Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.     

Effects of arecoline, safrole, and nicotine on collagen phagocytosis by human buccal mucosal fibroblasts as a possible mechanism for oral submucous fibrosis in Taiwan

BACKGROUND: Oral submucous fibrosis (OSF) is associated with the betel quid chewing habit, and 86% of betel quid chewers in Taiwan are also smokers. Arecoline and safrole are major principles in the composition of betel quid, and nicotine is the main toxic ingredient of cigarettes. METHODS: To explore the pathogenesis of OSF, flow cytometry was used to compare collagen phagocytosis by fibroblasts from the normal and the OSF region of the same 15 OSF patients. RESULTS: The results indicated that heterogeneity of fibroblasts existed because collagen phagocytosis by fibroblasts from the normal region was higher than from the OSF region in the same patient. The percentage of phagocytic cells was significantly inhibited by 10, 25 and 50 microg/ml arecoline, safrole and nicotine in normal fibroblast cultures, respectively, and the percentage of phagocytic cells was significantly reduced by 25, 25 and 50 microg/ml arecoline, safrole and nicotine in OSF fibroblast cultures, respectively. Collagen phagocytosis by fibroblasts exhibited prominent dose-dependent inhibition as the concentration of arecoline, safrole, and nicotine increased. Besides, nicotine had a synergistic effect on arecoline- or safrole-inhibited collagen phagocytosis. CONCLUSIONS: The present study concludes that even in the same person, the collagen phagocytosis by fibroblasts is different between normal and OSF region. The deficiency in collagen phagocytosis by fibroblasts of the lesion might participate in the pathogenesis of OSF. Arecoline, safrole and nicotine, which are released in saliva during BQ chewing plus cigarette smoking, inhibit collagen phagocytosis by fibroblasts in a dose-dependent manner and may induce OSF formation in Taiwan's patients.     

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

槟榔碱及机械刺激构建大鼠口腔黏膜下纤维化模型

目的 利用槟榔碱和机械刺激构建Sprague-Dawley（SD）大鼠口腔黏膜下纤维化（OSF）模型。方法 采用两因素析因实验设计将48只大鼠分为8个组,每组6只。分别用不同浓度（0、0.5、2、8 mg·mL ^-1）槟榔碱涂擦及机械刺激 (有或无毛刷涂擦)。处理16周后测量开口度;取局部颊黏膜行苏木精-伊红（HE）染色观察病理变化;并检测组织内Ⅲ型胶原、转化生长因子-β1（TGF-β1）和γ干扰素（IFN-γ）的表达情况。结果 2和8 mg·mL ^-1（中、高）浓度槟榔碱处理16周,颊黏膜出现了典型的OSF病理特征;开口度显著减小并且Ⅲ型胶原、TGF-β1表达显著增加（P<0.05）。机械刺激虽可导致黏膜Ⅲ型胶原、TGF-β1和IFN-γ表达增高（P<0.05）,但无病理学改变,开口度改变不显著。结论 中、高浓度槟榔碱有致OSF作用;机械刺激无法导致大鼠OSF的发生。     

槟榔碱及机械刺激构建大鼠口腔黏膜下纤维化模型

目的 利用槟榔碱和机械刺激构建Sprague-Dawley（SD）大鼠口腔黏膜下纤维化（OSF）模型。方法 采用两因素析因实验设计将48只大鼠分为8个组,每组6只。分别用不同浓度（0、0.5、2、8 mg·mL ^-1）槟榔碱涂擦及机械刺激 (有或无毛刷涂擦)。处理16周后测量开口度;取局部颊黏膜行苏木精-伊红（HE）染色观察病理变化;并检测组织内Ⅲ型胶原、转化生长因子-β1（TGF-β1）和γ干扰素（IFN-γ）的表达情况。结果 2和8 mg·mL ^-1（中、高）浓度槟榔碱处理16周,颊黏膜出现了典型的OSF病理特征;开口度显著减小并且Ⅲ型胶原、TGF-β1表达显著增加（P<0.05）。机械刺激虽可导致黏膜Ⅲ型胶原、TGF-β1和IFN-γ表达增高（P<0.05）,但无病理学改变,开口度改变不显著。结论 中、高浓度槟榔碱有致OSF作用;机械刺激无法导致大鼠OSF的发生。     

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

Chang YC, Tai KW, Cheng MH, Chou LSS, Chou MY: Cytotoxic and non-genotoxic effects of arecoline on human buccal fibroblasts in vitro. J Oral Pathol Med 1998; 27: 68–71. © Munksgaard, 1998.
Betel quid chewing has been linked to oral submucous fibrosis and oral cancer. Cytotoxicity and genotoxicity assays were used to investigate the pathobiologi-cal effects of arecoline on cultured human buccal fibroblasts. Arecoline increased double-stranded polynucleic acid at the concentration of 0.1 to 10 μg/ml in a concentration-dependent manner. At a concentration higher than 50 μg/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 μg/ml. On the other hand, 600 μg/ml glutathione (GSH) and 200 μg/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.     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although over-simplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.     

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

槟榔碱对口腔黏膜成纤维细胞分泌整合素α2的影响

目的：通过研究槟榔碱对体外培养的人口腔黏膜成纤维细胞（FB）表达整合素α2的影响,探讨整合素α2在口腔黏膜成纤维性变（OSF）发病机制中的可能作用。方法：体外培养人正常口腔黏膜成纤维细胞（FB）,培养基中加入不同浓度（0、10、20、40、80、160μg/ml）的槟榔碱孵育,MTT法12h后检测FB的增殖水平。RT-PCR法24h后检测0、10、20、40μg/ml浓度组整合素α2mRNA的表达水平。结果：槟榔碱浓度为20μg/ml时,FB增殖活性开始下降,与对照组相比无统计学差异（p〉0.05）,40μg/ml时下降明显与对照组相比有统计学差异（p〈0.05）。正常对照组整合素α2mRNA表达量低,随槟榔碱浓度增高其表达量呈增高趋势（p〈0.05）。结论：槟榔碱具有刺激FB表达整合素α2的作用,提示FB分泌整合素α2增多,进而诱导胶原合成增多,可能是OSF发生机制之一。     

Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan

Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5×105 cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-³H] -lysine labelled purified chick - embryo aorta elastin substrate. After incubation for 10 h at 37°C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84±1.15 mg/ml) and lysyl oxidase activity (3558.6±345.5 cpm/106 cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35±0.96 mg/ml and 2436.0±352.6 cpm/10⁶ cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.     

颊脂垫瓣在口腔黏膜下纤维性变术后缺损修复中的应用

目的：研究颊脂垫瓣在口腔黏膜下纤维性变（OSF）切除后组织缺损修复中的应用。方法：10例伴重度张口受限的OSF患者,应用颊脂垫瓣修复切除后遗留的组织缺损,观察颊脂垫瓣的愈合过程及修复效果。结果：术后9例患者张口度均大于3cm,颊脂垫瓣愈合良好,术后1周颊脂垫瓣明显水肿,组织瓣表面有薄层伪膜覆盖,1周后伪膜逐渐消失,2周后伪膜完全消失,2～3周后水肿明显消退,颊脂垫表面逐渐上皮化,6～8周再生的黏膜变得光滑,呈粉红色,类似于正常口腔黏膜。结论：颊脂垫瓣为修复OSF术后缺损提供了一种良好的手术方式,有利于改善张口受限症状。