葫芦素I对HaCaT细胞体外增殖及对角蛋白17、STAT3、VEGF表达的影响

Effects of cucurbitacin I on in vitro proliferation of HaCaT cells and the expression of keratin 17, signal transducer and activator of transcription 3 and vascular endothelial growth factor

目的探讨葫芦素I对HaCaT细胞体外增殖及角蛋白17（K17）、信号转导及转录激活子3（STAT3）、血管内皮生长因子（VEGF）表达的影响。方法采用不同浓度葫芦素I（0．0125、0．025、0．05、0．1μmol／L）、含与0．1μmol／L葫芦素I等体积DMSO的DMEM培养液（溶媒组）、DMEM培养液（阴性对照组）、10nmol／L卡泊三醇（阳性对照组）分别作用于HaCaT细胞。采用CCK8法检测葫芦素I作用12、24、36h后对HaCaT细胞体外增殖的影响，RT．PCR法检测葫芦素I作用24h对HaCaT细胞K17和VEGFmRNA表达的影响，Western印迹法检测葫芦素I作用24h对HaCaT细胞K17、STAT3、磷酸化STAT3（P-STAT3）和VEGF蛋白表达的影响。结果0．0125μmol／L葫芦素I作用12h即开始表现出对HacaT细胞体外增殖的抑制作用，当葫芦素I浓度增加到0．1μmol／L时，24h和36h的抑制率分别为43．00％4-2．11％和48．98％±2．27％。与阴性对照组相比，各浓度葫芦素I组对HaCaT细胞体外增殖均有抑制作用（均P〈0．05），且该抑制作用随葫芦素I浓度的增加及作用时间的延长逐渐增强。不同浓度葫芦素I作用于HaCaT细胞24h后，K17mRNA及其蛋白、P-STAT3蛋白、VEGFmRNA及其蛋白表达量均随葫芦素I浓度的增加而降低，差异有统计学意义（均P〈0．05）。结论葫芦素I可抑制HaCaT细胞的体外增殖，并可在mRNA水平下调K17、VEGF的表达，在蛋白水平下调K17、P，STAT3、VEGF的表达。

Objective To evaluate effects of cucurbitacin I on in vitro proliferation of HaCaT cells and the expression of keratin 17 （K17）, signal transducer and activator of transcription 3 （STAT3） and vascular endothelial growth factor （VEGF） in cultured HaCaT cells. Methods In vitro cultured HaCaT ceils were divided into 6 groups to be treated with cucurbitacin I at different concentrations of 0.0125, 0.025, 0.05 and 0.1 μmol/L （0.0125, 0.025, 0.05 and 0.1 μmol/L cucurbitacin I groups）, DMEM containing the same volume of DMSO as 0.1 μmol/L cucurbitacin I （DMSO group）, DMEM （negative control group） and 10 nmol/L calcipotriol （positive control group）, respectively. Cell counting kit- 8 （CCK8） assay was performed to assess in vitro cellular proliferative activity after 12-, 24-, 36-hour treatment, reverse transcription （RT） -PCR to measure the mRNA expression of K17 and VEGF in HaCaT cells after 24-hour treatment, and Western blot analysis to determine the protein expression of K 17, STAT3, phosphorylated-STAT3 （p-STAT3） and VEGF after 24-hour treatment. Statistical analysis was carried out by one-way analysis of variance （ANOVA）, repeated measures ANOVA, Student-Newman-Keuls （SNK）-q test and Pearson correlation analysis. Results The proliferative activity of HaCaT cells started to decrease after 12-hour treatment with cucurbitacin I at the concentration of 0.0125 txmol/L. When the concentration of cucurbitacin I increased up to 0. 1 μmol/L, the cell proliferation rates were inhibited by 43.00% ± 2.11% and 48.98% ± 2.27% after 24- and 36-hour treatment respectively. Compared with the negative control group, cucurbitacin I at different concentrations all could inhibit in vitro proliferation of HaCaT cells （all P 〈 0.05）, and the inhibitory effects increased gradually with the increase of cucurbitacin I concentration and treatment duration. After 24-hour treatment, the mRNA expression of K17 and VEGF and the protein expression of K17, VEGF and P- STAT3 were significantly decreased along with the increase of concentration of cueurbitacin ] （all P 〈 0.05 ）. Conclusion Cueurbitaein I can inhibit in vitro proliferation of HaCaT cells, and down-regulate the mRNA expression of K17 and VEGF and protein expression of K17, VEGF and P-STAT3.