活血解毒中药含药血清对氧化低密度脂蛋白诱导的内皮细胞损伤和凋亡的影响

Effects of Chinese herbal drug-containing serum on oxidative damage and apoptosis of umbilical vein endothelial cells induced by oxidized low-density lipoprotein

目的:比较单纯活血中药含药血清与活血解毒中药配伍含药血清对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)刺激的人脐静脉内皮细胞(human umbilical veinendothelial cell,HUVEC)活力、氧化损伤及凋亡的影响。方法:32只Wistar大鼠随机分为空白对照组(蒸馏水)、阳性对照组(辛伐他汀1.8mg/kg)、活血组(芎芍胶囊0.135g/kg)和活血解毒组(芎芍胶囊合用黄连胶囊,各0.135g/kg)。连续灌胃7d,末次给药后1h,腹主动脉采血制备含药血清。胶原酶消化法制备HUVEC,将ox-LDL(100μg/L)和(或)相应含药血清作用于HUVEC,24h后倒置显微镜下观察细胞形态,四氮唑蓝比色法观察含药血清对HUVEC活力的影响,乳酸脱氢酶(lactate dehydrogenase,LDH)漏出率检测法观察细胞膜损伤,比色法检测细胞内超氧化物歧化酶(superoxide dismutase,SOD)活性,硫代巴比妥酸法检测细胞丙二醛(malondialdehyde,MDA)水平,流式细胞仪结合Annexin V异硫氰酸荧光素/碘化丙啶双染色法测定HUVEC凋亡率。结果:与空白对照组比较,ox-LDL刺激24h后HUVEC活力和SOD活性降低,细胞内MDA含量升高,HUVEC早、晚期凋亡比例增加(P<0.01,P<0.05)。与单纯使用ox-LDL刺激相比,活血药、活血解毒药和辛伐他汀含药血清均能明显减轻ox-LDL对HUVEC的损伤,抑制HUVEC早期凋亡(P<0.01,P<0.05),但对LDH漏出无明显抑制作用;活血中药和辛伐他汀含药血清可明显降低HUVEC内MDA含量,提高SOD活性(P<0.01,P<0.05),活血解毒中药含药血清对上述指标无明显影响。活血药物血清与活血解毒药物血清比较差异无统计学意义。结论:活血及活血解毒中药配伍对ox-LDL导致的HUVEC氧化损伤和早期凋亡均具有一定的保护作用。

Objective：To investigate the effects of Chinese herbal drug-containing serum,prepared by administration of Chinese herbal medicine for activating blood （Xiongshao Capsule,XS） or for activating blood and detoxifying （Xiongshao Capsule plus Huanglian Capsule,XSHL） in rats,on cell viability,oxidative damage and apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized low-density lipoprotein （ox-LDL）.Methods：Thirty-two rats were randomly divided into 4 groups：control group,positive control group （simvastatin 1.8 mg/kg）,activating blood （XS,0.135 g/kg） group,and activating blood and detoxifying （XS Capsule 0.135 g/kg and Huanglian Capsule 0.135 g/kg,XSHL） group. Corresponding drugs were continuously administered to the rats for 7 days and then drug-containing serum was harvested 1 hour after the last administration. HUVECs isolated from newborn children by collagenase digestion were stimulated by ox-LDL （100 μg/L） and incubated with corresponding drug-containing serum for 24 hours. Untreated HUVECs were also used as a normal control. The morphology and structure of HUVECs were observed by an inverted microscope. Cell viability was measured by methyl thiazolyl tetrazolium method,and cell membrane damage was determined by lactate dehydrogenase （LDH） leakage. Activity of superoxide dismutase （SOD） was examined by spectrophotometry,and content of malondialdehyde （MDA） in the cell lysate was examined by thiobarbituric acid assay. HUVECs were stained with Annexin V-fluorescein isothiocyanate and propidium iodide and analyzed on a flow cytometry to determine apoptosis.Results：Compared with the normal HUVECs,the cell viability and the activity of SOD were significantly decreased while the content of MDA and apoptosis rate were significantly increased after 24-hour ox-LDL stimulation （P0.01,P0.05）. Simvastatin-,XS-,and XSHL-containing serum significantly promoted the ox-LDL-stimulated HUVEC viability and inhibited early apoptosis （P0.01,P0.05）,while had no significant effect on LDH leakage. Simvastatin-containing serum and XS-containing serum also showed significant decrease in MDA content and increase in SOD activity,while XSHL-containing serum showed no significant effects. There was no significant difference between the XS-containing serum group and the XSHL-containing serum group. Conclusion：Both sera containing XSHL and XS show protective action against the oxidative damage and apoptosis of HUVECs induced by ox-LDL.