黄芪汤含药血清对血管内皮生长因子诱导的大鼠肝窦内皮细胞增殖、迁移和成管能力的影响及其作用机制

Effect of serum containing Huangqi decoction on the proliferation,migration,and tubulogenesis of rat liver sinusoidal endothelial cells induced by vascular endothelial growth factor and its mechanism of action

目的 基于蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路探讨黄芪汤含药血清对血管内皮生长因子(VEGF)诱导的大鼠肝窦内皮细胞(LSEC)增殖、迁移和成管能力的影响。方法 制备黄芪汤含药血清,体外分离、培养SD大鼠LSEC,将大鼠LSEC随机分为空白组,VEGF组,血清对照组,低、中、高剂量黄芪汤含药血清组,利用噻唑蓝(MTT)比色法、Transwell实验、成管实验分别检测各组LSEC增殖、迁移和成管能力,通过Western Blot法检测血小板-内皮细胞黏附分子-1(CD31)、内皮素-1(ET-1)、内皮性一氧化氮合酶(eNOS)和AKT/mTOR信号通路中AKT、磷酸化(p)-AKT、mTOR、磷酸化(p)-mTOR蛋白表达水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用Tukey’s检验。结果 与空白组相比,VEGF组、血清对照组能够促进大鼠LSEC增殖、迁移和血管新生(P值均<0.05);Western Blot结果显示,CD31、ET-1、eNOS和AKT/mTOR信号通路相关蛋白表达显著升高(P值均<0.05)。VEGF组与血清对照组比较,上述指标差异均无统计学意义(P值均>0.05)。与血清组比较,黄芪汤中、高剂量组能显著抑制VEGF诱导的大鼠LSEC增殖、迁移和血管新生(P值均<0.05);Western Blot结果显示,CD31、ET-1、eNOS和AKT/mTOR信号通路相关蛋白表达均显著降低(P值均<0.05)。结论 较高剂量黄芪汤含药血清能抑制VEGF诱导的大鼠LSEC增殖、迁移和成管能力,其作用机制可能与调控AKT/mTOR信号通路有关。

Objective To investigate the effect of serum containing Huangqi decoction on the proliferation,migration,and tubulogenesis of rat liver sinusoidal endothelial cells(LSECs) induced by vascular endothelial growth factor(VEGF) and its mechanism of action based on the protein kinase B(AKT)/mammalian target of rapamycin(mTOR) signaling pathway.Methods The serum containing Huangqi decoction was prepared,and rat LSECs were isolated and cultured in vitro.Then rat LSECs were randomly divided into blank group,VEGF group,serum control group,and low-,middle-,and high-dose serum containing Huangqi decoction groups.MTT colorimetry,Transwell assay,and tubulogenesis assay were used to measure the proliferation,migration,and tubulogenesis abilities of LSECs in each group,and Western Blot was used to measure the protein expression levels of platelet endothelial cell adhesion molecule-1(CD31),endothelin-1(ET-1),and endothelial nitric oxide synthase(eNOS),as well as AKT,phosphorylated-AKT(p-AKT),mTOR,and phosphorylated mTOR(p-mTOR) in the AKT/mTOR signaling pathway.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the Tukey’s test was used for further comparison between two groups.Results Compared with the blank group,the VEGF group and the serum control group had significantly promoted proliferation,migration,and angiogenesis of rat LSECs(all P<0.05),and Western Blot showed significant increases in the expression levels of CD31,ET-1,eNOS,and AKT/mTOR signaling pathway-related proteins(all P<0.05).There were no significant differences in the above indices between the VEGF group and the serum control group(all P>0.05).Compared with the serum group,the middle-and high-dose serum containing Huangqi decoction groups had significantly inhibited proliferation,migration,and angiogenesis of rat LSECs induced by VEGF(all P<0.05),and Western Blot showed significant reductions in the expression levels of CD31,ET-1,eNOS,and AKT/mTOR signaling pathway-related proteins(all P<0.01).Conclusion A relatively high dose of serum containing Huangqi decoction can significantly inhibit the proliferation,migration,and tubulogenesis of rat LSECs induced by VEGF,possibly by regulating the AKT/mTOR signaling pathway.