Abstract:

Objective To preliminarily predict the potential pathways and targets of Xiaoban Tongmai Formula in anti-atherosclerosis （AS） by network pharmacology analysis， and to verify its possible mechanism combined with in vitro cell experiment. Method The databases including Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， GeneCards， Swiss Target Prediction， and Uniprot were used to collect the information on active compounds and corresponding targets of Xiaoban Tongmai Formula to construct the “compound-target-disease” network. The potential targets and pathways were predicted by protein-protein interaction （PPI） network， and the intersection targets were subjected to Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis.The human aortic vascular smooth muscle cells （HA-VSMCs） were cultured and identified in vitro， and the abnormal proliferation of HA-VSMCs were induced by oxidized low-density lipoprotein（ox-LDL） and identified； MTT method was used to detect the proliferation activities of the HA-VSMCs in various groups after treated with different concentrations of Xiaoban Tongmai Formula；the safety of Xiaoban Tongmai Fang was confirmed. The HA-VSMCs were divided into blank group， model group （the abnormal proliferation of HA-VSMCs was induced）， rosuvastatin group （treated with 4 μmol·L^-1 rosuvastatin after inducing the abnormal proliferation of HA-VSMCs）， and low， medium， and high doses of Xiaoban Tongmai Formula groups （treated with 0.025， 0.050， and 0.100 mg·L^-1 Xiaoban Tongmai Formula after inducing the abnormal proliferation of HA-VSMCs）；enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of monocyte chemotactic protein-1（MCP-1）， interleukin-6（IL-6）， and interleukin-8（IL-8） in supernatant of the HA-VSMCs in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of nuclear factor kappa-B（NF-κB） p65 mRNA and fibroblast growth factors 2（FGF2） mRNA in the HA-VSMCs in various groups； Western blotting method was used to detect the expression levels of NF-κB p65 and FGF2 proteins in the HA-VSMCs in various groups. Results Xiaoban Tongmai Formula contained 103 active ingredients that exert anti-AS effect by acting on 189 target genes. The potential targets included IL-6， IL-8， vascular endothelial growth factor A（VEGFA）， nuclear factor kappa B1（NF-κB1）， and RELA （NF-κB p65）. The GO functional analysis and KEGG pathway enrichment analysis results showed that Xiaoban Tongmai Formula exerted anti-AS effects by regulating lipid metabolism， hypoxia-inducible factor-1（HIF-1）， epidermal growth factor（EGF）， phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）， and NF-κB signaling pathways.The cell morphology and immunofluorescence staining results confirmed that the cells were HA-VSMCs. The oil red O staining results showed numerous red lipid droplets， indicating successful modeling. The MTT assay results showed that Xiaoban Tongmai Formula had no significant effect on the proliferation rate of HA-VSMCs within a certain dose range， indicating good safety. The ELISA results showed that compared with model group， the levels of MCP-1 and IL-6 in supernatant of the HA-VSMCs in rosuvastatin group and different doses of Xiaoban Tongmai Formula groups were decreased （P<0.05 or P<0.01）， and the levels of IL-8 in supernatant of the HA-VSMCs in 0.050 and 0.100 mg·L^-1 Xiaoban Tongmai Formula groups were decreased （P<0.01）； compared with rosuvastatin group， the levels of MCP-1 in supernatant of the HA-VSMCs in different doses of Xiaoban Tongmai Formula groups were decreased （P<0.01）， and the levels of IL-8 in supernatant of the HA-VSMCs in 0.050 and 0.100 mg·L^-1 Xiaoban Tongmai Formula groups were decreased （P<0.01）. Compared with model group， the expression levels of NF-κB p65 mRNA in the HA-VSMCs in rosuvastatin group and different doses of Xiaoban Tongmai Formula groups were decreased （P<0.01）， and the expression levels of FGF2 mRNA in the HA-VSMCs in rosuvastatin group and 0.050 and 0.100 mg·L^-1 Xiaoban Tongmai Formula groups were decreased （P<0.01）； compared with rosuvastatin group， the expression levels of NF-κB p65 and FGF2 mRNA in the HA-VSMCs in 0.050 and 0.100 mg·L^-1 Xiaoban Tongmai Formula groups were decreased （P<0.05 or P<0.01）. Compared with model group， the expression levels of NF-κB p65 and FGF2 proteins in the HA-VSMCs in rosuvastatin group and different doses of Xiaoban Tongmai Formula groups were decreased （P<0.01）； compared with rosuvastatin group， the expression levels of NF-κB p65 protein in the HA-VSMCs in 0.050 and 0.100 mg·L^-1 Xiaoban Tongmai Formula groups were decreased （P<0.01）， and the expression level of FGF2 protein in the HA-VSMCs in 0.100 mg·L^-1 Xiaoban Tongmai Formula group was decreased （P<0.01）. Conclusion Xiaoban Tongmai Formula has anti-inflammatory effect， inhibitory effect on the proliferation of HA-VSMCs， and anti-AS effect，and its mechanism may be related to the inactivation of NF-κB/FGF2 pathway.

Key words: Xiaoban Tongmai Formula, Atherosclerosis, Vascular smooth muscle cell, Network pharmacology, Cell proliferation