| ObjectiveThe impact of high iodine on artery vascular and mechanism were discussed via observing the proliferation or apoptosis of iodine with different concentration on Rat Thorax Aorta Vascular Smooth Muscle cells (VSMC) cultured in vitro.MethodsRat Thorax Aorta Vascular Smooth Muscle cells (A7r5) were cultured in37℃,5%CO2, saturated humidity. The experiment was performed during the logarithmic growth phase of cells. Experimental classification:the control group, iodized salt group. The latter was divided into22groups with the iodine concentration ranging from0.075mg/L to7500mg/L. And the blank group served as a control group. Cells were collected after the continuous cultivation of12h,24h and48h respectively. The amount of cells were detected by Cell counting method, the cell activity by Methyl Thiazolyl Tetrazolium (MTT), cell morphology by Wrigh-Giema staining, cell apoptosis morphology by the method of Acridine Orang staining, the apoptotic percent of cells by the method of flow cytometry, the expression level of baxmRNA and bcl-2mRNA related to the apoptosis of cultured cells by Reverse Transcription Polymersade Chain Reaction (RT-PCR), and the expression level of ASK-1and TRX-1by the method of Western Bloting. Comparison among many groups was performed by analysis of variance, comparison between two groups by Dunnett-test.Results1Cell CountingCells were cultured in culture solution contained iodine of different concentration for12h,24, and48h respectively. After culture of12h, compared with the control group, cells of the iodized salt group with the iodine concentration ranging from0.075to3750mg/L grew normally (P>0.05). The growth of cells cultured in the iodized salt group with the iodine concentration of5250mg/L was obviously restrained (P<0.01), and the inhibitory action was positively correlated with the iodine concentration. After culture of24h, compared with the control group, cells of the iodized salt group with the iodine concentration ranging from0.075to1500mg/L grew normally (P>0.05). The growth of cells cultured in the iodized salt group with the iodine concentration of3000mg/L was obviously restrained (P<0.01), and the inhibitory action was positively correlated with the iodine concentration. After culture of48h, compared with the control group, cells of the iodized salt group with the iodine concentration of0.075～1120mg/L grew normally (P>0.05). The growth of cells cultured in the iodized salt group with the iodine concentration of2250mg/L was obviously restrained (P<0.01), and the inhibitory action was positively correlated with the iodine concentration. With the iodine concentration increasing, the inhibitory action of iodine on cell growth was positively correlated with the iodine concentration.2Cell Activity by Methyl Thiazolyl Tetrazolium (MTT)Cells were cultured in culture solution contained iodine of different concentration for12h,24, and48h respectively. After culture of12h, compared with the control group, cells of the iodized salt group with the iodine concentration of2250mg/L grew normally (P>0.05). The growth of cells cultured in the iodized salt group with the iodine concentration of3750mg/L was obviously restrained (P<0.01), and the inhibitory action was positively correlated with the iodine concentration. After culture of24h, compared with the control group, cells of the iodized salt group with the iodine concentration of1500mg/L grew normally (P>0.05). The growth of cells cultured in the iodized salt group with the iodine concentration of3000mg/L was obviously restrained (P<0.01), and the inhibitory action was positively correlated with the iodine concentration. After culture of48h, compared with the control group, cells of the iodized salt group with the iodine concentration of600mg/L grew normally (P>0.05). The growth of cells cultured in the iodized salt group with the iodine concentration of1500mg/L was obviously restrained (P<0.01), and the inhibitory action was positively correlated with the iodine concentration. 3Cell Morphology Observation3.1Cell Morphology Observation by Wrigh-Giema StainingCompared with the control group, when the iodine concentration was less than or equal to1500mg/L, cell morphology was normal and no obvious changes could be found with cell morphology. And when the iodine concentration was equal to or more than3750mg/L, the amount of Rat Thorax Aorta Vascular Smooth Muscle cells of the iodized salt group decreased with concentration increasing. Cell shrinkage and distortion occurred and Intercellular space also increased. With the concentration increasing, cell membrane damage and cell nuclei pyknosis occurred, and cell showed star and polygon. At the same time, bare nucleus and even reticular chromatin structure also occurred.3.2Cell Morphology Observation by Acridine Orang StainingCompared with the control group, when the iodine concentration was less than or equal to1500mg/L, no obvious changes could be found with cell and cell structure was integrate. When the iodine concentration increased to3750mg/L or higher, cell morphology became irregular. Cell membrane was damaged. Cytoplasm decreased, got densified, lost contact with the surrounding cells and gathered around the cell edge. Cell nucleus got out of shape obviously. With the concentration increasing, fluorescence fragments gradually increased, and apoptotic bodies occurred with the iodine concentration increasing to certain value.4Apoptotic percent of Cells by the method of Flow CytometryCells were cultured in culture solution contained iodine of different concentration for48h. After culture of48h, compared with the control group, survival rate of cells of the iodized salt group with the iodine concentration of750mg/L decreased obviously(P<0.0/). And with the iodine concentration increasing, survival rate of cells decreased gradually. The apoptosis rate of cells cultured in the iodized salt group with the iodine concentration of750mg/L increased obviously (P<0.01). And with the iodine concentration increasing, survival rate of cells increased gradually.5Effect of Iodine on Expression of baxmRNA and bcl-2mRNA of A7r5cellCells were cultured in culture solution contained iodine of different concentration for48h. After culture of48h. compared with the control group, expression of baxmRNA and bcl-2mRNA of cells of the iodized salt group with the iodine concentration of750mg/L changed and statistic difference existed (P<0.05).When1500mg/L, expression of bcl-2mRNA gradually weakened, and statistic difference existed (P<0.05). And expression of bax mRNA gradually strengthened, and statistic difference existed (P<0.05)6Effect of Iodine on Expression of ASK-1and TRX-lof A7r5cellCells were cultured in culture solution contained iodine of different concentration for48h. After culture of48h, compared with the control group, expression level of ASK-1and TRX-1of cells of the iodized salt group with the iodine concentration of750mg/L changed significantly. And with the iodine concentration increasing, expression of ASK-1gradually weaken and statistic difference existed (P<0.01). And expression of TRX-1gradually strengthened and statistic difference existed (P<0.0J).Conclusion1. Iodine with certain concentration restrains the growth of Rat Thorax Aorta Vessel Smooth Muscle cells (VSMC), which reduced cell activity and showed a time-dose relationship.2. Iodine with high concentration can change morphology of Rat Thorax Aorta Vessel Smooth Muscle cells (VSMC) and lead to cell apoptosis.3. Iodine with high concentration can lead to the increase of cell apoptosis percent of Rat Thorax Aorta Vessel Smooth Muscle cells (VSMC) and promote cell apoptosis.4. Iodine with high concentration can not only stimulate expression of promoting apoptosis mRNA bax and promoting apoptosis protein TRX-1, but also restrain expression of restraining apoptosis mRNA bcl-2and restraining apoptosis protein ASK-1, which leads to cell apoptosis.So, the results above prompt that iodine with high concentration can damage Rat Thorax Aorta Vessel Smooth Muscle cells (VSMC). And that may be one of mechanisms leading to arterial sclerosis. |
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