基于网络毒理学及实验验证探讨槟榔对口腔黏膜下纤维化的影响

Effects of betel nut on oral submucosal fibrosis based on network toxicology and experimental verification

目的基于网络毒理学方法和体内实验验证探讨槟榔对口腔黏膜下纤维化的影响及作用机制。方法通过TCSMP和Swiss数据库筛选槟榔潜在活性成分对应靶点,利用GeneCards、Disgenet数据库得到口腔黏膜下纤维化(oral submucosal fibrosis,OSF)疾病相关靶点。将交集靶点上传至STRING数据平台构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,再采用Metascape数据平台进行基因本体(gene ontology,GO)功能和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。采用槟榔水提取液低、中、高浓度干预大鼠口腔黏膜,大鼠开口度、病理形态学、免疫组织化学等检测对网络毒理学结果进行验证。结果网络毒理学结果表明槟榔52个成分中含有587个相关靶点,OSF相关靶点761个,二者交集靶点72个。PPI网络分析结果显示,白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、细胞凋亡调节因子Bcl-2(apoptosis regulator Bcl-2,BCL2)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、细胞肿瘤抗原p53(cellular tumor antigen p53,TP53)可能是槟榔影响OSF的关键靶点。KEGG通路富集分析获得PI3K-Akt信号通路、AGE-RAGE信号通路、松弛素信号通路、EGFR酪氨酸激酶抑制剂耐药等信号通路。动物实验结果显示,中、高剂量组开口度值较对照组、低剂量组差异有统计学意义(P<0.05),HE染色、Masson染色表明中、高剂量组出现不同程度的纤维化病变,免疫组织化学结果提示槟榔水提取液上调IL-6、TNF-α等炎性因子,且呈一定的剂量相关性。结论槟榔可能通过上调IL-6、TNF-α等关键炎性因子以介导炎症反应诱导口腔黏膜下纤维化的发生。

Objective To investigate the effects of betel nut on oral submucosal fibrosis(OSF)and its mechanism of action based on network toxicology and in vivo experimental verification.Methods Targets corresponding to the potential active ingredients of betel nut were screened through TCSMP and Swiss databases,and OSF-related targets were obtained using GeneCards and Disgenet databases.The intersection targets were uploaded to the String data platform to construct a protein-protein interaction(PPI)network,and then the Metascape data platform was used to perform gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses.The oral mucosa of rats was treated with the aqueous extract of betel nut at low,medium,and high concentrations respectively,and the results of network toxicology were verified by the measurement of mouth opening degree,pathomorphological examination,and immunohistochemical analysis in the rats..Results The network toxicology analysis showed that there were 587 related targets in the 52 ingredients of betel nut and 761 OSF-related targets,of which there were 72 intersection targets.PPI network analysis indicated that interleukin-6(IL-6),tumor necrosis factor(TNF),matrix metalloproteinase-9(MMP-9),apoptosis regulator Bcl-2(BCL2),epidermal growth factor receptor(EGFR),and cellular tumor antigen p53(TP53)may be the key targets of betel nut affecting OSF.PI3K-Akt,AGE-RAGE,and relaxin signaling pathways,EGFR tyrosine kinase inhibitor resistance and other signaling pathways were obtained by KEGG pathway enrichment analysis.The animal experiments showed that the mouth opening degrees of the medium-and high-dose groups were significantly different from those of the control group and low-dose group(P<0.05).HE staining and Masson staining showed that the medium-and high-dose groups had different degrees of fibrotic lesions.Immunohistochemical analysis suggested that the aqueous extract of betel nut upregulated the levels of inflammatory factors such as IL-6 and TNF-α,and this upregulation exhibited a certain dose-dependent relationship.Conclusion Betel nut may induce OSF by upregulating key inflammatory factors such as IL-6 and TNF-α,thereby mediating the inflammatory response.