白芍总苷对HaCaT细胞外泌体的影响及机制研究

Effect of total glucosides of paeony on exosomes of HaCaT cells and its mechanism

目的 探讨白芍总苷对HaCaT细胞增殖、侵袭、迁移能力及外泌体释放的影响与可能机制。方法 采用50、100、200μg/L白芍总苷处理HaCaT细胞(分别设为低剂量组、中剂量组、高剂量组),同时设对照组(加入等量完全培养液)。采用四甲基偶氮唑蓝(MTT)法检测各组不同时点的细胞增殖能力;采用Transwell小室实验检测细胞迁移能力、侵袭能力;培养24 h,高速离心提取外泌体,采用蛋白质印迹法(Western Blot)检测细胞周期蛋白D1(CyclinD1)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、CD63及CD81的蛋白表达量,采用实时荧光定量聚合酶链反应(qRT-PCR)检测细胞中CyclinD1、Akt、p-Akt、PI3K、p-PI3K的mRNA表达量。结果 培养0、24 h时,4组细胞增殖抑制率比较,差异无统计学意义(P>0.05);培养48、72 h时,细胞增殖抑制率由高至低排列依次为高剂量组、中剂量组、低剂量组、对照组,各组间差异有统计学意义(P<0.05)。迁移细胞、侵袭细胞数量由低至高排列依次为高剂量组、中剂量组、低剂量组、对照组,各组间差异有统计学意义(P<0.05)。CyclinD1、CD63、CD81、Akt、p-Akt、PI3K、p-PI3K蛋白表达量和CyclinD1、Akt、p-Akt、PI3K、p-PI3K mRNA表达水平由低至高排列均依次为高剂量组、中剂量组、低剂量组、对照组,各组间差异有统计学意义(P<0.05)。结论 白芍总苷能够抑制HaCaT细胞外泌体分泌,并抑制HaCaT细胞增殖、侵袭及迁移能力,其作用机制可能与下调PI3K/Akt信号通路有关。

Objective To investigate effects and mechanism of total glucosides of paeony on the proliferation, invasion, migration of HaCaT cells and the release of exosomes. Methods HaCaT cells were treated with total glucosides of paeony at concentrations of 50, 100 and 200 μg/L, and were set as low dose group, medium dose group and high dose group, and cells added equal amount of complete culture medium was designed as control group. The proliferation ability was detected by methyl thiazolyl tetrazolium(MTT) method at different time points;the migration and invasion ability of cells were detected by Transwell chamber test;the exosomes were extracted by high-speed centrifugation;protein expressions of CyclinD1, protein kinase B(Akt), phosphorylated-Akt(p-Akt), phosphoinositide 3-kinase(PI3 K), CD63 and CD81 were detected by western blot;the expression of CyclinD1, Akt, p-Akt and PI3 K mRNA was detected by quantitative real-time PCR(qRT-PCR). Results The inhibition rate of cell proliferation showed no difference among four groups at 0 h and 24 h(P>0.05). At 48 and 72 h of culture, the inhibition rates of cell proliferation were the highest in the high dose group, followed by medium dose group, low dose group, and were the lowest in the control group(P<0.05). The high dose group had the largest number of migrating cells and invading cells, followed by medium dose group and low dose group, and the control group had the lowest(P<0.05). Protein expressions of CyclinD1, CD63, CD81, Akt, p-Akt, PI3 K and p-PI3 K as well as expressions of CyclinD1, Akt, p-Akt, PI3 K and p-PI3 K mRNA were the highest in the high dose group, followed by medium dose group, low dose group, and were the lowest in the control group(P<0.05). Conclusion Glucosides of paeony can inhibit the secretion of exosomes, and the proliferation, invasion and migration of HaCaT cells. Its mechanism may be related to down-regulation of PI3 K/Akt signaling pathway.