| ObjectiveTo investigate the transmembrane transport mechanism of gigantol and syringic acid in HLEC cells, Providing pharmacokinetic basis for drug discovery and laying a solid theoretical foundation for drug concentration to targeted cell and targeted therapy.Methods1. Drug target identification cells. Take fluorescence labeled gigantol and syringic acid as materials incubated with different kinds of cells, such as human lens epithelial cells (HLEC), human normal liver cell (L-02), human umbilical vein endothelial cell (EA.hy926), human lung carcinoma cell (A549), human colon cancer cell (SW480), to comparison the fluorescence intensity of different groups. Then determine whether gigantol and syringic acid has targeted to cells.2. The concentration dependence observation about the cellular uptake of gigantol and syringic acid., and using confocal laser scanning microscopy to observe cell fluorescence intensity on different concentrations for gigantol and syringic acid. Through quantitative analysis of the fluorescence intensity to reflect the amount of gigantol and syringic acid absorbed into HLEC cells.3. The time dependence observation about the cellular uptake of gigantol and syringic acid. Using LSCM to observe cell fluorescence intensity on different time periods for gigantol and syringic acid. Through quantitative analysis of the fluorescence intensity to reflect the amount of gigantol and syringic acid absorbed into HLEC cells.4. On the condition of different cell membrane inhibitors and specific temperature processing, after treated with gigantol and syringic acid for a certain time, using confocal laser scanning microscopy to detect cell fluorescence intensity change and inhibition rate of absorption. And determine the membrane transport mechanism of gigantol and syringic acid.5. Using atomic force microscopy to detect the roughness and ultrastructure changes of HLEC cell membrane after treated with gigantol and syringic acid.Results1. The absorption level of gigantol into HLEC was bigger than the amount of EA.hy926 cells, and only a small amount of gigantol were uptaked into L-02, A549 and SW480 cells. The absorption level of syringic acid into HLEC and L-02 cells were notablely higher than SW480 and EA.hy926 cells. Only a small amount of syringic acid were uptaked into A549 cells.2. The fluorescence intensity of intracellular was enhanced by the increased concentration when treated with gigantol. The cellular absorption was larger when treated with 1μg/ml.**P<0.01 when compared with the other groups. And drugs were mainly located in the cytoplasm. When the concentration increased to 10μg/ml, the intracellular fluorescence intensity reached the maximum and distributed throughout the cell range.The fluorescence intensity of intracellular was enhanced by the increased concentration when treated with syringic acid. In combination with the results determined by MTT, the cellular absorption was very weak when the concentration were less than 10μg/ml. When the concentration was greater or equal to 10μg/ml, the intracellular uptake was the biggest and distributed in the cytoplasm and nucleus.##P<0.01 when compared with the other groups.3. After HLEC cells were treated with gigantol and syringic acid for 0.5h, weak fluorescence appeared, the drug intake was low and then reached its peak at 2h. As time goes by, the fluorescence intensity falling quickly to ateady state after 6 hours incubation. The process has no fluorescence quenching phenomenon and the character of fluorescent tag drugs are stable.4. HLEC cells treated with gigantol:the fluorescence intensity were reduced by 64.30% and 48.82% respectivily after treated with energy inhibitor 2,4-DNPH and NaN3. The fluorescence intensity were reduced by 87.02% and 84.95% respectivily after treated with endocytosis inhibitors mβ-CD and CPZ. The fluorescence intensity was reduced by 67.96% after treated with Hep. The fluorescence intensity was reduced by 76.61% after treated with 4℃.HLEC cells treated with syringic acid:the fluorescence intensity was declined by 69.75% after treated with energy inhibitor 2,4-DNPH and another energy inhibitor NaN3 was not that obvious. The fluorescence intensity were declined by 88.81% and 72.03% respectivily after treated with endocytosis inhibitors m(3-CD and CPZ. The fluorescence intensity was reduced by 54.25% after treated with Hep. The fluorescence intensity was reduced by 83.26% after treated with 4℃.5. Ultrastructure changes on cell membrane during transport:when treated with gigantol, some pit appeared with a diameter rang of 150～300nm, height rang of 40～60nm, roughness increased from 17nm to 26nm. The main size distribution histogram of protrusions on HLEC rised from 320～350nm to 420～450nm after treated with gigantol. Some pit structure of endocytosis appeared on cell membrane surface.When handled with syringic acid, some pit arised with a diameter rang of 125～ 350nm, height rang of 45～75nm, roughness increased from 16nm to 28nm. The main size distribution histogram of protrusions on HLEC rised from 105nm to 210nm after treated with syringic acid. Some pit structure of endocytosis appeared on cell membrane surface.ConclutionThe characteristic of gigantol and syringic acid transmembrane transport into HLEC cells mainly distributed in cytoplasm. Both of them has a time and dose dependent mode, and targeting to HLEC cells. Membrane transport inhibitors and temperature have a great inhibitory effects on the absorption of giantol and syringic acid. Many tipical pit on the membrane surface appeared when treated with gigantol and syringic acid. Therefore, The membrane transport mechanism of gigantol and syringic acid are active both through energy and carrier dependent endocytosis. |
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