Nicotinic Activity of Arecoline,the Psychoactive Element of "Betel Nuts", Suggests a Basis for Habitual Use and Anti-Inflammatory Activity

Habitual chewing of "betel nut" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6–10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline''s activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts.     

Betel nut extract and arecoline block insulin signaling and lipid storage in 3T3-L1 adipocytes

According to several population-based studies, betel nut chewing is associated with metabolic syndrome and diabetes in British South Asians and Taiwanese. However, the underlying molecular mechanism is not yet clear. Arecoline is an alkaloid-type natural product found in betel nuts. Our aim was to clarify the influence of betel nut extract and arecoline on lipid accumulation and insulin signaling in adipocytes. We found that betel nut extract and arecoline blocked lipid storage in 3T3-L1 adipocytes. The possible mechanism may function by inhibiting the expression of the insulin receptor, glucose transporter-4, fatty acid synthase, and the lipid droplet proteins perilipin and adipophilin. In addition, betel nut extract and arecoline increased the basal level of IRS-1 serine³⁰⁷ phosphorylation and decreased insulin-stimulated IRS-1 tyrosine, Akt, and PI3 kinase phosphorylation. In conclusion, betel nut extract and arecoline have diabetogenic potential on adipocytes that may result in insulin resistance and diabetes at least in part via the obstruction of insulin signaling and the blockage of lipid storage.     

Arecoline induces TNF-alpha production and Zonula Occludens-1 redistribution in mouse Sertoli TM4 cells

Background

Arecoline, a major alkaloid in Areca nut has the ability to induce oxidative stress. The effect of Areca nut, arecoline on reducing sperm quality and quantity were documented previously using several animal models. Junction disruption by down-regulation of the junction-adhesive protein via oxidative stress is an important route mediating abnormal spermatogenesis. Therefore, in this present study, we investigated the functional role of arecoline on junctional proteins.

Results

To analyze direct effects of arecoline on testis cells, confluent mouse testicular Sertoli cell line TM4 was exposed to arecoline. Arecoline decreased insoluble zonula occludens-1 (ZO-1) protein expression in TM4 cells, however, arecoline treatment increased TNF-alpha production in both TM4 and monocytic THP1 cells. In addition, ERK1/2 inhibitor PD98059 reversed arecoline effects on TNF-alpha and ZO-1.

Conclusions

Arecoline increases the production of TNF-alpha and induces protein redistribution of ZO-1. All these results explain the role of arecoline in male reproductive dysfunction, besides its cytotoxic induction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0093-z) contains supplementary material, which is available to authorized users.     

Effects of betel chewing on the central and autonomic nervous systems

Betel chewing has been claimed to produce a sense of well-being, euphoria, heightened alertness, sweating, salivation, a hot sensation in the body and increased capacity to work. Betel chewing also leads to habituation, addiction and withdrawal. However, the mechanisms underlying these effects remain poorly understood. Arecoline, the major alkaloid of Areca nut, has been extensively studied, and several effects of betel chewing are thought to be related to the actions of this parasympathomimetic constituent. However, betel chewing may produce complex reactions and interactions. In the presence of lime, arecoline and guvacoline in Areca nut are hydrolyzed into arecaidine and guvacine, respectively, which are strong inhibitors of GABA uptake. Piper betle flower or leaf contains aromatic phenolic compounds which have been found to stimulate the release of catecholamines in vitro. Thus, betel chewing may affect parasympathetic, GABAnergic and sympathetic functions. Betel chewing produces an increase in heart rate, blood pressure, sweating and body temperature. In addition, EEG shows widespread cortical desynchronization indicating a state of arousal. In autonomic function tests, both the sympathetic skin response and RR interval variation are affected. Betel chewing also increases plasma concentrations of norepinephrine and epinephrine. These results suggest that betel chewing mainly affects the central and autonomic nervous systems. Future studies should investigate both the acute and chronic effects of betel chewing. Such studies may further elucidate the psychoactive mechanisms responsible for the undiminished popularity of betel chewing since antiquity.     

Ultrastructural and hormonal changes in the pineal-testicular axis following arecoline administration in rats

Arecoline is an alkaloid of betel nut of Areca catechu. Betel nut is chewed by millions of people in the world and it causes oral and hepatic cancers in human. It has therapeutic value for the treatment of Alzheimer and schizophrenia. Arecoline has immunosuppressive, mutagenic and genotoxic effects in laboratory animals. It also affects endocrine functions. The objective of this study was to investigate the effects of arecoline on pineal-testicular axis in rats. Since pineal activity is different between day and night, the current study is undertaken in both the photophase and scotophase. The findings were evaluated by ultrastructural and hormonal studies of pineal and testicular Leydig cells, with quantitations of fructose and sialic acid of sex accessories. Arecoline treatment (10 mg/kg body weight daily for 10 days) caused suppression of pineal activity at ultrastructural level by showing dilatation of the cisternae of the rough endoplasmic reticulum (RER), large autophagosome-like bodies with swollen mitochondrial cristae, numerous lysosomes, degenerated synaptic ribbons and reduced number of synaptic-like microvesicles. Moreover, pineal and serum N-acetylserotonin and melatonin levels were decreased with increased serotonin levels in both the gland and serum. In contrast, testicular Leydig cell activity was stimulated with abundance of smooth endoplasmic reticulum (SER), electron-dense core vesicles and vacuolated secretory vesicles, and increased testosterone level in the arecoline recipients. Consequently, the testosterone target, like prostate, was ultrastructurally stimulated with abundance of RER and accumulation of secretory vesicles. Fructose and sialic acid concentrations were also significantly increased respectively in the coagulating gland and seminal vesicle. These results were more significant in the scotophase than the photophase. The findings suggest that arecoline inhibits pineal activity, but stimulates testicular function (testosterone level) and its target organs presumably via muscarinic cholinergic receptor in rats.     

Chewing Betel Quid and the Risk of Metabolic Disease,Cardiovascular Disease,and All-Cause Mortality: A Meta-Analysis

Background

Betel nut (Areca nut) is the fruit of the Areca catechu tree. Approximately 700 million individuals regularly chew betel nut (or betel quid) worldwide and it is a known risk factor for oral cancer and esophageal cancer. We performed a meta-analysis to assess the influence of chewing betel quid on metabolic diseases, cardiovascular disease, and all-cause mortality.

Methodology/Principal Findings

We searched Medline, Cochrane Library, Web of Science, and Science Direct for pertinent articles (including the references) published between 1951 and 2013. The adjusted relative risk (RR) and 95% confidence interval were calculated using the random effect model. Sex was used as an independent category for comparison.

Results

Of 580 potentially relevant studies, 17 studies from Asia (5 cohort studies and 12 case-control studies) covering 388,134 subjects (range: 94 to 97,244) were selected. Seven studies (N = 121,585) showed significant dose-response relationships between betel quid consumption and the risk of events. According to pooled analysis, the adjusted RR of betel quid chewers vs. non-chewers was 1.47 (P<0.001) for obesity (N = 30,623), 1.51 (P = 0.01) for metabolic syndrome (N = 23,291), 1.47 (P<0.001) for diabetes (N = 51,412), 1.45 (P = 0.06) for hypertension (N = 89,051), 1.2 (P = 0.02) for cardiovascular disease (N = 201,488), and 1.21 (P = 0.02) for all-cause mortality (N = 179,582).

Conclusion/Significance

Betel quid chewing is associated with an increased risk of metabolic disease, cardiovascular disease, and all-cause mortality. Thus, in addition to preventing oral cancer, stopping betel quid use could be a valuable public health measure for metabolic diseases that are showing a rapid increase in South-East Asia and the Western Pacific.     

Triphala extract negates arecoline-induced senescence in oral mucosal epithelial cells in vitro

BackgroundArecoline found in areca nut causes oral submucous fibrosis. Triphala is an Ayurvedic medicinal preparation used to improve overall physical wellness that has also been shown to improve oral health.ObjectivesTo assess the activity of Triphala extract on arecoline-induced senescence in oral mucosal epithelial cells in vitro.Materials and methodsOral mucosal epithelial cells were isolated and cultured in vitro. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess the viability of treated cells, while senescence was assessed by senescence-associated-β-galactosidase staining. Cell surface marker expression was analyzed by flow cytometry. Finally, real-time quantitative polymerase chain reaction was performed to examine gene expression levels.ResultsTriphala extract (5 µg/mL) reversed the cell senescence activity of arecoline, as evidenced by reduced β-galactosidase activity, increased Ki-67 marker expression, and reduced expression of senescence-related genes p16 and p21.ConclusionTriphala extract helped to reduce the pathological effects of arecoline-induced pathogenesis.Clinical relevance.Arecoline found in the areca nut causes oral pathological conditions including oral submucous fibrosis. Our results showed that Triphala counteracted the adverse effects of arecoline, in particular, negating senescence in oral mucosal epithelial cells. As a translational effect, Triphala treatment could restore normal epithelial thickness in oral submucous fibrosis, thus reducing the clinical severity of the disease. This reestablishment of oral homeostasis would help to improve oral health-related quality of life in patients with oral submucous fibrosis.     

Transplacental micronucleus inducing ability of arecoline, a betel nut alkaloid, in mice

Arecoline, a major betel nut alkaloid, was tested for its effectiveness in inducing micronuclei in fetal mouse blood after transplacental exposure late in the gestation period. Positive results were obtained and a linear dose-response relationship was expressed when pregnant mice were treated with arecoline at dose levels of 20, 40 and 80 mg/kg and micronucleated polychromatic erythrocytes from fetal blood were subsequently analysed.     

Systematic Review and Meta-Analysis of Association of Smokeless Tobacco and of Betel Quid without Tobacco with Incidence of Oral Cancer in South Asia and the Pacific

Aim

This systematic review and meta-analysis aimed to critically appraised data from comparable studies leading to quantitative assessment of any independent association between use of oral smokeless tobacco in any form, of betel quid without tobacco and of areca nut with incidence of oral cancer in South Asia and the Pacific.

Methods

Studies (case control and/or cohort) were identified by searching Pub Med, CINAHL and Cochrane databases through June 2013 using the keywords oral cancer: chewing tobacco; smokeless tobacco; betel quid; betel quid without tobacco; areca nut; Asia, the Pacific and the reference lists of retrieved articles. A random effects model was used to compute adjusted summary OR_RE for the main effect of these habits along with their corresponding 95% confidence intervals. To quantify the impact of between-study heterogeneity on adjusted main-effect summary OR_RE, Higgins'' H and I₂ statistics along with their 95% uncertainty intervals were used. Funnel plots and Egger''s test were used to evaluate publication bias.

Results

Meta-analysis of fifteen case–control studies (4,553 cases; 8,632 controls) and four cohort studies (15,342) which met our inclusion criteria showed that chewing tobacco is significantly and independently associated with an increased risk of squamous-cell carcinoma of the oral cavity (adjusted main-effect summary for case- control studies OR_RE = 7.46; 95% CI = 5.86–9.50, P<0.001), (adjusted main-effect summary for cohort studies RR = 5.48; 95% CI = 2.56–11.71, P<0.001). Furthermore, meta-analysis of fifteen case control studies (4,648 cases; 7,847 controls) has shown betel quid without tobacco to have an independent positive association with oral cancer, with OR = 2.82 (95% CI = 2.35–3.40, P<0.001). This is presumably due to the carcinogenicity of areca nut. There was no significant publication bias.

Conclusion

There is convincing evidence that smokeless (aka chewing) tobacco, often used as a component of betel quid, and betel quid without tobacco, are both strong and independent risk factors for oral cancer in these populations. However, studies with better separation of the types of tobacco and the ways in which it is used, and studies with sufficient power to quantify dose-response relationships are still needed.     

Epidemiology of areca (betel) nut use in the mariana islands: Findings from the University of Guam/University of Hawai`i cancer center partnership program

BackgroundAreca (betel) nut is considered a Group 1 human carcinogen shown to be associated with other chronic diseases in addition to cancer. This paper describes the areca (betel) nut chewing trend in Guam, and health behaviors of chewers in Guam and Saipan.MethodsThe areca (betel) nut module in the Guam Behavioral Risk Factor Surveillance Survey was used to calculate the 5-year (2011–2015) chewing trend. To assess the association between areca (betel) nut chewing and health risks in the Mariana Islands, a cross-section of 300 chewers, ≥18 years old, were recruited from households in Guam and Saipan. Self-reported socio-demographics, oral health behaviors, chronic disease status, diet, and physical activity were collected. Anthropometry was measured. Only areca (betel) nut-specific and demographic information were collected from youth chewers in the household.ResultsThe 5-year areca (betel) nut chewing prevalence in Guam was 11% and increased among Non-Chamorros, primarily other Micronesians, from 2011 (7%) to 2015 (13%). In the household survey, most adult chewers (46%) preferred areca nut with betel leaf, slaked lime, and tobacco. Most youth chewers (48%) preferred areca nut only. Common adult chronic conditions included diabetes (14%), hypertension (26%), and obesity (58%).ConclusionThe 5-year areca (betel) nut chewing prevalence in Guam is comparable to the world estimate (10–20%), though rising among Non-Chamorros. Adult and youth chewers may be at an increased risk for oral cancer. Adult chewers have an increased risk of other chronic health conditions. Cancer prevention and intervention strategies should incorporate all aspects of health.     

Atherogenic effect of Arecoline: A computational study

There are over 600 million people worldwide covering Asian and Oceanic countries including India have the habit of chewing areca nut as masticator in different forms. Arecoline (C(8)H(13)NO(2)) has been reported as one of the abundant constituents of areca nut. A good number of scientific publications have made Arecoline responsible for oral cancer. Based on observation from clinical situation in North East India, one of the most betel quid chewing region of the country, we suspected a link between consumption of areca nut and Cerebro Vascular Disease like stroke. Therefore, we considered Low Density Lipoprotein (LDL) receptor as target and Arecoline as ligand and studied ligand -target interaction using computational tools. Also we considered High Density Lipoprotein (HDL) receptor as another target to see if Arecoline has any binding potential with it over and above LDL receptor. Docking result indicated that Arecoline and Cholesterol both, have affinity towards extracellular domain of Human LDL receptor but affinity of Arecoline is much higher (-12.3560.) than that of Cholesterol(-0.1810). Docking of Arecoline and 1, 2-Hexyl-1- cyclopentanone thiosemicarbazone (thiosemicarbazone) with Bovine HDL receptor showed that Arecoline also has the potential (Score, -6.2690Kcal/Mol) to block HDL receptor though its potential is less than that (score, -10.0509 Kcal/Mol) of control (thiosemicarbazone). We, therefore, suggest that by inhibiting endocytosis of LDL cholesterol because of blocking LDL receptor function and also by preventing LDL cholesterol uptake by liver from blood because of interference with HDL receptor, Arecoline may contribute to atherosclerosis. The study therefore, indicates a positive correlation between chewing of betel quid and Cerebro Vascular Disease.     

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.     

Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro.

Aqueous, acetic acid, hydrochloric acid and ethanol extracts of betel nut (Areca catechu L.) have been found to induce unscheduled DNA synthesis in Hep 2 cells obtained from human larynx carcinoma, in vitro. Different concentrations of extracts of betel nut induced dose-dependent unscheduled DNA synthesis in Hep 2 cells. Together with the viability of the Hep 2 cells, our results indicate that the aqueous and acetic acid extracts of betel nut induce relatively more unscheduled DNA synthesis than the hydrochloric acid and ethanol extracts and arecoline. The carcinogenic potency of raw and unprocessed betel nut of North-East India used in this study is discussed.     

Salivary Proteomic Analysis of Betel Nut (Areca catechu) Consumers by Mass Spectrometry Revealed Primary Indication of Oral Malignancies

Areca nut is the fourth most widely used addictive and psychoactive substance consumed by approximately 10% of the world’s population. The use of areca nut is estimated to account for up to 50% of oral cancer in the low-income, and middle-income countries. In the present study, the effect of betel nut chewing on saliva proteomics was investigated by using mass spectrometry. Matrix-assisted laser desorption ionization mass spectrometry was used to generate a profile of the peptides in betel nut consumers and control group. We found 13 peptide peaks which were significantly altered (p?<?0.05) in the betel nut addicts when compared with the control group. These significant peptides signals were corresponding to protein cystatin SN (CST1), cystatin S (CST4), alpha 2 macroglobulin (A2M), complement C3 (C3), apolipoprotein E (APOE), serum albumin (ALB), matrix metalloproteinase-9 (MMP-9), deleted in malignant brain tumor protein 1 (DMBT1), zinc-alpha-2-glycoprotein (ZAG), and protein S100A8. The correlation analysis of significant peptides intensities with the history of betel nut chewing was also performed. The peptides of CST1 and CST4 showed negative correlation, whereas the peptides of the MMP-9, DMBT1, APOE, and C3 showed positive correlation with significant differences. STRING analysis of these proteins revealed that most of these proteins are interacting with each other. The present study identifies a number of proteins in a significantly different abundance in the betel nut consumers group. Some of these proteins are the reported biomarkers of several oral malignancies, which implies that the usage of betel nut could lead to inflammation, and development of oral cancer.

     

Areca (betel) nut chewing practices of adults and health behaviors of their children in the Freely Associated States,Micronesia: Findings from the Children’s Healthy Living (CHL) Program

BackgroundChewing areca (betel) nut has been deemed carcinogenic. The practice has become a public health concern in Micronesia. The Children's Healthy Living (CHL) Program included an areca (betel) nut questionnaire in a survey of household characteristics in the Freely Associated States (FAS). This paper describes areca (betel) nut chewing practices of adults and the health behaviors of their children.MethodsA cross-section of 1200 children (2–8 year-olds) and their caregivers in Chuuk, Kosrae, Pohnpei, Republic of Palau, Republic of the Marshall Islands (RMI), and Yap were recruited. Socio-demographics, adult areca (betel) nut chewing practices, and other health behaviors of children and adults were assessed. Child anthropometric measurements were collected to estimate weight status.ResultsThe FAS areca (betel) nut chewing prevalence was 42%, ranging from 3% (RMI) to 94% (Yap). Among chewers, 84% added tobacco, 97% added slaked lime, 85% added betel leaf, and 24% mixed the components with alcohol. Among FAS children, 95% practiced daily teeth-brushing and 53% visited the dentist annually. Compared to non-chewing households, areca (betel) nut chewing households were more likely to have very young children enrolled, more highly educated adults, and members that used tobacco and alcohol.ConclusionThe FAS areca (betel) nut chewing prevalence (42%) is above the world prevalence of 10–20%, with wide variability across the islands. The oral health findings in this study may inform future oral cancer prevention programs or policies. Regular monitoring of areca (betel) nut use is needed to measure the impact of such programs or policies.     

Impact of VEGF-C Gene Polymorphisms and Environmental Factors on Oral Cancer Susceptibility in Taiwan

Background

Oral cancer, which is the fourth most common male cancer, is associated with environmental carcinogens in Taiwan. Vascular endothelial growth factor (VEGF)-C, an angiogenic/lymphangiogenic factor with high expression levels in tumor tissues, plays important roles in the development of several malignancies. This study was designed to examine associations of five VEGF-C gene polymorphisms with the susceptibility to and clinicopathological characteristics of oral squamous cell carcinoma.

Methodology/Principal Findings

Five single-nucleotide polymorphisms (SNPs) of VEGF-C were analyzed by a real-time polymerase chain reaction (PCR) in 470 male patients with oral cancer and 426 cancer-free controls. In this study, we found that the VEGF-C rs7664413 and rs2046463 polymorphisms were associated with oral-cancer susceptibility but not with any clinicopathological parameters. The GGACA or GACTG haplotype of five VEGF-C SNPs (rs3775194, rs11947611, rs1485766, rs7664413, and rs2046463) combined was also related to the risk of oral cancer. Among 611 male smokers, VEGF-C polymorphism carriers who also chewed betel quid were found to have a 14.5–24.2-fold risk of having oral cancer compared to the VEGF-C wild-type carrier who did not chew betel quid. Among 461 male betel-quid chewers, VEGF-C polymorphism carriers who also smoked had a 2.7–18.1-fold risk of having oral cancer compared to those who carried the wild type but did not smoke.

Conclusions

Our results suggest that the two SNPs of VEGF-C (rs7664413 and rs2046463) and either of two haplotypes of five SNPs combined have potential predictive significance in oral carcinogenesis. Gene-environmental interactions among VEGF-C polymorphisms, smoking, and betel-quid chewing might alter one''s susceptibility to oral cancer.     

Interactions between Environmental Factors and Melatonin Receptor Type 1A Polymorphism in Relation to Oral Cancer Susceptibility and Clinicopathologic Development

Background

The purpose of this study was to explore the combined effect of melatonin receptor type 1A (MTNR1A) gene polymorphisms and exposure to environmental carcinogens on the susceptibility and clinicopathological characteristics of oral cancer.

Methodology and Principal Findings

Three polymorphisms of the MTNR1A gene from 618 patients with oral cancer and 560 non-cancer controls were analyzed by real-time polymerase chain reaction (PCR). The CTA haplotype of the studied MTNR1A polymorphisms (rs2119882, rs13140012, rs6553010) was related to a higher risk of oral cancer. Moreover, MTNR1A gene polymorphisms exhibited synergistic effects of environmental factors (betel quid and tobacco use) on the susceptibility of oral cancer. Finally, oral-cancer patients with betel quid-chewing habit who had T/T allele of MTNR1A rs13140012 were at higher risk for developing an advanced clinical stage and lymph node metastasis.

Conclusion

These results support gene-environment interactions of MTNR1A polymorphisms with smoking and betel quid-chewing habits possibly altering oral-cancer susceptibility and metastasis.     

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-1β 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.