卡马拉素对HaCaT细胞体外增殖及对白细胞介素17C、CCL20、NF—KB表达的影响

Effects of rottlerin on in v/fro proliferation of and expressions of interleuldn-17C, CCL20 and nuclear factor-kB in a human keratinoeyte cell line HaCaT

目的探讨卡马拉素对HaCaT细胞体外增殖及对白细胞介素17C（IL-17C）、趋化因子CCL20、NF—KB表达的影响。方法用不同浓度卡马拉素组（0．5、1．0、2．0、4．0μmol／L）、含与4．0μmolIL卡马拉素等体积二甲基亚砜的RPMI1640培养液（溶媒组）、RPMI1640培养液（对照组）分别作用于HaCaT细胞。采用CCK8法检测卡马拉素作用于HaCaT细胞24、48、72h对细胞体外增殖的影响；RT—PCR法检测卡马拉素对HaCaT细胞IL-17C和CCL20mRNA表达的影响；Western印迹法检测卡马拉素对HaCaT细胞IL-17C、CCL20和NF—KB蛋白表达的影响。统计学处理采用重复测量的方差分析、单因素方差分析和Pearson相关分析。结果各浓度组卡马拉素对HaCaT细胞增殖的抑制作用有随时间变化的趋势（F=126．936，P〈0．05），药物作用时间越长，抑制作用越强；HaCaT细胞增殖的抑制率也随卡马拉素浓度的增加而升高（F=838．308，P〈0．05）；不同浓度卡马拉素组对HaCaT细胞体外增殖的抑制作用随时间变化的趋势不同，药物浓度与时间存在交互作用（F=15．961，P〈0．05）。不同浓度卡马拉素作用于HaCaT细胞48h后，IL-17CmRNA及其蛋白、CCL20mRNA及其蛋白、NF—KB蛋白表达量均随卡马拉素浓度的增加而不断降低，差异均有统计学意义（F值分别为206．041、233．887、143．883、162．431、577．915，均P〈0．05）。结论卡马拉素可抑制HaCaT细胞的体外增殖，并可在mRNA和蛋白水平下调IL-17C、CCL20的表达，而两者表达量的降低可能与NF—KB表达下调有关。

Objective To investigate the effects of rottlerin on in vitro proliferation of and expressions of interleukin （IL）-17C, CCL20 chemokine, and nuclear factor （NF）-KB in cultured human HaCaT keratinocytes. Methods Some HaCaT cells were divided into several test groups treated with rottlerin at concentrations of 0.5, 1.0, 2.0 and 4.0 μmol/L, a solvent group treated with RPMI 1640 culture solution containing the same volume of dimethyl sulfoxide （DMSO） as that of 4.0 μmol/L rottlerin, and a control group treated with RPMI 1640 culture solution. Cell counting kit-8 （CCKS） assay was conducted to estimate the proliferative activity of HaCaT cells after 24-, 48- and 72- hour culture, RT-PCR to determine the mRNA expressions of IL-17C and CCL20 after 48-hour culture, and Western blot to measure the protein expressions of IL-17C, CCL20 and NF-KB after 48-hour culture. Statistical analysis was carried out by using repeated-measures analysis of variance, one-way analysis of variance and Pearson correlation analysis with the SPSS16.0 software. Results Rottlerin showed an inhibitory effect on the proliferation of HaCaT cells, and the inhibitory effect increased over time （F = 126.936, P 〈 0.05） and with the increase of rottlerin concentrations （F = 838.308, P 〈 0.05）, with a significant interaction effect between rottlerin concentrations and treatment duration （F = 15.961, P 〈 0.05）. After 48-hour treatment, a significant decrease was observed in the mRNA and protein expressions of IL-17C （F = 206.041,233.887, respectively, both P 〈 0.05） and CCL20 （F = 143.883, 162.431, respectively, both P 〈 0.05）, as well as in the protein expression of NF-KB （F= 577.915, P〈 0.05） in the test groups with the increase in rottlerin concentrations. Conclusions Rottlerin can inhibit the proliferation of HaCaT cells in vitro, and decrease the mRNA and protein expressions of IL-17C and CCL20 likely by downregulating the protein expression of NF-KB.