保元汤活性成分4’,7,8-三羟基二氢黄酮抑制过氧化氢诱导人脐静脉内皮细胞损伤的作用机制研究

Active ingredient of Baoyuan Decoction, 4’,7,8-trihydroxydihydroflavonoid, improves H_(2)O_(2)-induced HUVEC cells damage

目的 研究保元汤中4’,7,8-三羟基二氢黄酮(4’,7,8-trihydroxydihydroflavonoid, TF)对过氧化氢(hydrogen peroxide, H_(2)O_(2))诱导人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)氧化损伤的改善作用,并进一步阐明其药理机制。方法 用不同浓度的TF处理HUVEC血管内皮细胞6小时后给予15μmol/L和30μmol/L H2O2刺激1小时,检测细胞存活率并确定TF起效剂量。利用2,2-二苯基-1-苦肼基自由基(2,2-dipheny 1-1-picrychydrazy 1,DPPH)分析实验、DCF-DA荧光探针和蛋白免疫印迹法(Western Blot)分别检测TF对自由基的清除作用,以及H_(2)O_(2)损伤后TF对HUVEC细胞内活性氧(reactive oxygen species, ROS)水平和细胞凋亡相关蛋白磷酸化蛋白激酶B(phosphorylated protein kinase B,P-Akt)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)以及腺苷二磷酸核糖聚合酶(poly adenosinediphosphate-ribose polymerase, PARP)表达的调控作用。结果 TF给药处理能显著提高H2O2损伤后HUVEC细胞的存活率(P<0.01)。机制研究表明,TF能显著加速自由基的清除,降低细胞中ROS水平,并通过调控P-Akt/PARP/Bcl-2通路抑制HUVEC细胞的凋亡。结论 TF作为保元汤的活性成分,能保护内皮细胞对抗H2O2诱导的氧化损伤,其作用机制可能与其加速自由基的清除、提高细胞清除ROS能力和抑制细胞凋亡有关。

Objective To study the effects of 4’,7,8-Trihydroxyflavanone(TF) in Baoyuan Decoction on the peroxidative damage of HUVEC vascular endothelial cells induced by hydrogen peroxide(H_(2)O_(2)), and to further clarify its pharmacological mechanism. Methods After 6 hours treatment of different doses of TF, the HUVEC cells were damaged by H2O2for 1 hours and the cell viability was investigated to determine the activity of TF. Moreover, the DPPH free radical experiment, DCF-DA fluorescent probe, and Western blot experiment were used to evaluate the effects of TF on free radical scavenging, the level of reactive oxygen(ROS), and the expression of apoptosis-related proteins P-Akt, Bcl-2 and PARP in HUVEC cells after H2O2insult, respectively. Results The 10 μmol/L and 20 μmol/L doses of TF can significantly increase the cell viabilities of HUVEC cells after 15 μmol/L and 30 μmol/L H_(2)O_(2) injury(P<0.01). Mechanism studies showed that TF significantly accelerated the elimination of free radicals, reduced the level of ROS in cells, and inhibited HUVEC cells apoptosis by regulating the P-Akt/PARP/Bcl-2 pathway. Conclusion Our results showed that TF, as the active ingredient of Baoyuan Decoction, protected endothelial cells against the oxidative damage induced by H_(2)O_(2), and its mechanism of action may be related to the acceleration of free radical scavenging, the improvement of the ability of cells to scavenging ROS, and the inhibition of cell apoptosis.