葛根素通过调控活性氧对缺氧诱导的PASMCs增殖的影响(1)
[摘要] 探讨葛根素(puerarin，Pue)对缺氧所引起的大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响，并探讨其机制是否通过调控活性氧来实现的。原代培养大鼠PASMCs，选用2～5代用于实验。利用体外噻唑蓝比色法(MTT)、蛋白免疫印记法、流式细胞、DCFH-DA荧光观察Pue对PASMCs增殖能力的影响。利用蛋白免疫印记法检测ROS是否参与Pue抑制PASMCs增殖的过程。实验分组为：常氧组、缺氧组、缺氧+Pue组、缺氧+Pue+Rotenone组、缺氧+Rotenone组。其中Rotenone为ROS阻断剂。结果显示，Pue对常氧条件下的PASMCs增殖无影响，可抑制缺氧引起的PASMCs增殖。在常氧和缺氧状态下Pue对PASMCs的肿瘤坏死因子-α(TNF-α)表达均无影响;可以使缺氧导致的细胞周期蛋白(Cyclin A)、细胞增殖核抗原(PCNA)表达下调;DCFH-DA探针证明Pue能逆转缺氧引起的ROS含量升高。Rotenone与Pue均可抑制因缺氧诱导的HIF-1α，Cyclin A，PCNA表达上调且存在协同作用。这些结果说明，Pue可抑制缺氧引起的PASMCs增殖，这一作用可能是通过调控ROS来完成的。
[关键词] 葛根素;PASMCs;增殖;PCNA;Cyclin A;HIF-1α;ROS
[收稿日期] 2015-01-09
[基金项目] 哈尔滨市科技创新人才项目(2013RFXXJ092);黑龙江省自然科学基金重点项目(2D201416)
[通信作者] *王力维，硕士，研究方向为心血管药理，E-mail：hrbcuwlw@163.com
[作者简介] 张晓丹，教授，博士生导师，E-mail：zhangxd85@163.com
Effect of puerarin on hypoxia induced proliferation of PASMCs
by regulating reactive oxygen
ZHANG Xiao-dan1， WANG Li-wei1*， WANG Shu-jing1， ZHU Da-ling2， YANG Yan-nan1， SHENG Jie-jing1， SONG Sha-sha2
(1.School of Pharmacy， Harbin University of Commerce， Harbin 150076， China;
2. Daqing Campus， Harbin Medical University， Daqing 163319， China)
[Abstract] To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether its mechanism is achieved by regulating reactive oxygen. PASMCs of primarily cultured rats (2-5 generations) were selected in the experiment. MTT， Western blot， FCM and DCFH-DA were used to observe Pue′s effect the proliferation of PASMCs. The Western blot was adopted to detect whether ROS participated in Pue′s effect in inhibiting PASMC proliferation. The PASMCs were divided into five groups： the normoxia group， the hypoxia group， the hypoxia+Pue group，the hypoxia+Pue+Rotenone group and the hypoxia+Rotenone group， with Rotenone as the ROS blocker. According to the results， under the conditions of normoxia， Pue had no effect on the PASMC proliferation; But， under the conditions of hypoxia， it could inhibit the PASMC proliferation; Under the conditions of normoxia and hypoxia， Pue had no effect on the expression of the tumor necrosis factor-α (TNF-α) among PASMCs， could down-regulate the expression of hypoxia-induced cell cycle protein Cyclin A and proliferative nuclear antigen (PCNA). DCFH-DA proved Pue could reverse ROS rise caused by hypoxia. Both Rotenone and Pue could inhibit the up-regulated expressions of HIF-1α， Cyclin A， PCNA caused by anoxia， with a synergistic effect. The results suggested that Pue could inhibit the hypoxia-induced PASMC proliferation. Its mechanism may be achieved by regulating ROS. (张晓丹 王力维 王淑静 朱大岭 杨艳南 盛洁静 宋莎莎)
[关键词] 葛根素;PASMCs;增殖;PCNA;Cyclin A;HIF-1α;ROS
[收稿日期] 2015-01-09
[基金项目] 哈尔滨市科技创新人才项目(2013RFXXJ092);黑龙江省自然科学基金重点项目(2D201416)
[通信作者] *王力维，硕士，研究方向为心血管药理，E-mail：hrbcuwlw@163.com
[作者简介] 张晓丹，教授，博士生导师，E-mail：zhangxd85@163.com
Effect of puerarin on hypoxia induced proliferation of PASMCs
by regulating reactive oxygen
ZHANG Xiao-dan1， WANG Li-wei1*， WANG Shu-jing1， ZHU Da-ling2， YANG Yan-nan1， SHENG Jie-jing1， SONG Sha-sha2
(1.School of Pharmacy， Harbin University of Commerce， Harbin 150076， China;
2. Daqing Campus， Harbin Medical University， Daqing 163319， China)
[Abstract] To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether its mechanism is achieved by regulating reactive oxygen. PASMCs of primarily cultured rats (2-5 generations) were selected in the experiment. MTT， Western blot， FCM and DCFH-DA were used to observe Pue′s effect the proliferation of PASMCs. The Western blot was adopted to detect whether ROS participated in Pue′s effect in inhibiting PASMC proliferation. The PASMCs were divided into five groups： the normoxia group， the hypoxia group， the hypoxia+Pue group，the hypoxia+Pue+Rotenone group and the hypoxia+Rotenone group， with Rotenone as the ROS blocker. According to the results， under the conditions of normoxia， Pue had no effect on the PASMC proliferation; But， under the conditions of hypoxia， it could inhibit the PASMC proliferation; Under the conditions of normoxia and hypoxia， Pue had no effect on the expression of the tumor necrosis factor-α (TNF-α) among PASMCs， could down-regulate the expression of hypoxia-induced cell cycle protein Cyclin A and proliferative nuclear antigen (PCNA). DCFH-DA proved Pue could reverse ROS rise caused by hypoxia. Both Rotenone and Pue could inhibit the up-regulated expressions of HIF-1α， Cyclin A， PCNA caused by anoxia， with a synergistic effect. The results suggested that Pue could inhibit the hypoxia-induced PASMC proliferation. Its mechanism may be achieved by regulating ROS. (张晓丹 王力维 王淑静 朱大岭 杨艳南 盛洁静 宋莎莎)