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To investigate the effects of atractylodin in mouse primary vascular smooth muscle cells(VSMCs).
Observe different concentrations (5,10,20,40,60 mg/l) of atractylodin dilutions impact in VSMCs at different time points, observe changes in cell morphology by microscope, cell proliferation assay by MTT, detected the changes in cell cycle and apoptosis percentage by flow cytometry, detect the changes in several specific proteins by Western blotting, and examine changes in cytokine secretion by liquid chip.
Repeated tests confirmed concentration impact on the effect of atractylodin acting on VSMCs. The concentration of 5 mg/l and 10 mg/l atractylodin dilutions promote VSMCs proliferation, concentrations greater than 20 mg/l inhibit VSMCs proliferation and exist in dependent relationship between time and concentration. With increasing concentration of atractylodin, cell proliferation inhibition rate was close to 100%. Morphological features of apoptosis were observed by microscope. In the treatment group, many VSMCs were be at the cell cycle G1 phase as indicated by flow cytometry, the quantity of apoptotic cells increased according to time and concentration increases. Western blotting analysis showed, after atractylodin acted on VSMCs, expression of PARP-1, p-IRE1 and GRP78 was significantly higher than the control group, and AIF was activated. However, cleaved-caspase 3, caspase 8 and caspase 9 precursor protein expression remained unchanged. Liquid chip tests confirmed that atractylodin inhibits VSMCs secreted IL-6, IL--8, and TNF-alpha.
Atractylodin may inhibit the proliferation of VSMCs in a dose and time dependent manner. Atractylodin blocks the cell cycle and the cell cycle is arrested in G1 phase. Atractylodin induces caspase-independent apoptosis in VSMCs via PARP-1/AIF apoptotic pathway.