桉树脑对HaCaT细胞水通道蛋白3和透明质酸表达的影响

Effects of 1,8-cineole on expression of aquaporin 3 and hyaluronan in HaCaT cells

目的:探讨桉树脑(Cin)对人永生化角质形成细胞(HaCaT)水通道蛋白(AQP)3及透明质酸(HA)转录和翻译的影响。方法:以倍他米松(BT)为对照,不同浓度Cin(0.01%、0.001%、0.0001%和0.00001%)分别孵育HaCaT细胞36 h后,采用实时荧光定量聚合酶链式反应(q-PCR)检测AQP3 mRNA表达水平,免疫印迹法(western blot)分析AQP3蛋白表达,以及酶联免疫吸附试验(ELISA)检测细胞外基质中HA的含量。结果:与空白对照组相比,qPCR结果示0.01%和0.001%Cin均显著增加了AQP3基因的转录(P<0.05);而10μmol/L BT明显下调AQP3基因转录(P<0.05);0.01%和0.001%Cin分别与10μmol/L BT共同孵育HaCaT细胞后,均可逆转10μmol/L BT引起的AQP3 m RNA表达下调(P<0.05)。Western blot结果表明,与空白对照组比较,0.01%Cin组和0.001%Cin组AQP3蛋白表达均显著增加(P<0.05);10μmol/L BT组AQP3蛋白表达明显下调(P<0.05)。与空白对照组比较,0.01%Cin+10μmol/L BT组和0.001%Cin+10μmol/L BT组AQP3蛋白表达均下降(P<0.05);与10μmol/L BT组比较,AQP3蛋白表达均明显提高(P<0.05)。ELISA结果表明,与空白对照组比较,10μmol/L BT组HA表达水平下调(P<0.05);与10μmol/L BT组比较,0.01%Cin+10μmol/L BT组和0.001%Cin+10μmol/L BT组HA表达水平均上调(P<0.05),但均低于空白对照组(P<0.05)。结论:Cin可不同程度逆转BT对角质形成细胞AQP3和HA的抑制作用。

Objective:To investigate the effects of eucalyptol(Cin)on the expression of aquaporin-3(AQP3)and hyaluronan(HA)in human keratinocytes(HaCaT).Methods:With betamethasone(BT)as control,HacaT cells were incubated with 1×10-2~1×10-5 diluted Cin for 36 hours.AQP3 gene and protein were detected by real-time fluorescence quantitative PCR(qPCR)and western blot.The expression of HA in extracellular matrix was detected by enzyme-linked immunosorbent assay(ELISA).Results:Compared with the control group,0.01%Cin and 0.001%Cin significantly increased the expression of AQP3 gene(P<0.05);and 10μmol/L BT significantly decreased the expression of AQP3 gene(P<0.05).HaCaT cells incubated with 0.01%Cin+10μmol/L BT or 0.001%Cin+10μmol/L BT both reversed the decrease of AQP3 induced by 10μmol/L BT group(P<0.05).Western blot results showed that both 0.01%Cin and 0.001%Cin significantly increased the expression of AQP3 protein compared to the control group(P<0.05);AQP3 protein was significantly down-regulated by 10μmol/L BT group compared to the control group(P<0.05);both 0.01%Cin+10μmol/L BT and 0.001%Cin+10μmol/L BT decreased expression of AQP3 protein significantly compared to the control group(P<0.05).Compared with 10μmol/L BT group,AQP3 protein was significantly in creased(P<0.05).ELISA results showed that the HA level was decreased in 10μmol/L BT group compared with the control group(P<0.05).The HA levels in 0.01%Cin+10μmol/L BT group and 0.001%Cin+10μmol/LBT group were higher than that in 10μmol/L BT group(P<0.05),but failed to reach the level of the control group.Conclusion:Cin can reverse the inhibition effects of BT on AQP3 and HA in HaCaT cells.