目的 探讨理气活血滴丸对体外低流体剪切力加载的人脐静脉内皮细胞凋亡的影响。方法 选取清洁级健康雄性SD大鼠40只，随机分成模型组及理气活血滴丸低、中、高剂量组共4组，每组各10只。理气活血滴丸低、中、高剂量组分别予43。75，87。5，175mg·kg-1·d-1理气活血滴丸，每12h灌胃1次;模型组予0。9％生理盐水10ml/(kg·d)，每12h灌胃一次。所有大鼠连续给药96h。并于最末1次给药后1h经腹主动脉取血，常规制作药物血清。体外培养的人脐静脉内皮细胞(HUVECs)分为空白组、模型组、理气活血滴丸低、中、高剂量组共5组，除空白组外，其余各组分别予以含对应组别药物血清的培养基培养48h后，均以低流体剪切力(4dynes/cm2)加载24h。利用EdU检测法检测不同组别HUVECs增殖时DNA渗入水平。利用TUNEL法检测不同组别HUVECs的凋亡情况。Western blotting法检测不同组别HUVECs中Yes相关蛋白(YAP)、含PDZ结合序列的转录活化共因子(TAZ)、JNK1/2应激活化蛋白激酶(stress activated protein kinase，SAPK)，以及天冬氨酸蛋白水解酶-3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)的表达情况。结果 与模型组比较，理气活血滴丸低、中、高剂量组细胞的凋亡率及YAP、TAZ、JNK1/2和Caspase-3蛋白表达水较低，细胞增殖率及Bcl-2蛋白表达量增高，差异具有统计学意义(P＜0。05)。结论 理气活血滴丸能抑制低流体剪切力加载的HUVECs的凋亡，其作用可能与下调相关力学-生物信号通路相关蛋白YAP、TAZ、JNK1/2和凋亡相关因子Caspase-3表达，以及上调Bcl-2表达有关。
Effect of Liqi Huoxue Dropping Pills on apoptosis of human umbilical vein endothelial cells under low fluid shear force through YAP/TAZ-JNK signaling pathway
Objective To investigate the effect of Liqi Huoxue dripping pills on the apoptosis of popular venous endothelial cells under low fluid shear force in vitro.Methods Forty healthy male SD rats of clean grade were randomly divided into model group and four groups of Liqi Huoxue dropping pills at low,medium and high doses,with 10 rats in each group.Low-dose,medium-dose and high-dose Liqi Huoxue dropping pills were given 43.75mg/(kg·d),87.5mg/(kg·d)and 175mg/(kg·d),re-spectively,once every 12 hours.The model group was given 0.9％normal saline 10ml/kg/d and gavage every 12h.All rats were given the drug continuously for96h.Blood was collected from abdominal aorta 1 hour after the last administration,and drug serum was routinely prepared.Human navus vein endothelial cells(HUVECs)cultured in vitro were divided into blank group,model group,Liqi Huoxue dropping pills low-dose,medium-dose and high-dose groups.Except the blank group,the other groups were cultured in the medium containing the corresponding group of drug serum for 48h,and then loaded with low fluid shear force(4dynes/cm2)for 24h.EdU assay was used to detect DNA penetration levels in different groups of HUVECs.Apoptosis of HU-VECs in different groups was detected by TUNEL method.Yes associated protein(YAP),transcription activation cofactor(TAZ)containing PDZ binding sequence,JNK1/2 stress activated protein kinase(SAPK)in HUVECs of different groups were detected by Western blotting.And the expression of Caspase-3 and B lymphoblastoma-2 gene(Bcl-2).Results Compared with model group,the apoptosis rate and the protein expression water of YAP,TAZ,JNK1/2 and Caspase-3 in Liqi Huoxue dropping pills at low,medium and high doses were lower,while the cell proliferation rate and the protein expression of Bel-2 were increased,with statistical significance(P＜0.05).Conclusion Regulating qi and activating blood can inhibit the apopto-sis of HUVECs under low fluid shear force loading,which may be related to the down-regulation of the mechano-biological sig-naling pathways related proteins YAP,TAZ,JNK1/2 and apoptosis-related factors Caspase-3,and the up-regulation of Bel-2 expression.
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