基于Akt/JNK/p38 MAPK信号通路探讨健脾活骨方对酒精致血管内皮细胞功能损伤的保护作用

Protective Effect of Jianpi Huogu Prescription on Functional Injury of Vascular Endothelial Cells Caused by Alcohol Based on Akt/JNK/p38 MAPK Signaling Pathway

目的:观察健脾活骨方(JPHGP)对酒精致血管内皮细胞功能损伤的保护作用,并基于蛋白激酶B/c-Jun氨基末端激酶/p38 MAPK(Akt/JNK/p38 MAPK)信号通路探索相关作用机制。方法:通过鸡胚尿囊膜实验、胸主动脉环实验和人脐静脉内皮细胞(HUVEC)的迁移、侵袭、黏附和管腔形成实验,在有或无酒精诱导条件下,观察JPHGP不同质量浓度8、16、32μg·L^(-1)对血管新生的影响;采用蛋白免疫印迹法(Western blot)检测HUVEC中Akt、JNK、p38 MAPK等磷酸化表达水平。结果:与正常组比较,模型组动脉环周围微血管数目及长度均减少,HUVEC的迁移、侵袭、和管腔形成能力降低(P<0.05,P<0.01);JPHGP16、32μg·L^(-1)组作用后能明显升高鸡胚尿囊膜新生血管长度(P<0.05,P<0.01),与模型组比较,JPHGP 8、16、32μg·L^(-1)组能浓度依赖地增加胸主动脉环周围微血管数量(P<0.05,P<0.01),JPHGP 32μg·L^(-1)组能升高胸主动脉环周围微血管长度(P<0.05);JPHGP 16、32μg·L^(-1)组均能增强HUVEC的迁移、侵袭、和管腔形成能力。Western blot检测结果表明,与正常组比较,模型组中的p-JNK/JNK、p-p38 MAPK/p38 MAPK、p-Akt/Akt蛋白表达水平均显著降低(P<0.01);与模型组比较,JPHGP 8、16、32μg·L^(-1)组的p-p38 MAPK/p38 MAPK、p-Akt/Akt蛋白表达水平均显著升高(P<0.01),JPHGP 16、32μg·L^(-1)组的p-JNK/JNK的蛋白表达水平显著升高(P<0.01)。结论:JPHGP对酒精致血管内皮细胞功能损伤具有保护作用,其机制可能与激活Akt/JNK/p38MAPK信号通路有关,相关研究结果将为JPHGP“健脾活血”功效阐明提供一定的科学依据。

Objective:To investigate the protective effect of Jianpi Huogu prescription(JPHGP)on the functional injury of vascular endothelial cells caused by alcohol and explore its mechanism based on protein kinase B/c-Jun amino-terminal kinase/p38 MAPK(Akt/JNK/p38 MAPK)signaling pathway.Method:Through chick embryo allantoic membrane,thoracic aortic ring,and migration,invasion,adhesion,and lumen formation of human umbilical vein endothelial cells(HUVEC),the effect of JPHGP with different concentrations(8,16 and 32μg·L^(-1))on angiogenesis was observed in the presence or absence of alcohol.The expression levels of phosphorylation of Akt,JNK,and p38 MAPK were determined by Western blot.Result:As compared with the normal group,the number and length of capillaries around the arterial ring in the model group were decreased,and the migration,invasion,and lumen formation capacity of HUVEC were decreased(P<0.05,P<0.01).After treatment with 16 and 32μg·L^(-1) JPHGP,the length of neovascularization in chick embryo allantoic membrane was significantly increased(P<0.05,P<0.01).Compared with the model group,the 8,16,and 32μg·L^(-1) JPHGP groups increased the number of capillaries around the thoracic aortic ring in a concentration-dependent manner(P<0.05,P<0.01),and the 32μg·L^(-1) JPHGP group increased the length of capillaries around the thoracic aortic ring(P<0.05).The 16 and 32μg·L^(-1) JPHGP groups enhanced the migration,invasion,and lumen formation capacity of HUVEC.The results of Western blot showed that,as compared with the normal group,the protein expression levels of p-JNK/JNK,p-p38 MAPK/p38 MAPK,and pAkt/Akt were significantly decreased in the model group(P<0.01),and as compared with the model group,the protein expression levels of p-p38 MAPK/p38 MAPK and p-Akt/Akt were significantly increased in the 8,16,and 32μg·L^(-1) JPHGP groups(P<0.01)and the protein expression level of p-JNK/JNK was increased significantly in the 16 and 32μg·L^(-1) JPHGP groups(P<0.01).Conclusion:JPHGP has a protective effect on the functional injury of vascular endothelial cells caused by alcohol,and its mechanism may be related to the activation of Akt/JNK/p38 MAPK signaling pathway.Relevant research results will provide certain scientific basis for clarifying the effect of JPHGP on'invigorating spleen and promoting blood circulation'.