diseases . In thisBesides the general features, exosomes released by neural cells also exhibit some specific characteristics depending on their cell origins. A substantial body of researches have explored the inducible factors and secretion mechanism of exosomes in CNS .The neuron is the basic structural and functional unit of the CNS. The main function of neurons is to receive, integrate, and transport information to maintain the proper activity of the CNS. The release of exosomes has been demonstrated both in developing neurons and differentiated mature neurons. These neuron-derived exosomes (NDEs) contain some neuron-specific markers, including L1 cell adhesion molecule (L1CAM) and the GluR2/3 subunits of glutamate receptors . Exosome+ stimulation could promote the secretion of NDEs, suggesting that exosome secretion can be modulated by depolarization. The team then confirmed that the release of exosomes from well-differentiated neurons was influenced by calcium influx and glutamatergic synaptic excitability  via Gαs proteins to regulate the generation of cAMP then the cAMP activates GEF1/2 to transmit signals to phospholipase C (PLC) via Rap1. While 5-HT2a,b transmit signals to PLC via Gαq proteins. PLC can further mediate the increase of cytosolic calcium, promoting the fusion of MVBs and cell membranes to release exosomes  and amyloid peptide can promote the secretion of ADEs, thus mediating cell damage of the CNS . In the Oligodendrocytes are responsible for myelin formation, maintenance, and nutritional support for axons . OligodeThe release of ODEs is closely relevant to the activity of neurons . The gluNeural stem/progenitor cells are self-renewal multipotent cells and are capable of generating neurons, astrocytes, and oligodendrocytes during the development of the CNS . In adulUnder physiological conditions, exosomes function as an important information carrier of the CNS, widely involving in the physiological processes of neural cells. This section focuses on the neural cell-derived exosomes and aims to describe their emerging role in the physiological cross-talk between neuron- neuron, neuron-glia, glia-glia, and NSC-neuron/glial cell . At presvia exosomes. Through the fusion of exosomes and neuronal PM, exosomal PRR7 is taken up by neurons, and subsequently inhibits Wnt signaling, activates GSK3β signaling, and induces specific removal of excitatory synapses to maintain the synapse numbers under normal conditions  are present in the exosomes of the supernatant of cultured primary embryonic cortical neurons. Let7c and miR21 may be internalized to the target neurons through exosomes and interact with Toll-like receptor 7 (TLR7), restricting dendritic growth during development in a cell-autonomous manner . Moreovenditions . Furtherc growth . These dThe neuron-microglia communication is crucial for the growth and development of synapses and functional synaptic transmission. Under physiological conditions, NDEs are putatively involved in the regulation of synaptic pruning by modifying the phagocytic activity of microglia. During neuronal remodeling, exosomes released following neuronal depolarization were demonstrated to up-regulate the levels of complement component 3 (C3) in microglia, thus enhancing the phagocytosis of microglia and facilitating the pruning of inactive synapses . In contin vivo, and increases GLT1 expression by suppressing the GLT1-inhibiting miRNAs (miR-132 and miR-218)  in astrocytes, which is believed to modulate extracellular glutamate concentration and synaptic transmission . The teamiR-218) .As mentioned before, neurons can modulate the secretion of ODEs by releasing neurotransmitters. And ODEs were found to be selectively internalized by neurons and microglia but rarely by oligodendrocytes and astrocytes. The uptake of oligodendrocyte exosomes occurs mainly in the dendrites and axons of neurons and depends on the endocytosis mediated by clathrin and dynamin . Althougin vivo keeps scarce. Exosomes can also cause the alteration of signal transduction pathways and the gene differential expression in recipient neurons  could promote the differentiation of OPCs into oligodendrocytes, while this effect was reduced with EVs from old astrocytes (cultured in vitro ≥16 weeks). Further proteomic analysis showed that the proteins of EVs released by astrocytes also changed with the increase of culture time. This study supports the idea that astrocytes can provide support for oligodendrocyte differentiation through exosomes, and aging may reduce their ability to provide homeostasis support -myosin signaling axis on the recipient oligodendrocytes, thus avoiding cell surface expansion and inhibiting the oligodendroglial differentiation and myelin formation. Interestingly, this study also showed that the secretion of self-inhibiting exosomes was significantly down-regulated by incubating oligodendrocytes with conditioned neuronal medium, indicating that neurons possibly participate in the regulation of oligodendrocyte differentiation and myelin formation by controlling the release of ODEs, which may contribute to the maintenance of brain homeostasis .via exosomes, activating the STAT1 signaling pathway and mediating the activation of pro-inflammatory signaling in target cells , which has L-asparaginase catalytic activity and can increase the level of aspartate in the extracellular microenvironment, thereby regulating the energy metabolism of surrounding cells . Proinflet cells . Stevanain vitro study has shown that exosomes released from NSCs can promote neuronal axonal elongation and neural network reconstruction under both normal and hypoxia-reperfusion injury conditions  is characterized by the deposition of the toxic beta-amyloid (Aβ) protein in the extracellular matrix between neurons, and the formation of neurofibrillary tangle due to the abnormal phosphorylation of tau protein, which could further induce neuroinflammation, oxidative stress, neurodegeneration and even neuronal apoptosis . Many revia MDEs was proved to be more efficient than the naked form in vivo, and the microglial depletion and inhibition of MDEs synthesis can significantly reduce tau proliferation both in vivo and in vitro  among neural cells, and further inducing apoptosis and promote the development of AD. For example, it has been confirmed that NDEs are implicated in the transmission of Aβ oligomers (oAβ) between neurons, and blocking the formation and secretion of NDEs can suppress the spread of oAβ . Trans-sin vitro . Taken tNeuroinflammation is viewed as a key mechanism of AD, and recent studies highlighted the role of MDEs and ADEs in AD-related neuroinflammation. Glutaminase C (GAC) was found to be abnormally elevated in the brain tissues of early AD mice. With the stimulation of pro-inflammatory factors , GAC can be specifically up-regulated in microglia, thus inducing cell activation, exosome secretion, as well as the content modification of exosomes. The released MDEs contain up-regulated pro-inflammatory miRNAs and down-regulated anti-inflammatory miRNAs, thereby facilitating the transmission of neuroinflammation in AD . A1-typeBesides the propagation of toxic proteins and neuroinflammation, neural cell-derived exosomes can also contribute to the neurodegeneration of AD through other pathways. It has been demonstrated that amyloid peptide-treated astrocytes can release exosomes containing proapoptotic proteins. These ADEs accumulated in the medium and bound to other astrocytes after reaching the proapoptotic concentration, thus promoting apoptosis by activating caspase 3. This study revealed a new mechanism of exosome-mediated astrocytes apoptosis under pathological conditions .However, some other evidence indicated that the release of NDEs can also serve as a neuroprotective role to maintain neuronal homeostasis by promoting the clearance of toxic proteins. It was discovered that NDEs could promote the conformational transition of Aβ in the extracellular matrix and form non-toxic amyloid fibrils on the exosomal surface. Following the internalization of NDEs, the Aβ amyloid fibrils on the exosomal surface were internalized by microglia and further transported to lysosomes for degradation. Sphingomyelin synthase 2 (SMS2) has been shown to up-regulate the secretion of NDEs during this process . MoreoveMultiple studies have revealed that neural-derived exosomes detected from peripheral body fluids carry specific proteins and genetic cargos, enabling to realize the early identification of AD at the preclinical stage. Regarding the exosomal protein biomarker of AD, the initial studies mainly focused on Aβ and Tau. It has been demonstrated that the levels of Aβ1–42, p-Tau 181, and p-Tau 396 in blood NDEs significantly increased in AD than healthy individuals and can predict the progression of AD 10 years before the clinical stage . These pGene cargo carried by exosomes also serves as biomarkers for early recognition of AD, in which exosomal miRNAs have been extensively studied and well described in some reviews . At presThe hallmarks of Parkinson’s disease (PD) pathology are the degeneration of dopamine neurons in the substantia nigra pars compacta and the progressive accumulation of α-synuclein (α-syn) and the formation of oligomers in neurons . Neural It is increasing evidence that the aggregation and propagation of α-syn are at least partially dependent on exosomes. A recent study reported that although α-syn can be released directly by neurons, pathogenic forms of α-syn may be more commonly secreted through exosomes and other EVs. EVs carrying α-syn may be more prone to internalized by the recipient cells, highlighting the unique advantage of EVs in the transmission of toxic proteins . Neuronsvia exosomes , a neurotoxin that could induce cell death, is widely used in models of PD both in vivo and in vitro. Exosomes released from normal astrocytes were found to significantly reduce MPP+-induced apoptosis of hippocampal neurons. This may be because miR-200A-3p in the ADEs can down-regulate the mitogen-activated protein kinase kinase 4 (MKK4) expression in MPP+-induced neurons and further inhibit the activation of c-jun n-terminal kinase (JNK), which is essential in the apoptotic signaling pathway , mutations of copper-zinc superoxide dismutase (SOD1) are one of the important causes of inherited ALS. Exosomes from neural cells seem to serve as an important part of the pathological development of ALS. Several researches point to a role for exosome-carried proteins as biomarkers of ALS . SimilarCollectively, these studies demonstrated that exosomes from neurons, microglia, and astrocyte are involved in the occurrence and development of neurodegenerative diseases and this role is twofold, involving both nerve damage and neuroprotection. In the future, how to distinguish the beneficial or harmful exosomes under different physiological and pathological conditions is crucial for the development of new exosome therapies for neurodegenerative diseases. Oligodendrocytes are responsible for CNS myelination and provide nutritional support to axons and neurons. Dysfunction of oligodendrocytes and myelin may increase the vulnerability of neuronal degeneration . HoweverApplications of exosomes in the therapy of neurodegenerative diseases are embodied in three aspects: one is as a promising target for disease therapy, the second is the use of stem cell-derived exosomes, and the third is to design exosomes as drug delivery vehicles.Exosomes secreted by neural cells perform an increasingly vital role in spreading toxic proteins and mediating neuroinflammation, contributing to the occurrence and development of neurodegenerative diseases. Therefore, blocking the formation of neural exosomes or their internalization by target cells may be emerging targets for the therapy of neurodegenerative diseases. As an example, a recent study has shown that inhibiting P2RX7 can suppress ATP-induced secretion of tau-containing MDEs, thus significantly decreasing the accumulation of tau protein and improving the working memory of AD mice. This study suggests that P2RX7 may be a novel therapeutic target of AD by inhibiting exosome secretion . Howeverin vitro BBB model of AD, Most current research on exosome-mediated therapy has focused on the use of exosomes from stem cells. Stem cell-derived exosomes have similar active components and functional characteristics to stem cells and have lower immunogenicity and almost zero possibility of malignant transformation. Therefore, stem cell exosomes may serve as a safer and more effective therapeutic strategy for neurodegenerative diseases. A great number of studies highlighted the great potential of stem cell exosomes in the therapy of neurodegenerative diseases. Human mesenchymal stem cells (MSCs) secreted exosomes contain a range of therapeutic nucleic acids, proteins, and lipids that have been widely believed to reduce neuroinflammation and improve clinical symptoms of AD and PD . ExosomeEspecially, exosomes are potential drug delivery vehicles. Dependent on transferrin-transferrin receptor interactions, exosomes in the blood appears to have good brain targeting capabilities that allow drugs to cross the BBB and perform their pharmacological effects more efficiently . Wang etin vitro and animal experiments, with a lack of clinical trials. These issues need to be further overcome in future studies. In addition, further research is needed to deepen our understanding of neural exosomes in neurodegenerative diseases and clarify whether they have a potential advantage in designing new therapeutic strategies.Consequently, it is becoming increasingly evident that exosomes have great capacity in the therapy of neurodegenerative diseases. However, there are also many challenges. First, exosomes injected peripherally may be cleared by immune cells. How to safely and efficiently deliver designed exosomes to specific targets in CNS still needs further research. Secondly, exosomes carrying specific therapeutic active molecules may elicit immune responses in patients. Furthermore, most of the current research comes from in vitro) or by injecting or adding exosomes obtained from cell culture into animal models. Whether these studies can reflect the characteristics and function of exosomes in vivo needs to be further clarified. More animal and clinical researches are needed to verify the neural exosome-mediated physiological and pathological processes in vivo. Moreover, because of the overlap of EVs in size, composition, and biochemical markers, the isolation and purification of exosomes remain a challenge, so some of the results discussed in this article may reflect the function of exosomes mixed with other EVs. The development of techniques to accurately isolate these vesicle subtypes will help us better understand the unique properties of exosomes in neural cell communication. In addition, to fully understand the role of exosomes in the intercellular and diseases of the CNS, more basic work is needed to elucidate the biological characteristics of neural cell-derived exosomes, such as the mechanisms of biogenesis and content sorting, how to target recipient cells (whether the targeting is specific and the specific mechanisms), and how to participate in complex biological processes in recipient cells. Given the dual effect of exosomes in the CNS, recognition of conditions that induce beneficial or harmful exosome secretion, and research of related intracellular mechanisms are beneficial for disease intervention from the exosome level.In the last decade, exosomes have attracted extensive attention as important carriers of intercellular information exchange. This review provides preliminary evidence for the function of neural exosomes in CNS intercellular communication in both health and neurodegenerative diseases. However, the field of neural cell-derived exosomes in the CNS is still in its infancy and faces many problems and challenges. So far, most researches on the neural exosomes are performed in cell culture (In summary, as a novel information transfer carrier, neural cell-derived exosomes perform an increasingly vital role in the intercellular communication of the CNS, mediating the regulation of physiological activities, as well as the damaged or protective effect under neurodegenerative diseases. The study of neural exosomes in the CNS may contribute to the development of new diagnostic and therapeutic strategies for neurodegenerative diseases.YX, SL, and LH provided the theme and ideas of this manuscript. LH, XD, and XL consulted the relevant literature. LH and XD wrote the manuscript. LH, XD, SL, XL, and YX revised the manuscript. All authors approved the final manuscript and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Life on earth has evolved under the influence of regularly recurring changes in the environment, such as the 24 h light/dark cycle. Consequently, organisms have developed endogenous clocks, generating 24 h (circadian) rhythms that serve to anticipate these rhythmic changes. In addition to these circadian rhythms, which persist in constant conditions and can be entrained to environmental rhythms, light drives rhythmic behavior and brain function, especially in nocturnal laboratory rodents. In recent decades, research has made great advances in the elucidation of the molecular circadian clockwork and circadian light perception. This review summarizes the role of light and the circadian clock in rhythmic brain function, with a focus on the complex interaction between the different components of the mammalian circadian system. Furthermore, chronodisruption as a consequence of light at night, genetic manipulation, and neurodegenerative diseases is briefly discussed. Mus musculus and Rattus norwegicus, the most commonly used mammals in the laboratory, are nocturnal. However, the aspect of different temporal niches is often not sufficiently taken into consideration when translating basic research in rodents into human applications. The aim of this broad review article is to highlight the role of light on rhythms in physiology and behavior, especially in nocturnal rodents from a neuroanatomical point of view, and to emphasize the important distinction between light-driven/time-of-day-dependent and endogenously driven/circadian rhythms.Life on earth has evolved under the influence of rhythmic changes in the environment, such as the 24 h light/dark cycle. Living organisms have developed internal circadian clocks, which allow them to anticipate these rhythmic changes and adapt their behavior and physiology accordingly. This is most obvious for plants in which the anticipation of the time window for photosynthesis, the light phase, provides a selection advantage over plants that simply react to the onset of the light phase. Moreover, for the early cold-blooded terrestrial animals, anticipating the light phase and, thus, the time with higher ambient temperature and better availability of visual cues has been a selection advantage. In contrast, for the early (warm-blooded) mammals, which developed in the Mesozoic (about 250 million years ago), anticipating the twilight and the dark phase, thus the time window for avoiding the diurnal predatory dinosaurs was crucial. Presumably, the early mammals gradually extended their behavior from the nocturnal towards the twilight phases of the day, resulting in activation of both cone- and rod-based vision . ConsequIn mammals, the circadian clock is hierarchically organized in a circadian system. The central circadian rhythm generator is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Rhythmic output of the SCN governs subsidiary circadian oscillators in the brain and the periphery. The SCN and the subsidiary oscillators consist of more or less strongly coupled cellular oscillators, each comprising a molecular clockwork composed of transcriptional/translational feedback loops of clock genes (reviewed in ). The SCTwo main mechanisms help to specialize in a nocturnal or a diurnal niche . In the Per1 . N. NPer2 sthe CREB ,142. In the CREB , indicatthe CREB . This inthe CREB . In addithe CREB , regulatthe CREB , which ithe CREB ,146,147)the CREB , a mechathe CREB ). Microdthe CREB . These nthe CREB ,152,153.the CREB  and the the CREB ). FurtheA brief light pulse applied during the dark phase enhances the consolidation of contextual fear conditioning and CA1 LTP , indicatChronic disruption of circadian rhythms in humans, for example in shift and night work, has aversive effects on health in general (reviewed in ) and mayRhythmic brain function is controlled by light and the circadian system. The SCN and its rhythmic output govern endogenous/circadian rhythms, which are entrained to the environmental light/dark cycle by retinal input. Direct and indirect retinal input is provided to many parts of the brain, contributing to light-dependent responses in a wide range of neuronal networks. At the cellular level, synaptic plasticity is modulated by a molecular clockwork in neurons and glia, which are modulated by various rhythmic neuronal, glial, and endocrine signals.In recent decades, great advances have been made in understanding the molecular basis of circadian time-keeping mechanisms and circadian light perception in the mammalian brain. The greatest challenge for the future will be to decipher the complex interactions and connectivity between the various components of the circadian system at the level of complex neural networks. This is mandatory for understanding not only rhythmic basic brain function, such as sleep but also higher cognitive function such as learning and memory under physiological and pathological conditions. The cholinergic ascending reticular activating system (ARAS) is a key element in the control of wakefulness and sleep–wake transition. It activates the thalamus, which controls general cortical activity and transmission of sensory information to the cerebral cortex. By interacting with other brain stem reticular nuclei, the ARAS also modulates muscle tone as well as autonomic functions during wake and sleep. In addition, cortical activity is indirectly (via the ARAS) and directly (not shown) modulated by the noradrenergic locus coeruleus, the dopaminergic ventral tegmental area, and the serotoninergic raphe nuclei. Sleep- and wake-inducing hypothalamic nuclei control ARAS activity. During sleep and wake, the ARAS is inhibited and activated by a system of GABAergic neurons in the ventrolateral preoptic nucleus (VLPO) and of histaminergic neurons in the tuberomammillary nucleus (TMN), respectively. Orexinergic neurons in the lateral hypothalamus (LH) contribute to arousal projections into the TMN, the forebrain, the thalamus, and the brain stem. The circadian rhythm in sleep/wakefulness is controlled by the suprachiasmatic nucleus (SCN) which projects to the dorsomedial hypothalamus (DMH) via the subparaventricular zone (SPZ). DMH neurons project to the VLPO and the lateral hypothalamus using inhibitory and excitatory neurotransmitters orchestrating rhythmic changes in sleep–wake and wake–sleep transitions. Light information reaches the SCN, the SPZ, the LH, and the VLPO by direct innervation from the retina. Diencephalic, mesencephalic and telencephalic brain regions are assembled in red, grey, and green boxes, respectively. ACh, acetylcholine. Based on ,105,113.


HCC is one of the most common malignant tumors and has an extremely poor prognosis. Accumulating studies have shown that noncoding RNA (ncRNA) plays an important role in hepatocellular carcinoma (HCC) development. However, the details of the related mechanisms remain unclear. The heterogeneity of the tumor microenvironment (TME) calls for ample research with deep molecular characterization, with the hope of developing novel biomarkers to improve prognosis, diagnosis and treatment. ncRNAs, particularly microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), have been found to be correlated with HCC neogenesis and progression. In this review, we summarized the aberrant epigenetic and genetic alterations caused by dysregulated ncRNAs and the functional mechanism of classical ncRNAs in the regulation of gene expression. In addition, we focused on the role of ncRNAs in the TME in the regulation of tumor cell proliferation, invasion, migration, immune cell infiltration and functional activation. This may provide a foundation for the development of promising potential prognostic/predictive biomarkers and novel therapies for HCC patients. Protein-coding genes have been well studied, but protein-coding regions account for only 1.5% of the whole human genome . MultituWith advancement in research, ncRNAs were found to exert biological functions at the RNA level and were identified as vital regulators in several physiological and pathological processes, especially in cancer –9. The rIn this review, we summarized the aberrant epigenetic and genetic alterations that cause aberrant ncRNA expression. We also discussed the functional mechanism of classical ncRNAs in the regulation of gene expression and provide examples of the far-reaching influence that these molecules have in affecting HCC processes. Furthermore, we summarized the effect of ncRNAs on the TME and laid the foundation for their applications in targeted therapy. In particular, we emphasized the role of ncRNAs in regulating tumor-associated macrophages (TAMs) in human HCC. Finally, we discussed the potential of ncRNAs as prognostic and diagnostic biomarkers and therapeutic targets in the future.As documented in the previous section, various ncRNAs were aberrantly expressed in HCC. Aberrant expression of ncRNAs can arise through many different mechanisms. The section reviews mainly focused on aberrant epigenetic mechanisms  , 22, miRDNA methylation (including DNA hypomethylation and/or promoter gene CpG hypermethylation), an epigenetic modification, is a common factor that regulates the expression of ncRNAs in HCC , 45–47. In recent years, the intriguing features of lncRNAs and circRNAs and DNA methylation have also been explored. For example, lncRNA SRHC was found to be downregulated in HCC tissues and serum samples compared with normal liver tissues and matched serum samples . The dowRecently, RNA methylation of ncRNAs has gained much attention –54. For Although ncRNAs do not encode proteins, they regulate gene expression levels at various levels  in the form of RNA. The functional mechanism of ncRNAs is complex and is not yet fully understood. Previously published studies mainly focused on the following mechanism of gene expression regulation.via the binding and recruitment of epigenetic modifiers to specific genomic regions in HCC  mechanism is a crucial mechanism by which lncRNAs indirectly regulate gene expression , 68. LncIn the nucleus, lncRNAs can modulate transcriptional regulators that have activating or repressive activities . For exaGenerally, lncRNAs indirectly regulate the stability of mRNAs. For example, UFC1 promotes the stability of β-catenin mRNA by binding with the mRNA stabilizing protein HuR, increasing the levels of β-catenin and promoting HCC progression . lncRNA miRNAs can act as miRNA sponges, participate in the splicing of target genes, translate genes into proteins and interact with RNA binding proteins (RBPs) . The submiRNAs can exert both tumor-suppressive and oncogenic effects in HCC progression , 87. miRMost studies have focused on the role of ncRNAs in the regulation of HCC cell functions and target genes. With advances in bioinformatics and next-generation sequencing, an increasing number of studies have shown that ncRNAs participate in cell-to-cell communication and regulate tumor immune cell activation, proliferation and cytokine secretion , therebyT cells play an important role in the immune response against various pathogens and cancer cells . The T cSome studies have also explored the function of miRNAs in Th17 cells in the TME of HCC. During Th17 cell differentiation, treatment with the miR-132 mimic promoted the differentiation of Th17 cells, leading to a higher percentage of Th17+ cells and higher secretion of IL-17 and IL-22 . FurtherRegulatory T cells (Tregs) normally play an immunosuppressive and tolerogenic role in the immune system and have been found to be coopted by tumor cells to escape immune surveillance . Yang etTAMs play an essential role in the inflammatory microenvironment and mediate the initiation and progression of HCC according to their polarization state (M1/M2) . The miRThe expression of miR-615-5p is upregulated in NK cells of HCC patients compared with those of healthy controls. Silencing the expression of miR-615-5p impaired NK cytotoxicity and forced the expression of cytotoxic markers NKG2D, TNF-α and perforin . Bian etIncreasing evidence has demonstrated that lncRNAs are involved in HCC progression and immune escape. For instance, lncRNA fetal-lethal noncoding developmental regulatory (FENDRR) acts as a miR-423-5p sponge to inhibit the immune-suppressive capacity of Tregs, therefore impairing the immune escape of HCC . ConversMacrophages respond to environmental signals in two polarized ways: classical proinflammatory activation (M1) and alternative anti-inflammatory activation (M2). Many lncRNAs have been found to participate in the regulation of macrophage M1/M2 polarization, such as lnc TUC339 , MALAT1 An increasing number of studies have indicated the important role of lncRNAs in NK cell development and function , 126. FoEmerging evidence demonstrates that specific circRNAs play regulatory roles in innate and adaptive immune pathways , 121. FoHCC is one of the most common malignancies and ranks as the fourth leading cause of cancer-related death globally. The major reason is that most of the patients are diagnosed at an advanced stage. Therefore, exploration of diagnostic and predictive biomarkers for HCC patients in the early stage is needed. Specific ncRNAs released from cancer cells and tissues exist in exosomes, which are small membrane-derived vesicles. Interestingly, circulating exosomes can serve as vesicles loaded with ncRNAs –131. PreFurthermore, an increasing number of studies have confirmed that several ncRNAs might serve as promising prognostic biomarkers and therapeutic targets for the treatment of HCC. Numerous circRNAs, lncRNAs and miRNAs have been found to be dysregulated in tumor tissues, tumor cells, immune cells, plasma, and exosomes and to be markedly correlated with the prognosis of HCC patients, even with the activation of immune cell function involving the remodeling of TAMs. Therapeutically targeting tumor-promoting ncRNAs or restoring the function of ncRNAs with tumor-suppressive function might be promising methods. In fact, the regulatory networks of ncRNAs are extremely complicated because ncRNAs work with other biomolecules, such as coding RNAs, ncRNAs, DNAs and proteins . A betteVarious ncRNAs have been found to be aberrantly expressed in HCC and are regulated by different mechanisms. Here, we mainly focused on the dysregulation of epigenetic mechanisms and genetic alterations, such as CNVs, histone modification, DNA methylation, and RNA methylation. Although ncRNAs do not encode proteins, they regulate gene expression levels at various levels  in the form of RNA. Additionally, ncRNAs play important roles in the TME and are involved in the regulation of tumor cell proliferation, invasion, migration, immune cell infiltration and functional activation. Because ncRNAs serve as either tumor suppressors or oncogenes in the progression of HCC, a better understanding of the role and related regulatory mechanism is necessary for the development of promising prognostic/predictive biomarkers and potential targeted treatments.LL and JL designed and guided the study. CX and XG wrote and edited the manuscript. ZB and YS helped with reference collection. All authors read and approved the final manuscript.This work was funded by the National Key Research and Development Program of China , and the National Nature Science Foundation of China (U20A20343).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biological tissues are not uniquely composed of cells. A substantial part of their volume is extracellular space, which is primarily filled by an intricate network of macromolecules constituting the extracellular matrix (ECM). The ECM serves as the scaffolding for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. Understanding the intimate interaction between the cells and their structural microenvironment is central to our understanding of the factors driving the formation of normal versus remodelled tissue, including the processes involved in chronic fibrotic diseases. The visualization of the ECM is a key factor to track such changes successfully. This review is focused on presenting several optical imaging microscopy modalities used to characterize different ECM components. In this review, we describe and provide examples of applications of a vast gamut of microscopy techniques, such as widefield fluorescence, total internal reflection fluorescence, laser scanning confocal microscopy, multipoint/slit confocal microscopy, two-photon excited fluorescence (TPEF), second and third harmonic generation , coherent anti-Stokes Raman scattering (CARS), fluorescence lifetime imaging microscopy (FLIM), structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), ground-state depletion microscopy (GSD), and photoactivated localization microscopy (PALM/fPALM), as well as their main advantages, limitations. The extracellular matrix (ECM) is a three-dimensional structure of fibers, gels, and minerals (such as the hydroxyapatite found in bone ) that unHowever, despite the advances in several areas, little is known regarding the mechanisms by which specific structural and mechanical properties of the ECM influence its interaction with cells, especially within a tissue-like context or in tissue models, such as organoids and 3D-bioprinted models. This lack of knowledge prevents the understanding of cellular microenvironments regarding effective tissue repair, remodelling, and even the action of therapies. The visualization of key ECM components has been the subject of studies for several years. Every time a new imaging technology emerges, several applications focused on the characterization of cellular responses and the distribution, morphology, and changes within the ECM closely follow. In this review, we present an overview of the main components of the ECM and an extensive description of several optical microscopy techniques that have been used to track changes in the cellular microenvironment.The fibrous proteins of the ECM consist of collagens ,8,9,10, Elastin is a unique protein that forms the bulk of elastic fibers, which impart elasticity and resilience to tissues . Much liFibronectin (FN), much like elastin, is a unique, dimerized protein that forms fibrillar networks within the ECM . The FN Laminins are noncollagenous proteins found as a major component of basement membranes, a specialized form of ECM that anchors cells and binds tissues . GeneralWhile collagens, elastins, and other fibrous proteins give structure and mechanical properties to the ECM, the space between them is filled with proteoglycans. Proteoglycans are composed of glycosaminoglycan (GAG) chains attached to a protein core . They arAs with the fibrous proteins of the ECM, different types and proportions of proteoglycans can impact the form and function of the tissue in which it is found. For example, versican is a proteoglycan found predominantly in the brain and blood vessels and has implications in tissue formation, cancer, and inflammation , or aggrProteoglycans can be categorized into four main families: small leucine-rich proteoglycans (SLRPs), hylectans, pericellular and basement membrane proteoglycans, and cell-surface proteoglycans . A schemThe last major extracellular glycosaminoglycan found in sizable amounts within the ECM is hyaluronic acid (HA) or hyaluronan. HA is found in many different tissues and has many roles , rangingWhile different configurations and proportions of the macromolecules of the ECM can drastically change the form and function of a tissue, changes in the state of the ECM also have implications for the health of the tissue. As tissue ages, it stiffens, a process mediated by the incorrect crosslinking of collagen fibers . While tHowever, should the injury become chronic or feedback mechanisms fail, the ECM will continue to be changed. The continuous remodelling of the ECM eventually alters the properties of the tissue and can lead to scar formation . TherefoRecent advances in the imaging of ECM components using different microscopy techniques have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. In addition, the visualization of key ECM proteins is crucial to reveal how changes in the cell microenvironment promote cellular responses leading to differentiation. In the next section, we discuss a set of optical imaging techniques for visualization of biochemically specific tissue features, including modalities that allow the visualization of ECM components such as collagen and elastin without the need for staining or sample preparation ,38,39,40The need to integrate new tools to better investigate the underlying mechanisms of diseases and characterize the various stages of tissue remodelling has received researchers’ attention for several decades. Optical microscopy has been a valuable tool for studying key facets of ECM remodelling. Some modalities and their label-free characteristics, combined with high sensitivity and specificity for major extracellular molecules, make them an attractive alternative  to conventional histology in studying tissue structures and ECM composition ,41.For example, fluorescence as an optical effect has been studied since the late 1800s, when visible light was observed emanating from objects that did not appear to reflect or have any apparent source of light . While iWith specific probes for a plethora of compounds, chemicals, proteins, and processes found within living cells, fluorescence microscopy became a powerful tool for cell and molecular biologists  and remaClassic light microscopy is limited in what resolution it can resolve by diffraction. This limit is defined by a point spread function (PSF), which is associated with the optical properties of the microscope. The PSF is the 3D diffraction pattern of light emitted from a very small point in the sample and transmitted to the image plane through the microscope objective. The PSF scales in size directly with the wavelength of light used and inversely to the numerical aperture of the microscope . Thus, aWhile the resolution of classic light microscopy has been limited by diffraction to objects greater than a few hundred nanometers, obscuring the details of most cellular and acellular processes, several methods have been developed that circumnavigate this restriction in resolution, placed under the term ‘super-resolution microscopy’. Super-resolution techniques have allowed the imaging of fine details of the ECM, such as collagen fibrils, down to single-molecule imaging . They caCombining different immunohistochemical stainings with specific microscopy modalities, various ECM components, and cells associated with it can be successfully visualized, including their 3D structure. To illustrate the capability of different modalities in imaging different ECM components, in Linear optical methods rely on the absorption of single excitation light photons and the emission of single emission light photons; nonlinear methods, however, do not. The development boom in laser technology, which happened in the 1990s, allowed us to start exploring and applying the principles of nonlinear interactions between light and biological tissue ,54. In tOther imaging modalities, such as electron microscopy or scanning probe microscopy, provided higher spatial resolution. However, optical microscopy modalities offered unique advantages to investigate biological structures, including the ECM. Nonlinear optical processes are harnessed in the following modalities: two-photon excitation (TPEF), second-harmonic generation (SHG), triple-harmonic generation (THG), and coherent anti-Stokes Raman scattering (CARS).These modalities are often referred to as multiphoton microscopy in biomedical sciences, and some of their main advantages and limitations are summarized in In In the next sections, we discuss the various optical microscopy modalities and highlight their use in characterizing proteins of the extracellular matrix. Starting with linear optical methods, we present their use in imaging both intrinsic fluorophores of the ECM, such as elastin, and extrinsic fluorophores, such as Hoechst. Super-resolution techniques are also discussed as well as their use in imaging proteins found in association with the ECM, such as integrins. Nonlinear modalities are among the most powerful in imaging structural proteins found within the ECM, such as collagen and elastin. Finally, Raman-based modalities and their use in imaging some more exotic intrinsic fluorophores are also described. A summary of some representative works focused on imaging ECM components using different optical microscopy modalities is shown in Light interaction with biological samples  is complex. The different composition, layers, and optically inhomogeneous properties of biological samples allow light to be reflected at a material interface, refract when light enters a tissue structure that has a different refractive index, have the photon energy absorbed by the material, or have photons scattered in the material, as shown in Even simply considering the ECM, the different fibrous proteins and components have different optical properties. However, in biological tissues, the variation in the refractive index is often small, and the tissues are, in many cases, transparent. One of the most significant interactions is the absorption of light. It determines how far it can penetrate a specific tissue and depends strongly on wavelength. The different absorption coefficients dictate how much energy a specific tissue can absorb from a particular optical source. Once the light is absorbed, it can be converted into heat, be radiated (fluorescence), or be consumed in photochemical reactions.On the other hand, scattering is a phenomenon experienced by light as it travels through an inhomogeneous medium, such as biological tissues. While photons travel in a straight line from their source, small irregularities in the media through which they travel force them to deviate from this path. Light is scattered when induced dipoles within the media are inhomogeneous, deviating the photon from its path and producing different types of scattering, dependent on the wavelength of the incident light and the size of the scatterer .While many types of scattering exist, the three most often seen are Rayleigh, Mie, and Raman . RayleigWhile scattering changes the vibrational state of a molecule upon absorption of the incident light, fluorescence changes the electronic levels of a molecule . Upon abTaking advantage of most of these interactions, optical microscopy allows us to investigate changes at the molecular, cellular, and tissue levels. As shown in In the next subsections, we present a description of each of the most common linear optical microscopy modalities, as well as examples of applications focused on imaging ECM components.The most basic form of fluorescence microscopy, widefield fluorescence microscopy (WFFM), bathes the entire sample in light, filtering excitation and emission light and observing through a microscope . While tWFFM has been used extensively in viewing the ECM. From viewing collagen fibril assembly  to viewiTIRF Microscopy is a method very similar to that of WFFM but allows imaging of fluorophores very close to a surface while preventing imaging of fluorophores further from that surface . Using aDue to its narrow depth of excitation, TIRF microscopy is ideal for imaging cell-substrate interfaces, as mentioned previously. It has been used to map cell-substrate separation distances , showingVega et al. explored the effect of acetylation on the ECM kidney . Using TTIRF microscopy has also found use complementing other technologies, such as its use in guiding atomic force microscopy (AFM) in testing the biomechanical properties of the pericellular matrix of chondrocytes found in cartilages. Chery et al. used TIRF microscopy to explore the pericellular matrix (PCM), a narrow, specialized band of ECM found around chondrocytes of cartilage . Using TIn 2020, Umana-Diaz et al. demonstrated the use of time-lapse TIRF microscopy in exploring the role of Lysyl Oxidase-Like 2 (LOXL2), an enzyme responsible for the crosslinking of fibrillar ECM proteins, in the vascular basement membrane . Using tAt its most basic form, confocal microscopy is very similar to widefield microscopy. The major difference between this technique and WFFM is that excitation light is passed through a pinhole aperture before reaching the sample, and a pinhole aperture is situated before the detector. This has the advantage of removing any emitted light that is out of focus . As a reDue to its selectivity in only imaging the focal plane and relative ease of use, LSCM has found extensive use in imaging and characterization of the ECM. From the early use of LSCM in the exploration of the expression of fibronectins and laminins in the ECM, postimplantation of grafts in mice  to the cLSCM has continued to be a valuable tool in the characterization of ECMs, especially in vivo and in artificial matrix constructs. In 2021, Pínter et al. used LSCM in their study of proteoglycan distribution in the adult rat brain . Using LHayes et al. also used LSCM in localizing the proteoglycan perlecan in intervertebral disks . With LSWhile LSCM is the most common form of confocal microscopy used today , other vSDCM is faster than LSCM, making it suitable for imaging live cells  but can In imaging live cells, Doyle et al. demonstrated the use of SDCM in their study of the migration of fibroblast and mesenchymal cells . By imagSIM is a super-resolution microscopy technique that uses a structured pattern of excitation light to illuminate the sample, usually a series of parallel lines, but other patterns may also be used . As the SIM has found extensive use in imaging of cells and cellular processes due to its suitability in the imaging of live cells ,119, imaAs focal adhesions are relevant in the ability of a cell to respond to mechanical stress and generate mechanical stresses on the ECM, investigation of the structure of focal adhesions is critical in understanding how mechanical stresses and cells interact . DzyubenSTED Microscopy is another super-resolution microscopy technique used to break the diffraction limit. It relies on the depletion of fluorophores in a torus around the focal point, leaving fluorophores at the focal point free to fluoresce upon excitation . Using aMuch like other super-resolution methods, STED and GSD have been used extensively in the past and have found ongoing use in imaging live cells ,127 and STED microscopy has recently been used in the characterization of extracellular fluid flow in the extracellular space (ECS) of the brain. In 2018, Tønnesen et al. used 3D-STED to image diffusible fluorescent markers within the ECS, a method they termed ‘SUSHI’, producing clear images in a tissue that has defied conventional light microscopy in the past . Using tSTED has also found use in imaging integrins, cell-surface receptors that directly communicate with extracellular components such as fibronectin. Spiess et al. demonstrated the use of STED in direct imaging of integrins, comparing images produced by STED microscopy to those produced by another super-resolution technique, STORM . Their wPALM is a super-resolution technique able to target and image single molecules . The funPALM has found use predominantly in the imaging of the internal workings of the cellular environment. PALM has been used in imaging the proteins of organelles, such as mitochondrial proteins , cell-suMore recently, PALM has been used in further elucidation of the structure and function of integrins found in focal adhesions. Moore et al. used a variation on PALM known as iPALM, combining interferometry with PALM modalities . Using iAnother similar method to PALM is stochastic optical reconstruction microscopy (STORM). Whereas PALM relies on the photobleaching of fluorophores, STORM relies on fluorophores that are reversibly switchable between on and off states . FluoropAs with PALM, STORM has considerable use in imaging cellular processes and structures. STORM has been used in imaging mitochondrial dynamics, targeting proteins within the mitochondria . ImagingLike PALM, STORM has been recently used in imaging integrins. Fan et al. used STORM to image integrins on the surface of neutrophils . They deSTORM has also been used in the characterization of bone mineralization. Zhou et al. used STORM in their study of bone mineralization . Using SFLIM is an imaging technique that relies on the difference in the decay rate of fluorescence . FLIM isFLIM has found recent use in imaging collagen and elastin of the ECM. In 2018 Li et al. used FLIM to study the extracellular matrix architecture of bovine pericardium under the effects of collagenase activity . FLIM waVazquez et al. demonstrated the use of FLIM as a nondestructive assessment of the mechanical properties of hydrogel . HaudensFLIM has also been used in imaging nonproteinous components of the ECM. In 2020, Okkelman et al. used FLIM to image the distribution of extracellular calcium ions, an important signalling ion . The useFluctuation-based super-resolution microscopy (FSM) is a collection of techniques that rely on the small fluctuations in emitted light of fluorophores. Capturing these fluctuations over time, with tens to hundreds of images, and performing higher-order statistical analysis of those fluctuations produce images with improved resolution , and an Pixel reassignment super-resolution microscopy uses one or multiple focal points to scan across the sample, much like confocal microscopy . FluoresNLOM, also known as multiphoton microscopy, uses nonlinear interactions between light and matter to generate images in contrast with conventional microscopy, which typically uses linear interactions . NLOM is a group of techniques that rely on nonlinear optical effects to image proteins, cells, and other endogenous and exogenous fluorophores.In imaging and characterization of the ECM, NLOM has found significant use. NLOM has been used in the imaging of the ECM of embryoid bodies, exocrine tissue, cartilage, and heart valves . NLOM haUsing NLOM, Cavinto et al. (2021) were able to explore how the ECM changes over time, targeting the collagen and elastin fibres of the murine aorta, to understand how aging affects the aorta and how biaxial loading changes the microstructure of the ECM . They deOne of the most well known nonlinear optical microscopy modalities is two-photon excitation fluorescence (TPEF), a technique used in life science for several decades after Denk et al. first demonstrated the use of two-photon excitation in living cells . Since tThe nonlinear effect upon which TPEF relies is similar to one-photon excitation fluorescence (OPEF), found in conventional fluorescence microscopy. While one photon is absorbed and emitted in OPEF, more than one photon is absorbed in TPEF. While only one photon is emitted, multiple photons may be absorbed by the fluorophore. As multiple photons are absorbed, lower intensities of light may be used in the excitation of fluorescence . TPEF ofTPEF continues to be a powerful tool in the quantification of the ECM and has found several recent uses in mapping the ECM. In 2021, Benbouja and Hartnick used TPEF to quantify the morphology of the ECM in human vocal cords . TPEF waWhile TPEF relies on the absorption of two or more photons to change the energy state of a fluorophore to an excited state, multiharmonic generation (MHG) relies on the absorption of two or more photons to bring the fluorophore to a virtual state . The phoSecond harmonic generation (SHG) relies on the doubling of the frequency of excitation photons and finds particular use in the imaging of noncentrosymmetric structures, such as fibrillar collagen . SHG hasSHG has found considerable use in imaging of fibrillar collagen found in the ECM since Campagnola et al. demonstrated the use of SHG in the imaging of fibrillar collagen, acto-myosin, and tubulin  in bioloIn 2020, Atkuru et al. explored the effect of cellular aging on oral extracellular matrix organisation . Using SThe role of collagen fiber morphology on the migration of ovarian cancer cells was explored by Alkmin et al. Using SHG imaging, they analysed and characterized the morphology of collagen fibers in normal ovarian tissues, high-risk tissues, and benign and high-grade tumors . Once imFinally, a variant of SHG imaging, known as polarization-dependent SHG imaging has recently been used in the characterization of ECM. Polarization-dependent SHG uses polarized excitation light and can be used to detect the orientation of collagen fibrils . PendletCoherent anti-Stokes Raman scattering (CARS) as a physical phenomenon has been harnessed as an imaging modality since the first CARS microscope was constructed in 1982 . This moCARS microscopy is commonly used as a label-free, noninvasive technique for imaging live cells and internal cellular processes, as it is well suited to generating and receiving signals from the phospholipids that surround cells and cell organelles ,186,187.Recent uses of CARS microscopy have targeted a wider range of biomolecules than lipids, such as GAG chains, commonly found to be attached to proteins in proteoglycans. Lima et al. explored the use of GAG probes for use in CARS microscopy . UsuallyThe ECM plays an important role in regulating tissue and cellular functions in the body. ECM homeostasis is essential for wound healing, development, and normal organ functioning. Different chronic and inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and even some cancers present significant changes in the ECM structure. Therefore, having specific tools to image the ECM components as well as cells specific to tissues is essential. Optical microscopy provides insights into the spatial, temporal, and molecular changes happening in the ECM as well as regarding the cellular adhesion points, cytoskeleton morphology, and other features of such a complex microenvironment. Recent developments and applications of optical imaging have been used to address increasingly complex biological questions related to ECM remodelling, and a collection of representative studies was presented in this review. We discussed the main advantages and limitations of the mains optical microscopy modalities and provided an overview on how these techniques can support advances in biology by allowing us to visualize structural changes and mechanisms crucial to tissue remodelling. While imaging techniques and technologies improve, image analysis algorithms represent another area of continued interest. From deep-learning models to neural networks, improvements in ECM image analysis furthers our ability to explore and characterize the fundamental structures of tissues and organs ,193. The


Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment.  The transparency of the cornea allows light to enter the eyeball. In addition, the smooth surface and convex curve of the cornea support the focusing of images of the outside world on the retina. The loss of these properties of the cornea therefore impairs visual acuity and diminishes quality of life. As a consequence of its location at the ocular surface, the cornea is exposed to pathogens such as bacteria in the external environment. However, infection of the cornea occurs only rarely as a result of the operation of various protective mechanisms. Physically, the corneal surface is protected by the eyelids and is covered by tear fluid . Tear flPseudomonas aeruginosa and the Gram-positive bacterium Staphylococcus aureus are the two most frequent causative agents of infectious keratitis [Infectious keratitis caused by bacteria, fungi, protozoa, or viruses is a leading cause of corneal blindness, despite the development of various antimicrobial drugs ,18. Bacteratitis .Despite the recent emergence of drug-resistant strains of bacteria, which is a serious clinical problem, the administration of antimicrobial agents is the mainstay of treatment for bacterial keratitis at its early stages. The development of new drugs with different mechanisms of action is an urgent need; however, in order to avoid the increasing problem of drug resistance. With regard to the advanced stage of bacterial keratitis, which manifests as corneal ulcer and subsequent corneal perforation, there is currently no treatment modality available to arrest the progression of corneal stromal melting. Although the excessive degradation of stromal collagen that gives rise to corneal ulcer has been found to be mediated by a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade, no treatment targeted to this cascade has yet been established.Excellent reviews on the epidemiology, bacteriology, and clinical treatment of infectious keratitis have been published ,31,32,33The onset and course of bacterial keratitis can be divided into various phases . Events The onset of bacterial keratitis (phase 1) begins with the penetration of bacteria into the corneal epithelium and superficial portion of the stroma and their subsequent replication in the host tissue. This penetration of the corneal epithelium can result in the release of alarmins by damaged epithelial cells. At this stage, the infection can be diagnosed on the basis of clinical signs and symptoms, smear culture, and detection of bacterial DNA by the polymerase chain reaction, and it can be halted by the administration of appropriate antimicrobial agents. Prompt diagnosis and treatment can thus lead to the resolution of most cases of bacterial keratitis.P. aeruginosa, together with LBP and sCD14, induces the production of uPA and MMPs in addition to expression of the chemokine IL-8 and the adhesion molecule ICAM-1 (intercellular adhesion molecule-1) by corneal fibroblasts [In infectious keratitis, keratocytes of the corneal stroma, rather than corneal epithelial cells, are responsible for innate immune responses. Keratocytes or corneal fibroblasts (activated keratocytes) recognize bacterial components through members of the Toll-like receptor (TLR) family of proteins ,42,43. Lroblasts ,45,46,47roblasts .Phase 2 of bacterial keratitis is characterized by the further penetration of actively replicating bacteria into the corneal stroma and the onset of corneal ulceration, which is defined as the destruction of the normal architecture of the corneal stroma associated with the melting of stromal collagen. Bacteria themselves secrete collagen-degrading enzymes that mediate direct collagen degradation by the bacterial cells. However, it is corneal fibroblasts, whose activation is triggered by factors released from the bacteria, that are largely responsible through the production of collagen-degrading MMPs for the destruction of extracellular collagen associated with corneal ulcer. Corneal fibroblasts thus serve as sentinel cells, local immune modulators, and mediators of collagen degradation in infectious keratitis ,35,36. MThe further infiltration and activation of inflammatory cells in the infected cornea mediated by chemokines, adhesion molecules, and other cytokines contribute to phase 3 of bacterial keratitis. These inflammatory cells—in particular, PMNs—release additional cytokines such as IL-1 that further stimulate corneal fibroblasts. The interaction of PMNs and corneal fibroblasts results in the operation of a vicious cycle , with thWith regard to therapeutic strategies for bacterial keratitis, elimination of the causative agent with antimicrobials is thus the first choice for cases at phase 1 or 2. Prompt presentation to an ophthalmologist, proper diagnosis, identification of the causative bacterial species, and administration of treatment are essential to avoid progression to corneal ulcer. No reliable drug treatment is available for cases that have progressed to phase 3, with a poor prognosis characterized by the loss of corneal transparency, and visual acuity being the likely outcome. Regardless of the causative bacteria, the first 24 to 48 h after the onset of bacterial keratitis correspond to phase 1 and early phase 2. However, further progression depends on the causative agent and host factors including predisposing conditions. The duration of phases 2 and 3 thus varies among individuals, with detailed clinical observation being important to identify disease stage.In the corneal stroma, keratocytes are embedded in a tightly packed extracellular matrix consisting mostly of type I collagen, with cellular functions being continuously modulated by interaction with this matrix. Keratocytes in the healthy cornea manifest a flattened and stellate morphology. They elaborate cytoplasmic processes that are connected to those of neighboring cells by gap junctions, thereby forming a three-dimensional network structure ,54. AlthIn pathological conditions, keratocytes transform into corneal fibroblasts, and activation of the uPA-plasmin-MMP cascade is central to the associated degradation of stromal collagen. The homeostatic balance of collagen maintenance is thus tilted in favor of collagen degradation with the onset of bacterial keratitis. The components of the uPA-plasmin-MMP cascade as well as upstream regulatory factors have been well investigated at the molecular level in vitro, including analyses of their expression at the mRNA and protein levels and of their activity. Furthermore, insight into the pathobiology of corneal ulceration in phases 2 and 3 of bacterial keratitis has been provided by measurement of collagen degradation in collagen gel cultures. Infiltrated PMNs also come into contact with resident corneal fibroblasts and stromal collagen during bacterial keratitis. The functions of PMNs are also affected by this extracellular matrix, with the release of IL-1 and IL-8 by these cells having been found to be stimulated and inhibited, respectively, by culture in a collagen gel ,57.P. aeruginosa and S. aureus also enhance fibroblast-mediated collagen degradation [The collagen-degrading activity of corneal fibroblasts cultured in a three-dimensional collagen matrix can be determined directly by measurement of collagen breakdown products . The advradation .Uncontrolled degradation of collagen in the corneal stroma underlies the development of corneal ulcer. The degradation of collagen is mediated by cleavage of collagen fibers by extracellular proteases, endocytosis of collagen fragments as a result of their interaction with collagen receptors, and further intracellular proteolysis within lysosomes ,61. Two Pseudomonas aeruginosa and S. aureus are the most common bacteria isolated from infectious corneal ulcer [P. aeruginosa and S. aureus are able to cause infectious corneal ulcer is essential to devising new treatment strategies  to stimulate the production of MMPs by corneal fibroblasts and that of factors (including elastase and thermolysin) to activate pro-MMPs.With the use of the three-dimensional gel culture system, it was shown that sterile culture broth of roblasts . These rS. aureus, cysteine proteases known as staphopains are secreted by the bacterial cells and are capable of degrading collagen [P. aeruginosa, sterile culture broth of S. aureus manifested direct collagenolytic activity in a three-dimensional collagen gel only in the presence of plasminogen. Another factor released by S. aureus is staphylokinase (SAK), which binds to trace amounts of plasmin to form an SAK–plasmin complex that manifests uPA-like activity. The direct collagen-degrading activity of S. aureus culture broth thus appears to be actually mediated by plasmin generated from plasminogen by the SAK–plasmin complex. The uPA-like activity of this complex also seems to be responsible in part for the promotion by the sterile culture broth of collagen degradation mediated by corneal fibroblasts [In the case of collagen . Howeverroblasts .Corneal fibroblasts play a central role as both sentinel cells and modulators of local immune reactions during bacterial keratitis . They alAt the onset of bacterial keratitis, infection damages corneal epithelial cells and triggers the infiltration of inflammatory cells including macrophages and PMNs. The damaged epithelial cells release various endogenous molecules known as alarmins that signal danger to surrounding tissue . AlarminBoth IL-1α and IL-1β bind to the same IL-1 receptor and show identical biological activities . The proBacterial infection triggers the recruitment of PMNs to the infection site by activation of an innate immune reaction. The infiltrated PMNs then secrete IL-1, which, as mentioned above, is also released by damaged epithelial cells as an alarmin. IL-1 plays a key role in regulation of the host response to bacterial infection including that associated with corneal ulcer. IL-1 contributes to corneal tissue destruction in part by stimulating the production by corneal fibroblasts of chemokines such as IL-8 that prolong the infiltration of PMNs in the cornea . The mutPseudomonas aeruginosa releases both bacterial collagenase, which directly degrades collagen [P. aeruginosa is recognized by TLR4 expressed at the surface of corneal fibroblasts [P. aeruginosa thus promotes collagen degradation by corneal fibroblasts through pathways both dependent on and independent of the uPA-plasmin system.The role of the interaction of corneal fibroblasts with pathogen-derived factors in collagen degradation has been investigated with the use of the collagen gel culture system . Pseudomcollagen , as wellcollagen ,59,66. Iroblasts ,88 and iroblasts . InfectiS. aureus also stimulates collagen degradation by corneal fibroblasts. Sterile culture broth of S. aureus thus up-regulates the production of pro-MMPs and uPA by corneal fibroblasts [S. aureus stimulates collagen degradation by corneal fibroblasts are thus dependent on plasminogen.In addition to the direct collagenolytic activity of secreted enzymes, roblasts , with thEffective drug treatment for bacterial corneal ulcer remains to be established. The preferred treatment for the initial stages of bacterial keratitis is the administration of appropriate antibiotics. Eyedrops containing broad-spectrum antibiotics are thus often applied, although no such antibiotic is effective against all potential pathogens. One disadvantage of antibiotic therapy is that it can induce the rapid release of LPS from Gram-negative bacteria . MoreoveMMPs are a potential therapeutic target to prevent collagen degradation by corneal fibroblasts. However, whereas drugs that are able to inhibit MMP activity in vitro have been developed, none to date has shown any clinical benefit with regard to halting the progression of corneal ulcer. Agents including citrate , the cheDoxycycline is a long-acting tetracycline antibiotic but also possesses MMP-inhibitory activity ,97,98,99Nocardia infection as well as no unfavorable reactions [Steroids are currently the main class of drugs administered for amelioration of corneal ulcer . If bacteactions .Tripterygium wilfordii Hook F and has been used in Chinese herbal medicine for the treatment of inflammatory diseases. Triptolide was also found to inhibit LPS-induced chemokine and adhesion molecule expression in, as well as IL-1-induced collagen degradation by, corneal fibroblasts [Triptolide is a compound produced by the plant species roblasts ,47,82, eAnother possible therapeutic target for corneal ulcer is the uPA-plasmin system. Epigallocatechin-3-gallate (EGCG) is the principal polyphenol in extracts of green tea and suppresses the IL-1β-induced up-regulation of uPA expression in and collagen degradation by corneal fibroblasts  Figure . The tarFinally, self-retaining cryopreserved amniotic membrane grafts are being pursued as another potential modality for the treatment of corneal ulcer associated with bacterial keratitis ,111.The future development of drugs for the treatment of corneal ulcer, which is characterized by the uncontrolled degradation of stromal collagen, will thus likely entail investigation of agents that target corneal fibroblasts themselves or individual components of the uPA-plasmin-MMP cascade that underlies collagen degradation by these cells. Bacterial infection of the cornea remains a serious medical problem. If the condition is diagnosed promptly and appropriate antibacterial treatment initiated, the clinical outcome is satisfactory in most cases. However, the emergence of drug-resistant bacteria poses a threat to this treatment approach.The progression of bacterial keratitis to corneal ulcer also presents a therapeutic challenge. Once initiated, the degradation of corneal stromal collagen by corneal fibroblasts is able to proceed even after elimination of infecting bacteria. Furthermore, whereas the inflammatory reaction to invading bacteria is initially beneficial to the host, the persistence of this response can contribute to destruction of the normal structure and function of the cornea, with the resulting corneal opacity negatively affecting visual acuity and quality of life. Further insight into the pathobiology of infectious corneal ulcer is likely to inform the development of new treatment options. With regard to the study of collagen degradation, the culture system based on the embedding of corneal fibroblasts in a three-dimensional collagen gel remains an important experimental model that allows investigation of the interaction of various molecular factors as well as bacteria and inflammatory cells with corneal fibroblasts.


Circulating protein-based biomarkers are well-established tools for the diagnosis, stratification, and management of heart failure. Despite having a certain difference in biomarker panels that are recommended by the American Heart Association/American College of Cardiology and European Cardiology Society to routine clinical practice, cardiac biomarkers, mainly natriuretic peptides, demonstrate high discriminative ability to rule out the diagnosis of HF, and predict the risk of mortality/hospitalization among patients with overt HF. Although there are several challenges regarding the economic burden with biomarkers, their reproducibility, affordability, and interpretations of the results in serial measures, biomarker-based guided HF management, and prediction appear to be promising. However, there is no agreement in the design of various biomarker strategies , which could best fitted to each phenotype of HF and provide strong evidence for personified therapy of HF. We included four articles in the special issue “Prognostication of Heart Failure Evolution: From Circulating Biomarkers to Genetic Risk Predictive Score,” and these will be attractive for readers who are interesting in this topic.Topf et al. reported a narrative review of 159 high-quality articles in the field of multiple biomarkers approaches to the management of HF with the aim to decrease mortality and improve quality of life. Authors provided in-depth analysis of the ability of several models consisted of biomarkers of the inflammatory axis, extracellular matrix remodeling, fibrosis, and oxidative stress to be a powerful diagnostic and therapeutic tool for patients having HF and found that multiple biomarker-based therapy has promising potential for providing tailored treatments among HF individuals.Berezin et al. depicts the challenging pathogenetic role of adipose tissue accumulation in the occurrence and development of HF among patients having several metabolic conditions. The wide spectrum of underlying molecular mechanisms, such as insulin resistance, oxidative stress, altered cardiac and vascular reparation, microvascular inflammation, and metabolic memory phenomenon, corresponded well with adverse cardiac remodeling, endothelial dysfunction, accelerating atherosclerosis, renal injury, and muscle weakness. Moreover, HF with preserved ejection fraction (HFpEF) rather than HF with reduced ejection fraction (HFrEF) can be concisely predicted through imbalance between pro-  and anti-inflammatory  adipocytokines. Yet, dysregulation in natriuretic peptides and neprilysin synthesis occurred in obese patients and diabetics closely affecting adipocytokine signaling and local pro-inflammatory adipocytokine production may be a crucial mechanism, by which a switch of HF phenotypes from HFpEF to HFrEF becomes possible.The article provided by Shrivastava et al. concentrated on several circulating protein-based , genetic-based, and transcriptomics-based biomarkers, including non-coding RNAs, to develop polygenic risk scores for prediction of HF outcomes. The authors emphasized that genetic biomarkers did not significantly improve the discriminative value of classic cardiovascular risk factors and protein-based biomarkers. In addition, genetic risk scores appear to be time-consuming and expensive for clinical routine. Finally, a complementary panel constructed from combined circulating protein-based and transcriptomics-based biomarkers is a promising platform to improve individualized prediction and risk stratification of HF.Zerbo et al. performed a systematic review of 24 articles to elucidate whether HF is the most important comorbidity that may be remarkably related to poor clinical outcomes and hematological alteration that occurred in cardiac implantable electronic device infection. Until recently, surgical interventions, such as cardiac implantable electronic device removal, and antimicrobial therapy in patients with infections had been considered as the gold standard. Investigators found that high pre-operative white blood cell count and elevated levels of C-reactive protein increased the risk of right ventricular failure development, and also increased red blood cell distribution width value or decreased platelet count was associated with poor clinical outcomes.We believe that the topic of circulating biomarkers and multiple biomarker models offers a great opportunity for readers to find out more current challenges, benefits, and discoveries in the field of HF biomarkers. We hope that the special issue will open up for new prospective for investigations and attract new ideas for consideration in the future.AB and ML have prepared the editorial with the consent from BP and IM. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Atherosclerosis is a complex disease closely related to the function of endothelial cells (ECs), monocytes/macrophages, and vascular smooth muscle cells (VSMCs). Despite a good understanding of the pathogenesis of atherosclerosis, the underlying molecular mechanisms are still only poorly understood. Therefore, atherosclerosis continues to be an important clinical issue worthy of further research. Recent evidence has shown that long non-coding RNAs (lncRNAs) and RNA-binding proteins (RBPs) can serve as important regulators of cellular function in atherosclerosis. Besides, several studies have shown that lncRNAs are partly dependent on the specific interaction with RBPs to exert their function. This review summarizes the important contributions of lncRNAs and RBPs in atherosclerosis and provides novel and comprehensible interaction models of lncRNAs and RBPs. Atherosclerosis is the main cause of large-artery atherosclerotic (LAA) stroke . While iin vitro after ox-LDL treatment, and downregulation of RNCR3 accelerated the progress of atherosclerosis, exacerbated hypercholesterolemia and inflammatory factor release, and decreased ECs and VSMCs proliferation , which exert significant biological roles in multiple diseases. Although, the function of lncRNA is complex and still controversial, there is sufficient evidence to suggest that many lncRNAs have important cellular functions . Signififeration . Furtherferation . Some smferation . HoweverSeveral studies have supported that lncRNAs perform many different functions by directly interacting with RNA-binding proteins (RBPs) . In the lncRNAs and RBPs are a major area of interest within the field of atherosclerosis. At present, the mechanism of lncRNAs and lncRNAs-RBPs interaction in atherosclerosis remains an area of high research interest. Therefore, it is important to elucidate these molecular interactions to better understand the underlying mechanisms of atherosclerosis. In this review, we first briefly introduce the biology of lncRNA and RBPs. Next, we discuss how lncRNAs and RBPs regulate endothelial cells, macrophages, and vascular smooth muscle cells in atherosclerosis. Finally, we provide three novel and simple interaction models between lncRNAs and RBPs.lncRNAs are defined as non-coding transcripts of more than 200 nucleotides in length, that do not translate into functional proteins , and theAccumulated evidence shows that lncRNAs can bind to DNA, RNA, and proteins; change the stability and translation of cytoplasmic mRNAs; and interfere with signaling pathways . AccordiRNA-binding proteins are proteins that bind RNA through one or more RNA-binding domains (RBDs) and alter the fate or function of the bound RNA, its activity, or the expression of the target gene . The strSimilar to lncRNAs, RBP regulatory function in atherosclerosis has also garnered attention. Therefore, understanding the regulatory molecules of lncRNAs and RBPs in atherosclerosis as well as the interaction mode between lncRNA and RBP will lay a foundation for future understanding and prevention and treatment of atherosclerosis from the perspective of molecular biology.There is increasing evidence that lncRNAs and RBPs can serve as important regulators of cellular function in atherosclerosis. It is reported that lncRNA ANRIL was the first non-coding RNA identified to be associated with atherosclerosis and expressed in endothelial cells, smooth muscle cells, and immune cells . NeverthThe vascular endothelium is a single layer of ECs that constitute the intima of arteries, veins, and capillaries . It is wMore recently, several published studies have described that lncRNAs may contribute to their role in inflammation, proliferation, migration, and apoptosis. For example, Hu et al. observed that the expression of NEXN was upregulated by lncRNA NEXN-AS1 . Prior sSimilar to lncRNAs, evidence has shown that RBPs play important roles in EC function. For example, the RBP QKI-7 promotes degradation of CD144/NLGN1/TSG6 mRNA, which plays a negative role in EC barrier function and angiogenesis, thus aggravating the progress of atherosclerosis . More reDuring the development of atherosclerosis, macrophages differentiate from monocytes entering the intima and become macrophage-derived foam cells by taking up ox-LDL . At the When macrophages are activated, they trigger an innate immune response. IFN-γ which belongs to Th1 cytokines and lipopolysaccharides can activate M1 macrophages to make pro-inflammatory cytokines. These proinflammatory cytokines contain TNF-α, IL-1β, IL-6, IL-12, and IL-23 . M2 macrvia the TGF-β1/Smad pathway  , 130. VSvia ROR2  as a novel intergenic lncRNA activated by VSMCs proliferation. Recent evidence suggests that SMILR interacts with CENPF mRNA and STAU1 in the Cell Cycle Network . BecauseTaken together, these studies show that lncRNAs are crucially important in the proliferation, migration, apoptosis, and senescence of ECs, VSMCs, and monocytes/macrophages, resulting in two different outcomes of promoting the progression of atherosclerosis or protecting from atherosclerosis. Given the recent curiosity in relating proliferative pathways to plaque development, lncRNAs, and RBPs may be a hopeful direction.Thus far, few RBPs associated with atherosclerosis have been found to interact with lncRNAs to exert regulatory functions. Although these RBPs are not particularly well-reported, it is worthwhile to further investigate the related aspects of the mechanism, which will be beneficial to provide a basis for future disease treatment through etiology. Collectively, the interaction of lncRNA and RBP in atherosclerosis is shown in HuR  is an RBP that is essential in cellular responses to the immune system and cell cycle  and has The hnRNPs are a type of ribonucleoprotein (RNP) and belong to a large family of RBPs that play an essential role in the cellular nucleic acid metabolism. Their function varies depending on the cellular localization , 141. Thin situ hybridization (FISH) and detected hnRNPL by RNA pull-down assay and confirmed its activation of macrophages and promotion of immune response gene expression by linc-AAM silencing or knockout (KO) and overexpression of lncRNA  RNA-binding protein known to be involved in mRNA decay. It binds dsRNA structures that are formed not only by intramolecular base-pairing of 3′UTR sequences but also by intermolecular base-pairing of 3'UTR sequences with a lncRNA elements . The lncelements .via which RNA-binding proteins interact with transcripts are varied and complex. Not to mention the binding to lncRNAs, because lncRNAs are involved in a variety of biological functions.The structures and mechanisms Degradation, repression and overexpression of lncRNAs are important for the regulation of biological messaging; however, in addition to physiological factors  , 148, phTaken together, the expression of RBPs effect the level of lncRNAs, but the mechanism is yet unclear and needs further investigation, the same RBPs can regulate different lncRNAs, by contrast, the same lncRNA can be regulated by different RBPs in a different environment.In addition to binding to lncRNAs to affect the expression of lncRNAs, RBPs can also affect the expression of lncRNA target genes. For example, Chen et al. found that exosomal lncRNA LNMAT2 recruited hnRNPA2B1 to the PROX1 promoter to upregulate PROX1 expression by directly interacting with hnRNPA2B1, leading to lymphangiogenesis and lymphatic metastasis in bladder cancer . The exovia the PI3K/AKT and Ras/MAPK pathways  complexes formed with proteins to connect multiple proteins to regulate the expression of their target genes. For instance, hnRNPK and YBX1 are lncRNA SCAT7-interacting proteins, and recruitment of the FGFR2 and FGFR3 promoter regions SCAT7-hnRNPK-YBX1 RNP complexes promotes transcriptional activation of the FGF/FGFR pathway, resulting in sustained cell proliferation pathways . Furtherpathways .The binding of lncRNA to RBP can regulate the expression of lncRNA and affect the expression of the target gene, and it can also affect the activity of the binding proteins; the outcomes are similar, in that all of them will affect the corresponding signaling pathway and thus change the regulatory outcome of the molecule. For example, the interaction between lncRNA and UPFI can regulate the expression of other mRNA but also decrease the stability of the lncRNAs. In some cases, the binding of lncRNA and UPF1 will affect the expression of UPF1, though the specific mechanisms involved need to be further investigated . AdditioThe limitation is that there were few extensive studies so far on the mechanism of lncRNA and RBP interaction in the field of atherosclerosis; most of the model construction in this review is based on studies on tumor-specific conditions.Taken together, the studies discussed in this review show that lncRNAs and RBPs play biological functions in atherosclerotic ECs, SMCs, and macrophages; simultaneously, this review provided novel and understandable interaction models of lncRNAs and RBPs. Further extensive research is needed in the future to understand the functions and mechanisms of lncRNAs and RBPs, and provide novel and effective methods for the diagnosis, prevention and treatment of atherosclerosis.YD drafted the manuscript. RY, SZ, and QX provided insightful discussions and comments on the manuscript. HZ, XP, and XZ discussed and revised the manuscript. All authors read and approved the final manuscript.This work was supported by the National Natural Science Foundation of China (No. 81771259) and the Natural Science Foundation of Shandong Province (ZR2020MH138).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The gut microbiota is an important regulator for maintaining the organ microenvironment through effects on the gut-vital organs axis. Respiratory tract infections are one of the most widespread and harmful diseases, especially in the last 2 years. Many lines of evidence indicate that the gut microbiota and its metabolites can be considered in therapeutic strategies to effectively prevent and treat respiratory diseases. However, due to the different gut microbiota composition in children compared to adults and the dynamic development of the immature immune system, studies on the interaction between children’s intestinal flora and respiratory infections are still lacking. Here, we describe the changes in the gut microbiota of children with respiratory tract infections and explain the relationship between the microbiota of children with their immune function and disease development. In addition, we will provide perspectives on the direct manipulation of intestinal microbes to prevent or treat pediatric respiratory infections. The intestinal tract is home to about 40 trillion microbiota, and the total number of its genes is about 150 times that of human genes. Given the size of the intestinal flora, it is also known as the “second genome”, “second brain”, or “gut brain” of the human body. Compared with the microbiota in other parts of the human body, the intestine contains huge bacterial groups and rich species. Therefore, it influences other organs and human body functions in diverse ways. It will soon be commonplace to treat various diseases with consideration of the intestinal microbiota.The gut microbiota is an increasingly recognized component of the human body that shows dynamic changes with age, region, diet, and medication  Figure, regulatin situ, but they also pass through intestinal tract into capillaries and transfer to major organs in the circulation. SCFAs have critical functions in the systemic action of the gut microbiota and play important roles in defense against infection, mitigation of autoimmune diseases, and anti-tumor therapy. In recent years, the description of the gut microbiota and its metabolites involved in respiratory diseases has gradually become more elaborate. With regard to the microbes themselves, microbial surface molecules like lipopolysaccharide (LPS) and lipoteichoic acids can be recognized by the immune system and induce corresponding humoral and cellular immune processes that can improve the host immune system. In controlled experiments on specific pathogen-free (SPM) mice and bacteria-free mice, SPF mice exhibited increased lymphatic tissue activity, more intestinal Peyer’s patches, and greater antibody secretion with less susceptibility to systemic inflammation , which can induce the development of gut-associated immune tissues (GALT). Peyer’s patches, mesenteric lymph nodes, and isolated lymphoid follicles (ILFs) are important secondary lymphoid tissues of GALT and vital sites for the B cells that produce IgA to neutralize pathogenic microorganisms , 14. TheActinobacteria, Bacilli, Clostridium cluster IV, and Bacteroidetes phylum-like groups , Bifidobacterium-dominant profile (21%), Enterobacter/Veillonella-dominant profile (22%), and Bacteroides-dominant profile (28%). However, the predominant taxa in children with bronchitis changed to: Enterobacter/Veillonella-dominant profile (15%), and Bacteroides-dominant profile (44%). Researchers hypothesized that the Bacteroides-dominant profile may put infants at a higher risk of developing bronchitis. This lays a theoretical foundation for modifying the gut microbiota to treat or prevent bronchitis. Lei Li and colleagues  can effectively lower the infection rate of the upper respiratory tract ; Scientific Research Project of Shanghai Hongkou District Health Commission (HKQ-ZYY-2020-01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.