| Objective1. To investigate the effects of phosphatidylinositol-3 kinase(PI3Ks) signaling pathway in the migration of vascular smooth muscle cells(VSMCs) induced by epidermal growth factor(EGF) and in regulating actin binding protein Twinfilin-1 together with the reorganization of actin filament in response to EGF.2. To explore the effects of Ruanmailing on Twinfilin-1 and reorganization of actin filament in response to platelet-derived growth factor(PDGF).MethodsPart1VSMCs were isolated from thoracic aortae of Sprague-Dawley rats. Cells between 3-5 passage were used for experiment. Cells were incubated respectively with 0μmol/L, 5μmol/L, 25μmol/L and 50μmol/L LY294002 1 hour before 25ng/ml EGF treatment. Cells incubated with DMEM of 1% FBS were regarded as control. The migration rates of VSMCs were detected by wound healing assay at the 6th hour and 12 th hour by measuring the relative bare area produced by scribing. 6 hours after EGF incubation, expression of twinfilin-1 in cells was evaluated by Western-blot. Structure of microfilament and redistribution of twinfilin-1 in cells were detected by confocal microscopy with FITC-phalloidine and Alexa647 staining, respectively.Part2Ruanmailing serum was prepared from rats after 5 days of drug gavage. VSMCs were randomly divided as follows: control, PDGF(10ng/ml), 10% normal serum + PDGF, 10% Ruanmailing serum + PDGF,LY294002 + PDGF. In the latter three groups, cells were incubated respectively with 10% normal serum, 10% Ruanmailing serum, 50μmol/L LY294002 1hour before 10ng/ml PDGF treatment. 6 hours after treatment, twinfilin-1 in cells was detected by confocal microscopy with Alexa647 staining. Structure of microfilament was detected by FITC-phalloidine labeling under confocal microscopy.ResultsPart 1The migration of VSMCs was obviously increased by EGF(the relative bare area(%) between cells were 73.33±2.83 vs Ctrl 92.14±4.34 at the 6th hour and 44.77±4.23 vs Ctrl 82.96±2.67 at the 12 th hour after scratch, P<0.01). Migration of VSMCs was inhibited by LY294002 in a concentration-depended manner. The relative bare area(%) between cells incubated with different concentration of 5μmol/L, 25μmol/L, 50μmol/L LY294002 were 78.93±2.52, 85.86±3.16, 90.27±2.78 at the 6th hour and 54.63±3.30, 70.59±2.91, 81.13±3.03 at the 12 th hour respectively(all P<0.01 vs EGF).Expression of twinfilin-1 was upregulated by EGF in VSMCs. However it was inhibited by LY294002 in a concentrate-depended manner. Redistribution of twinfilin-1 was detected in cells stimulated by EGF together with the rearrangement of F-actin. However, expression and redistribution of twinfilin-1 by EGF could be blocked by LY294002, with less amount and loosely-arranged of stress fibers in cells.Part2In quiescent cells without any stimulants in medium, twinfilin-1 was localized in cytoplasm prominently around the nucleus with no apparent stress fibers in cells. Compared with the control, after PDGF(10ng/ml) stimulation, twinfilin-1 was upregulated and redistributed mainly from peri-nucleus to the whole cytoplasm, especially lamellipodia and actin rich filopodias. Actin cytoskeleton was rearranged with a cluster of stress fibers intensely distributed in cytoplasm. Interestingly however, after treatment with 10% Ruanmailing containing serum, both expression and redistribution of twinfilin-1 induced by PDGF were suppressed. Twinfilin-1 scarcely localized to the lamellipodia and filopodias. The stress fiber was markedly reduced and loosely arranged simultaneously. Treatment of 50μmol/l LY294002 leaded to the same change of twinfilin-1 and cytoskeleton to that of 10% Ruanmailing containing serum.Conclusion5 PI3 Ks signaling pathway plays an important role in the migration of VSMCs.Inhibition of PI3 Ks signaling pathway may suppress the expression and redistribution of twinfilin-1, resulting in less amount of stress fibers. Ruanmailing may suppress the expression and redistribution of twinfilin-1 induced by PDGF, with decreased and loosely-arranged stress fibers. |
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