Advances in cellular and molecular mechanisms of oral submucosal fibrosis

LIN Ya-nan, WANG Tao?

1.School of Stomatology, Hainan Medical University, Haikou 571199, China

2.Department of Stomatology, Hainan General Hospital, Haikou 570311, China

Keywords:

ABSTRACT Oral submucosal fibrosis (OSF) is a pathological process in which the oral mucosa forms abnormal repair in persistent chronic injury.It is characterized by inflammatory reactions,massive extracellular matrix deposition and vascular loss, ultimately leading to structural and functional damage of the oral mucosa.The infiltration of inflammatory cells is the fuse of oral submucosal fibrosis, suggesting that the immune microenvironment of local tissues plays an important role in its occurrence and development.Activated myofibroblasts can secrete large amounts of extracellular matrix, which reassemble and remodel, thereby resisting collagenase degradation, while the loss of blood vessels can significantly aggravate the progression of fibrosis.This article reviews the etiology of oral submucosal fibrosis and the effects of local tissue immune environment imbalance, extracellular matrix deposition and vascular loss on the progression of oral submucosal fibrosis.

1.Introduction

Oral submucous fibrosis (OSF) is a general term for a series of symptoms caused by excessive deposition of submucosal collagen, which mainly includes oral ulcers, dry mouth, burning sensation and limited mouth opening, which will affect the oral function and quality of life of patients.In addition, OSF is also a precancerous lesion, and its malignant conversion rate is as high as 7% to 13%, which significantly increases the mortality of patients[1].Etiologically, it is generally accepted that OFS is associated with areca nut chewing, but in recent years, relevant studies have found that epigenetic inheritance is also involved in this lesion.Pathogenetically, oral submucosal fibrosis is a disease in which healing fails after mucosal injury.The initial stage of oral submucosal fibrosis allows inflammatory cells to accumulate,induces the production of profibrotic cytokines and growth factors by related cells, and activates myofibroblasts to secrete large amounts of extracellular matrix deposition and lead to vascular loss.At present, there are various treatment methods, of which vasodilator therapy is the main treatment, but the total effective rate is caused by vascular loss.Therefore, in order to further understand the occurrence and development of this disease and further develop new therapeutic targets, this article reviews the research progress of the etiology, immune environment changes, extracellular matrix deposition and vascular loss of oral submucosal fibrosis.

2.Pathogenic factors of oral mucosal fibrosis

2.1 Local Factors

Epidemiology suggests that areca nut chewing is one of the most dangerous causative factors for OSF.Relevant studies have shown that, in China, almost all patients with OSF have the habit of chewing areca nut and OSF There is a direct linear correlation between incidence and frequency of betel quid chewing[2].

Studies have shown that arecoline (ANE), the most abundant alkaloid in areca nut, plays an important role in OSF pathogenesis, ANE triggers inflammation of the oral mucosa , which in turn secretes inflammatory factors and cytokines to initiate fibrosis,and activates myofibroblasts and secretes large amounts of extracellular matrix[3], at the same time, Downregulation of matrix metalloproteinases(MMP) by ANE, and up-regulation of matrix metalloproteinases - 1 (TIMP-1) of tissue inhibitors, thereby reducing collagen degradation and inducing extracellular matrix(ECM) deposition[4].In addition, ANE has antiproliferative and cytotoxic effects on endothelial cells, which may lead to impaired vascular function, reduce local vascularity, and trigger hypoxia,which is involved Development of OSF[5].

A study showed that a significant proportion of betel quid chewers were also smokers (86%) or drinkers (74%), which may suggest that habits such as smoking and alcohol consumption will increase Risk of OSF.The results also indicated a significant increase in areca nut chewing and tobacco use OSF of morbidity and OSF induction has an additive effect[6].

2.2 Systemic Factors

2.2.1 Nutritional imbalance

Malnutrition (e.g., vitamin deficiency), changes in serum iron, zinc,and copper are related Development of OSF.Studies have found serum β-carotene and vitamins in patients with OSF Significant reduction in C, E levels , which disturbs the inflammatory repair response in the lamina propria, leading to healing and scratch defects, which are ultimately aggravated Development of OSF[7].Iron and serum ferritin levels are decreased in patients with OSF,total iron binding capacity (TIBC) Increase, which is similar to Fig.The clinical stage and histological grade of OSF are closely related,the decrease in serum iron can also change epithelial structure,increase mucosal permeability, and inhibit its barrier protection[8].Current studies have also found that zinc has the ability to resist fibrosis and can induce superoxide dismutase Activation of SOD,thereby inhibiting reactive oxygen species Generation of (ROS), the reduction of zinc can reduce the activity of the peroxidase system in the oral mucosa and remove certain harmful substances produced by arecoline stimulation.In contrast, patients also had higher serum copper levels, which enhanced collagen fiber cross-linking and lysyl oxidase of elastin (LOX), activity, increased collagen production and stabilization of collagen structure, in addition, increased salivary copper concentration is associated with increased clinical grade[9].

2.2.2 Genetic susceptibility

OSF has a certain genetic susceptibility, which is related to a variety of chromosomal, genetic and molecular changes.The results showed that the allele and genotype frequencies of collagen, MMPs,TGF-β1 and LOX were found to be higher in patients with OSF,which may change the transcriptional activity and the function of the corresponding proteins, leading to the genetic susceptibility of OSF[10].

Epigenetic versus genetic is that epigenetic changes are heritable and reversible.Epigenetics includes DNA methylation, histone modification and chromatin remodeling complexes and MicroRNA.A total of 3 294 differentially methylated regions were identified when DNA methylation changes were analyzed in 7 OSF tissues and 5 control tissues, and 38 hypermethylated down-regulated genes and 55 hypomethylated up-regulated genes were found compared with the transcriptome, and this study highlighted epigenetic global dysregulation in oral tissues of OSF patients[11].Dickkopf-1 is an inhibitor of the Wnt/β-catenin pathway, which is expressed at lower levels in OSF than in healthy tissue, but the gene is methylated at higher levels than in healthy tissue, while it is high.The level of dickkopf-1 methylation may be reduced Dickkopf-1 Of expression,which may lead to Aberrant activation of the Wnt/β-catenin pathway and development of OSF[12].At present, there are few reports on the role of epigenetic changes in the development of oral submucosal fibrosis at home and abroad, which may become the focus of future research due to reversible epigenetic symptoms.

3.Oral mucosal injury and changes in the immune microenvironment

Chewing areca nut can lead to mucosal tissue damage, which in turn allows inflammatory cells to accumulate and secrete profibrotic cytokines such as Toll-like receptors, interleukins, and chemokines,resulting in changes in the immune microenvironment, which affects the activation of myofibroblasts (MFB) and extracellular matrix deposition.

Toll-like receptors (TLRs) are a class of mediating innate immune responses It causes the activation of inflammatory cells and releases a series of cytokines, thus initiating or amplifying the inflammatory response, which is one of the important recognition receptors involved in the immune response, and is involved in the evolution and development of a variety of fibrosis.TLR2 may act through activation Stat3 pathway promotion Expression of TGF-β, promotes fibroblast proliferation, increases collagen synthesis, and aggravates Progression of OSF, in addition, it is associated with monocyte chemotactic proteins - 1 (MCP-1) and inflammatory cells synergize with each other , involved in the development of fibrosis[13].

Interleukins play an important role in the process of fibrosis.Interleukin action is complex and forms a network-like overlap.It has been shown that in In buccal mucosa of patients with OSF The levels of IL-1α, IL-1β, IL-6, IL-8, and IL-17 were significantly higher than those in normal mucosa.IL-I It increases collagen gene transcription in a dose-dependent manner and promotes ECM Accumulation[14].In a study SNAIL-IL-6 In Fig.Inhibition was found when the role in the differentiation activity of OSF myofibroblasts IL-6 not only downregulated many fibrosis markers such as α-SMA and Type I collagen, and it also inhibits Expression of Snail, indicating that IL-6 And Snail may mutually enhance or amplify each other in the regulation of myofibroblast activation,forming a profibrotic effect[15].Cytokines secreted by Th17 cells such as IL-17 in OSF have powerful proinflammatory effects,can induce the release of inflammatory cytokines and produce an inflammatory response in a variety of cells[16].

Chemokines are the largest subfamily of cytokines that can drive inflammatory cells to accumulate to the site of injury and synergize with profibrotic factors to promote the progression of fibrosis.CCL2 is involved in the pathogenesis of fibrosis in animal models, such as fibrous diseases such as atherosclerosis, cirrhosis, pulmonary fibrosis or glomerulosclerosis, and a recent study found that the expression of CCL2 in the late stage of OSF was increased compared with that in the early stage, and was associated with MFB Of the number is directly proportional to, possibly with OSF-associated fibroblasts actively participate in the secretion of CCL2, leading to the recruitment of myofibroblasts at this site of disease and promoting fibrosis progression[17].

The persistence of inflammation after chronic injury is a driver of fibrogenesis because it leads to a vicious cycle of inflammation,tissue damage, and fibrosis.The use of antagonists of the inflammatory response to control the activation and migration of inflammatory cells or even regress fibrosis may be a new direction of immunomodulatory therapy.

4.Oral submucosal extracellular matrix deposition

The main collagen types in oral submucosal fibrosis are Ⅰ, ⅢCollagen type, immunohistochemical analysis showed There was a significant correlation between the expression of type Ⅰ collagen and its partner collagen/heat shock protein in oral submucosal fibrotic lesions, and with the development of the disease, Type Ⅲcollagen is almost completely replaced by type Ⅰ collagen.Since collagens play a crucial role in the folding and assembly of collagen, so the increase in collagen content in oral submucosal fibrotic tissue may be due to excessive stabilization of collagenin[18].Fibroblasts in OSF tissues have been reported to synthesize more collagen than normal fibroblasts , which may be due to phenotypic shifts in these cells and irreversible transformation MFB, this myofibroblast can participate in the repair of wounds and fibrosis of organs by secreting collagen and reconstituting the extracellular matrix It has a stronger ability to synthesize collagen and secrete cytokines than fibroblasts,thus this so-called “activated fibroblast” controls Process of OSF[19].Myofibroblasts are widely sourced, mainly from fibroblast proliferation, epithelial-to-mesenchymal transition and endothelialto-mesenchymal transition[20], its pair The contribution of OSF is not known.If we can inhibit the activity of myofibroblasts or reduce the source of myofibroblasts, it may delay the development of fibrosis.Another major mechanism promoting fibrosis is reduced collagen degradation, resulting in a stable state of collagen bundle formation.Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases, can degrade collagen, while tissue inhibitor of matrix metalloproteinases (TIMPs) are inhibitors of collagen degradation.The imbalance between MMPs and TIMPs in OSMF has been found to occur with reduced expression of MMPs.On the one hand , confirmed Decreased MMP expression in OSF ,resulting in reduced collagen degradation, on the other hand, i.e.Overexpression of TIMPs , inhibits collagen degradation, so it causes excessive and persistent collagen deposition[21], while the imbalance between collagen synthesis and degradation may lead to the proportion of the triple helical structure of collagen molecules from Fig.Change from 2:1 to 3:1 , thus resisting the degradation of proteases[22].In addition, flavonoids (catechins and tannins) reduce collagenase activity, increase the cross-linking of collagen fibers,and force them to be less susceptible to collagenase degradation[23].All of the above can lead to reduced collagen degradation so that extracellular matrix deposition, aggravating fibrosis.

Collagen production and degradation are also controlled by the balance of profibrotic and antifibrotic cytokines, and if they are dysregulated they can alter the homeostasis of collagen production and promote the formation of various forms of fibrotic diseases.Among profibrotic cytokines, transforming growth factor - Β(TGF-β) plays a key role , studies have confirmed Activation and involvement of TGF-β pathway in oral submucosal fibrosis, which was significantly increased in areas with fibrosis, while in experiments on Fig.Inhibition by TGF-β can significantly weaken the development of fibrosis , visible TGF-β is closely associated with fibrosis[24].Another most important finding is the downregulation of anti-fibrotic genes, particularly bone morphogenetic proteins (BMP) -7.Bone morphogenetic protein is a multifunctional protein It belongs to the transforming growth factor-β superfamily,which mainly regulates the development and differentiation of tissues and organs.One of the functions of bone morphogenetic proteins is to regulate fibrosis, which has been reported BMP-7 can inhibit TGF-β-mediated renal fibrosis[25]And pulmonary fibrosis[26], Study Finding BMP-7 It has shown significant efficacy in reducing renal fibrosis, and these indicate that B MP-7 has an anti-fibrotic effect ,and may have the potential to treat oral submucosal fibrosis[25].

5.missing vessels

Vascular loss and increased vascular permeability are one of the pathological features of oral submucosal fibrosis.The increased permeability of blood vessels leads to the exudation of some molecules and triggers an inflammatory response, and with the progression of the disease, extracellular matrix deposition increases and leads to fibrosis, further leading to the occlusion and reduction of vascular vessels[27], the inability to induce reparative angiogenesis during this process is one of the reasons for the angiopenia.In fact, endothelial cells do not have the same regenerative capacity as epithelial cells, and they hardly proliferate in response to injury,followed by the loss of endothelial cells.In addition, arecoline is cytotoxic to endothelial cells, can arrest the cell cycle, and can easily lead to vascular endothelial cell injury or even apoptosis, therefore,consumption of areca nut can further lead to reduced vascularity of OSF[28].

Similar to epithelial cells, endothelial cells can also undergo phenotypic conversion into myofibroblasts, a phenomenon known as interendothelial conversion Endothelial-Mesenchymal Transition,EndMT).One study indicated that microvascular injury is prevalent in OSF, which is characterized by production EndMT, the main mechanism is that arecoline stimulates mitochondria, leading to Increased ROS, activates the ER stress-related PERK pathway and activates YAP When YAP translocation into the nucleus causes the expression of EndMT-related genes and enhances collagen secretion,leading to ECM accumulation and myofibroblast activation, has been shown VP reversed this process by inhibiting YAP-TEAD.In addition to the generation of small numbers of myofibroblasts,EndMT also resulted in the loss of endothelial cells, is considered to be the main cause of angiopenia[29].

It may be a promising strategy if fibrosis can be improved or even reversed by increasing therapeutic angiogenesis.Vascular endothelial growth factor (VEGF) is a functional glycoprotein with high activity It has the functions of inducing the proliferation, migration,new angiogenesis of vascular endothelial cells and maintaining the integrity of capillary endothelial cells[30].Confirmed by basic research Transfection of adenoviral vectors with VEGF can induce the formation of new blood vessels in fibrotic mucosa of mice[31], the same has been reported in renal fibrosis VEGF induces angiogenesis and /or capillary repair stabilizes renal function and slows the progression of renal fibrosis[30].Therefore, in Fig.It seems feasible that induction of VEGF expression on the basis of OSF prompts angiogenesis to improve fibrosis.

6.Prospects

In summary, oral submucosal fibrosis is a complex dynamic process, which involves multiple cells and cytokines.Recent studies have allowed us to more comprehensively understand the effects of inflammatory infiltration, extracellular matrix deposition, and microangiopathy on OSF.This paper expounds the etiology, cellular and molecular mechanisms of OSF, which is helpful to develop new therapeutic targets and carry out early diagnosis.There are still many key problems to be solved urgently in the complex process, but we still believe that the study of OSF mechanism is translated into an effective method for clinical treatment.

Author’s contribution:

The first author (Yanan Lin) wrote and the corresponding author(Tao Wang) reviewed.

All authors declare no conflict of interest.