甘草酸二铵对人毛囊生长作用及对Wnt／β连环蛋白信号通路的影响

In vitro effect of diammonium glycyrrhizinate on the growth of human hair follicles and Wnt/β-catenin signaling pathway

目的 探讨甘草提取物甘草酸二铵对体外培养人毛囊生长的作用并检测Wnt/β连环蛋白信号通路相关分子的表达。方法 分别将体外分离人毛囊培养于0.1、0.01、0.001、0.000 1 μmol/L浓度的甘草酸二铵中10 d，并设置不添加甘草酸二铵Williams E培养基组为对照组，显微镜下每日测量并记录其生长长度，观察毛囊形态改变并拍照。用免疫荧光评估毛母质细胞增殖情况及毛母质细胞中Wnt/β连环蛋白信号通路各主要分子β连环蛋白、GSK3β、p？GSK3β、Lef1表达情况。采用重复测量方差分析及单因素方差分析进行统计学分析。结果 重复测量方差分析显示，与对照组相比，仅0.01 μmol/L甘草酸二铵对毛囊生长的促进作用有统计学意义（P 〈 0.05），其余各组与对照组相比差异无统计学意义（均P 〉 0.05）。与对照组相比，0.01 μmol/L甘草酸二铵组毛囊进入退行期时间推迟，而其余各组与对照组相比均无明显变化。免疫荧光显示，与对照组相比，0.1、0.01、0.001 μmol/L浓度组毛母质细胞中ki67阳性细胞较多，而0.000 1 μmol/L浓度组无明显区别。单因素方差分析显示，β连环蛋白、p？GSK3β、Lef1在各组中的表达量差异有统计学意义（F =12.604、16.65、15.266，均P 〈 0.05），而GSK3β在各组中的表达差异无统计学意义（F =1.472，P 〉 0.05）。LSD？t检验显示，与对照组相比，0.1、0.01、0.001 μmol/L浓度组毛母质细胞中β连环蛋白、p？GSK3β、Lef1表达量较多（均P 〈 0.05），而0.000 1 μmol/L浓度组与对照组差异无统计学意义（P 〉 0.05）。结论 0.01 μmol/L甘草酸二铵具有明显促进人毛囊生长的作用，可增强毛母质细胞增殖活性，延迟毛囊进入退行期，该作用可能与其激活Wnt/β连环蛋白信号通路相关。

Objective To evaluate the effect of diammonium glycyrrhizinate extracted from the Chinese traditional medicine licorice root on the growth of human hair follicles cultured in vitro, and to detect the of wnt/β-catenin signaling pathway-related molecules. Methods Isolated hair follicles were cultured with diammonium glycyrrhizinate at different concentrations of 0.1, 0.01, 0.001 and 0.000 1 μmol/L for 10 days, and the hair follicles cultured in Williams′ E medium without diammonium glycyrrhizinate served as a control group. The length of hair follicles was measured under a microscope every day, the morphologic changes of hair follicles were observed, and photos were taken. Immunofluorescence assay was performed to assess the proliferation of hair matrix cells, as well as to determine the of β-catenin, glycogen synthase kinase 3β （GSK3β）, phosphorylated GSK3β （p-GSK3β） and lymphoid enhancer factor-1 （Lef1） in the Wnt/β-catenin signaling pathway. Statistical analysis was carried out by repeated-measures analysis of variance and one-way analysis of variance. Results As repeated-measures analysis of variance showed, only 0.01 μmol/L diammonium glycyrrhetate showed significantly promotive effect on the growth of hair follicles compared with the medium alone （P 〈 0.05）, and there were no significant differences in the length of hair follicles between the other concen-tration groups and the control group. Compared with the control group, the transition to the catagen phase of human hair cycle was delayed in the 0.01-μmol/L diammonium glycyrrhetate group, while it did not change in the other diammonium glycyrrhetate groups and control group. Immunofluorescence assay showed that the number of ki67-positive hair matrix cells was obviously increased in the 0.1-, 0.01-, 0.001-μmol/L diammonium glycyrrhizinate groups compared with the control group, while there was no difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group. One-way analysis of variance revealed that the of β-catenin, p-GSK3β and Lef1 significantly differed among all the groups （F =12.604, 16.65, 15.266 respectively, P 〈 0.05）, while no significant difference in the of GSK3β was found among these groups （F =1.472, P 〉 0.05）. Least significant difference （LSD）-t test revealed that the expre-ssion of β-catenin, p-GSK3β and Lef1 in the hair matrix cells was significantly higher in the 0.1-, 0.01-, 0.001-μmol/L diammonium glycyrrhizinate groups than in the control group （all P 〈 0.05）, but there was no significant difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group （P 〉 0.05）. Conclusion Diammonium glycyrrhetate at the concentration of 0.01 μmol/L shows markedly promotive effect on the in vitro growth of hair follicles, and can increase the proliferative activity of hair matrix cells and delay the transition to the catagen phase, which may be associated with the activation of Wnt/β-catenin signaling pathway.