OBJECTIVE: To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophage-secreted exosomes to induce the transformation of human oral mucosal fibroblasts (HOMFs) into fibroblast phenotype. METHODS: Exosomes were harvested from human monocytic cell line THP-1 with or without arecoline treatment. The effects of arecoline-treated THP-1 cell culture supernatant (CS), THP-1-derived exosomes (EXO), exosome-depleted THP-1 cell supernatant (NES), miR-155-5p overexpression, and miR-155-5p inhibitor on migration ability of arecoline-treated HOMF cells were examined using Transwell migration assay. The polarization of THP-1 cells was detected using flow cytometry. DCFH-DA was used to detect the level of oxidative stress in the cells with different treatments. The mRNA and protein expressions of α- SMA, type I collagen and SOCS1 in the cells were detected with qRT-PCR and Western blotting. RESULTS: Flow cytometry showed that arecoline-treated THP-1 cells exhibited obvious polarization from M₀ to M₁. Both the supernatant and exosomes from arecoline-treated THP-1 cells significantly enhanced the migration ability of HOMF cells, increased intracellular oxidative stress, up-regulated the expressions of miR-155- 5p and the mRNA and protein levels of α-SMA and type I collagen, and lowered the mRNA and protein expressions of SOCS1. In HOMF cells treated with exosomes from arecoline- treated THP-1 cells, overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions of α-SMA and type I collagen, and miR-155-5p inhibitor caused the opposite changes. CONCLUSION Arecoline can up-regulate miR-155-5p expression in THP-1 cells and inhibit the expression of SOCS1 protein in HOMF cells via the exosome pathway, thus promoting the fibrotic phenotype transformation of HOMF cells.
Humans ; Exosomes ; Arecoline/pharmacology* ; Collagen Type I ; Fibroblasts ; Macrophages ; MicroRNAs

In this study, low-field nuclear magnetic resonance(LF-NMR) and magnetic resonance imaging(MRI) were employed to analyze the water distribution, status, and migration in the moistening process of Arecae Semen. Peleg model was adopted to study the water absorption kinetics of Arecae Semen moistened at different water temperatures(10, 30, and 50 ℃). The Arecae Semen samples soaked at different water temperatures all contained four water states: binding water T_(21), non-flowing water T_(22), free water T_(23), and unbound water T_(24). Non-flowing water had the largest increase in peak area during the moistening process, followed by free water. The peak areas of non-flowing water, free water, and total water were correlated with the water content(P<0.01). Therefore, LF-NMR can quickly and non-destructively predict the water content of Arecae Semen during moistening. The peak area of non-flowing water and the content of free water were correlated with the content of arecoline in the soaking solution(P<0.01), which indicated that the faster flow of non-flowing water and more free water corresponded to more arecoline dissolved. The MRI images showed that the water migration pathway varied at different soaking temperatures, and the moistening degree obtained by this means was consistent with that obtained based on traditional experience. The rate constant K_1 fitted by Peleg model decreased with the increase in water temperature, while the capacity constant K_2 showed an opposite trend. The Arrhenius equation fitting of K_1 with temperature showed that the activation energy of Arecae Semen in the moistening process was 32.98 kJ·mol~(-1). LF-NMR/MRI can be used to analyze the water status and content and determine the end moisturing point of Arecae Semen. Peleg model can accurately describe the water absorption properties of Arecae Semen in the moistening process. The findings of this study can guide the moistening optimization and mechanism research of other seed Chinese medicinal materials.
Areca ; Arecoline/analysis* ; Drugs, Chinese Herbal/analysis* ; Kinetics ; Seeds/chemistry* ; Water/analysis*

OBJECTIVE: The aim of this study was to induce oral submucous fibrosis (OSF) in Sprague-Dawley(SD) rat models by arecoline and mechanical stimulation. METHODS: Two factors factorial design was used to divide 48 rats into 8 groups (n=6). Different concentrations of arecoline (0, 0.5, 2, and 8 mg·mL⁻¹) and mechanical stimulation (with or without brush) were treated. After 16 weeks of treatment, the mouth opening was measured, the pathological changes of the buccal mucosa were observed, and the expressions of type Ⅲ collagen, transforming growth factor β1 (TGF-β1), and interferon-γ (IFN-γ) were detected. RESULTS: In rats with moderate and high concentrations of arecoline, typical OSF pathological features were observed in the buccal mucosa, the mouth openings were significantly reduced, and the expression levels of type Ⅲ colla-gen and TGF-β1 were significantly increased (P<0.05). Although mechanical stimulation can increase the three indexes of mucosa (P<0.05), no pathological change and difference in the mouth opening was observed (P>0.05). CONCLUSIONS Moderate and high concentrations of arecoline can induce OSF in SD rats, but mechanical stimulation cannot induce OSF.
Animals ; Arecoline ; pharmacology ; Fibroblasts ; Mouth Mucosa ; Oral Submucous Fibrosis ; Rats ; Rats, Sprague-Dawley

The regulation mechanism of arecoline on rat hepatic CYP2E1 was studied in vivo. After oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg x kg(-1) x d(-1)) to rats for one week, the hepatic CYP2E1 mRNA level remained unchanged, but the hepatic CYP2E1 protein content was dose-dependently increased. Additionally, although the hepatic CYP2E1 activity was induced by AH treatment, the induction was attenuated with the increase in dosage. The results indicate that the effect of arecoline on rat hepaticdoes not involve transcriptional activation of the gene, but largely involves the stabilization of CYP2E1 protein against degradation or increased efficiency of CYP2E1 mRNA translation, and additionally involve the post- ranslational modification of CYP2E1 protein. Furthermore, the CYP2E1 response is fairly equal among the different species, the induction of rat hepatic CYP2E1 by arecoline suggests that there is a risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid use human.
Animals ; Arecoline ; pharmacology ; Cytochrome P-450 CYP2E1 ; metabolism ; Cytochrome P-450 CYP2E1 Inducers ; pharmacology ; Humans ; Liver ; drug effects ; metabolism ; RNA, Messenger ; Rats

Streptococcus mutans (S. mutans) is a facultative anaerobic bacterium mainly found in the oral cavity and is known to contribute to tooth decay and gingivitis. Recent studies on intestinal microbiota have revealed that microorganisms forming a biofilm play important roles in maintaining tissue homeostasis through their own metabolism. However, the physiological roles of oral microorganisms such as S. mutans are still unclear. In our current study, we identified that constituents released from S. mutans (CR) reduce arecoline-mediated cytotoxicity without producing toxic effects themselves. Arecoline, as a major alkaloid of areca nut, is known to mediate cytotoxicity on oral epithelial cells and induces a sustained intracellular Ca2+ ([Ca2+]i) increase that is cytotoxic. The exposure of human gingival fibroblast (HGF) cells to CR not only inhibited the sustained [Ca2+]i increase but also the initial [Ca2+]i elevation. In contrast, CR had no effects on the gene regulation mediated by arecoline. These results demonstrate that S. mutans has physiological role in reducing cytotoxicity in HGF cells and may be considered a novel pharmaceutical candidate.
Areca ; Arecoline ; Biofilms ; Epithelial Cells ; Fibroblasts ; Gingivitis ; Homeostasis ; Humans ; Metabolism ; Microbiota ; Mouth ; Nuts ; Streptococcus mutans* ; Tooth

OBJECTIVETo explore the effects of arecoline on hepatic insulin resistance in type 2 diabetes rats and to elucidate its possible mechanism.

METHODSForty five Wistar rats were fed with high fructose diet for 12 weeks to induce type 2 diabetic rat model. rats were randomly divided into 5 groups (n = 8): control group, model group and model group were treated with different dose (0, 0.5, 1, 5 mg/kg) of arecoline. After 4 weeks, the fasting blood glucose, blood lipid and insulin level measured , mRNA expression of liver constitutive androstane receptor (CAR), pregnane X receptor (PXR), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR), the protein expression of p-AKT and glucose transporter4 (GLUT4) were detected by Western blot.

RESULTS1.5 mg/kg arecoline could significantly decrease the level of fasting blood glucose, blood lipid, blood insulin level and liver G6Pase, PEPCK, IL-6, TNF-alpha mRNA level in type 2 diabetes rats. 1.5 mg/kg arecoline also could significantly increase CAR, PXR mRNA level and p-AKT and GLUT4 protein expression.

CONCLUSIONArecoline improved hepatic insulin resistance in type 2 diabetes rats by increasing the mRNA levels of CAR and PXR leading to the creased glucose metabolism and inflammation related genes expression.

Animals ; Arecoline ; pharmacology ; Diabetes Mellitus, Experimental ; metabolism ; Diabetes Mellitus, Type 2 ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Glucose-6-Phosphatase ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Wistar ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Receptors, Steroid ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effects of non-neuronal muscarinic receptors (NNMR) stimulation on atherosclerosis and endothelial cells activation.

METHODSAtherosclerosis model was established in ApoE-/- mice by a high fat diet for 7 weeks. During the experimental periods, animals were received a low (7 mg/kg/d) or a high (21 mg/kg/d) dose of arecoline by gavage. At the termination of the treatments, serum total cholesterol and NO levels were measured, and the aorta morphology was analyzed by hematoxylin and eosin staining. The gene expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in the thoracic aortas was determined by RT-PCR, and the MCP-1 protein expression and NF-κB activity were detected by Western blot analysis. NO production, MCP-1 secretion in cultured rat aortic endothelial cells (RAECs), and monocyte-endothelium adhesion assay were also performed after arecoline treatments.

RESULTSArecoline efficiently decreased atherosclerotic plaque areas, increased serum nitric oxide (NO) content, suppressed the mRNA and protein expression of MCP-1, and modulated the IκB-α degradation and P65 phosphorylation in the aortae of ApoE-/- mice. Furthermore, arecoline promoted NO production and suppressed MCP-1 secretion in cultured RAECs after ox-LDL exposure, and either atropine or NG-nitro-L-arginine methylester could abrogate these effects. Arecoline also significantly inhibited the adherence of U937 monocytes to the ox-LDL injured human umbilical vein endothelial cells, which could be abolished by atropine.

CONCLUSIONOur results indicate that arecoline attenuates the progression of atherosclerosis and inhibits endothelial cells activation and adherence by stimulating endothelial NNMR. These effects, at least in part, are due to its modulation on NF-κB activity.

Animals ; Aorta ; cytology ; Apolipoproteins E ; Arecoline ; pharmacology ; Atherosclerosis ; physiopathology ; prevention & control ; Cell Adhesion Molecules ; metabolism ; Chemokine CCL2 ; metabolism ; Cholesterol ; blood ; Disease Progression ; Endothelial Cells ; cytology ; drug effects ; Endothelium, Vascular ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; I-kappa B Proteins ; metabolism ; Lipoproteins, LDL ; Mice ; Mice, Knockout ; Monocytes ; cytology ; NF-KappaB Inhibitor alpha ; Nitric Oxide ; blood ; Nitroarginine ; pharmacology ; Rats ; Receptors, Muscarinic ; physiology ; Transcription Factor RelA ; metabolism

Arecoline is a major alkaloid of areca nuts which are widely chewed by southeast Asian and it manifests various toxic effects in different organs of human and animals. In this work, mature mice were treated by vitamins C plus E, arecoline, or both daily for four weeks. The results showed that arecoline significantly increased the levels of serum alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and significantly decreased the levels of reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) in the liver tissues. Additionally, the body weight, testis weight, sperm counts, motility and normal sperms also were significantly decreased. The supplement of vitamins C and E can bring the activities of ALP and GPT to normal levels and partially restore the sperm counts compared to the arecoline-treated group but have no other positive effects. In conclusion, the vitamins C and E partially attenuated the arecoline-induced hepatotoxiciy but basically had on protective effects against the arecoline-induced testicular toxicity.
Alkaline Phosphatase ; Animals ; Areca ; Arecoline* ; Asian Continental Ancestry Group ; Body Weight ; Catalase ; Glutamic Acid ; Glutathione ; Humans ; Liver ; Mice* ; Nuts ; Oxaloacetic Acid ; Pyruvic Acid ; Sperm Count ; Spermatozoa ; Superoxide Dismutase ; Testis ; Vitamins*

OBJECTIVE: To explore and analyze the the expression change of miRNA associated with oral submucous fibrosis (OSF) treated by the Salvia combined with law-dose prednisolone. METHODS: Ten pairs of tissues from patients with typical early or advanced stage clinical pathological features of OSF and their paired normal tissues (internal control), were selected respectively. The miRNA expression profiles between the OSF and its paired controls were compared by the Affymetrix analysis. The primary normal oral mucous cells were cultured in arecoline (50 μg/mL) for 3, 6, 12 d (0 d ser ved as cont rol), and the primary OSF-fibroblast cells were cultured with Salvia (90 mg/mL) combined with low-dose prednisolone for 12, 24, 36 h (0 h served as control). The differential expression of miRNA was detected. RESULTS: Arecoline induced the expression changes of miRNAs in normal mucosal cells. Salvia combined with low doses of prednisolone reversed the related miRNA expression. CONCLUSION MiRNAs play an essential role in the occurrence and development of OSF. Salvia combined with low-dose prednisolone can reverse the expression of related miRNAs in OSF cells.
Arecoline ; Cells, Cultured ; Coculture Techniques ; Fibroblasts ; cytology ; drug effects ; Humans ; MicroRNAs ; metabolism ; Mouth Mucosa ; cytology ; drug effects ; Oral Submucous Fibrosis ; metabolism ; Prednisolone ; pharmacology ; Salvia ; chemistry ; Transcriptome

OBJECTIVEEndothelial apoptosis plays an important role in the initiation of atherosclerosis. It would be useful to clarify whether activation of non-neuronal muscarinic receptor (NNMR) could prevent endothelial apoptosis and atherosclerosis. We investigated the effects of NNMR activation on regulating rat aortic endothelial cells (RAECs) apoptosis induced by homocysteine, an independent risk factor of atherosclerosis, and further studied its molecular mechanism.

METHODSRAECs were incubated using homocysteine at the concentration of 2.7 mmol/L for 36 h. RAECs were also pre-treated with carbachol or arecoline to examine their effects. RT-PCR was used to assess changes in the gene expression related to cell apoptosis.

RESULTSIncubation of RAECs with homocysteine at the concentration of 2.7 mmol/L resulted in morphologic changes, such as cellular shrinkage, membrane blebbing, chromatin condensation and margination. These could be attenuated by pretreatment with carbachol and arecoline at the concentration of 10 micromol/L for 12 h. Homocysteine induced apoptosis in RAECs and the molecular mechanisms were associated with the regulation of fas, fas-L and caspase-8 in the death receptor pathway, bcl-2, bcl-xL and bax in the mitochondrial pathway, caspase-12 in the endoplasmic reticulum pathway and caspase-3, caspase-6 and p53 as downstream effectors. Carbachol and arecoline attenuated the effects of homocysteine on genes in the death receptor pathway, in the mitochondrial pathway and in the downstream pathway. Atropine could reverse all of the effects of arecoline.

CONCLUSIONActivation of NNMR by carbacol and arecoline inhibits homocysteine-induced endothelial cell apoptosis mainly through regulation of death receptor pathway, mitochondrial pathway and downstream effectors.

Animals ; Aorta ; cytology ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Arecoline ; Carbachol ; Cell Cycle ; Endoplasmic Reticulum ; metabolism ; Endothelial Cells ; cytology ; drug effects ; Homocysteine ; adverse effects ; Mitochondria ; metabolism ; Rats ; Receptors, Muscarinic ; metabolism