活血补肾方含药血清对人脐静脉内皮细胞增殖作用及对丝裂原活化蛋白激酶信号通路的影响

摘要

目的 用人脐静脉内皮细胞(HUVEC)为模型观察活血补肾含药血清对增殖作用及对p42/44丝裂原活化蛋白激酶信号通路的影响. 方法 噻唑蓝染色观察20％、10％、5％含药血清对HUVEC细胞的增殖作用.检测一氧化氮合酶(eNOS)活力和NO含量.反转录PCR法检测eNOSmRNA表达,免疫印迹法检测蛋变化. 结果 含药血清有效促进HUVEC细胞的增殖,经过24 h含药血清处理,含药血清呈现浓度依耐性促进HUVEC细胞增殖,增殖率分别为10.8％(t=6.82,P＜0.05)、14.7％(t=6.25,P＜0.05)、33.3％(t=12.16,P＜0.01).含药血清处理组eNOS活力增强,明显高于未含药血清;20％含药组NO含量与对照组比较,差异有统计学意义(P＜0.05);20％和10％含药对eNOS mRNA的表达有促进作用(P＜0.05).活血补肾方能提高P-p42／44蛋白表达水平(P＜0.01). 结论 补肾活血方促进内皮细胞的增殖,提高内皮细胞NO分泌,其机制是通过p42/44丝裂原活化蛋白激酶信号通路增加eNOS活力和mRNA的表达.%Objective To observe the drug-containing Huoxue Bushen (HXBS) serum on the proliferation of human umbilical vein endothelial cells (HUVECs) and p42/44MAPK pathways by using human umbilical vein endothelial cells (HUVECs).Methods The proliterative effects of 20％,10％,5％ drug-containing HXBS serum on HUVECs were observed by MTT.Nitric oxide synthase (NOS) activity was detected by endothelial nitric oxide synthase (eNOS) activity kit.The content of nitric oxide (NO) was determined by NO kit.The mRNA expression of eNOS was detected by real-time reverse transcription polymerase chain reaction (RT-PCR).The protein expression of pP42/44 was detected by Western blotting.Results HUVECs proliferation was effectively promoted by drug-containing HXBS serum.The proliferation rate of HUVECs was increased along with the drug-containing HXBS serum concentration increasing after 24 h treatment (10.8 ％,14.7 ％,33.3 ％,t=6.82,6.25,12.16,respectively,P＜0.05 or 0.01).eNOS activity was significantly increased in the drug-containing HXBS serum-treated group as compared with the control group.There was a significant difference in NO content between 20％ drug-containing HXBS serum treated group and control group (P＜0.05).The expression of eNOs mRNA was significantly promoted in 20％ and 10％ drug-containing HXBS serum groups (P ＜ 0.05).P-p42/44 protein level was significantly increased by drug-containing HXBS serum (P ＜ 0.01).Conclusions Conclusions HXBS can promote endothelial cell proliferation and improve endothelial cell NO secretion by increasing the activity and mRNA expression of eNOS through p42/44 signaling pathway,which provides a theoretical basis for improving cardiovascular disease in postmenopausal women.