| Objective We study the inhibition effect of Tanshione IIA on the proliferation of PDGF-BB-induced HAVSMC cells by applying the state-of-the-arts biomedical technology. Two-dimensional electrophoresis and matrix assisted laser desorption ionization time-of-flight mass spectrometry were used to analysis and identify the proteins differentially expressed after Tanshinone IIA treatment of the cells. The study provided basic information for the further clinical application of Tanshinone II A and clarified the multi-targets of Chinese traditional medicine against atherosclerosis.Methods The HAVSMC cells were seeded in 96-well plate, and the cells were treated with different concentrations of Tanshione IIA with 10 μg/L PDGF-BB for 24h. The proliferation and cell cycle of PDGF-BB-induced HAVSMC were analyzed by Flow cytometry. The differentially expressed were identified by 2D-DIGE (Two dimension difference gel electrophoresis) associate with Mass Spectrometry Identification. Finally, function of the differential proteins was analysis.Results 1) According to MTT assay, Tanshion IIA at low concentration (at 0.5 μg/mL or below) didn’t affect the proliferation of HAVSMC cells induced by PDGF-BB(P> 0.05) Tanshion IIA at higher concentration (more than 1 μg/mL) significantly inhibited the proliferation of PDGF-BB-induced HAVSMC cells (P<0.01). We choose 1 μg/mL,2 μg/mL and 4 μg/mL of Tanshion IIA to conduct the subsequent experiments.2) Compared with the control, ① The numbers of G1/M cells were decreased and the cells with S/M and G2/M cycle were increased after treated with PDGF-BB, which suggested that PDGF-BB can promote the proliferation of the HAVSMC cells. ② After exposure to 2 and 4 μg/mL of Tanshione IIA, the number of G1/M cells were increased and the numbers of S/M and G2/M were decreased, which indicated that Tanshione IIA suppressed the cell proliferation by arrest cell cycle in G1 phase. Compared with PDGF-BB-induced group, groups co-cultured with Tanshione IIA both at 1 μg/mL and 2 μg/mL has no significant effect on cell cycle. However,4 μg/mL Tanshione IIA can induce an incensement of G1/M cells and a dramatically drop in the number of S/M and G2/M cells. Our results suggested that Tanshione IIA suppressed the proliferation of PDGF-BB-induced HAVSMC cells by cell cycle arrest in G1 phase.3) According to the proteomic results, there were 46 differential proteins with 44 down-regulated and 2 up-regulated. The down-regulated proteins were listed as follows: Protein disulfide-isomerase.60 kDa heat shock protein, mitochondrial, T-complex protein 1 subunit epsilon, Heterogeneous nuclear ribonucleoprotein K, Keratin, type I cytoskeletal 9, E3 ubiquitin-protein ligase MIB1 Leucine-zipper-like transcriptional regulator 1, Zinc finger FYVE domain-containing protein 19, Vimentin, Unconventional myosin-ⅩⅧb, Protein disulfide-isomerase A3, Mineralocorticoid receptor, Kelch-like ECH-associated protein 1, T-complex protein 1 subunit beta, Maestro heat-like repeat-containing protein family member 2A, Cytochrome b-c1 complex subunit 1, mitochondrial, Interferon alpha-8, Hypoxanthine-guanine phosphoribosy Ltransferase, Zinc finger protein 229, NADH dehydrogenase flavoprotein complex 1 assembly factor,Smoothelin, Zinc finger protein 394, Kinesin-like protein KIF1C, Zinc finger protein 618, cytoplasmic Ras and EF-hand domain-containing protein, Solute carrier family 25 member 47, Actin, cytoplasmic 2, Glycyl-tRNA synthetase, ATP synthase subunit beta, mitochondrial, Tektin-4,78 kDa glucose-regulated protein.Tropomyosin beta chain, Tropomyosin alpha-4 chain, Ras-related protein Rab-39A, Tropomyosin alpha-3 chain, LIM domain onlyprotein 7, Zinc finger imprinted 3, Microtubule-associated protein 6. Transmembrane protein 2, NADH dehydrogenase flavoprotein2, mitochondrial, Septin-14, Nesprin-2, Stress-70 protein, mitochondrial stress protein, Myosin light polypeptide 6. While the expression of cysteine ligase and tyrosine protein kinase was up-regulated. The functions of these differential proteins are mainly involved in oxidative stress, cellular energy metabolism substances, cell components and cytoskeleton, signal transduction and genetic expression regulation.Conclusion① By treating with 10 μg/L PDGF-BB for 24h, we successfully established cell proliferation model of HAVSMA ② Tanshione IIA can significantly inhibit the proliferation of PDGF-BB-induced HAVSMC cells by arresting cellular cycle at G1 phrase. ③ Tanshione IIA inhibite the proliferation of PDGF-BB-induced HAVSMC and played as the role of anti-atherosclerotic by various signaling pathways involving cytoskeleton structure, signal transduction, energy metabolism, oxidative stress to name a few. |
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