Li Ming, Deng Zhiyuan, Xie Changqin, Chen Juan, Yuan Zhenying, Rahhal Omar, Tang Zhangui
Hunan Key Laboratory of Oral Health Research, Hunan Clinical Research Center of Oral Major Diseases and Oral Health, Xiangya Stomatological Hospital, Xiangya School of Stomatology, Central South University, Changsha, China.
Changsha Stomatological Hospital, Changsha, China.
Oral Dis. 2024 Apr;30(3):1252-1263. doi: 10.1111/odi.14602. Epub 2023 Jun 25.
Fibroblast activating protein (FAP) is associated with various organ fibrosis. However, the expression and molecular function of FAP in oral submucous fibrosis (OSF) is still unclear.
The high-performance liquid chromatography was used to detect the presence of alkaloids in areca nut extract (ANE). Real-time qPCR, Western blot, and Immunohistochemistry assay were used to analyze the expression of FAP mRNA or protein in OSF and normal oral tissue. A chi-squared test analyzed the relationship between FAP protein expression and clinicopathological data of OSF patients. CCK-8, Wound-healing, and Transwell migration assay were employed to assess the effect of the proliferation and migration ability of hOMF cells with FAP overexpression or knockdown. The expression level of a-SMA, FSP1, and P13K-Akt signaling pathways-related protein in hOMF cells transfected with FAP overexpression or knockdown plasmid was verified by western blot assay.
The four specific areca alkaloids (Arecoline, Guvacine, Arecaidine, and Guvacoline) were successfully detected in the ANE. The viability of hOMF cells was significantly improved in the 50 μg/mL ANE group and was inhibited in the 5 and 50 mg/mL ANE groups. The expression of FAP was upregulated in OSF tissues, and hOMF cells treated with 50 μg/mL ANE and was related to pathology grade, clinical stage, and history of chewing betel nut. Additionally, FAP may promote the proliferation, migration, and activation of hOMF cells through the P13K-Akt signaling pathway.
This study found that ANE had a bidirectional effect on the viability of hOMF cells, and the FAP gene was a potential therapeutic target in OSF.
成纤维细胞活化蛋白（FAP）与多种器官纤维化相关。然而，FAP在口腔黏膜下纤维化（OSF）中的表达及分子功能仍不清楚。
采用高效液相色谱法检测槟榔提取物（ANE）中生物碱的存在情况。运用实时定量聚合酶链反应、蛋白质免疫印迹法和免疫组织化学分析法分析FAP mRNA或蛋白在OSF组织及正常口腔组织中的表达。采用卡方检验分析FAP蛋白表达与OSF患者临床病理数据之间的关系。运用细胞计数试剂盒-8法、伤口愈合实验和Transwell迁移实验评估FAP过表达或敲低对人口腔黏膜成纤维细胞（hOMF细胞）增殖和迁移能力的影响。通过蛋白质免疫印迹法验证转染FAP过表达或敲低质粒的hOMF细胞中α-平滑肌肌动蛋白（α-SMA）、成纤维细胞特异性蛋白1（FSP1）及磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-Akt）信号通路相关蛋白的表达水平。
在ANE中成功检测到四种特定的槟榔生物碱（槟榔碱、古液碱、槟榔次碱和去甲槟榔碱）。50μg/mL ANE组hOMF细胞活力显著提高，而5mg/mL和50mg/mL ANE组细胞活力受到抑制。FAP在OSF组织中表达上调，50μg/mL ANE处理的hOMF细胞中FAP表达上调，且与病理分级、临床分期及咀嚼槟榔史有关。此外，FAP可能通过PI3K-Akt信号通路促进hOMF细胞的增殖、迁移和激活。
本研究发现ANE对hOMF细胞活力具有双向作用，FAP基因是OSF潜在的治疗靶点。
