丹参红花提取物保护内皮细胞免受氧化损伤的体外实验

In vitro experiment of danshen root and carthamus tinctorius extract in the protection of endothelial cells from oxidative damage

目的:观察低密度脂蛋白和无血清培养刺激下人脐静脉内皮细胞内活性氧、NOX4mRNA水平和细胞凋亡的变化,以及丹参红花提取物的干预作用。方法:实验于2005-11/2006-05在卫生部老年医学重点实验室完成。取人新鲜脐带分离培养人脐静脉内皮细胞,分为:①正常对照组。②无血清培养组:用无血清培养基培养24h。③低密度脂蛋白处理组:以含有800mg/L低密度脂蛋白的培养基培养16h。④丹参红花提取物预处理组:用丹参红花提取物(商品名丹红滴注液,由陕西步长集团提供浓缩液,国药准字号Z20026866/Z20026867)预处理24h后加入800mg/L低密度脂蛋白继续培养16h或在无血清培养基中培养24h。以MTT法观察丹参红花提取物对人脐静脉内皮细胞生长的影响;应用反转录-聚合酶链反应检测NOX4mRNA水平;用流式细胞仪分析细胞凋亡率;并以DCFH-DA法检测细胞内活性氧生成量。结果:①低密度脂蛋白处理组NOX4mRNA表达、细胞内活性氧生成量和细胞凋亡为正常对照组的1.3倍,1.2倍和4倍。②无血清培养组NOX4mRNA表达、细胞内活性氧生成量和细胞凋亡分别为正常对照组的1.4倍、1.6倍和3倍。③丹参红花提取物预处理组NOX4mRNA表达、细胞内活性氧生成量和细胞凋亡分别为低密度脂蛋白处理组的0.4倍,0.5倍和0.2倍。④丹参红花提取物组NOX4mRNA表达、细胞内活性氧生成量和细胞凋亡分别为无血清培养组的0.2倍、0.5倍和0.6倍。结论:丹参红花提取物能够有效抑制高脂及缺血状态下NOX4mRNA表达水平,从而抑制人脐静脉内皮细胞内活性氧的产生及细胞凋亡,对内皮细胞起到很好的保护作用。

AIM： To investigate the production of reactive oxygen species （ROS）, expression of NADPH oxidase 4 （NOX4） mRNA and apoptotic rate in human umbilical vein endothelial cells （HUVECs） treated with low density lipoprotein or serum-free, and interventional effect of danshen root and carthamus tinctorius extract. METHODS： The experiment was performed at the Key Laboratory of Geriatric Medicine of Ministry of Health from November 2005 to May 2006. Fresh human umbilical cord was obtained to culture HUVECs. The cells were assigned into ①normal control group, ②serum-free group： cultured for 24 hours in serom-free medium; ③low density lipoprotein group： cultured with 800 mg/L low density lipoprotein medium for 16 hours; ④ danshen root and carthamus tinctorius extract pretreatment group： After pretreatment with danshen root and carthamus tinctorius extract （brand name： Danhong injection, concentrated solution provided by Shanxi Buchang Group, number Z 20026866/Z 20026867） for 24 hours, 800 mg/L low density lipoprotein was added to culture for 16 hours or culture in serom-free medium for 24 hours, The effect of danshen root and carthamus tinctorius extract on HUVECs was observed with MTT method. NOX4 mRNA level was analyzed with reverse transcription-polymerase chain reaction （RT-PCR）. Apoptotic rate was measured with flow cytometry and ROS production was detected with DCFH-DA method. RESULTS： ①NOX4 mRNA expression, ROS production and apoptotic rate in the low density lipoprotein group was 1,3 times, 1,2 times and 4 times of those in the normal control group. ②NOX4 mRNA expression, ROS production and apoptotic rate in the serom-free group was 1,4 times, 1.6 times and 3 times of those in the normal control group, respectively. ③ NOX4 mRNA expression, ROS production and apoptotic rate in the danshen root and carthamus tinctorius extract pretreatment group was 0,4 times, 0.5 times and 0.2 times of those in the low density lipoprotein group, respectively, ④NOX4 mRNA expression, ROS production and apoptotic rate in the danshen root and carthamus tinctorius extract pretreatment group was 0.2 times, 0.5 times and 0,6 times of those in the serum-free group, respectively. CONCLUSION： Danshen root and carthamus tinctorius extract can effectively prevent the NOX4 mRNA expression under high-lipid and ischemic status so as to inhibit ROS production and apoptotic rate in HUVECs, and has effectively protective effect on endothelial cells.