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.

Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation.

OBJECTIVES: Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation. MATERIALS: Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2', 7'-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-L-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. RESULTS: TGM-2 expression was significantly higher in OSF specimens than normal specimens (p < 0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p < 0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p < 0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p < 0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p < 0.05). CONCLUSIONS: Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation. CLINICAL RELEVANCE: TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

Lycopene: features and potential significance in the oral cancer and precancerous lesions.

Data from epidemiological studies have indicated that diets rich in fruits and vegetables are likely to benefit many aspects of the prevention of oral malignancy. Lycopene is a red-coloured carotenoid predominantly accumulated in tomatoes as well as other fruits and vegetables. It has been claimed to alleviate chronic diseases such as cancers and cardiovascular disease. Hence, the aim of this review is to summarize the features and its potential significance of lycopene in the development, prevention and treatment of oral premalignant lesions and oral cancer. Studies showed that lycopene might have beneficial effects in the management of some premalignant lesions in the oral cavity including oral submucous fibrosis and oral leukoplakia and may be an adjunct in the prevention and therapy of oral cancer. However, more mechanistic studies and randomized controlled trials of large sample size are necessary to further confirm these effects and to eventually make lycopene to be used in the community prevention and clinically routine management of these diseases.

Low-power laser irradiation inhibits arecoline-induced fibrosis: an in vitro study.

Oral submucous fibrosis (OSF) is a potentially malignant disorder that is characterized by a progressive fibrosis in the oral submucosa. Arecoline, an alkaloid compound of the areca nut, is reported to be a major aetiological factor in the development of OSF. Low-power laser irradiation (LPLI) has been reported to be beneficial in fibrosis prevention in different damaged organs. The aim of this study was to investigate the potential therapeutic effects of LPLI on arecoline-induced fibrosis. Arecoline-stimulated human gingival fibroblasts (HGFs) were treated with or without LPLI. The expression levels of the fibrotic marker genes alpha-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF/CCN2) were analysed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blots. In addition, the transcriptional activity of CCN2 was further determined by a reporter assay. The results indicated that arecoline increased the messenger RNA and protein expression of CCN2 and α-SMA in HGF. Interestingly, both LPLI and forskolin, an adenylyl cyclase activator, reduced the expression of arecoline-mediated fibrotic marker genes and inhibited the transcriptional activity of CCN2. Moreover, pretreatment with SQ22536, an adenylyl cyclase inhibitor, blocked LPLI's inhibition of the expression of arecoline-mediated fibrotic marker genes. Our data suggest that LPLI may inhibit the expression of arecoline-mediated fibrotic marker genes via the cAMP signalling pathway.

Establishing a normal range for mouth opening: its use in screening for oral submucous fibrosis.

The maximal mouth opening of 700 healthy Nepalese adults, age range of 18-68 years, measured by paraclinical workers was determined as there are no data available for an Asian population. The mean value of the inter-incisal distance was 47.1 mm (range 33.7-60.4 mm) and 98% of the population surveyed fell within this range. The minimum limit of normal oral opening was determined to be 34 mm. Ten out of 13 patients with histologically confirmed oral submucous fibrosis (OSF) had a maximal oral opening of less than 34 mm. It was concluded that reduced oral opening measured by paraclinical workers as a single screening test for oral submucous fibrosis, has a sensitivity of only 77% and will detect only advanced cases. However, each of the 3 subjects from the healthy population found to have restricted mandibular opening and who agreed to be examined further had significant oral conditions, namely oral submucous fibrosis (2) and pericoronitis (1). The study confirms the value of this measurement as a screening procedure for significant oral disease by paraclinical staff particularly in developing countries where trained dental and medical personnel are scarce. Because of its limited sensitivity, measurement of mouth opening is unsatisfactory as a single screening test for OSF, as it will not detect early stages of the disease.

Early human safety study of turmeric oil (Curcuma longa oil) administered orally in healthy volunteers.

OBJECTIVE: Turmeric extract and turmeric oil have shown chemoprotective effect against chemically-induced malignancies in experimental animals. They can reverse precancerous changes in oral submucous fibrosis in humans. The use of turmeric or Curcuma longa Linn as a spice and household remedy has been known to be safe for centuries. In view of the long term administration required for cancer prevention a Phase I clinical trial of turmeric oil (TO) was designed to study the safety and tolerance of TO in volunteers for a period of 3 months. MATERIAL AND METHODS: Nine healthy volunteers between 20 and 33 years of age were tested for haemoglobin, blood counts, liver and kidney functions, bleeding and clotting time and serum electrolytes initially and at 1 and 3 months of treatment. They were administered 0.6 ml of TO three times a day for 1 month and 1 ml in 3 divided doses for 2 months. The acute tolerability study on Day 1 was conducted in a Clinical Pharmacology daycare Unit. Blood pressure and pulse were recorded frequently on Day 1 and at 24, 48, 72 and 96 hours and fortnightly till 12 weeks. Volunteers were daily supervised for TO intake as well as for any side effects throughout the study period. RESULTS: Nine volunteers were enrolled for the study. One discontinued on 3rd day for allergic skin rashes which, on discontinuation of TO, gradually disappeared by two weeks. Another discontinued on 7th day for intercurrent fever requiring antibiotic treatment. Seven volunteers completed the study. There was no effect of TO, in two doses, on pulse and blood pressure and no side effects in acute tolerability study on Day 1. There was no effect of TO intake on weight, blood pressure, symptoms and signs upto 12 weeks. There was no clinical, haematological, renal or hepatic-toxicity of TO at 1 month and 3 months. Serum lipids did not show significant change except in one volunteer (reversible). CONCLUSIONS: In view of the potential for reversing oral submucous fibrosis, a precancerous condition for oral cancer, TO, can be recommended directly for a Phase II trial in patients.

Effect on the incidence of oral submucous fibrosis of intervention in the areca nut chewing habit.

Incidence of oral submucous fibrosis was calculated from a 10-yr prospective intervention study of 12,212 individuals with a strong component of health education on tobacco and area nut chewing. Based on 11 new cases among 6341 chewers, the annual incidence was 8.0 per 100,000 among men and 29.0 for women. An earlier 10-yr follow-up study, with no intervention component, served as control. Based on 11 new cases among 3,809 chewers, the annual incidence was 21.3 per 100,000 for men and 45.7 for women controls. Although the decrease in the incidence in the intervention cohort was not statistically significant due to small number of cases, the results underscored the causal role of areca nut chewing and indicated the potential for primary prevention of oral submucous fibrosis.

Cytotoxic and non-genotoxic effects of arecoline on human buccal fibroblasts in vitro.

Betel quid chewing has been linked to oral submucous fibrosis and oral cancer. Cytotoxicity and genotoxicity assays were used to investigate the pathobiological effects of arecoline on cultured human buccal fibroblasts. Arecoline increased double-stranded polynucleic acid at the concentration of 0.1 to 10 micrograms/ml in a concentration-dependent manner. At a concentration higher than 50 micrograms/ml, arecoline was cytotoxic to cultured fibroblasts and the cytotoxicity was dose-dependent. No genotoxicity for arecoline was found even at a concentration of 400 micrograms/ml. On the other hand, 600 micrograms/ml glutathione (GSH) and 200 micrograms/ml glycyrrhizin could prevent the arecoline-induced cytotoxicity. These results indicate that arecoline is a cytotoxic agent and no genotoxicity was found to human buccal fibroblasts. Furthermore, increasing consumption of GSH- and glycyrrhizin-rich foods may reduce the oral diseases associated with betel quid chewing.