Abstract:

Objective To investigate the inhibitory effect of ginseng-lithospermum erythrorhizon extract on the proliferation and invasion of human keloid fibroblasts （HKF） based on network pharmacology， molecular docking technology and in vitro cell experiments， and to clarify its mechanism. Methods Traditional Chinese Medicine Systems Pharmacology （TCMSP） database， Traditional Chinese Medicine Integrated Database （TCMID）， Swiss Target Prediction database， and UniProt database were used to retrieve the active components of ginseng-lithospermum extract and their corresponding target genes. GeneCards database and Online Mendelian Inheritance in Man （OMIM） database were used to screen keloid （KD）-related targets. STRING online platform and Cytoscape 3.10.3 software were used to construct the protein-protein interaction （PPI） network and identify core targets. Metascape database was used to perform Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis on the above core targets. CB-Dock2 website was used to conduct molecular docking analysis and visualize the results. HKF cells were divided into blank group （100 μL complete medium）， control group （100 μL complete medium supplemented with equal volume of solvent）， and low dose， medium dose， and high dose of ginseng-lithospermum erythrorhizon extract groups （100 μL complete medium supplemented with ginseng-lithospermum extract at final concentrations of 10， 20， and 40 mg·L^-1， respectively）. Cell counting kit-8 （CCK-8） method was used to detect the survival rates of HKF cells in various groups. Flow cytometry method was used to detect the percentages of cells in different cell cycle phases in various groups. Transwell chamber assay was used to detect the numbers of invasion cells in HKF cells in various groups. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of serine/threonine protein kinase 1 （AKT1）， interleukin-1β（IL-1β）， and prostaglandin-endoperoxide synthase 2 （PTGS2） mRNA in HKF cells in various groups. Results A total of 27 active components of ginseng-lithospermum extract and 31 intersection targets between the drug and KD were screened out， among which 9 core targets were identified， including IL-1β， AKT1 and PTGS2. GO functional enrichment analysis mainly involved biological processes including cell proliferation， apoptosis， and oxidative stress response. KEGG signaling pathway enrichment analysis mainly involved phosphatidylinositol 3-kinase/protein kinase B （PI3K-Akt） signaling pathway， mitogen-activated protein kinase （MAPK） signaling pathway， Janus kinase/signal transducer and activator of transcription （JAK/STAT） signaling pathway， and cancer-related pathways. The binding energies between PTGS2 and the five active components were all lower than -7 kcal·mol^-1. Compared with control group， the survival rates of HKF cells in low dose， medium dose， and high dose of ginseng-lithospermum erythrorhizon extract groups were significantly decreased （P<0.05）， the percentages of cells in G₀/G₁ phase were significantly increased （P<0.05）， and the numbers of invasion cells were significantly decreased （P<0.05）. Compared with control group， the expression levels of AKT1 and IL-1β mRNA in HKF cells in low dose， medium dose， and high dose of ginseng-lithospermum erythrorhizon extract groups were significantly decreased （P<0.05）， while the expression levels of PTGS2 mRNA were significantly increased （P<0.05 or P<0.01）. Conclusion Ginseng-lithospermum erythrorhizon extract inhibited the proliferation and invasion of HKF cells and induced cell cycle arrest. Its mechanism may be associated with regulating the core targets AKT1， IL-1β， and PTGS2， and mediating PI3K-Akt， MAPK， and JAK/STAT signaling pathways.

Key words: Keloid, Ginseng, Lithospermum erythrorhizon, Network pharmacology, Molecular docking, Human keloid fibroblast