Network pharmacology and experimental validation to explore the anti-aging mechanism of action of betulinic acid

This subject aims to investigate the potential targets and mechanism of action of betulinic acid in anti-aging through network pharmacology, molecular docking technique combined with in vitro validation. After mining betulinic acid action targets through the Swiss Target Prediction database, we predicted the targets related to anti-aging by TTD, GAD, and PharmGkb databases and used common targets to construct a protein–protein interaction (PPI) network. The core targets were visualized by Cytoscape 3.7.2 software, and perform core targets by Cytoscape 3.7.2 software with GO and KEGG enrichment analysis. Using Cytoscape constructs, the “active ingredient-target-disease-pathway” network to obtain the core components and validate the binding mode of core components and core targets by molecular docking. The effects of betulinic acid on the proliferation and senescence of HK-2 cells, the expression of senescence-related secretory proteins, and the expression of key targets were investigated by constructing an in vitro cell senescence model. The PPI network screened ten genes, including SRC, MAPK1, PIK3R1, HSP90AA1, GRB2, PTPN11, ESR1, MAPK8, MAPK14, and EGFR, working as the core targets of betulinic acid anti-aging. They exerted anti-aging effects by participating in the PI3K–Akt signaling pathway, MAPK signaling pathway, Ras signaling pathway, estrogen receptor signaling pathway, FoxO signaling pathway, etc. Among them, TP53 and SIRTl are two central genes in the PPI network that interact. Molecular docking results showed that betulinic acid and core targets can bend freely. In vitro experiments showed that betulinic acid could increase HK-2 cell proliferation, inhibit cellular senescence, decrease the expression of TGF-β and IL-6, and significantly down-regulate the expression of p53 and p21. Among them, TP53 and SIRTl are two central genes in the PPI network interaction. This study confirmed that betulinic acid can exert anti-aging effects through multiple targets and pathways. The mechanism of action may be related to the regulation of the SITR1–p53 pathway.

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