Development of Epigallocatechin 3-gallate-Loaded Hydrogel Nanocomposites for Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is a chronic progressive disease associated with increased collagen deposition and TGF-ß1 release. The current therapy and management have been a limited success due to low efficacy and adverse drug reactions. This study aimed to evaluate epigallocatechin 3-gallate (EGCG) encapsulated nanoparticles loaded mucoadhesive hydrogel nanocomposite (HNC) for OSF. Developed HNC formulations were evaluated for their permeation behaviour using in vitro as well as ex vivo studies, followed by evaluation of efficacy and safety by in vivo studies using areca nut extract-induced OSF in rats. The disease condition in OSF-induced rats was assessed by mouth-opening and biochemical markers. The optimized polymeric nanoparticles exhibited the required particle size (162.93 ± 13.81 nm), positive zeta potential (22.50 ± 2.94 mV) with better mucoadhesive strength (0.40 ± 0.002 N), and faster permeation due to interactions of the positively charged surface with the negatively charged buccal mucosal membrane. HNC significantly improved disease conditions by reducing TGF-ß1 and collagen concentration without showing toxicity and reverting the fibroid buccal mucosa to normal. Hence, the optimized formulation can be further tested to develop a clinically alternate therapeutic strategy for OSF.

Antifibrotic effect of silymarin on arecoline-induced fibrosis in primary human buccal fibroblasts: an in silico and in vitro analysis.

BACKGROUND: This study aimed to assess silymarin's anticancer and antifibrotic potential through in silico analysis and investigate its impact on in vitro arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS & RESULTS: The study utilized iGEMDOCK for molecular docking, evaluating nine bioflavonoids, and identified silymarin and baicalein as the top two compounds with the highest target affinity, followed by subsequent validation through a 100ns Molecular Dynamic Simulation demonstrating silymarin's stable behavior with Transforming Growth Factor Beta. HBF cell lines were developed from tissue samples obtained from patients undergoing third molar extraction. Arecoline, a known etiological factor in oral submucous fibrosis (OSMF), was employed to induce fibrogenesis in these HBFs. The inhibitory concentration (IC50) of arecoline was determined using the MTT assay, revealing dose-dependent cytotoxicity of HBFs to arecoline, with notable cytotoxicity observed at concentrations exceeding 50µM. Subsequently, the cytotoxicity of silymarin was assessed at 24 and 72 h, spanning concentrations from 5µM to 200µM, and an IC50 value of 143µM was determined. Real-time polymerase chain reaction (qPCR) was used to analyze the significant downregulation of key markers including collagen, epithelial-mesenchymal transition (EMT), stem cell, hypoxia, angiogenesis and stress markers in silymarin-treated arecoline-induced primary buccal fibroblast cells. CONCLUSION: Silymarin effectively inhibited fibroblast proliferation and downregulated genes associated with cancer progression and EMT pathway, both of which are implicated in malignant transformation. To our knowledge, this study represents the first exploration of silymarin's potential as a novel therapeutic agent in an in vitro model of OSMF.

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.

Polyphenol-based targeted therapy for oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic, progressive, and precancerous condition mainly caused by chewing areca nut. Currently, OSF therapy includes intralesional injection of corticosteroids with limited therapeutic success in disease management. Therefore, a combined approach of in silico, in vitro and in vivo drug development can be helpful. Polyphenols are relatively safer than other synthetic counterparts. We used selected polyphenols to shortlist the most suitable compound by in silico tools. Based on the in silico results, epigallocatechin-3-gallate (EGCG), quercetin (QUR), resveratrol, and curcumin had higher affinity and stability with the selected protein targets, transforming growth factor beta-1 (TGF-ß1), and lysyl oxidase (LOX). The efficacy of selected polyphenols was studied in primary buccal mucosal fibroblasts followed by in vivo areca nut extract induced rat OSF model. In in vitro studies, the induced fibroblast cells were treated with EGCG and QUR. EGCG was safer at higher concentrations and more efficient in reducing TGF-ß1, collagen type-1A2 and type-3A1 mRNA expression than QUR. In vivo studies confirmed that the EGCG hydrogel was efficient in improving the disease conditions compared to the standard treatment betamethasone injection with significant reduction in TGF-ß1 and collagen concentrations with increase in mouth opening. EGCG can be considered as a potential, safer and efficient phytomolecule for OSF therapy and its mucoadhesive topical formulation help in the improvement of patient compliance without any side effects. Highlights Potential polyphenols were shortlisted to treat oral submucous fibrosis (OSF) using in silico tools Epigallocatechin 3-gallate (EGCG) significantly reduced TGF-ß1 and collagen both in vitro and in vivo EGCG hydrogel enhanced antioxidant defense, modulated inflammation by reducing TGF-ß1 and improved mouth opening in OSF rat model.

A biomaterial-based therapy using a sodium hyaluronate/bioglass composite hydrogel for the treatment of oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic, inflammatory and potentially malignant oral disorder. Its pathophysiology is extremely complex, including excessive collagen deposition, massive inflammatory infiltration, and capillary atrophy. However, the existing clinical treatment methods do not fully take into account all the pathophysiological processes of OSF, so they are generally low effective and have many side effects. In the present study, we developed an injectable sodium hyaluronate/45S5 bioglass composite hydrogel (BG/HA), which significantly relieved mucosal pallor and restricted mouth opening in OSF rats without any obvious side effects. The core mechanism of BG/HA in the treatment of OSF is the release of biologically active silicate ions, which inhibit collagen deposition and inflammation, and promote angiogenesis and epithelial regeneration. Most interestingly, silicate ions can overall regulate the physiological environment of OSF by down-regulating α-smooth muscle actin (α-SMA) and CD68 and up-regulating CD31 expression, as well as regulating the expression of pro-fibrotic factors [transforming growth factor-ß1 (TGF-ß1), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and tissue inhibitors of metalloproteinase-1 (TIMP-1)] and anti-fibrotic factors [interleukin-1ß (IL-1ß)] in macrophage. In conclusion, our study shows that BG/HA has great potential in the clinical treatment of OSF, which provides an important theoretical basis for the subsequent development of new anti-fibrotic clinical preparations. STATEMENT OF SIGNIFICANCE: : Oral submucous fibrosis (OSF) is a chronic, inflammatory and potentially malignant mucosal disease with significant impact on the quality of patients' life. However, the existing clinical treatments have limited efficacy and many side effects. There is an urgent need for development of specific drugs for OSF treatment. In the present study, bioglass (BG) composited with sodium hyaluronate solution (HA) was used to treat OSF in an arecoline-induced rat model. BG/HA can significantly inhibit collagen deposition, regulate inflammatory response, promote angiogenesis and repair damaged mucosal epithelial cells, and thereby mitigate the development of fibrosis in vivo.

A novel approach to develop an animal model for oral submucous fibrosis.

Epidemiological data have proved the association of consumption of areca nut with the causation of oral submucous fibrosis (OSF). OSF is a chronic inflammatory disease with the potential for malignant transformation from 7 to 13%. The establishment of animal models makes it easier for researchers to focus on the therapeutic options to combat this disease further. We developed and compared two areca nut extract (ANE) administration methods in Swiss albino mice to establish OSF. This study compared an invasive intrabuccal injection technique with a non-invasive intraoral droplet administration. The duration of induction was around 12 weeks. Histopathology (H&E, Masson's trichrome staining) and gene expression analysis (COL-I, COL-II, and α-SMA) were performed using RT-PCR to confirm the OSF in animals. Our study showed that ANE administration through the intraoral droplet method exhibited significantly higher fibrosis than the intrabuccal injections, as evidenced by the H&E and Masson's trichrome staining. Furthermore, intraoral administration of ANE significantly upregulated the mRNA expression of COL-I, COL-II, and α-SMA, as revealed by the RT-PCR analysis. The non-invasive droplet method could simulate the absorption of areca nut seen in humans through daily dosing. This study establishes the intraoral droplet method as an efficient and non-invasive method to administer the ANE to develop OSF. These findings will aid in the efficient development of OSF animal models for interventional studies, including screening novel drugs in the reversal of the OSF.

Inhibition of miR-497 Attenuates Oral Submucous Fibrosis by Inhibiting Myofibroblast Transdifferentiation in Buccal Mucosal Fibroblasts.

PURPOSE: Oral submucous fibrosis (OSF) is a common chronic condition with poor prognosis, and existing therapies for OSF are limited in effectiveness. This study was designed to explore the role of miR-497 in arecoline (AR)-induced OSF. MATERIALS AND METHODS: After miR-497 was silenced or overexpressed in buccal mucosa fibroblasts (BMFs), different concentrations of AR (5-200 µg/ml) were applied to incubate BMFs, and 50 µg/ml of AR was chosen for subsequent experiments. Thereafter, collagen gel contraction assay was used to detect the contractile capacity of BMFs. Transwell assay and wound healing assay were applied to detect migration and invasiveness of the cells. In addition, immunofluorescence staining, qRT-PCR and western blot were conducted to measure the expression of miR-497, collagen I and α-SMA, as well as the phosphorylation of Smad2 and Smad3. RESULTS: After successful inhibition or overexpression of miR-497 in AR-induced BMFs, the results showed that miR- 497 inhibition suppressed the contractility, migration and invasiveness of AR-induced BMFs, whereas overexpression of miR-497 produced the opposite. In addition, miR-497 inhibition down-regulated the expression level of collagen I and α-SMA in AR-exposed BMFs. Furthermore, TGF-ß1 expression, Smad2 and Smad3 phosphorylation were also repressed in AR-induced BMFs after miR-497 inhibition. Correspondingly, overexpression of miR-497 reversed the expression of the aforementioned proteins. CONCLUSION: miR-497 inhibition may attenuate OSF by inhibiting myofibroblast transdifferentiation in BMFs via the TGF-ß1/Smads signaling pathway, indicating that miR-497 might represent an underlying target for treating OSF.

Are There Betel Quid Mixtures Less Harmful than Others? A Scoping Review of the Association between Different Betel Quid Ingredients and the Risk of Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is a potentially malignant condition of the oral cavity characterized by progressive fibrosis of the submucosal tissues. OSF is typically associated with the use of betel quid (BQ), a chewing package made of natural products (e.g., areca nut, betel leaves), with or without smokeless tobacco. BQ ingredients contain pro-carcinogenic bioactive compounds, but also potentially protective biomolecules, and we have shown recently that the chemical properties of different BQ recipes vary, which may explain the unequal prevalence of OSF and oral cancer in BQ users in different geographical regions. Hence, this scoping review was aimed at evaluating the existing literature regarding different BQ compounds and their association with OSF. The repository of the National Library of Medicine (MEDLINE/PubMed), medRxiv databases, Google scholar, Baidu scholar, CNKI, and EBSCO were used to search for publications that investigated the association between BQ chewing and OSF up to November 2021. The search terminology was constructed using the keywords "betel quid" and "oral submucous fibrosis", and their associated terms, with the use of Boolean operators. The search was conducted under Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines, together with clear inclusion and exclusion criteria. The review showed that the risk of developing OSF varied between different BQ recipes, and that chewing BQ mixtures containing betel inflorescence (BI) significantly increased the risk of OSF, as did the addition of tobacco. Conversely, the use of betel leaf in the mixture was likely to be protective, which may be due to the presence of polyphenols. Although further research is needed to determine the effect of individual BQ ingredients in the development of OSF, our pilot results provide the scope and rationale for informing future chemopreventive strategies for OSF and oral cancer in BQ chewers.

N6-methyladenosine modification contributes to arecoline-mediated oral submucosal fibrosis.

BACKGROUND: Oral submucosal fibrosis (OSF) is a precancerous condition that closely related to the habit of chewing betel nut. The OSF patients of 3%-19% may develop cancer, and this probability is increasing year by year. Epigenetics modifications have been reported as part of the pathogenesis of OSF. However, in OSF field, the role and mechanism of arecoline-induced activation of transforming growth factor ß (TGF-ß) signaling on N6-methyladenosine (m6A) modification remain unclear. In this study, we investigated the effect and mechanism of arecoline on m6A modification. METHODS: MeRIP-Seq and RNA-seq were performed in arecoline-stimulated cells. Quantitative polymerase chain reaction and western blot were performed to detect the expression of m6A writers and erasers. CCK-8 and flow cytometry analyses were performed to measure the cell viability and apoptosis. RESULTS: m6A level was increased in OSF tissues compared to normal tissues; arecoline promoted the m6A methyltransferase Mettl3 and Mettl14 through TGF-ß. MeRIP-seq and RNA-seq analyses found that MYC was the target gene of Mettl14. In addition, Mettl14 silence reversed the effects of arecoline on cell proliferation and apoptosis in Hacat cells. CONCLUSION: TGF-ß-METTL14-m6A-MYC axis was crucially implicated in arecoline-mediated OSF and may be an effective therapeutic strategy for OSF treatment.

Molecular Mechanisms of Malignant Transformation of Oral Submucous Fibrosis by Different Betel Quid Constituents-Does Fibroblast Senescence Play a Role?

Betel quid (BQ) is a package of mixed constituents that is chewed by more than 600 million people worldwide, particularly in Asia. The formulation of BQ depends on a variety of factors but typically includes areca nut, betel leaf, and slaked lime and may or may not contain tobacco. BQ chewing is strongly associated with the development of potentially malignant and malignant diseases of the mouth such as oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively. We have shown recently that the constituents of BQ vary geographically and that the capacity to induce disease reflects the distinct chemical composition of the BQ. In this review, we examined the diverse chemical constituents of BQ and their putative role in oral carcinogenesis. Four major areca alkaloids-arecoline, arecaidine, guvacoline and guvacine-together with the polyphenols, were identified as being potentially involved in oral carcinogenesis. Further, we propose that fibroblast senescence, which is induced by certain BQ components, may be a key driver of tumour progression in OSMF and OSCC. Our study emphasizes that the characterization of the detrimental or protective effects of specific BQ ingredients may facilitate the development of targeted BQ formulations to prevent and/or treat potentially malignant oral disorders and oral cancer in BQ users.

Honokiol inhibits arecoline-induced oral fibrogenesis through transforming growth factor-ß/Smad2/3 signaling inhibition.

BACKGROUND/PURPOSE: The habit of areca nut chewing has been regarded as an etiological factor of precancerous oral submucous fibrosis (OSF). In the present study, we aimed to evaluate the anti-fibrosis effect of honokiol, a polyphenolic component derived from Magnolia officinalis. METHODS: The cytotoxicity of honokiol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the expression of TGF-ß/Smad2 signaling as well as α-SMA and type I collagen were measured as well. RESULTS: Honokiol exerted higher cytotoxicity of fBMFs compared to normal cells. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility and wound healing capacities were all suppressed by honokiol treatment. In addition, the expression of the TGF-ß/Smad2 pathway was downregulated along with a lower expression of α-SMA and type I collagen in honokiol-receiving cells. CONCLUSION: Our data suggest that honokiol may be a promising compound to alleviate the progression of oral fibrogenesis and prevent the transformation of OSF oral epithelium into cancer.

Research Achievements of Oral Submucous Fibrosis: Progress and Prospect.

Oral submucous fibrosis (OSMF) is a kind of chronic, insidious disease, and it is categorized into potentially malignant disorders (PMD), which poses a global and regional problem to public health. It is considered to be a multifactorial disease, such as due to areca nut chewing, trace element disorders, and genetic susceptibility. However, there is still no unanimous conclusion on its pathogenesis, diagnosis, and treatment strategies. Hence, this article provides a comprehensive review and prospect of OSMF research, providing scholars and clinicians with a better perspective and new ideas for the research and treatment of OSMF.

Arecoline suppresses epithelial cell viability by upregulating tropomyosin-1 through the transforming growth factor-ß/Smad pathway.

CONTEXT: Oral submucous fibrosis (OSF) is a chronic and progressive disease. Arecoline, present in betel nuts, has been proposed as a vital aetiological factor. However, the underlying mechanism remains unclear. OBJECTIVES: This research elucidates the expression of tropomyosin-1 (TPM1) and its regulation mechanism in HaCaT cells treated with arecoline. MATERIALS AND METHODS: HaCaT cells were assigned into three groups: (1) Control; (2) Treated with arecoline (0.16 mM) for 48 h (3) Treated with arecoline (0.16 mM) and transfected with small interfering RNA (siRNA) for TPM1 (50 nM) for 48 h. CCK8, cell cycle, and apoptosis phenotypic analyses were performed. PCR and western blot analyses were performed to detect the expression level of TPM1 and examine the related signalling pathway. RESULTS: The IC50 of arecoline was approximately 50 µg/mL (0.21 mM). The arecoline dose (0.16 mM) and time (48 h) markedly increased TPM1 expression at the mRNA and protein levels in HaCaT cells. Arecoline suppressed the cell growth, caused cell cycle arrest at the G1 phase, and induced cell apoptosis in HaCaT cells. siRNA-mediated knockdown of TPM1 attenuated the effect of arecoline on cell proliferation, apoptosis, and cell cycle arrest at the G1 phase. Furthermore, blocking of the transforming growth factor (TGF)-ß receptor using SB431542 significantly suppressed TPM1 expression in the cells treated with arecoline. DISCUSSION AND CONCLUSIONS: Arecoline suppresses HaCaT cell viability by upregulating TPM1 through the TGF-ß/Smad signalling pathway. This research provides a scientific basis for further study of arecoline and TPM1 in OSF and can be generalised to broader pharmacological studies. TPM1 may be a promising molecular target for treating OSF.

Oral Submucous Fibrosis: A Review on Biomarkers, Pathogenic Mechanisms, and Treatments.

Oral submucous fibrosis (OSF) is a collagen deposition disorder that affects a patient's oral function and quality of life. It may also potentially transform into malignancy. This review summarizes the risk factors, pathogenic mechanisms, and treatments of OSF based on clinical and bio-molecular evidence. Betel nut chewing is a major risk factor that causes OSF in Asia. However, no direct evidence of arecoline-induced carcinogenesis has been found in animal models. Despite identification of numerous biomarkers of OSF lesions and conducting trials with different drug combinations, clinicians still adopt conservative treatments that primarily focus on relieving the symptoms of OSF. Treatments focus on reducing inflammation and improving mouth opening to improve a patient's quality of life. In conclusion, high-quality clinical studies are needed to aid clinicians in developing and applying molecular biomarkers as well as standard treatment guidelines.

Expression of Salivary miRNA 21 in Oral Submucous Fibrosis (OSMF): An Observational Study.

OBJECTIVE: To observe the expression patterns of salivary mRNA 21 in different stages and grades of OSMF and also in habitual areca nut chewers without OSMF. SUBJECTS AND METHODS: The study consisted of a total of 185 samples, where 61 patients had chewing habits (chewing gutkha and other forms of areca nut) and had OSMF (Group 1). 61 patients had chewing habits but did not have OSMF (Group 2), and 63 were normal healthy patients (control group) without any chewing habits (Group 3). Unstimulated saliva samples were collected from patients following the standard operating procedures. miRNA 21 was isolated and purified from saliva samples using the miRNeasy Mini Kit, Qiagen. The primers for miRNA relative quantification analysis were designed using the Primer Express software of Applied Biosystems. Quantification of all the samples was carried out using SYBR chemistry in an Applied Biosystems Real-Time PCR. RESULTS: There was no statistically significant difference between the demographic characteristics of patients. There was a statistically significant difference between the expressions of miRNA 21 amongst the three groups noted in Kruskal Wallis test. (<0.001*) A post hoc test was perfomed to confirm the statistical difference between patients within all three groups. There was no statistically significant difference noted between the OSMF group and patients with chewing habits group (G1 vs. G2 p: 0.10), but there was a significant difference when compared with normal patients. (G1 vs. G3 p: <0.001*) and (G2 vs. G3 <0.001*). CONCLUSION: This study concludes that miRNA 21 is overexpressed in OSMF and chewing habit patients. But the expression levels were not significantly associated with the severity of the disease process. A long term and large scale studies are required to assess its application as a diagnostic profibrotic marker in OSMF.

E3 ligase carboxyl-terminus of Hsp70-interacting protein (CHIP) suppresses fibrotic properties in oral mucosa.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) represents a precancerous lesion of oral mucosa that may progress into oral cancer and its major etiological factor is areca nut chewing. Carboxyl-terminus of Hsp70-interacting protein (CHIP) functions as an ubiquitin E3 ligase and is associated with fibrosis diseases. In the current study, we sought to investigate whether CHIP participated in the areca nut-mediated OSF development. METHODS: The mRNA expression of CHIP in arecoline-stimulated buccal mucosal fibroblasts (BMFs) and OSF tissues was determined by qRT-PCR. Collagen gel contraction, migration and invasion assays were carried out to evaluate the myofibroblast activation. The protein expression levels of α-SMA and transglutaminase 2 (TGM2) were assessed by Western blot. RESULTS: The expression level of CHIP was reduced in BMFs following arecoline treatment in a dose-dependent manner, which was consistent with the observation of lower CHIP expression in OSF specimen compared to the normal counterparts. Ectopic expression of CHIP mitigated the myofibroblast activities, including elevated collagen gel contractility and cell motility. In addition, we showed that overexpression of CHIP downregulated the α-SMA and TGM-2 expression, which may lead to less fibrosis alteration. CONCLUSION: CHIP may not only function as a key regulator of protein quality control but also a critical deciding factor to oral fibrogenesis. Our findings suggested that CHIP possesses the anti-fibrotic effect, which may be mediated by TGM2 regulation. Restoration of CHIP could be a therapeutic direction to help OSF patients.

Evaluation of mucoadhesive dexamethasone sodium phosphate gel in the treatment of arecoline-induced oral submucous fibrosis in wister albino rats: A cross-sectional study.

AIM: The present work aimed to prepare an oral mucoadhesive gel of dexamethasone sodium phosphate to serve the purpose of treating oral submucous fibrosis by incorporating the drug in a polymeric matrix to facilitate the localisation of the drug at the absorption site, to prolong drug delivery and to provide patient convenience. MATERIALS AND METHODS: The formulations F1, F2 and F3 were prepared using 2, 2.5 and 3% of carboxymethyl cellulose sodium, formulations F4, F5 and F6 were prepared using 2, 2.5 and 3% of hydroxypropyl methylcellulose, respectively, and formulations F7, F8 and F9 were prepared using equal mixtures of carboxymethyl cellulose sodium and hydroxypropyl methylcellulose in the concentrations of 1, 1.25 and 1.50%, respectively. The prepared formulations were subjected for screening of physicochemical parameters, viz, homogeneity, grittiness, viscosity studies, spreadability, extrudability, mucoadhesive strength, pH, drug content uniformity, in vitro drug diffusion, Fourier transform infrared spectroscopy spectral analysis and stability studies. RESULTS: Among the nine formulations prepared, the formulation F8 containing 1.25% carboxymethyl cellulose sodium, 1.25% hydroxypropyl methylcellulose having a mucoadhesive strength of 12.600 ± 0.01 g and drug release of 88.473 ± 0.457% was considered as the promising one and was further used for in vivo study. CONCLUSION: Oral application of the gel for 4 months in arecoline-induced oral submucous fibrosis rats showed more than 80% reduction in fibrosis. The histopathological results supported these findings.

Angiotensin (1-7) inhibits arecoline-induced migration and collagen synthesis in human oral myofibroblasts via inhibiting NLRP3 inflammasome activation.

Arecoline induces oral submucous fibrosis (OSF) via promoting the reactive oxygen species (ROS). Angiotensin (1-7) (Ang-(1-7)) protects against fibrosis by counteracting angiotensin II (Ang-II) via the Mas receptor. However, the effects of Ang-(1-7) on OSF remain unknown. NOD-like receptors (NLRs) family pyrin domain containing 3 (NLRP3) inflammasome is identified as the novel mechanism of fibrosis. Whereas the effects of arecoline on NLRP3 inflammasome remain unclear. We aimed to explore the effect of Ang-(1-7) on NLRP3 inflammasome in human oral myofibroblasts. In vivo, activation of NLRP3 inflammasomes with an increase of Ang-II type 1 receptor (AT1R) protein level and ROS production in human oral fibrosis tissues. Ang-(1-7) improved arecoline-induced rats OSF, reduced protein levels of NADPH oxidase 4 (NOX4) and the NLRP3 inflammasome. In vitro, arecoline increased ROS along with upregulation of the angiotensin-converting enzyme (ACE)/Ang-II/AT1R axis and NLRP3 inflammasome/interleukin-1ß axis in human oral myofibroblasts, which were reduced by NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine, and NOX4 small interfering RNA (siRNA). Furthermore, arecoline induced collagen synthesis or migration via the Smad or RhoA-ROCK pathway respectively, which could be inhibited by NLRP3 siRNA or caspase-1 blocker VX-765. Ang-(1-7) shifted the balance of RAS toward the ACE2/Ang-(1-7)/Mas axis, inhibited arecoline-induced ROS and NLRP3 inflammasome activation, leading to attenuation of migration or collagen synthesis. In summary, Ang-(1-7) attenuates arecoline-induced migration and collagen synthesis via inhibiting NLRP3 inflammasome in human oral myofibroblasts.

A comparative study to evaluate the efficacy of lycopene and curcumin in oral submucous fibrosis patients: A randomized clinical trial.

BACKGROUND: : Oral submucous fibrosis (OSMF) is a most prevalent potentially malignant disorder associated with betel quid chewing frequently observed in the Indian population. The present study conducted is much of a keen interest because there is much new information, both in the press and the medical literature, about the benefits of fresh fruits and vegetables and antioxidants (such as lycopene and curcumin) for both prevention and treatment of diseases. As clinicians, we often prescribe medications with significant adverse effects, and certainly, if armed with evidence to support using such antioxidants as safer therapeutic alternatives for treatment of OSMF. AIMS AND OBJECTIVE: The aim of the study was to compare and evaluate the efficacy of lycopene and curcumin given orally in clinically diagnosed OSMF patients. MATERIALS AND METHODS: Sixty patients were divided randomly into two groups Group A and Group B. After fulfilling the eligibility criteria, sixty patients were randomly allotted based on fishbowl method into thirty each. This technique eliminated the selection bias arising in the study. Group A individuals were treated with 4 mg of lycopene and Group B individuals were given 300 mg of curcumin thrice daily for 3 months. Both the groups were assessed in terms of mouth opening and burning sensation. The statistical analysis was done using SPSS Version 16.0 statistical Analysis Software. RESULTS: In Group A, the initial burning sensation was 65.83 ± 3.98%, and in Group B, it was 62.33 ± 5.22% (visual analog scale). After 3 months, there was complete cessation of burning sensation in both the groups. Burning sensation between the groups was statistically nonsignificant (P > 0.05). In Group A, mean mouth opening at baseline (1st visit) observed was 3.17 ± 0.08 cm which improved to 3.52 ± 0.07 cm after 3 months of the treatment period. In Group B, mean mouth opening at baseline (1st visit) observed was 3.32 ± 0.07 cm which improved to 3.52 ± 0.08 cm after 3 months of the treatment period. On comparing intergroup, the difference was statistically nonsignificant (P > 0.05). However, on comparing intergroup, average percent change in mean mouth opening from 1st visit to subsequent time intervals across the time period was found to be statistically significant (P < 0.05). Group A showed 11.1 ± 1.0% improvement in mean mouth opening and Group B showed 6.2 ± 0.4% improvement in the mean mouth opening from the 1st visit till the posttreatment period. The change in the mean mouth opening from 1st visit till posttreatment in Group A was 0.35 ± 0.14, and in Group B, it was 0.20 ± 0.09. CONCLUSION: Lycopene showed better results than curcumin in improving mouth opening; both the drugs were equally effective in decreasing burning sensation in OSMF patients.

miR-200b ameliorates myofibroblast transdifferentiation in precancerous oral submucous fibrosis through targeting ZEB2.

Oral submucous fibrosis (OSF) is a progressive scarring disease. MicroRNA-200b (miR-200b) has been reported as a tumour suppressor, but its role in the precancerous OSF remains unknown. In this study, we investigated the impact of miR-200b on myofibroblastic differentiation activity. Arecoline is a major areca nut alkaloid and has been employed to induce the elevated myofibroblast activity in human buccal mucosal fibroblasts (BMFs). Treatment of arecoline in BMFs dose-dependently reduced gene expression of miR-200b, which corresponded with the decreased expression of miR-200b in fBMFs. The arecoline-induced myofibroblast activities were abolished by overexpression of miR-200b in BMFs, and the same results were observed in fBMFs. In addition, α-SMA was inhibited by an increase in miR-200b. We further demonstrated that miR-200b-mediated decrease in ZEB2 led to down-regulation of α-SMA, vimentin. Loss of miR-200b resulted in enhanced collagen contraction and migration capabilities, and knockdown of ZEB2 reversed these phenomena. Lastly, we showed the expression of miR-200b was significantly less and ZEB2 was markedly higher in OSF tissues. These results suggested that down-regulation of miR-200b may contribute to the pathogenesis of areca quid-associated OSF through the regulation of ZEB2 and myofibroblast hallmarks.