Mucus and mucin: changes in the mucus barrier in disease states

ABSTRACT

In this review we discuss mucus, the viscoelastic secretion from goblet or mucous producing cells that covers and protects all non-keratinized wet epithelial surfaces. In addition to the surface of organs directly contacting with the external environment such as the eyes, this layer provides protection to the underlying gastrointestinal, respiratory and female reproductive tracts by trapping pathogens, irritants, environmental fine particles and potentially harmful foreign substances. Mucins, the primary structural components of mucus, form structurally different mucus layers at different sites in a process regulated by a variety of factors. Currently, more and more studies have shown that the mucus barrier is not only closely related to various intestinal mucus diseases, but also involved in the occurrence and development of various airway diseases and mucus-related diseases, thus it may become a new target for the treatment of various related diseases in the future. Since the dysfunction of the mucous layer is closely related to various pathological processes, in-depth understanding of its molecular mechanism and physiological role is of great theoretical and practical significance for disease prevention and treatment. Here, we discuss different aspects of the mucus layer by focusing on its chemical composition, synthetic pathways, and some of the characteristics of the mucus layer in physiological and pathological situations.

Introduction

Mucus produced by secretory epithelial cells is a complex viscoelastic gel, which forms a barrier on the epithelial surface as a defense mechanism to protect the body from external harmful factors and constitutes the first line of defense against foreign harmful substances.¹ This important barrier can be viewed as a semi-permeable membrane, distributing over organs, including the intestines, stomach, ears, eyes, airways, and reproductive tract. The barrier allows penetration of nutrients, water, gases, hormones, etc., while at the same time restricts the entry of most pathogens, as well as foreign particles, due to their site-barrier and adhesion properties.^2,3 In addition, many defensive compounds, are secreted by the interaction between mucosal epithelial cells interact and leukocytes in the basement, such as mucins, antibodies, defensins, proteases, lysozymes, etc., which collectively build an antimicrobial barrier contributing to regulated mucosal homeostasis.⁴ The regulation of this seemingly “paradoxical” task is achieved through interactions between mucus structural components and molecules, which operate in a highly dynamic manner to maintain the integrity of the mucus barrier and the balance of immune homeostasis.⁵

The thickness of the mucus layer varies considerably within and between different organ systems.⁶ For example, the thickness of the tear film is about 3–5 µm.⁷ The average thickness of the vaginal mucosa is about 1.4 mm, and the thickness and viscosity of the mucus in the female genital tract varies with the ovulatory cycle.⁸ The mucus layer of the respiratory tract is relatively thin and mobile (nasal cavity: 5–15 µm,⁹ trachea: 10–30 µm,¹⁰ bronchi: 2–5 µm¹⁰). In contrast, the thickness of the mucus layer of the gastrointestinal tract varies along its length, with the small intestine being the thinnest (200–250 µm),¹¹ the stomach the second thinnest (450–500 µm),¹¹ and the colon the thickest (450–700 µm).¹² These differences in mucus thickness may reflect the various protective functions and challenges of mucus in its specific organs. The mucogel layer on the mucosa of the gastrointestinal tract has a dual function. On one hand, it acts as a barrier to effectively block the invasion of acids, enzymes and microorganisms into the mucosa, thereby protecting epithelial cells from attack. On the other hand, it acts as a lubricant to reduce the mechanical damage to the mucosa by reducing the shear forces associated with the mechanical action during digestion.¹³ Respiratory tract mucus prevents dehydration of the lung epithelial surface while also protecting the lower airways by trapping and clearing inhaled particles and pathogens through mucociliary action and coughing.¹⁴ The tear film lubricates the surface of the eye, prevents drying of the cornea conjunctiva, maintains the optical properties of the cornea, as well as flushes and defends the surface of the eye against foreign bodies and microorganisms.¹⁵ The mucus plug at the mouth of the cervix protects against the entry of bacteria and facilitates the movement of sperm during the mid-cycle.¹⁶

In this article, we will explore how mucins, the building blocks of mucus, are synthesized, how their expression is regulated and achieved, and how they are altered in the course of disease (Figure 1).

Figure 1.

Schematic diagram of the mechanism of the mucus layer in physiological defense and diseases.

Mucus formation and structure

Mucins

Mucus is a complex gel composed primarily of water, mucins, lipids, electrolytes, salts, immune molecules, DNA, and antibacterial peptides. Among these components, mucins, accounting for 1%–5% of the total composition, serve as the key functional elements.¹⁷ Mucin is mainly classified into two categories: transmembrane mucins and gel-forming mucins.¹⁸ Transmembrane mucins bind to the cell membrane through their transmembrane regions and exhibit different structures and functions inside and outside the cell, creating a stable environment for the epithelial cell surface.¹⁹ As listed in Table 1, transmembrane mucins play a key role in hindering foreigners, cells, or pathogens from adhering to mucosal surfaces and thus avoiding infection, and they are essential for maintaining the integrity of the mucosal barrier.¹⁸ The gel-forming mucins listed in Table 2 are a class of highly O-glycosylated glycoproteins. These mucins have cysteine-rich regions and repetitive PTS domains, which play a vital role in building mucus barriers, protecting mucous membranes from damage, and defending against pathogenic microorganisms.⁵⁴

Table 1.

Transmembrane mucin types.

Types	Distribution	Physiological function	References
MUC1	Gastrointestinal tract, mammary gland, gallbladder, cervix, respiratory tract, kidneys, eyes, middle ear epithelium, B-cells, T-cells	Involved in lubrication and moisturization of normal cells; Signal transduction regulation, immunomodulation; Anti-adhesion and involvement in tumor development;	20,21
MUC3A	Intestine, gallbladder, middle ear epithelium, airway epithelium	Provides a protective lubricating barrier against particles and infectious agents on mucosal surfaces; Promotes cell migration and inhibits apoptosis;	22,23
MUC3B	Intestine, gallbladder, middle ear epithelium, airway epithelium	Protects mucosal surfaces from particles and pathogens.; May be a potential biomarker for disease;	23,24
MUC4	Gastrointestinal tract, respiratory tract, eye, middle ear epithelium, cervix	Involved in the formation of a mucus barrier; Regulates signaling pathways such as cell growth, differentiation and apoptosis; Promoting tumor growth;	25,26
MUC12	Gastrointestinal tract, pancreas, respiratory tract, kidneys, prostate, uterus	Involved in epithelial cell protection, adhesion regulation and signaling; Regulation of epithelial cell growth and invasion;	27
MUC13	Gastrointestinal tract, respiratory tract, kidney, appendix, middle ear epithelium	Protective and lubricating action on mucosal surfaces; Regulation of apoptosis and proliferation;	28,29
MUC14	Heart, kidneys, lungs	Involved in the formation of the mucus layer; Involved in the regulation of inflammation and immune cells; The most promising protective regulator of breast cancer;	30
MUC15	Spleen, thymus, prostate, testes, ovaries, intestines, bone marrow, lymph nodes, Middle ear epithelium	Creates a selective molecular barrier in the epithelium and mediates signaling; Affects cell growth, adhesion, invasion, and metastasis; immunomodulation;	31,32
MUC16	Genital tract, respiratory tract, eye, middle ear epithelium	Immunomodulatory effects; Promotes cancer cell proliferation and suppresses anti-cancer immune responses; Certain cancer treatment markers;	33,34
MUC17	Gastrointestinal tract, middle ear epithelium	Limits bacterial adhesion and invasion of superficial epithelial cells; Closely associated with tumor development;	35,36
MUC20	Kidney, gastrointestinal tract, lung, prostate, liver, middle ear epithelium	Regulation of cell migration and invasion; Physical protection; Immunomodulation; Cell signaling;	37,38
MUC21	Lungs, intestines, thymus, testes	Regulation of cell adhesion; Resistance to apoptosis; Affects cell metastasis and invasion;	39,40
MUC22	Lungs, placenta, testes	Identify a subtype of lung cancer;	41

Table 2.

Gel-forming mucin types.

Types	Distribution	Physiological function	References
MUC2	Gastrointestinal tract, respiratory tract, middle ear epithelium, eye, cells	Involved in the formation of a mucus barrier; Shaping microbes; Regulation of the immune system; Involved in signaling;	42,43
MUC5AC	Respiratory, gastrointestinal, cervical, eye, middle ear epithelium	Formation of a respiratory mucus layer; Increases the viscosity of the mucus; Remove foreign objects; A potential marker for certain diseases;	44,45
MUC5B	Respiratory tract, salivary glands, cervix, gallbladder, pancreas, semen, middle ear epithelium	Formation of respiratory mucus; Fending off pathogens; Regulation of the physical properties of mucus; Biomarkers for certain diseases;	44
MUC6	Gastrointestinal tract, gallbladder, pancreas, semen, cervix, middle ear epithelium	Formation of the gastric mucus layer; Immunohistochemical markers of gastric origin; Protects the gastric mucosa;	46
MUC7	Salivary glands, respiratory tract, middle ear epithelium, eye	Provides lubrication and moisturization of the oral mucosa; Possesses powerful antimicrobial activity;	47,48
MUC8	Trachea, cervix, testes, endometrium, placenta	Maintaining the stability of the internal environment; Involvement in the inflammation;	49,50
MUC9	Fallopian tube, middle ear epithelium	Maintenance of mucosal barrier integrity; Participates in fertilization and promotes early embryonic development; Maintaining local immune homeostasis;	51,52
MUC19	Lungs, bone marrow, lymph nodes, thymus, eyes, stomach	Involved in the immune response; Potential biomarkers for certain cancers;	53

Mucin structure

Although mucus is often regarded as a viscous substance covering the inner walls of the nasal cavity and digestive tract. In fact, there are many types of mucins in the human body, and they play their own roles in different physiological parts. In mammals, there is a specific group of mucin polymers (such as MUC2, MUC5AC, MUC5B, MUC6, and MUC19), and these molecules are similar in domain arrangement and overall configuration (Figure 2).⁵⁵ We will explore in detail the secretory properties of gelatinous MUC2–a substance that contains up to 80% carbohydrates and forms a protective layer in the intestines that covers an area of up to 185 square meters.³

Figure 2.

Structural building blocks of polymeric mucins. The figure illustrates domain structures of different gel-forming mucins. The tandem repeat region is rich in proline, threonine and serine (PTS domain) which is heavily glycosylated and forms the mucin-like domain; amino and carboxy-terminal cysteine-rich domains (vWD, vWC and vWF domains); and cysteine-rich structural domains within the PTS region (cys D); the amino and carboxy-terminal structural domains are essential for polymer formation. Mucin polymers are stabilized by tail-to-tail disulfide bonding through the carboxyl-terminal CK structural domain and the amino-terminal D3 structural domain.

Structurally, MUC2 is a major component of intestinal mucus, present in the small and large intestine, forming the mucus skeleton.⁵⁶ The human MUC2 consists of approximately 5200 amino acids in a number of different structural domains, the most important of which are tandem repeats of proline (Pro), threonine (Thr), and serine (Ser) centers, termed the PTS region. The exposed hydroxyl groups on Thr and Ser act as potential sites for O-glycosylation and give mucus its “bottle-brush” structure, while Pro ensures that the structure of the mucin remains unfolded in the Golgi apparatus, permitting its O-glycosylation process (Figure 3).¹¹ In these PTS structural domains, glycan structures are not only abundant but also diverse.⁵⁷ The mucin contains an N-terminal region with four cysteine-rich von Willebrand factor type D (vWD) domains (vWD1, vWD2, and vWD3) on one side of the PTS domain, along with a truncated domain (vWD’) located between vWD2 and vWD3. On the other side, the D4 structural domain containing the vWC structure, the vWB structural domain, the vWC structural domain and the CK structural domain together form the carboxyl-terminal region. The central mucin structural domain is usually interrupted by a 100 amino acid long structural domain called the CysD structural domain.⁵⁸

Figure 3.

O-glycosylation process of MUC2. The specific structure of MUC2 includes the first glycosylation by a peptidyl-GalNAc transferase that adds the first sugar GalNAc residues to ser and thr in the PTS region. Subsequent addition of galactose, GlcNAc, NeuAc, fucose, sialic acid and sulphate residues by at least 30 different glycosyltransferases to extend and terminate the o-glycan chains.

Mucin biosynthesis

The production of mucin polymers occurs primarily in specialized secretory cells, which face multiple challenges, such as building polymer chains, managing glycosylation reactions, and dealing with the size of molecules. While there is a general understanding of the stages involved in mucin biosynthesis and its cellular location (Figure 4), the comprehensive mechanistic details of any particular mucin remain largely unexplored.

Figure 4.

Mucin biosynthesis and mucus formation. Identification of the steps of goblet cell mucus production and its secretion and expansion within the lumen (from 1 to 6). Firstly, MUC2 monomers form dimers in the endoplasmic reticulum (ER), which are then o-glycosylated in the Golgi, and the assembled polymers are then stored in secretory granules via Ca²⁺ and PH-dependent mechanisms. Mucus secretion is a complex process: the goblet cells fill their secretory vesicles with MUC2 while migrating from the crypt bottom and contain other components, such as the Fc fragment of IgG-binding protein (FCGBP), chloride channel accessory 1 (CLCA1), zymogen granule protein 16 (ZG16) and the trefoil factor peptide group (TFF3). The secretory vesicles extrude their content after their fusion with the apical membrane of the goblet cells, by exocytosis, allowing mucus secretion, and it expands upon release, which is dependent on HCO₃⁻ secretion to alter the intestinal lumen environment.

During its formation, mucin monomers are translated in the coarse endoplasmic reticulum (rER) of goblet cells. rER is rich in a variety of modifying enzymes, these enzymes are responsible for participating in the folding process of newly synthesized proteins, N-glycosylation, C-mannosylation, and disulfide bond formation.⁵⁹ AGR2, a protein homologous to the anterior gradient protein, is an endoplasmic reticulum (ER)-resident protein that is specifically expressed in the secretory cells of the intestine. Its main function is to ensure proper pairing and formation of disulfide bonds in mature proteins.⁶⁰ Here, the mucin monomers polymerize to form a dimer by non-covalent distortion of the cysteine-rich CK structural domain downstream of the PTS via disulfide bonds. In addition, folding of the nascent MUC2 polypeptide also occurs in the ER. However, when ER homeostasis is disturbed, unfolded or misfolded mucin monomers accumulate within the endoplasmic reticulum would lead to endoplasmic reticulum stress (RES), which ultimately allows mucus and goblet cells depletion.⁶¹ These MUC2 monomers are modified within the ER by n-glycosylation and C-mannosylation, a process that allows MUC2 to fold correctly to form stable and mature dimers, thereby reducing RES and inflammation.⁶²

Subsequently, the newly synthesized dimer is transported from the ER to the Golgi apparatus and undergoes complete glycosylation modification as it travels through the Golgi body. In this process, O-glycosylation in the Golgi apparatus is the most common form of post-translational modification of MUC2 protein, and its sugar chain accounts for more than 80% of the total mass of MUC2 molecules.⁴² These glycans can bind water and form hydrated gel-like properties. The production of glycan chains is a stepwise process, and the order in which the glycans meet the different glycosyltransferases plays a key role in O-glycosylation, coordinating their initiation, extension and termination. In cis-Golgi, the synthesis of glycan chains begins with the polypeptide N-acetylgalactosamine (GalNAc) transferase, which ligates GalNAc to the hydroxyl group of serine or threonine residues to generate Tn antigens.⁶³ As the O-glycosylated mucin dimer moves from the cis-Golgi to the post-Golgi, the glycan chain is extended by the addition of residues such as galactose and N-acetylglucosaminoglucose (GlcNAc). Finally, termination of extension in trans-Golgi by addition of charged sugars (fucose, sialic acid residues) or sulfates.^3,11 The terminal sialic acid residues confer a net negative charge on the mucin, which leads to charge and steric repulsion between the oligomers. This situation causes the mucin molecules to extend and harden, forming extended rod-like structures. Eventually, the oligomers are arranged around the mucin core in a “bottle brush” shape.⁶⁴

After polymerization, MUC2 is sorted and stored in secretory granules regulated by goblet cells along with other major mucin proteins (e.g., FCGBP, CLCA1, TFF3 and ZG16).⁶⁵ MUC2 accumulation and release is a complex process controlled by pH and calcium ion concentration. First, folded MUC2 accumulates densely in secretory granules at acidic pH and high Ca²⁺ concentration.⁶⁶ Then the secretory granules enter the intestinal lumen at neutral pH and low Ca²⁺ concentration to release MUC2. There are two main routes of release of MUC2: a basal type dependent on cytoskeletal motility of the secretory granules, which presents continuous low-dose secretion, and a modulated type involving cytotoxic effects stimulated by external active factors, such as cholinergic agonists, hormones, microorganisms, microbial products, toxins, and inflammatory cytokines, reactive oxygen and nitrogen species, etc.⁶⁷ The released MUC2 polymer swells rapidly in the intestinal lumen. It increases in size by more than 1000-fold through depolymerization and hydration and unfolds to form a loosely structured reticulated laminar structure in the presence of an alkaline environment generated by HCO3–. Ultimately, a gradient mucus layer of reduced density is formed, with increased permeability from the inner to the outer layer.⁶⁸ As a result, newly secreted mucus continuously pushes previous mucus from the inner layer into the lumen, renewing the mucus layer.

Mucins and intercellular junctions

Mucins, as a class of highly glycosylated macromolecular proteins, play a crucial role in maintaining mucosal barrier function and intercellular homeostasis. Recent studies show that mucins do not just act as lubricants or protective molecules. They also interact with key intercellular junctions – tight junctions (TJs), adherens junctions (AJs), desmosomes/hemidesmosomes, and gap junctions (GJs) – to jointly maintain epithelial barrier integrity and function.⁶⁹ The gel-forming mucins (such as MUC2 and MUC5AC) form a mucus layer that not only provides physical protection for epithelial cells but also maintains barrier integrity by reducing direct damage to TJs from pathogens and mechanical stimuli.^70,71 Transmembrane mucins (e.g., MUC1, MUC4) regulate the expression and function of junctional proteins through their intracellular domains. For instance, MUC1 modulates the expression of E-cadherin, thereby influencing the stability of AJs, and serves as a critical regulator of epithelial polarity and cell-cell adhesion during epithelial-mesenchymal transition (EMT).⁷² Furthermore, using bioinformatics tools (cBioPortal and SurvExpress), researchers identified 187 co-expressed genes significantly associated with MUC4 expression. Gene Ontology analysis revealed that these genes are closely linked to biological processes such as cell adhesion, cell-cell junctions, glycosylation, and cell signaling. Additionally, MUC4 expression was found to correlate with MUC16 and MUC20, suggesting potential functional interactions among these mucins.⁷³ Under pathological conditions, aberrant mucin expression is frequently associated with compromised intercellular junction integrity. Notably, MUC1 overexpression can modify the cell surface microenvironment, potentially impairing desmosomal adhesion mediated by Desmoglein-3 (DSG3) and consequently facilitating tumor metastasis.⁷⁴ Meanwhile, MUC2 may disrupt the anchoring function of hemidesmosome by interfering the interactions between integrin α6β4 and laminin, which holds significant clinical implications for inflammatory bowel disease progression and metastatic dissemination.⁷⁵ The dynamic equilibrium between mucins and intercellular junctions not only sustains normal physiological functions of epithelial tissues, but its dysregulation has emerged as a pivotal factor in the pathogenesis of various diseases. Delineating this intricate regulatory network will provide novel therapeutic targets for related disorders.

Intestinal diseases

The intestinal tract is one of the largest and most complex organs in mammals. It not only has the function of digestion and absorption, but its mucus barrier is also the biggest barrier to prevent bacterial translocation and endotoxin invasion in the intestinal lumen, which can maintain the stability of the internal environment and the normal life activities of the organism.⁷⁶ Intestinal barriers usually include a chemical barrier in the mucus layer, a mechanical barrier in the epithelial cell layer and an immune barrier in the lamina propria.^77,78 The barriers in the gut do not function independently, but are related in a way that creates a complex dynamic barrier system. Increased intestinal mucosal permeability, decreased goblet cells, altered intestinal cell TJs, abnormal mucin expression, decreased mucus layer thickness, and altered intestinal microbiota can be seen as manifestations of gut barrier damage.^79,80

The maintenance of intestinal homeostasis and its normal physiological function is highly dependent on intact barrier function. When the intestine or other organs are inflammatory, the structure and function of the gut barrier may be impaired, leading to a variety of diseases, including gastrointestinal disorders, as well as extraintestinal complications. Therefore, it is essential to prevent intestinal injury and protect the integrity of the intestinal mucosal barrier (Table 3).

Table 3.

Characteristics of mucus in intestinal mucosal diseases.

Name of diseases	Model	Mucus characteristics	Therapeutic options	References
Ulcerative colitis	Animal	Reduced MUC2 expression and thinning of mucus layer thickness; Decreased number of goblet cells; Down-regulation of the expression of TJ proteins; Altered intestinal flora, Listeria, Alistipes, P. copri Enterobacteriaceae and Veillonella gradually became dominant bacteria in UC mice.	Puerarin: Through the process of anti-IL-1β, IL-6 and TNF-α, goblet cells proliferation and mucus healing were facilitated; Increased thickness and decreased permeability of the colonic mucus layer; alleviate dysbiosis by reducing the abundance of Alistipes, P. copri and Veillonella, and increasing the level of Desulfovibrionacea.	81,82
Crohn’s disease	Animal	The mucin layer shows disruption and abnormal MUC2 glycosylation; Increased number of goblet cells; Altered intestinal flora, Pasteurellaceae, Enterobacteriaceae, Fusobacteriaceae, and Veillonella-ceae were predominant, the abundance of Bacte-roidales, Erysipelotrichales, and Clostridiales decreased.	Cinnamon: Downregulation of NLRP3 inflammatory vesicle activation and reduction of pro- inflammatory mediator expression in the colon; A modified community composition with a decrease in Helicobacter and Bacteroides and an increase in Bacteroidales, Alloprevotella and Lachnospiraceae.	83,84
Necrotizing enterocolitis	Animal	Immature intestinal mucus barrier with reduced antioxidant capacity; Decrease in the number of goblets and thinning of the mucus layer thickness; Altered intestinal flora, increased relative abundance of Bifidobacteria, Lactobacillus, Klebsiella and Enterococcus	R. chinensis fruits: Upregulation of ZO-1 and Occludin Protein Expression to Maintain Intestinal Barrier Integrity; Amelioration of oxidative stress, inflammation and apoptosis in intestinal epithelial cells by down-regulation of TLR4, p-NF-κB, iNOS, cleaved Caspase-3 and Bax.	85,86
Celiac disease	Human	Increased intestinal permeability and defective TJ proteins; Substantial reduction in intestinal absorptive surface area, intestinal villi atrophy; Altered intestinal flora, The proportion of bifidobacteria decreased and the proportions of anaplasmosis and E. coli increased.	the only treatment for celiac disease is a life-long, strict gluten-free diet leading to improvement in quality of life, ameliorating symptoms, and preventing the occurrence of refractory celiac disease, ulcerative jejunoileitis, and small intestinal adenocarcinoma and lymphoma.	87,88
Irritable bowel syndrome	Animal	Decrease in the number of goblet cells and changes in the structure of mucin O-glycans; Decrease in MUC2 content in the intestine and decrease in the thickness of the mucus layer; The increase of pathogenic bacteria, such as Veillonellaceae, Pasteurellacaeae, Enterobacteriaceae and Fusobacteriaceae, and the decrease of Bacteroidales, Bifidobacterium Erysipelotrichales, Lattobacillus Fecalibacterium and Clostridiales	Atractylodes lancea: Reduces intestinal damage by anti-TNF-α, IL-6, 5-HT, VIP, SP; Upregulation of TJ proteins; reduced harmful bacteria (Turicibacter, Parasutterella, and Erysipelatoclostridium) and enhanced beneficial bacteria (Enterorhabdus, Parvibacter, and Akkermansia).	89,90
Hirschsprung’s disease	Human	Decreased number of goblet cells, increased permeability of the intestinal epithelial barrier and disruption of TJ proteins; Increased relative abundance of Akkermansia and Veillonella parvula, decreased relative abundance of Dysgonomonas and Clostridium cluster XI Va.	Most surgical treatments, including transanal endorectal traction, laparoscopic access, as a way to improve bowel function.	91,92
Colon cancer	Animal	Decreased number of goblet cells, decreased mucus secretion, increased intestinal permeability; Decrease in TJ proteins; Increased relative abundance of Enterotoxigenic Bacteroides fragilis Fusobacterium nucleatum, Peptostreptococcus anaerobius, Enterococcus faecalis, and Escherichia coli.	Ganoderma: Amelioration of A microbiota dysbiosis by inhibiting the TLR4/MyD88/NF-κB signaling pathway; Improved intestinal barrier function, increased goblet cells, increased MUC2 secretion and increased expression of TJ proteins; Downregulation of IL-1β, i NOS and COX-2 expression and inhibition of macrophage infiltration.	70,93
Non-alcoholic fatty liver disease	Animal	Intestinal barrier damage with reduced mucus layer thickness and increased permeability; Increased secondary bile acid levels; Relatively rich in potentially pathogenic taxa, mainly Enterobacteriaceae, Staphylococcaceae and Enterococcaceae and a relative decrease in potentially beneficial commensal autochthonous taxa, particularly Lachnospiraceae, Ruminococcaceae and Clostridiales XIV.	Gynostemma: Promoted proliferation of goblet cells and mucus healing by down-regulating IL-1β, IL-6, TLR4 and TNF-α levels; decreased the ratio of Firmicutes to Bacteroidetes, Increased the abundance of beneficial bacteria (Lactococcus spp.) and inhibited the abundance of pathogenic bacteria (Ruminococcus spp.) in the intestinal.	94,95
Acute pancreatitis	Animal	Down-regulation of TJ protein expression and increased intestinal permeability;Impaired function of goblet cells and Paneth cells; Decrease the number of good bacteria such as Mycobacterium phylum, Lactobacillus and Bifidobacterium, and increase the number of pathogenic bacteria such as Enterobacteriaceae, Clostridium and Bacteroidetes.	Biochanin A: Protection against AP and associated intestinal damage by inhibiting activation of the TLR4-MAPK/NF-κB signaling pathway and NLRP3 inflammatory vesicles; Up-regulation of the TJ proteins attenuate the associated barrier damage; Reduced translocation of pathogenic Escherichia coli to the pancreas.	96,97
Diabetes	Animal	Increased intestinal permeability; down-regulation of TJ proteins; Decreased number of goblet cells, mucosal atrophy, loss of intestinal villi; Compared with the normal people, the number of Bifidobacteria, Clostridium and Firmicutes in the intestinal flora of diabetics decreased significantly, the number of Bacteroides and β-proteus increased significantly.	Vaccarin: Protecting the intestinal barrier by inhibiting the ERK/MLCK signaling pathway and regulating intestinal flora composition; Elevated expression of TJ proteins and a marked increase in goblet cells; Decreased relative abundance of Lactobacillaceae, Lachnospiraceae and Desulfovibrio and increased relative abundance of Rikenellaceae, Bacteroides and Muribaculaceae.	98,99
Alzheimer’s disease	Animal	The goblet cells were significantly reduced and mucus secretion was reduced; Decreased levels of TJ proteins and increased intestinal permeability; Decreased function of Paneth cells; Increased abundance of Escherichia, Blautia, Bifidobacterium, Streptococcus, Lactobacillus, Dorea, and decreased abundance of Alistipes, Bacteroides, Parabacteroides, Sutterella, Paraprevotella.	Sinomenine: Inhibition of TLR4/NF-κB activation through modulation of cholinergic anti-inflammatory pathways protects brain and intestinal homeostasis and attenuates cognitive impairment; Upregulation of TJ proteins expression; Increased abundance of Odoribacter, Mucispirillum, Muribaculaceae and decreased abundance of pro-inflammatory strains of Erysipelotrichaceae, Bacilli, and Firmicutes.	100,101

Annotation: NLRP3, NOD-like receptor thermal protein domain associated protein 3; TLR4, Toll-like receptor 4; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated b cells; p-NF-κB, Phosphorylated nuclear factor kappa-light-chain-enhancer of activated b cells; iNOS, Inducible nitric oxide synthase; Caspase-3, Cysteine aspartate specific protease-3; Bax, Bcl-2 associated X protein; 5-HT, 5-Hydroxytryptamine; VIP, Vasoactive intestinal polypeptide; SP, Surfactant protein; MyD88, Myeloid differentiation primary response protein 88; MAPK, Mitogen-activated protein kinase; ERK, Extracellular regulated kinase; MLCK, Myosin light chain kinase.

Mucus barrier and intestinal diseases

Inflammatory bowel disease

Inflammatory Bowel Disease (IBD) is a chronic autoimmune mediated inflammatory disease consisting mainly of two subtypes, ulcerative colitis (UC) and Crohn’s disease (CD), affecting more than 6 million people worldwide.¹⁰² Patients with IBD have similar pathological and clinical symptoms such as recurrent bleeding, abdominal pain, diarrhea, loss of appetite, weight loss and fatigue.¹⁰³ Both UC and CD exhibit a disruption of the mucus barrier, a phenomenon that is the result of a combination of an imbalance in the gut mucosal immune system and dysbiosis in commensal microbiota.¹⁰⁴

Studies have shown that the mucus layer of the colon is more easily penetrated in patients with UC.¹⁰⁵ Further analysis of the mucus layer in patients with UC revealed a range of glycosylation abnormalities,¹⁰⁶ including shortened oligosaccharide chain length, reduced sulfation, reduced fucoidan glycosylation, and increased salivary acidification.⁶² Furthermore, this glycosylation abnormalities would result in the thinner mucus layer due to the collapse of the mucus gel caused by reduced negative charge on the surface of the mucin fibers.¹⁰⁷ In addition, electron microscopy further showed that accumulation of partially synthesized or misfolded mucins can be found in the endoplasmic reticulum of goblet cells active UC.¹⁰⁸ The accumulation of these misfolded mucins in the endoplasmic reticulum will reduce the secretion of mature mucins, deplete goblet cells, slow down the secretion of mucus, and thin the mucus layer.¹⁰⁹ Simultaneously, mucus composition is altered, e.g., glycosylation products, TFF3, and defensins are reduced.^110,111 This results in increased mucus permeability, allowing bacteria to come into direct contact with epithelial cells, which leads to microbiota translocation and immune system activation.^112,113

For CD patients, intestinal inflammation is often chronic and prolonged, and the scope of the lesion is extensive. A large number of inflammatory factors stimulate intestinal mucus secretion, resulting in a significant thickening of the mucus layer. Although the mucus layer thickens, there are still many bacteria that can penetrate this barrier and come into direct contact with intestinal epithelial cells. This penetration may be related to the decrease in glycosylation and sulfation levels in the mucus barrier and the increase in sialic acidation levels. These changes weaken the defense function of the mucus barrier and make it easier for bacteria to penetrate.^62,114–116

In the pathogenesis of IBD, the structure and secretion of mucin, and thickness and permeability of the mucin layer are disrupted to varying degrees, which ultimately leads to the persistence of intestinal inflammation. These alterations suggest that the mucus barrier plays an important role in the pathogenesis of IBD, and may provide new avenues of treatment for the disease.

Necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a gastrointestinal emergency characterized by an inflammatory response of the intestinal tissue and intestinal ischemic necrosis.¹¹⁷ NEC occurs predominantly in preterm and low-birth-weight infants,¹¹⁸ and is characterized by submucosal edema, hemorrhage and intestinal villi damage, which in severe cases can be complicated by total intestinal mucosal necrosis or intestinal perforation.¹¹⁹ Compared to healthy infants, patients with NEC have an underdeveloped intestinal mucus barrier with significantly fewer goblet cells and Paneth cells. And these infants typically exhibit low mucin production and insufficient mucus thickness, which hampers their ability to effectively trap bacteria and toxic macromolecules.^120,121 TFF3, secreted by goblet cells, is part of the protective mechanism of the intestinal mucosa. TFF3 is considered to act synergistically with mucins to enhance the protective barrier properties of the mucus layer against bacterial toxins, thus playing an important role in epithelial protection, repair and restoration.¹²² In addition, Paneth cells and individual immune cells in the gastrointestinal tract produce lysozyme. Lysozyme secreted by Paneth cells into the intestinal lumen, is the main source of lysozyme in the intestines, which is in direct contact with the intestinal flora, and increases resistance to infection.¹²³ The premature deficiency of TFF3 and lysozyme in patients with NEC reduces mucus viscosity and contributes to intestinal necrosis.¹²⁴

Celiac disease

Celiac disease (CeD) is a chronic immune disease characterized by lesions of the small intestinal mucosa caused by gluten intolerance, with an average prevalence of up to 1% globally, and its typical symptoms include diarrhea, abdominal pain, anemia, osteoporosis, etc.¹²⁵ As gluten contains many repeated amino acid sequences (e.g. glutamine, proline), it cannot be effectively degraded by digestive enzymes in the human gastrointestinal tract, resulting in the formation of long, digestion-resistant oligopeptides. These oligopeptides accumulate in the mucosal epithelium of the small intestine, which are prone to inducing both innate and acquired immune responses.¹²⁶ Gluten contains glutelin, and under normal conditions there is limited translocation of the toxic gliadin fraction of gluten to the lamina propria of the gut.¹²⁷ However, due to the inhibition of tight junction proteins (TJ proteins) in CeD patients, increasing intestinal permeation, may allow for a higher interaction of the gliadin peptides with the immune system. This activates and triggers a series of acquired immune responses, resulting in atrophy of the villi of the small intestinal mucosa, crypt hyperplasia and infiltration of inflammatory cells.^128–130 These factors can result in the compromise of intestinal barrier function, facilitating the translocation of microbial antigens and endotoxins from the lumen to the lamina propria, and consequently inducing a pro-inflammatory microenvironment.

Irritable bowel syndrome

Irritable bowel syndrome (IBS) is a clinical syndrome of brain-gut axis interaction disorders with abdominal pain and distension accompanied by changes in feces character and bowel habit as the main symptoms.¹³¹ It can be clinically divided into three subtypes: IBS-C (constipation-based category), IBS-D (diarrhea-based) and IBS-M (mixed bowel movements with constipation and diarrhea).¹³² Intestinal barrier damage is prevalent in patients with IBS, and there is a positive correlation between the degree of abdominal pain and the extent of the damage.¹³³ Researchers have proved that IBS patients have a reduced goblet cells, changes in the structure of mucin O-glycans, decreased levels of MUC2 in the gut, and a reduced thickness of the mucus layer compared to healthy controls.⁸⁹ Meanwhile, PAR2, a G protein-coupled receptor,¹³⁴ is widely expressed in the gut and plays an important role in immune inflammation. Some animal experiments have demonstrated that PAR-2 expression and serine protease expression are higher in the IBS mouse model than in the control group, combined with intestinal barrier dysfunction. The mechanism of which may be that serine proteases inhibit the expression of TJ proteins through the activation of PAR2 receptor, causing a higher intestinal permeability and impaired barrier performance.^135,136

Hirschsprung’s disease

Hirschsprung’s disease (HSCR) is a rare congenital disorder of the intestines characterized by a lack of ganglion cells in the distal rectum that extend a variable distance into the proximal bowel. This lack of ganglion cells leads to a functional blockage of the bowel, which results in symptoms such as constipation, bloating and vomiting.¹³⁷ HSCR causes significant mortality and morbidity, but its pathogenesis remains unclear. Some studies suggest that changes in colonic epithelial surface goblet cells and the luminal mucus layer may play a critical role.¹³⁸ Related studies have proved that HSCR patients have reduced mucus turnover rates, lower mucin concentrations, abnormal ratios, and greater differences in the size and proliferation of goblet cells compared to the healthy state.^139,140 Meanwhile, the expression of TFF3, SPDEF, and KLF4 was significantly reduced in the ganglia and ganglionic colon of HSCR patients.^85,124 Secretory cell progenitor cells are the origin of goblet cells. SPDEF, a transcription factor belonging to the ETS family, and KLF4, a specific goblet cell differentiation factor in the colon, work synergistically to promote secretory progenitor cells to terminal differentiation in the direction of goblet cells and reach a mature state, while initiating mucin synthesis activities.^141,142 Reduced expression of goblet cell populations and insufficient mucin secretion can lead to a thinner and more permeable mucus layer, making the host susceptible to the attachment and invasion of enteropathogenic organisms, ultimately leading to intestinal barrier dysfunction.

Colon cancer

Colorectal cancer(CC)is the third most common cancer in the world, with about 1.9 million new cases and 900,000 deaths worldwide in 2020, and about 3.3 million new cases are expected by 2040.¹⁴³ The pathogenesis of CC remains incompletely understood and is currently thought to be the result of a combination of environmental and genetic factors. In addition, IBD such as UC and CD also contribute to the progression of CC.¹⁴⁴ Dysregulated expression of mucin, and abnormal patterns of its glycosylation have a close association with property and extent of CC.¹⁴⁵ Compared to non-neoplastic mucus, colon cancer mucus overexpresses MUC1, MUC5AC, and MUC17. While in contrast, MUC2 and MUC4 are progressively depleted during carcinogenesis. These aberrant expressions impair the barrier and regulate the level of tumor proto-oncogenes and oncogenes.^143,146 Overexpression and aberrant glycosylation of MUC1 promotes the proliferation of tumor cells. Its associated sialic acid antigen binds to a variety of receptors present on dendritic cells, macrophages and natural killer cells, which contributes to the escape of tumor cells from immunosurveillance by inhibiting the anti-tumor effects of immune cells, thereby promoting cancer cell differentiation, proliferation, invasion and metastasis.^147–149 Meanwhile, MUC2 deficiency leads to damaged barrier performs, dysbiosis of the gut microbiota and spontaneous intestinal inflammation.

Mucus barrier and extraintestinal diseases

Non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, is one of the most common chronic liver injury diseases, with an incidence rate of 25.2% in the world.¹⁵⁰ NAFLD includes simple NAFLD, nonalcoholic steatohepatitis (NASH) and cirrhosis, and once NAFLD progresses to NASH, the risk of going on to hepatocellular carcinoma (HCC) increases dramatically.^151,152 As an essential organ, the liver participates in axunge, proteide, and carbohydrate metabolism, enzyme activation, and glycogen reservoir, which intensely associated with the intestines in a bidirectional intestinal-liver-axis relationship.¹⁵³ As a core structural element of the gut-liver axis, the intestinal shield constitutes a physical and functional barrier separating the intestinal microbial community form the liver.¹⁵⁴ NAFLD patients are often accompanied by altered gut barrier function, in the way that inflammatory factors, microorganisms, and exogenous substances in the intestine can affect the physio pathological state of the liver through the portal vein.¹⁵⁵ It has been shown that rising inflammation of the gut mucosa and damage of the epithelial shield, which increases the likelihood of microbial product translocation, are extently associated with the progression of NAFLD. In NAFLD patients, the expression of TJ proteins was lower than healthy volunteers. This down-regulation directly leads to corruption to the gut mechanical shield, increasing the permeation of the gut lining. As permeability increases, microbes and exogenous substances in the gut more easily cross the intestinal barrier and enter the blood circulation. At the same time, inflammatory mediators released by the liver may also flow back to the intestine through the bloodstream, further exacerbating the damage to the intestinal barrier.^156,157 In addition, farnesoid X receptor (FXR) is a bile acid-activated receptor that enhances epithelial barrier properties, repairs intestinal vascular barrier damage, and controls metabolic syndrome.¹⁵⁸ Some studies have revealed reduced bile acid flow, insufficient bile acid lumen levels and reduced gut motility in the intestines of patients with NAFLD.⁹⁴ Under these conditions, the gut microbiome undergoes significant changes, with large numbers of bacteria producing secondary bile acids. For example, deoxycholic acid, an FXR-antagonistic bile acid that inhibits FXR-mediated signaling, thereby impairing mucus and antimicrobial peptide synthesis as well as the integrity of the intestinal vascular barrier.¹⁵⁹

Acute pancreatitis

Acute pancreatitis (AP) is edema and hemorrhage of pancreatic tissue caused by various etiologies, accompanied by necrosis of the pancreas or peripancreatic tissues and organ failure.¹⁶⁰ Although the level of clinical diagnosis and treatment has been improving in recent years, the mortality rate is still as high as 20% to 40% due to early multi-organ failure and late infectious complications in the pancreas.^161,162 The gut barrier dysfunction is one of the common complications in patients with AP, with 59% of them presenting with varying degrees of intestinal mucosal barrier dysfunction in the early course of AP.¹⁶³ Release of inflammatory mediators, enterocyte apoptosis, microcirculatory disorders and ischemia-reperfusion injury at the early stage of acute pancreatitis can cause intestinal mucosal barrier damage.¹⁶⁴ When the intestinal mucosal barrier becomes dysfunctional, intestinal bacteria are prone to translocation, which can lead to enterogenic infections and endotoxemia. In this case there is an increased risk of systemic inflammatory response syndrome and possibly even multiple organ failure, which can exacerbate acute pancreatitis.¹⁶³ During the course of AP, due to capillary leakage, a large amount of fluid enters the tissue interstitial leading to a reduction in effective circulating blood volume. Subsequently the blood redistributions throughout the body via neurohumoral regulation reduce intestinal perfusion, the intestinal mucosa becomes ischemic and hypoxic. And after a large amount of rehydration therapy, the intestinal mucosa undergoes reperfusion injury.¹⁶⁵ The xanthine oxidase and hypoxanthine produced in its process release a large number of oxygen free radicals, causing lipid peroxidation damage to intestinal epithelial cells, destroying the intercellular TJs structure and increasing the permeability of the intestinal mucosal barrier.^166,167 Phosphoenolpyruvate carboxykinase 1 (PCK1) is the first rate-limiting enzyme of the gluconeogenesis pathway in organisms, which is regulated by a variety of stress signals such as inflammation, hypoxia, and oxidation.¹⁶⁸ Yin et al.¹⁶⁹ found that PCK1 expression was significantly elevated in AP mice and AP patients. It was also demonstrated that inhibition of Pck1 attenuates pancreatic and intestinal injury and maintains intestinal homeostasis by improving intestinal permeability, lysozyme secreted by Paneth cells, MUC2 in goblet cells, and intestinal immune responses.

Diabetes

Diabetes mellitus is a chronic hyperglycemic disorder caused by insufficient insulin secretion or defective insulin action, often accompanied by disturbances in carbohydrate, lipid, and protein metabolism. In recent years, the incidence of diabetes mellitus has gradually increased, with type 2 diabetes mellitus(T2DM) accounting for more than 90% of the prevalence rate.¹⁷⁰ T2DM is a disease state characterized by persistent hyperglycemia, and its onset is closely related to obesity. The high intake of high-fat and high-cholesterol foods in Western dietary habits has become an important driving factor for the widespread epidemic of obesity and the occurrence and development of metabolic syndrome. Research evidence shows that such dietary patterns increase the permeation of the gut barrier and promote the leakage of lipopolysaccharide (LPS) into the blood, which in turn triggers the pathological process of metabolic endotoxemia.^171,172 Continuous infusion of low-dose LPS to mimic metabolic endotoxemia results in the progression of T2DM and atherosclerosis, highlighting the causal relationship between gut barrier dysfunction and the development of metabolic disease.¹⁷³ There is a significant correlation between T2DM and impaired intestinal barrier, so the mechanism of impaired intestinal barrier function in the development of T2DM is worthy of further exploration. It has been clinically demonstrated that patients with T2DM have markedly increased serum LPS, zonula occludens (ZO) and intestinal fatty acid-binding protein(IFABP) levels, indicating damage to intestinal epithelial cells and their TJs structures.¹⁷⁴ LPS is an essential element of the outer membrane of all G-bacteria, which can enter the blood across the gut when its permeability is increased.¹⁷⁵ A meta-analysis concluded that LPS levels were 64% higher in T2DM patients compared to non-diabetic groups.¹⁷⁶ Similar results were found in animal experiments, where structural abnormalities and altered permeability of the TJ proteins claudins-1, −2, −3, and ZO-1 in the duodenum and jejunum were observed in mice induced to enter the prediabetic stage by a high-fat diet.¹⁷⁷ In addition, myosin light chain kinase (MLCK), an key regulator of TJ proteins, is significantly expressed in T2DM model mice.⁹⁸ MLCK induces filament contraction of actin at the peripheral border, resulting in structural and functional dysregulation of TJ proteins, cell gap formation, and increased barrier permeability.¹⁷⁸ Also, in the context of insulin resistance, MLCK stimulates impairment of insulin sensitivity by promoting the level of inflammatory factors, such as TNF-α, IL-6, and IL-1β, which further exacerbates the onset and development of T2DM.¹⁷⁹

Diseases of the central nervous system

The microbe-gut-brain axis is a transmission system that builds two-way bridge between the brain and the gastrointestinal tract.¹⁸⁰ It consists of the central nervous system, the autonomic nervous system, the hypothalamic-pituitary-adrenal axis, the enteric nervous system, and the gut microbiota.¹⁸¹ On one hand, the cranial neural system can indirectly induce changes in the structure of intestinal microbial communities by adjusting the peristaltic rhythm, secretion behavior and intestinal wall permeability characteristics of the gastrointestinal tract. On the other hand, the intestinal microbial community and its metabolic by-products can exert influence on the central regulatory system with the help of the neuromodulation system, endocrine regulatory mechanism and immune response system in the brain-gut axis pathway, thereby regulating the higher functions of the brain.^180,182,183 The enteric nervous system is the body’s “second brain” and exhibits complex interactions with other organs, tissues and systems in the body.¹⁸⁴ From a mechanistic point of view, it communicates with the brain through a variety of neurological, endocrine and immune system pathways. It not only affects the brain’s cognitive, emotional and psychiatric symptoms, but also induces neurodegenerative diseases (NDDs) including Parkinson’s disease, Alzheimer’s disease, autism spectrum disorders and depression.¹⁰⁰ Simultaneously, studies have shown that NDDs all exhibit altered integrity of the intestinal barrier as well as dysbiosis of the intestinal flora.^185,186 In a study, excessive Aβ deposition was found in the intestinal epithelium of mice with increased intestinal permeation, inflammatory changes and reduced TJ proteins in a transgenic mouse model of Alzheimer’s disease (AD).¹⁸⁷ Surprisingly, there have also been reports of plasma LPS concentrations in AD patients that are 3 times higher than in the healthy population, suggesting that the intestinal barrier may be compromised. At the same time, these observations further support the importants that gut microbes may act in the pathological mechanism of AD.¹⁷⁵ In addition, metabolites produced by gut microbes, such as short-chain fatty acids (e.g. butyrate), help maintain the integrity of the intestinal epithelial barrier and host immunity.¹⁸⁸ Butyrate enhances the shield integrity by engaging various G protein-coupled receptors, which in turn activate intracellular pathways and upregulate the expression of genes responsible for TJ proteins.¹⁸⁹ Butyrate also promotes IL-22 by innate lymphoid cells and CD4+ T cells to maintain gut homeostasis through activation of free fatty acid receptor 3 and inhibition of histone deacetylase.¹⁹⁰ Liu et al.¹⁹¹ also concluded that butyrate significantly blocked cognitive impairment and anxiety, along with a neuroprotective effect by decreasing neuroinflammation and oxidative stress in the brains of AD mice. Therefore, short-chain fatty acids are critical in the onset and development of AD.

Airway disease

The airway epithelium consists of various cell types that, along with the immune system, create a robust barrier to prevent the invasion of foreign particles and pathogens. There are 14 mucins associated with the human respiratory system, with MUC5AC and MUC5B being the most prevalent. Together, these two mucins account for over 75% of the total mucin composition. These mucins are classified as gel-forming mucins and play a key role in determining the volume and viscosity of airway mucus.¹⁷ In the proximal airway, MUC5AC is mainly produced in the upper respiratory tract by surface epithelial goblet cells, whereas MUC5B is mainly secreted by mucous cells of submucosal glands. In the distal airway, both MUC5AC and MUC5B are produced by secretory cells (Club cells) on the epithelial surface. The development of most respiratory diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, asthma, and chronic rhinosinusitis, is associated with airway mucus plugs composed of MUC5B and MUC5AC (Table 4).²⁰²

Table 4.

Characteristics of mucus in airway mucosal diseases and other diseases.

Name of diseases	Model	Mucus characteristics	Therapeutic options	References
Chronic obstructive pulmonary disease	Animal	Increased mucus viscosity; Increased concentrations of MUC5AC and MUC5B; Surface epithelial mucous metaplasia and some hyperplasia; Presence of Achromobacter and Klebsiella.	Phloretin: Through inhibition of the EGFR/ERK/P38 signaling pathway, Reduced mucus secretion, inflammatory cell infiltration and inflammatory cytokine release, inhibition of MUC5AC and IL-1β expression in bronchial epithelial cells.	192,193
Cystic fibrosis	Animal	Hyperplasia of goblet cells and submucosal glands; Increased mucus viscosity; Increased concentrations of MUC5B and MUC5AC; S. aureus and P. aeruginosa, thought the major infectious factors of CF lung damage.	Naringin: Reduction of fluid viscosity by lowering MUC5AC and total protein secretion; Increased CFTR, AQP1, and AQP5 mRNA and protein expression, positively regulate apical CFTR insertion and promote CFTR activation by increasing intracellular cAMP.	194,195
Asthma	Animal	Hypertrophy and hyperplasia of goblet cells, increased airway mucin secretion and storage; Surface epithelial mucosal chemosis and indeterminate hyperplasia; Increased expression of MUC5AC gene and decreased expression of MUC5B gene.	Lonicerin: Through inhibiting the activation of Src/EGFR pathway, reduced the number of hyperresponsive inflammatory cells (especially eosinophils); Significantly attenuated inflammatory infiltrates and mucus secretion in lung tissue, as well as MUC5AC mRNA levels.	17,196
Chronic rhinosinusitis	Human	Hyperplasia of goblet cells and submucosal glands; Sustained increase in mucus secretion and highly dehydrated mucus with increased mucus viscosity; The expression levels of MUC5AC mRNA and MUC5B mRNA were significantly higher.	Surgical treatment is primarily performed. Medication-assisted treatment with drugs that reduce inflammation, decrease bacterial or pathogen load, and promote drainage of mucus or pus from the nose, such as antibiotics, topical intranasal steroids.	197,198
Dry eye disease	Animal	Decreased number of goblet cells, squamous metaplasia of the conjunctiva; Significantly reduced MUC5AC concentration.	β-aminoarteether: Reducing dry eye disease symptoms by regulating the TLR4/NF-κB/NLRP3 signaling pathway; Increased tear secretion, maintained the number of conjunctival goblet cells, reduced corneal damage, and lowered levels of conjunctival inflammatory mediators (TNF-α, IL-6, IL-10, or IL-1β).	199,200
Otitis media with effusion	Animal	Significant thickening of the middle ear mucosa, increased number of goblet cells, and increased mucus secretion; Loss or shortening of cilia; The expression level of MUC5AC was significantly highe.	Astaxanthin: Attenuates OME by inhibiting the Notch1/Hes1/mTORC1/S6K1 signaling pathway; Attenuates mucosal cell proliferation and reduces mucus secretion from goblet cells; Significantly reduced the synthesis and release of pro-inflammatory cytokines (IL-17, IL-6, TNF-α) and down-regulated MUC5A and TFF3 genes.	201

Annotation: EGFR, Epidermal growth factor receptor; ERK, Extracellular regulated kinase; P38, P38 Mitogen-activated protein kinase; AQP1, Aquaporin 1; AQP5, Aquaporin 5; cAMP, Cyclic adenosine monophosphate; Src, Non-receptor tyrosine kinase; Notch1, Neurogenic locus notch homolog protein 1; Hes1, Hairy and enhancer of split-1; mTORC1, Mechanistic target of rapamycin complex 1; S6K1, Ribosomal protein s6 kinase beta-1.

Chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a slow respiratory disease characterized by persistent irreversible obstruction of airflow. Common symptoms include dyspnea, cough, and sputum production. Over time, the symptoms of dyspnea gradually worsen, and during the exacerbation of the condition, the respiratory symptoms worsen, leading to further decline in lung function.²⁰³ The pathophysiological of airway mucus hypersecretion (AMH) is intricate, with one of the main pathophysiological features of COPD. Some studies suggest that it is closely related to epidermal growth factor receptor (EGFR), inflammatory responses, and tobacco smoke exposure (CSE).^204,205 Studies have shown that under the stimulation of various pathogenic factors such as smoking, infection, oxidative stress, air pollutants, etc., the respiratory tract produces a large number of pro-secretory factors and inflammatory mediators such as neutrophil elastase (NE), IL-13, TNF-α, LPS, EGFR, and so on. These irritating factors cause airway submucosal gland hypertrophy and goblet cells chemotaxis, up-regulation of the expression of MUC genes, mainly MUC5AC and MUC5B, and excessive mucin synthesis and secretion, which ultimately leads to AMH.^202,206,207 Bronchoscopy showed that compared with the healthy control group, patients with COPD showed significant pathological features such as decreased cilia number, shortened cilia length, and decreased cilia swing rate, which exacerbated mucus clearance disorders, resulting in worsening of clinical symptoms, recurrent infections, and accelerated decline of lung function.²⁰⁸ In addition, airway epithelial cells form an intact barrier by TJ proteins and adhesion junction proteins such as occludin and ZO.²⁰⁹ Pathogenic microorganisms or harmful gases, such as cigarette smoke extracts, can directly induce an inflammatory response, which in turn compromises the integrity of the epithelial barrier. During this course, inflammation-mediated loss of TJ and adhesion junctions makes the epithelial tissue susceptible to deeper infection, exacerbating the slow inflammation condition that persists in COPD patients.²¹⁰ Excessive mucus accumulation produces pathological consequences such as airway obstruction, airflow limitation, impaired gas exchange, ventilation/blood flow imbalance, recurrent respiratory infections, and even the formation of sputum plugs that obstruct the airway, and eventually life-threatening.

Cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene encoded by chromosome 7. The pathological changes and clinical manifestations can occur when CFTR deficiency is expressed in the epithelium of respiratory tract, pancreas, gastrointestinal tract, bile duct, sweat gland and some reproductive organs.²¹¹ Lung lesions are the main manifestation and its leading cause of death, and almost all patients develop COPD co-infections, leading to progressive loss of lung function.²¹² CFTR is an anionic channel for chloride, bicarbonate, the antioxidant thiocyanate, and glutathione.²¹³ Dysfunction of the CFTR primarily results in decreased chloride secretion and increased sodium absorption, as well as decreased bicarbonate secretion.²¹⁴ In the absence of a bicarbonate- and sodium-rich environment to remove and displace calcium, mucins are unable to undergo normal post-translational processing and amplify to their linear form, but remain compactly cross-linked, and the mucus becomes thicker and adheres to surface epithelial cells.¹⁰⁷ One study found that MUC5AC and MUC5B concentrations were elevated 30-fold and 8-fold, respectively, in CF patients compared to sputum from healthy controls, and that increased expression of MUC5AC in the airway surface epithelium and MUC5B in submucosal glands led to an increased rate of secretion of mucin into the respiratory tract, which led to mucus plug formation, infection and inflammation.²¹⁵ Meanwhile, β-tubulin expression is reduced in CF patients, which expression suggests cilia/ciliated cell defects, making mucus more difficult to remove.²¹⁶ These changes result in a decreased efficiency of mucus clearance, which creates a pernicious cycle of infection, inflammation, and injury that ultimately cause progressive deterioration of lung function.

Asthma

Asthma is a slow airway inflammatory characterized by hyperplasia of airway basal cells and goblet cells, hypersecretion of mucus and infiltration of the airways by mast cells, eosinophils, and lymphocytes. Clinical manifestations such as coughing, wheezing, chest tightness and reversible airflow limitation.^217–219 One study has shown that asthmatics store three times the normal amount of mucin in the airway epithelium.²²⁰ In healthy populations, a good balance exists between the production and clearance of MUC5AC and MUC5B. However, this balance is disrupted in asthmatics, as evidenced by goblet cell hyperplasia, increased secretion of MUC5AC, and a relative decrease in MUC5B, leading to altered mucus properties and decreased mucus clearance.^44,221 Ciliary clearance is an active defense mechanism of the airway epithelium. The mucus contains antimicrobial proteins such as lysozyme, immunoglobulin and antimicrobial peptides except mucins, with the ability of capturing inhaled particles, which are then cleared from the body by the regular oscillation of the cilia.²²² Impaired ciliary clearance of airway epithelial, observed in mild stable asthma, and further damaged in acute exacerbations will deeply exacerbate airway mucus obstruction, which leads to persistent airway inflammation in asthma.²²³

Chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is a common disease in otorhinolaryngology, which is characterized by runny nose, nasal congestion, impaired sense of smell, headache and facial pain.²²⁴ Mucus hypersecretion is common in patients with chronic sinusitis. Under normal circumstances, the mucous glands of the nasal mucosa and the goblet cells in the epithelium secrete mucus, which keeps the nasal mucosa moist, maintains the physiological function of the nose and prevents it from disease. When chronic inflammation occurs in the nasal mucosa, mucous glands and goblet cells are hypersecretory, producing mucous rhinorrhea, which affects the mucous cilia clearance rate, leading to mucous retention, aggravating inflammation, forming a vicious circle and resulting in respiratory complications.²²⁵ In the histological assessment of chronic rhinosinusitis (CRS), patients exhibit characteristics such as dysfunction of the nasal mucosal barrier, compromised mucociliary clearance, presence of inflammatory cell infiltration, hypersecretion of mucus, and remodeling of tissue architecture.^226,227 In CRS patients, mucins, particularly MUC5AC and MUC5B, constitute the primary components of airway mucus and significantly contribute to the pathophysiological alterations observed in the condition.²²⁸ Several studies have investigated the expression patterns of mucins in normal sinus mucosa and CRS, which have shown that the expression levels of MUC5AC mRNA and MUC5B mRNA are significantly higher in CRS compared to normal sinus mucosa.^229,230 MUC2 is a major component of intestinal mucin, which forms small molecular complexes in healthy human airway mucus. Given the altered mucus elasticity in chronic sinusitis, it has been reported that MUC2 mRNA levels are significantly higher in patients with chronic sinusitis. Therefore, the up-regulation of MUC2 expression in chronic sinusitis may play an important role in the pathogenesis of hypersecretion of CRS.²³¹

Other diseases

Dry eye disease

As an ophthalmic disease with a high incidence, the symptoms of dry eye syndrome (DED) are mainly dryness, sensation of foreigners or burning, sensitivity to light, indistinct vision, etc., and in serious cases, it will even affect vision and normal life.²³² Destabilization of the tear film is the central mechanism causing dry eye. The normal tear film consists of an aqueous layer, a lipid layer, and a mucin layer, of which the mucin layer, located in the innermost layer, is an important component of the tear film.²³³ There are several common histopathological manifestations of ocular surface epithelial tissue in patients with DED, including loss of conjunctival goblet cells, abnormally enlarged epithelial cells (squamous metaplasia), and an abnormal pattern of mucin glycosylation.^199,234 These alterations lead to increased tear evaporation and elevated tear osmolarity, which in turn triggers altered osmolarity of epithelial cells and an inflammatory response that disrupts the normal ocular surface environment.^15,199 On the ocular surface, the main gel-forming mucin expressed by conjunctival goblet cells is MUC5AC.²³⁵ MUC5AC aggregates and enhances the removal of pathogenic microorganisms and cellular debris from the mucosal surface and lubricates the ocular surface in a hydrated gel state to protect the ocular surface environment. It has been reported that conjunctival MUC5AC expression is reduced in patients with DED, and the normal concentration of MUC5AC in tear fluid is 8.2–32.4 ng/mg, whereas the concentration in patients with DED is only about 3.5 ng/mg.²³⁶ Tear mucin and tear osmolarity play an important role in the pathogenesis of dry eye, but their molecular weights are much lower than those of other tissues, and simple and rapid clinical measurements are still lacking.

Otitis media with effusion

Otitis media with effusion (OME) is a non-purulent inflammatory disease of the middle ear characterized by middle ear effusion and conductive hearing loss.²³⁷ Under normal conditions, mucin is synthesized and secreted to protect the middle ear epithelium and trap a variety of microorganisms. However, if the middle ear is stimulated to produce large quantities of mucin, it increases the consistency of the mucus layer and middle ear effusion, leading to dysfunction of the mucus cilium transport system in the middle ear and Eustachian tube, and the formation of a viscous middle ear effusion.²³⁸ The development of OME is characterized by neutrophilic infiltration, mucosal thickening, submucosal glandular hyperplasia, goblet cells hyperplasia, and cilia loss and shortening.^239,240 At least five mucins have been shown to have an important role in the pathogenesis of otitis media. The tympanic chamber expresses predominantly MUC5B, the eustachian tube expresses MUC5AC, MUC5B, MUC2, and MUC1, and the middle ear produce mucus mainly MUC5AC and MUC5B.^238,241 Some studies have revealed elevated level of MUC2, MUC5AC, and MUC5B genes of the middle ear in OME patients who are more likely to produce more viscous mucus, leading to a higher incidence of mucosal ciliary gap abnormalities, stagnation of fluid in the middle ear, and ultimately, chronic disease.^242–244

Conclusions and perspectives

The variety of functions of mucus is due to the complexity of mucin structure and the adaptability of mucus gels to different environments and needs. The proper functioning of the mucus barrier depends on the number of goblet cells, mucin expression levels, modification processes, secretory activity, and the rheological properties of the mucus. Mucins, as the major macromolecules in human mucus, are critical to the integrity of the mucus layer. And a large body of research has demonstrated the unique properties of mucins that can potentially be beneficial or detrimental to pathogens by facilitating intra-host binding and sequestration. However, many questions remain about our understanding of the details of the mechanisms by which mucus, and in particular mucins, regulate disease progression within and outside the host. For example, the glycosylation pattern of mucin-glycans has been shown to be critical for a variety of diseases, but it is not clear exactly how this is regulated. Similarly, the maintenance of homeostasis in the human body relies on a complex and dynamic balance and interaction between mucins, microbial communities, and host immune defenses, which together have an impact on human health, although the specific associations and regulatory mechanisms between them are not well understood. In the human mucus system, mucin variation is closely related to physiological and pathological changes. With the increasing recognition of its physiological function and clinical value, mucin is gradually established as a key molecule, biomarker and potential therapeutic target for regulating homeostasis. Deepening the understanding of the mechanism of action of mucin in a healthy state will provide new insights and strategies for understanding and treating mucinous dysfunction-related diseases.

In conclusion, the mucus barrier, once seen primarily as a lubricant, insulator, and humectant, is now recognized for its critical “dynamic” role in infection resistance, immune modulation, and the pathogenesis of various diseases. Since multiple mucins have become potential therapeutic targets for a variety of diseases, in-depth research on their functions and regulatory pathways may provide new directions for new treatments for related diseases.

Funding Statement

Major Programs of Natural Science Research in Colleges and Universities in Anhui Province [No. 2023AH040108]; Scientific Research Team Program of Anhui Colleges and Universities [Grant no.2022AH010036].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ contributions

Fangfang Fan and Xiaoqin Chu wrote the manuscript. Ruihan Guo prepared the figures and tables; Kun Pan designed and revised the paper; HongYe Xu controlled the language editing. All authors read and approved the final manuscript.

Availability of data and materials

The date used to support the findings of this study are available from the corresponding author upon reasonable request.