中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (2): 216-223.doi: 10.12307/2023.991

• 皮肤粘膜组织构建 skin and mucosal tissue construction • 上一篇    下一篇

β-谷甾醇对增生性瘢痕成纤维细胞作用机制的网络药理学分析

左  俊，马少林   

  1. 新疆医科大学第一附属医院整形科，新疆维吾尔自治区乌鲁木齐市  830000
  • 收稿日期:2022-11-02 接受日期:2022-12-12 出版日期:2024-01-18 发布日期:2023-06-30
  • 通讯作者: 马少林，医学硕士，主任医师，教授，博士生导师，新疆医科大学第一附属医院整形科，新疆维吾尔自治区乌鲁木齐市 830000
  • 作者简介:左俊，男，1985年生，湖南省衡阳市人，汉族，新疆医科大学第一临床医学院在读博士，主治医师，主要从事整形科临床与科研工作。
  • 基金资助:
    国家自然科学基金地区基金项目(81760345)，项目负责人：马少林：湖南省自然科学基金青年基金项目(2021JJ40487)，项目负责人：左俊

Mechanism of beta-sitosterol on hypertrophic scar fibroblasts: an analysis based on network pharmacology

Zuo Jun, Ma Shaolin   

  1. Department of Plastic Surgery, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • Received:2022-11-02 Accepted:2022-12-12 Online:2024-01-18 Published:2023-06-30
  • Contact: Ma Shaolin, Master, Chief physician, Professor, Doctoral supervisor, Department of Plastic Surgery, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • About author:Zuo Jun, MD candidate, Attending physician, Department of Plastic Surgery, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    Regional Foundation of National Natural Science Foundation of China, No. 81760345 (to MSL); Youth Program of Natural Science Foundation of Hunan Province, No. 2021JJ40487 (to ZJ)

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摘要：


文题释义：

增生性瘢痕：增生性瘢痕是由胶原蛋白等细胞外基质成分过度沉积以及成纤维细胞增殖和凋亡失衡引起的一种皮肤纤维化疾病。皮肤外伤后增生性瘢痕的总体发生率为40%-70%，烧伤后增生性瘢痕的发生率甚至高达80%。
β-谷甾醇：是植物界中含量最丰富、分布最广的植物甾醇之一。它除具有免疫调节作用外，还具有抗肿瘤、抗炎和抗纤维化等多种药理活性，其中促进细胞凋亡和细胞周期阻滞是β-谷甾醇最常见的抗肿瘤效应。


背景：目前针对增生性瘢痕有效的预防和治疗措施仍然有限。而大多数植物中草药不良反应小且来源丰富，为预防和治疗增生性瘢痕提供了新的思路和方法。 

目的：通过网络药理学和分子对接技术探讨植物来源β-谷甾醇作用于增生性瘢痕成纤维细胞的潜在分子机制，并通过细胞学实验进行初步验证。
方法：通过网络药理学方法，利用相关数据库及软件筛选β-谷甾醇的药物靶点，获取增生性瘢痕相关疾病靶点，取交集得到β-谷甾醇作用于增生性瘢痕的潜在作用(交集)靶点，使用Cytoscape软件和STRING数据库构建“药物-靶点-疾病”网络和蛋白质互作网络(PPI)，同时筛选出PPI中核心作用靶点。通过DAVID数据库对交集靶点进行GO生物学功能和KEGG通路富集分析，结合文献分析进一步确立与交集靶点关系密切的信号通路及核心靶点基因，应用AutoDock软件对β-谷甾醇和核心靶点蛋白进行分子对接。采用体外细胞实验验证β-谷甾醇对人增生性瘢痕成纤维细胞增殖、凋亡、细胞周期分布和核心靶点基因mRNA表达的影响。  

结果与结论：①β-谷甾醇和增生性瘢痕的交集靶点共56个，PPI网络中筛选出10个核心靶点：酪氨酸激酶(SRC)、丝裂原活化蛋白激酶3(MAPK3)、半胱氨酸蛋白酶3(CASP3)、载脂蛋白E(APOE)、雌激素受体1(ESR1)、固醇调节元件结合转录因子1 (SREBF1)、过氧化物酶体增殖物激活受体α(PPARα)、C-反应蛋白(CRP)、细胞间黏附分子1(ICAM1)和过氧化氢酶(CAT)。②结合文献报道和KEGG通路、GO功能分析结果，进一步认定MAPK信号通路与交集靶点关系密切，确定MAPK3(即为ERK1-MAPK)、CASP3、P53和肿瘤坏死因子为其核心靶点。③分子对接结果提示β-谷甾醇与核心靶点蛋白结合良好。④细胞实验结果表明，100 μmol/L的β-谷甾醇能抑制增生性瘢痕成纤维细胞增殖，降低线粒体膜电位、诱导细胞凋亡(P < 0.01)，使G1期细胞占比增加，S期细胞占比减少(P < 0.05)，同时上调CASP3、P53和肿瘤坏死因子mRNA表达(P < 0.05)，并下调MAPK3 mRNA表达(P < 0.001)。⑤上述数据证实，β-谷甾醇可能通过激活肿瘤坏死因子通路，上调CASP3和P53的表达，诱导增生性瘢痕成纤维细胞凋亡，同时抑制ERK-MAPK通路使细胞周期阻滞从而降低增生性瘢痕成纤维细胞增殖活性。

https://orcid.org/0000-0002-8962-616X(左俊) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 增生性瘢痕, 成纤维细胞, β-谷甾醇, 细胞凋亡, MAPK3(ERK1-MAPK), 肿瘤坏死因子, CASP3, P53, 网络药理学, 细胞周期

Abstract: BACKGROUND: At present, effective preventive and therapeutic measures for hypertrophic scar are still limited. In contrast, most of botanical herbs have few side effects and abundant sources, offering new ideas and approaches for the prevention and treatment for hypertrophic scar.
OBJECTIVE: To explore the potential molecular mechanism of plant-derived β-sitosterol on hypertrophic scar fibroblasts by network pharmacology and molecular docking techniques and to initially verify it by cytological experiments.
METHODS: Through the network pharmacology, the relevant database and software were used to screen the drug targets of β-sitosterol and obtain the hypertrophic scar-related disease targets. The potential (intersection) targets of β-sitosterol on hypertrophic scar were obtained. Cytoscape software and STRING database were used to construct the “drug-target-disease” network and protein-protein interaction network, and screen out the core targets in the protein-protein interaction network. Gene ontology (GO) biological function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of intersection targets were conducted through the DAVID database, and the signaling pathways and core target genes closely related to the intersection targets were further identified through literature analysis. AutoDock software was used to perform the molecular docking of β-sitosterol and core target proteins. In vitro cellular assays were used to verify the effects of β-sitosterol on proliferation, apoptosis, cell cycle distribution and mRNA expression of core target genes in human hypertrophic scar fibroblasts.

RESULTS AND CONCLUSION: There were 56 intersection targets of β-sitosterol and hypertrophic scar and 10 core targets were identified in the protein-protein interaction network, including tyrosine kinase, mitogen-activated protein kinase 3 (MAPK3), cysteine protease 3 (CASP3), apolipoprotein E, estrogen receptor 1, sterol regulatory element-binding transcription factor 1, peroxisome proliferator-activated receptor alpha, C-reactive protein, intercellular adhesion molecule 1, and catalase. Combined with the literatures and the functional analysis of the KEGG and GO, the MAPK signaling pathway was further identified to be closely related to the intersection targets, and MAPK3 (ERK1-MAPK), CASP3, P53 and tumor necrosis factor were identified as the core targets. The molecular docking results indicated that β-sitosterol was well bound to the core target proteins. Cellular assays showed that 100 μmol/L β-sitosterol inhibited hypertrophic scar fibroblast proliferation, decreased mitochondrial membrane potential and induced apoptosis (P < 0.01), increased the proportion of G1-phase cells and decreased the proportion of S-phase cells (P < 0.05), upregulated the mRNA expression of CASP3, P53 and tumor necrosis factor (P < 0.05), and downregulated the mRNA expression of MAPK3 (P < 0.001). To conclude, β-sitosterol may induce cell apoptosis in hypertrophic scar fibroblasts by activating the tumor necrosis factor pathway and upregulating the expression of CASP3 and P53, while inhibiting the ERK-MAPK pathway to arrest cell cycle and thus reduce the proliferation of hypertrophic scar fibroblasts.

Key words: hypertrophic scar, fibroblast, β-sitosterol, apoptosis, MAPK3 (ERK1-MAPK), tumor necrosis factor, CASP3, P53, network pharmacology, cell cycle

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