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