紫草素对脑胶质瘤细胞氨肽酶N活性的影响及机制

Inhibitory effect of shikonin on aminopeptidase N of glioma cells and its mechanism

目的探讨紫草素对脑胶质瘤细胞氨肽酶N活性的影响及机制。方法本文采用酶抑制动力学方法,研究了紫草素对氨肽酶N的抑制作用、抑制类型和抑制动力学常数,并进行了脑胶质瘤U251细胞的凋亡生长抑制实验及不同浓度紫草素溶液在氨肽酶N溶液中的荧光光谱测定、圆二色光谱测定。结果紫草素能可逆的竞争性抑制氨肽酶N的活性(半数抑制率IC50为93.59μmol·L^(-1),抑制常数Ki为32.74μmol·L^(-1));随着孵化时间的增加,不同浓度紫草素对氨肽酶N的相对活性无影响,其对脑胶质瘤U251细胞的半数抑制浓度为66.12μmol·L^(-1);紫草素可浓度依赖性的促进氨肽酶N内源性荧光发生猝灭效应,两者的结合常数Ka为4.12×104 L·mol-1,热力学参数ΔH和ΔS分别为38.64 k J·mol-1和179.21 J/(mol·K);紫草素还可诱导氨肽酶N构象的解折叠,使α-螺旋含量增加到32.1%,β-折叠含量减小到22.9%。结论紫草素是一种可逆的竞争型氨肽酶N抑制剂,其可迅速降低酶的活性,并诱导脑胶质瘤U251细胞的凋亡。紫草素的作用可能通过猝灭氨肽酶N的内源性荧光并诱导氨肽酶N构象的解折叠,使α-螺旋含量增加而实现的。

Objective To determine the effect and mechanism of shikonin on aminopeptidase N（APN） of alioma cells. Methods Enzyme inhibitory kinetics method was adopted to analyze the inhibitory effect, inhibition type and the kinetic constant of shikonin on APN. Growth inhibition test was conducted to assess the apoptosis of U251 glioma cell. The fluorescence spectra and circular dichroism spectra of different concentrations of shikonin in APN solution were also measured. Results Shikonin was a competitive inhibitor, and its half inhibition concentration（IC50） and inhibition constant（Ki） on APN were 93.59 μmol·L^（-1） and 32.74 μmol·L^（-1）, respectively. The effect of different concentrations of shikonin on APN didn＇t show dramatic change with the increase of incubating time. The IC50 of shikonin on U251 glioma apoptosis was 66.12 μmol·L^（-1）. Shikonin also quenched the fluorescence of APN in a concentration dependent manner. The binding constant was 4.12×104L·mol^-1, and ΔH and ΔS were 38.64 k J·mol- 1 and 179.21 J/（mol·K）. Shikonin could also induceing the unfold of APN conformation. The α-helix content increased to 32.1%, and the β-sheet content decreased to 22.9%. Conclusion Shikonin is a competitive APN inhibitor, which reduces the activity of APN quickly, and induces the apoptosis of U251 glioma cells. These effects may be through quenching the fluorescence of APN, inducing the unfolding of APN conformation, and increasing the α-helix content.