System Pharmacology-Based Anti-proliferation and Anti-angiogenesis Mechanisms of Oridonin in Psoriasis Keratinocytes by Blocking PI3K/AKT Signaling Pathway.

To study the anti-proliferative and anti-angiogenic mechanism of oridonin in psoriatic keratinocytes by blocking phosphatidylinositol 3-kinase (PI3K/Akt) signaling pathway based on system pharmacology. Protein targets of oridonin and psoriasis-related genes were predicted and their overlap was determined using the Venny package in R. The DAVID database was used for enrichment analysis. The oridonin-psoriasis-target and oridonin-target-pathway (OTP) networks associated with psoriasis, as well as a protein-protein interaction, were constructed using Cytoscape. Molecular docking with AutoDock was used to verify the relationships between oridonin and core targets, and ZDOCK was used to assess interactions between core proteins. All bioinformatics results were verified empirically. The mRNA levels of core targets in oridonin-treated cells were measured using real-time q-PCR. The HaCaT keratinocytes were divided to 4 groups: control, low-, medium- and high-concentration groups and treated with different concentrations of oridonin (1, 5 and 10 µmol/L) for 18 h, and then stimulated with tumor necrosis factor (TNF)-α (20 ng/mL) for 6 h. Western blot was used to detect PI3K/Akt pathway protein levels. Seventy-three targets were found to be strongly associated with the effects of oridonin on psoriasis. The Cluster ONE algorithm was used to extract 6 kernel clusters from the PPI network, and 9 PPI interaction pairs comprising 13 targets showed combined scores ⩾0.999. The OTP network, molecular docking, and protein interaction simulations indicated that oridonin targeted Sirtuin 1 (SIRT1), cyclin dependent kinase 2 (CDK2), Src tyrosine protein kinase (SRC), proteintyrosine phosphatase, nonreceptor-type (PTPN), and SIRT1-tumor protein p53 (TP53), with the CDK2-TP53, SRC-heat shock protein 90 alpha family class a member 1 (HSP90AA1), and PTPN1-epidermal growth factor receptor (EGFR) interaction pairs regulating the PI3K-Akt signaling pathway. Oridonin was observed to block hyperplasia in HaCaT keratinocytes, accompanied by reduced levels of TNF-α, interleukin (IL-)-6, and IL-1 β (P<0.05 or P<0.01). qPCR measurements showed that oridonin significantly inhibited the expression of angiogenesis-related genes, including C-X-C chemokine receptor type 7, C-X-C motif chemokine ligand 12, and vascular endothelial growth factor (P<0.05 or P<0.01). In addition, the qPCR results showed significant changes in the mRNA levels of SIRT1, CDK2, SRC, PTPN1, TP53, HSP90AA1, and EGFR, and Western blot indicated marked changes in the protein levels of cleaved caspase-3, BAD, Bcl-2, p-ERK1/2, and p-p38 after oridonin treatment (P<0.05 or P<0.01). The study identified the primary oridonin targets during psoriasis treatment using network pharmacology. Bioinformatics analysis with empirical verification demonstrated strong associations between SIRT1, CDK2, SRC, PTPN1, TP53, HSP90AA1, and EGFR and oridonin. These interactions were positively correlated with an inhibition of proliferation and angiogenesis mediated by blocking PI3K-Akt signaling pathway.