Serum and salivary interleukin-1ß level in oral precancer: An observational study.

BACKGROUND AND AIM: Precancer biomarkers help in early detection and management of oral potentially malignant disorders (OPMDs). Interleukin-1ß (IL-1ß), a biomarker, is known to be altered in oral submucous fibrosis (OSMF) and oral leukoplakia (OL). Therefore, we evaluated and compared the serum and salivary IL-1ß levels in patients with OSMF/oral leukoplakia and in gender- and age-matched healthy individuals. MATERIALS AND METHODS: An in vivo, prospective, observational study was conducted on 40 subjects. Subjects were divided into two groups with 20 individuals in each group, that is, Group I: OSMF/oral leukoplakia and Group II: control group. Salivary and serum IL-1ß levels were quantitatively estimated using enzyme-linked immunosorbent assay (ELISA). The statistical tests used were unpaired t-test and Chi-square test. RESULTS: The serum IL-1ß levels were significantly (P 0.001) lesser in Group I in comparison to Group II. The salivary IL-1ß levels remained insignificant between both the groups. However, in both the groups, the salivary IL-1ß levels were significantly higher compared to the serum IL-1ß levels. CONCLUSION: We found that the serum IL-1ß level can be considered as a prospective biomarker for dysplasia, whereas salivary IL-1ß alone needs more elaborated studies to account for its application as a potential biomarker in OPMD.

Alteration of salivary LPO, MDA, LDH, glutathione, GPx, SOD and vitamins in oral submucous fibrosis: A three-level meta-analysis study.

This study aims to investigate the alteration of salivary biomarker profiling in the development of oral submucous fibrosis (OSMF) and to explore the influence of saliva in the diagnosis of OSMF. A systematic search of published articles using the PRISMA guidelines was conducted to identify relevant studies on OSMF and saliva. All eligible studies, including case-control, cross-sectional studies, cohort, and pilot studies, contained the evaluation of salivary biomarker profiling in patients with OSMF. Salivary biomarker data from 28 selected articles were categorized into nine groups, and their mean values were determined. A three-step meta-analysis was performed by grouping salivary biomarker profiling into more heterogeneous categories based on OSMF classification, considering functional, histological, and clinical grading. The salivary biomarker profiling analysis revealed significant alterations in all markers, indicating their efficacy in OSMF diagnosis. Subgroup analyses highlighted significant associations in oxidative stress and protein with increased mean values, particularly emphasizing lipid peroxidase (LPO), malondialdehyde (MDA), and lactate dehydrogenase (LDH). Conversely, decreased mean values were observed in glutathione, glutathione peroxidase (GPx), superoxide dismutase (SOD), and vitamins. Notably, OSMF grading analysis demonstrated a significant difference in weighted effect sizes for histological grading, particularly in stage IV. The study underscores the alteration of specific salivary biomarkers, particularly those associated with LPO, MDA, LDH, glutathione, GPx, SOD, and vitamins, in diagnosing and grading OSMF.

Expression profile of diagnostic genes in oral submucous fibrosis.

Oral Submucous Fibrosis (OSMF) is a chronic precancerous disorder of the oral mucosa caused by chewing of areca nut and its other variants. Chewing of areca nuts leads to dysregulated expression of specific genes, leading to various premalignant or malignant disorders. This study aimed to determine the differential expression of the diagnostic genes (MYH6, TNNT3, MYL1, and TPM2) in healthy controls and OSMF patients using saliva and tissue samples, determining the histopathological grade of the clinical samples. A total of 20 patients were included in the study and were divided into two groups: Group I consisted of 10 healthy patients (control group) and Group II consisted of 10 OSMF patients. Unstimulated whole saliva samples were collected from both groups, and the tissue samples were divided into two parts: one for RT-qPCR analysis and the other for histopathological assay. The expression profile of genes concerning OSMF saliva and tissue samples was significantly upregulated compared to the healthy control, and all the clinical samples of the study were categorized into histopathological grade 1. The findings of this study concluded that these genes can be referred to as diagnostic genes for OSMF in early and very early clinical samples, and saliva can be used as a promising diagnostic tool for early OSMF studies.

Role of Stem Cells in the Pathogenesis and Malignant Transformation of Oral Submucous Fibrosis.

BACKGROUND: Pathogenesis and malignant potential of Oral submucous fibrosis(OSMF) have always been a topic of interest among the researchers. Despite OSMF being a collagen metabolic disorder, the alterations occurring in the connective tissue stroma affects the atrophic surface epithelium in later stages and progresses to malignant phenotypes. The present review aims to summarize the role of stem cells in the pathogenesis and malignant transformation of oral submucous fibrosis. MATERIALS AND METHODS: A literature search was carried out using data banks like Medline and Embase, google scholar and manual method with no time frame, pertinent to the role of mucosal stem cells in OSMF and its malignisation. The relevant literature was reviewed, critically appraised by all the authors and compiled in this narrative review. RESULTS: Critical appraisal and evaluation of the data extracted from the selected articles were compiled in this review. The collated results highlighted the upregulation and downregulation of various stem cell markers during the progression and malignisation of OSMF were depicted in a descriptive and detail manner in the present review. CONCLUSION: We highlight the potential of mucosal stem cells in the regulation and malignisation of OSMF. However, future large-scale clinical studies will be needed to support whether manipulation of this stem cells at molecular level will be sufficient for the treatment and preventing the malignant transformation of OSMF.

Oropharyngeal stenosis in patient with oral submucous fibrosis: a case report with 8-year follow-up.

Oral submucous fibrosis (OSF) is a chronic, progressive condition affecting the oral mucosa associated with areca nut consumption. It leads to restricted tongue movement, loss of papillae, blanching and stiffening of the mucosa, difficulty in opening the mouth, and challenges in eating due to inflammation and fibrosis. This report presents a rare case of oropharyngeal stenosis secondary to OSF in a 43-year-old male with a history of chewing betel nut. A surgical procedure similar to Uvulopalatopharyngoplasty was performed to excise the submucous oropharyngeal stenosis and to reconstruct the uvula, palatoglossal arch, and palatopharyngeal arch. At 8 years postoperatively, the patient exhibited a normal mouth opening and oropharyngeal aperture.

Correlation between Vascularity and Advancing Histological Grades of Oral Submucous Fibrosis with a Plausible Role in Malignisation: Systematic review of a persisting matter of conflict.

Objectives: This study aimed to quantify the vascularity in histological grades of oral submucous fibrosis (OSMF) and to determine if there is any connection between vasculogenesis and malignisation. Recent studies show no significant change in vascularity as the stage advances as opposed to the conventional concept. Methods: A comprehensive database search until December 2022 was conducted for published articles on vascularity in OSMF following preferred reporting items for systematic reviews and meta-analyses guidelines. Results: A total of 98 articles were screened of which 13 were included for systematic evaluation. The study included 607 cases, with a definite predilection for the male gender. Of the 13 studies, 11 evaluated mean vascular density. In more than half of the studies, the vascularity decreased as the stage advanced. Similar results were obtained for endothelial cells/µm2, mean vascular area percentage and mean vascular area. Conclusion: The present review supports the prevailing concept that vascularity decreases with the advancement of the OSMF stage. This denies the systemic absorption of carcinogens into the circulation with resultant longer exposure of compromised epithelium and malignisation.

Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis.

Oral submucous fibrosis (OSF) is a chronic and inflammatory mucosal disease caused by betel quid chewing, which belongs to oral potentially malignant disorders. Abnormal fibroblast differentiation leading to disordered collagen metabolism is the core process underlying OSF development. The epithelium, which is the first line of defense against the external environment, can convert external signals into pathological signals and participate in the remodeling of the fibrotic microenvironment. However, the specific mechanisms by which the epithelium drives fibroblast differentiation remain unclear. In this study, we found that Arecoline-exposed epithelium communicated with the fibrotic microenvironment by secreting exosomes. MiR-17-5p was encapsulated in epithelial cell-derived exosomes and absorbed by fibroblasts, where it promoted cell secretion, contraction, migration and fibrogenic marker (α-SMA and collagen type I) expression. The underlying molecular mechanism involved miR-17-5p targeting Smad7 and suppressing the degradation of TGF-ß receptor 1 (TGFBR1) through the E3 ubiquitination ligase WWP1, thus facilitating downstream TGF-ß pathway signaling. Treatment of fibroblasts with an inhibitor of miR-17-5p reversed the contraction and migration phenotypes induced by epithelial-derived exosomes. Exosomal miR-17-5p was confirmed to function as a key regulator of the phenotypic transformation of fibroblasts. In conclusion, we demonstrated that Arecoline triggers aberrant epithelium-fibroblast crosstalk and identified that epithelial cell-derived miR-17-5p mediates fibroblast differentiation through the classical TGF-ß fibrotic pathway, which provided a new perspective and strategy for the diagnosis and treatment of OSF.

circMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

BACKGROUND: circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. METHODS: Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). RESULTS: circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. CONCLUSION: circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.

Panax notoginseng saponins improve oral submucous fibrosis by inhibiting the Wnt/ß-catenin signal pathway.

OBJECTIVE: Oral submucous fibrosis (OSF) is a chronic, insidious, progressive mucosal disease that may be affected by mutations in the Wnt/ß-catenin signaling pathway. Panax notoginseng saponins (PNS) is a powerful anti-fibrosis agent; however, its effect and mechanism in treating OSF remain unclear. This study investigated the effect and mechanism of PNS treatment for OSF. STUDY DESIGN: Arecoline was used to induce OSF models in vivo and in vitro, which were then treated with PNS. Hematoxylin-eosin (HE) and Masson trichrome staining were used to observe histopathology changes; E-cadherin and ß-catenin were detected by Immunohistochemical assay, and type Ⅰ collagen (CollA1) and ß-catenin were detected by immunofluorescent staining. The Wnt/ß-catenin pathway and fibrosis signs were assessed using Western Blot and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: The expression of CollA1, Wnt1, and ß-catenin were increased, and E-cadherin, GSK-3ß, and ß-catenin expression were decreased in OSF models. PNS and inhibitor intervention increased E-cadherin, Wnt1, and ß-catenin and decreased CollA1 and GSK-3ß in a dose-dependent manner. CONCLUSION: PNS can improve OSF by inhibiting the Wnt/ß-catenin signal pathway and thus may be used as a potential medicine for the treatment of OSF.

DPSCs regulate epithelial-T cell interactions in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion characterized by fibrous tissue deposition, the incidence of which correlates positively with the frequency of betel nut chewing. Prolonged betel nut chewing can damage the integrity of the oral mucosal epithelium, leading to chronic inflammation and local immunological derangement. However, currently, the underlying cellular events driving fibrogenesis and dysfunction are incompletely understood, such that OSF has few treatment options with limited therapeutic effectiveness. Dental pulp stem cells (DPSCs) have been recognized for their anti-inflammatory and anti-fibrosis capabilities, making them promising candidates to treat a range of immune, inflammatory, and fibrotic diseases. However, the application of DPSCs in OSF is inconclusive. Therefore, this study aimed to explore the pathogenic mechanism of OSF and, based on this, to explore new treatment options. METHODS: A human cell atlas of oral mucosal tissues was compiled using single-cell RNA sequencing to delve into the underlying mechanisms. Epithelial cells were reclustered to observe the heterogeneity of OSF epithelial cells and their communication with immune cells. The results were validated in vitro, in clinicopathological sections, and in animal models. In vivo, the therapeutic effect and mechanism of DPSCs were characterized by histological staining, immunohistochemical staining, scanning electron microscopy, and atomic force microscopy. RESULTS: A unique epithelial cell population, Epi1.2, with proinflammatory and profibrotic functions, was predominantly found in OSF. Epi1.2 cells also induced the fibrotic process in fibroblasts by interacting with T cells through receptor-ligand crosstalk between macrophage migration inhibitory factor (MIF)-CD74 and C-X-C motif chemokine receptor 4 (CXCR4). Furthermore, we developed OSF animal models and simulated the clinical local injection process in the rat buccal mucosa using DPSCs to assess their therapeutic impact and mechanism. In the OSF rat model, DPSCs demonstrated superior therapeutic effects compared with the positive control (glucocorticoids), including reducing collagen deposition and promoting blood vessel regeneration. DPSCs mediated immune homeostasis primarily by regulating the numbers of KRT19 + MIF + epithelial cells and via epithelial-stromal crosstalk. CONCLUSIONS: Given the current ambiguity surrounding the cause of OSF and the limited treatment options available, our study reveals that epithelial cells and their crosstalk with T cells play an important role in the mechanism of OSF and suggests the therapeutic promise of DPSCs.

Evaluation of stromal myofibroblasts in oral submucous fibrosis and its malignant transformation: An immunohistochemical study.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion, with oral squamous cell carcinoma (OSCC) being the most prevalent malignancy affecting the oral mucosa. The malignant transformation of OSF into OSCC is estimated to occur in 7-13% of cases. Myofibroblasts (MFs) play pivotal roles in both physiological and pathological processes, such as wound healing and tumorigenesis, respectively. This study aimed to explore the involvement of MFs in the progression of OSF and its malignant transformation. MATERIALS AND METHODS: In total, 94 formalin-fixed paraffin-embedded tissue blocks were collected, including normal oral mucosa (NOM; n = 10), early-moderate OSF (EMOSF; n = 29), advanced OSF (AOSF; n = 29), paracancerous OSF (POSF; n = 21), and OSCC (n = 5) samples. Alpha-smooth muscle actin was used for the immunohistochemical identification of MFs. RESULTS: NOM exhibited infrequent expression of MFs. A higher staining index of MFs was found in AOSF, followed by EMOSF and NOM. Additionally, a significant increase in the staining index of MFs was found from EMOSF to POSF and OSCC. The staining index of MFs in NOM, EMOSF, AOSF, POSF, and OSCC was 0.14 ± 0.2, 1.69 ± 1.4, 2.47 ± 1.2, 3.57 ± 2.6, and 8.86 ± 1.4, respectively. All results were statistically significant (P < 0.05). CONCLUSIONS: The expression of MFs exhibited a gradual increase as the disease progressed from mild to malignant transformation, indicating the contributory role of MFs in the fibrogenesis and potential tumorigenesis associated with OSF.

Expression Profile of Circulating Exosomal microRNAs in Leukoplakia, Oral Submucous Fibrosis, and Combined Lesions of Leukoplakia and Oral Submucous Fibrosis.

OBJECTIVES: This study aims to elucidate the expression of circulating exosomal miRNAs miRNA 21, miRNA 184, and miRNA 145 in the studied groups, including patients with (i) leukoplakia; (ii) oral submucous fibrosis; (iii) oral submucous fibrosis with leukoplakia; (iv) oral squamous cell carcinoma; and (v) healthy individuals. STUDY DESIGN: An observational study was conducted among 54 patients who reported to the outpatient department of Saveetha Dental College and Hospitals. The patients were divided into three groups: Group I healthy individuals (n = 18), Group II: case group (leukoplakia, OSMF, and leukoplakia and OSMF) (n = 18), and Group III: OSCC (n = 18). Real-time polymerase chain reaction analysis was carried out to assess the expression profiles of miRNA 21, miRNA 184, and miRNA 145. The statistical analysis was calculated using SPSS software version 23. RESULTS: All three miRNAs showed a statistically significant difference in the one-way ANOVA test between the case group (leukoplakia, OSMF, and leukoplakia and OSMF), healthy group, and OSCC group (p < 0.005). The case group (leukoplakia, OSMF, leukoplakia and OSMF) showed upregulated expression of miRNA 21 and miRNA 184 with threefold change and fourfold change and downregulated expression of miRNA 145 with 1.5-fold change when compared to apparently healthy individuals. CONCLUSION: Plasma circulating exosomal miRNAs miRNA 21, miRNA 145, and miRNA 184 expression could be a novel panel of plasma biomarkers to categorise case group (leukoplakia, OSMF, leukoplakia and OSMF) patients with a high risk of malignant transformation.

Salivary transforming growth factor beta in oral submucous fibrosis: A diagnostic and predictive marker.

CONTEXT: Growth factors and cytokines like transforming growth factor beta (TGF-ß) play a key role in the pathogenesis of oral submucous fibrosis. AIMS: To elucidate the role of Salivary TGF-ß isoforms as a predictive and diagnostic marker for oral submucous fibrosis. SETTINGS AND DESIGN: A total of 30 OSMF and 10 control patients were included in this study, and their clinic-epidemiological data was recorded. METHODOLOGY: The expression of TGF-ß genes-TGF-ß1, TGF-ß2, TGF-ß3-was studied by a real-time polymerase chain reaction in tissue and saliva. Patients were given medicinal intervention for 12 weeks along with jaw-opening exercises. Expression of salivary TGF-ß genes was studied at 12 weeks. STATISTICAL ANALYSIS USED: SPSS software version 20. RESULT: Expression of salivary TGF beta isoforms in OSMF was more than in the control group. There was an increase in salivary TGF-ß1, ß2, ß3 expressions with increasing clinical grades of OSMF and advancing the stage of the disease. Expression of all the TGF beta isoforms was decreased after treatment with statistically significant results. Statistically significant correlations were found between the mean difference of TGF-ß1 and the mean difference between mouth opening and tongue protrusion. CONCLUSION: Salivary TGF-ß isoforms may be used in diagnosis, risk assessment, and screening of the entire population at risk of OSMF after its clinical validation. However, adequate sample size and segmental assessment of the expression of TGF-ß isoforms are needed for further evaluation.

Aberrantly downregulated FENDRR by arecoline elevates ROS and myofibroblast activation via mitigating the miR-214/MFN2 axis.

Long non-coding RNA FENDRR possesses both anti-fibrotic and anti-cancer properties, but its significance in the development of premalignant oral submucous fibrosis (OSF) remains unclear. Here, we showed that FENDRR was downregulated in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs), and overexpression of FENDRR mitigated various myofibroblasts hallmarks, and vice versa. In the course of investigating the mechanism underlying the implication of FENDRR in myofibroblast transdifferentiation, we found that FENDRR can directly bind to miR-214 and exhibit its suppressive effect on myofibroblast activation via titrating miR-214. Moreover, we showed that mitofusin 2 (MFN2), a protein that is crucial to the fusion of mitochondria, was a direct target of miR-214. Our data suggested that FENDRR was positively correlated with MFN2 and MFN2 was required for the inhibitory property of FENDRR pertaining to myofibroblast phenotypes. Additionally, our results showed that the FENDRR/miR-214 axis participated in the arecoline-induced reactive oxygen species (ROS) accumulation and myofibroblast transdifferentiation. Building on these results, we concluded that the aberrant downregulation of FENDRR in OSF may be associated with chronic exposure to arecoline, leading to upregulation of ROS and myofibroblast activation via the miR-214-mediated suppression of MFN2.

[Oral squamous cell carcinoma in the background of oral submucous fibrosis - a systematic review]. / Skvamozno-kletochnaya kartsinoma polosti rta na fone podslizistogo fibroza polosti rta - sistematicheskii obzor.

Oral squamous cell carcinoma (OSCC) in the background of oral submucous fibrosis (OSMF) is one of the most common presentations of oral cancer among Asian population. OSCC arising in the background of OSMF (OSCC with OSMF) has been a topic of interest among researchers recently and a few studies have considered this to be a distinct clinicopathological entity. This systematic review analyses the demographic and clinicopathological variations of OSCC with OSMF from conventional OSCC to evaluate the distinctiveness of OSCC with OSMF. A comprehensive search from PubMed, Google scholar and manual search were carried out and 4 articles were retrieved and analysed systematically. Out of the total 377 OSCC with OSMF cases and 542 conventional OSCC, males were found to be predominantly affected (82.7% and 73.6%). 47% of the OSCC with OSMF cases were well differentiated squamous cell carcinomas as against 33.4% in conventional OSCC. Lymph node metastases were seen predominantly in conventional OSCC (49.1%) than OSCC with OSMF cases (40.7%). OSCC with OSMF were more prevalent in males and showed better tumour differentiation and lesser lymph node metastasis. Even though the present results inculpate OSCC with OSMF as a distinct clinicopathological entity, there is a dire need for thorough investigation.

Areca nut-induced oral fibrosis - Reassessing the biology of oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a pathological condition characterized by excessive tissue healing resulting from physical, chemical, or mechanical trauma. Notably, areca nut consumption significantly contributes to the development of oral fibrosis. The current definition of OSF, recognizing its potential for malignant transformation, necessitates a more comprehensive understanding of its pathophysiology and etiology. HIGHLIGHTS: Areca nut induces fibrotic pathways by upregulating inflammatory cytokines such as TGF-ß and expressing additional cytokines. Moreover, it triggers the conversion of fibroblasts to myofibroblasts, characterized by α-SMA and γSMA expression, resulting in accelerated collagen production. Arecoline, a component of areca nut, has been shown to elevate levels of reactive oxygen species, upregulate the expression of various cytokines, and activate specific signaling pathways (MEK, COX2, PI3K), all contributing to fibrosis. Therefore, we propose redefining OSF as "Areca nut-induced oral fibrosis" (AIOF) to align with current epistemology, emphasizing its distinctive association with areca nut consumption. The refined definition enhances our ability to develop targeted interventions, thus contributing to more effective prevention and treatment strategies for oral submucous fibrosis worldwide. CONCLUSION: Arecoline plays a crucial role as a mediator in fibrosis development, contributing to extracellular matrix accumulation in OSF. The re-evaluation of OSF as AIOF offers a more accurate representation of the condition. This nuanced perspective is essential for distinguishing AIOF from other forms of oral fibrosis and advancing our understanding of the disease's pathophysiology.

Evaluation of possible role of fluoride in pathogenesis of oral submucous fibrosis: A pilot study.

BACKGROUND: Oral submucous fibrosis (OSMF) is a potentially malignant disorder. Although areca nut chewing is an established risk factor, its low prevalence among nut chewers indicates additional factors likely facilitates pathogenesis. We recently demonstrated high fluoride levels in smokeless tobacco products and hypothesized a potential pathological role of fluoride in OSMF. Further exploring this novel role, this study compared fluoride levels in tissue, serum, and saliva samples from OSMF patients and healthy controls. METHODS: The ethically approved study included 25 clinically confirmed OSMF patients and 25 healthy matched controls. OSMF cases underwent buccal mucosal incisional biopsy, while controls had buccal mucosa tissue sampling during third molar removal. Fasting venous blood and unstimulated saliva were collected. Fluoride levels were analysed using ion chromatography and expressed as median (IQR). RESULTS: OSMF cases showed significantly higher fluoride concentrations compared with controls in tissue biopsies (30.1 vs. 0 mg/kg, p < 0.0001), serum (0.4 vs. 0 mg/L, p = 0.005) and saliva (1.3 vs. 0 mg/L, p < 0.0001). Majority (68%) of controls had undetectable fluoride levels across all samples. Tissue fluoride weakly correlated with OSMF severity (r = -0.158, p = 0.334). CONCLUSION: The preliminary findings demonstrated increased tissue fluoride levels in OSMF patients compared with healthy controls. Along with a previous study showing high fluoride content in smokeless tobacco products, these findings provided early evidence suggesting fluoride could play a contributory role in OSMF pathogenesis. Further large-scale investigation is warranted to definitively establish whether the association between fluoride exposure and OSMF is indicative of causation.

Stromal thrombospondin 1 suppresses angiogenesis in oral submucous fibrosis.

A decline in mucosal vascularity is a histological hallmark of oral submucous fibrosis (OSF), a premalignant disease that is largely induced by betel quid chewing. However, the lack of available models has challenged studies of angiogenesis in OSF. Here, we found that the expression of thrombospondin 1 (THBS1), an endogenous angiostatic protein, was elevated in the stroma of tissues with OSF. Using a fibroblast-attached organoid (FAO) model, the overexpression of THBS1 in OSF was stably recapitulated in vitro. In the FAO model, treatment with arecoline, a major pathogenic component in areca nuts, enhanced the secretion of transforming growth factor (TGF)-ß1 by epithelial cells, which then promoted the expression of THBS1 in fibroblasts. Furthermore, human umbilical vein endothelial cells (HUVECs) were incorporated into the FAO to mimic the vascularized component. Overexpression of THBS1 in fibroblasts drastically suppressed the sprouting ability of endothelial cells in vascularized FAOs (vFAOs). Consistently, treatment with arecoline reduced the expression of CD31 in vFAOs, and this effect was attenuated when the endothelial cells were preincubated with neutralizing antibody of CD36, a receptor of THBS1. Finally, in an arecoline-induced rat OSF model, THBS1 inhibition alleviated collagen deposition and the decline in vascularity in vivo. Overall, we exploited an assembled organoid model to study OSF pathogenesis and provide a rationale for targeting THBS1.

PDE12 disrupts mitochondrial oxidative phosphorylation and mediates mitochondrial dysfunction to induce oral mucosal epithelial barrier damage in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic oral mucosal disease. The pathological changes of OSF include epithelial damage and subepithelial matrix fibrosis. This study aimed to reveal the epithelial injury mechanism of OSF. A histopathological method was used to analyze oral mucosal tissue from OSF patients and OSF rats. The expression of PDE12 in the oral epithelium was analyzed by immunohistochemistry. The epithelial-mesenchymal transition (EMT) and tight junction proteins in arecoline-treated HOKs were explored by western blotting. Epithelial leakage was assessed by transepithelial electrical resistance and lucifer yellow permeability. The expression of PDE12 and the mitochondrial morphology, mitochondrial permeability transition pore opening, mitochondrial membrane potential, and mitochondrial reactive oxygen species (mtROS) were evaluated in arecoline-induced HOKs. Oxidative phosphorylation (OXPHOS) complexes and ATP content were also explored in HOKs. The results showed significant overexpression of PDE12 in oral mucosal tissue from OSF patients and rats. PDE12 was also overexpressed and aggregated in mitochondria in arecoline-induced HOKs, resulting in dysfunction of OXPHOS and impaired mitochondrial function. An EMT, disruption of tight junctions with epithelial leakage, and extracellular matrix remodeling were also observed. PDE12 overexpression induced by PDE12 plasmid transfection enhanced the mtROS level and interfered with occludin protein localization in HOKs. Interestingly, knockdown of PDE12 clearly ameliorated arecoline-induced mitochondrial dysfunction and epithelial barrier dysfunction in HOKs. Therefore, we concluded that overexpression of PDE12 impaired mitochondrial OXPHOS and mitochondrial function and subsequently impaired epithelial barrier function, ultimately leading to OSF. We suggest that PDE12 may be a new potential target against OSF.