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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 compare the activities of human umbilical veinendothelial cell (HUVEC) stimulated by oxidized low-density lipoprotein (ox-LDL) with Chinese medicinal serum containing traditional Chinese medicine and promoting blood circulation and detoxifying herbs. , Oxidative damage and apoptosis. Methods: Thirty-two Wistar rats were randomly divided into blank control group (distilled water), positive control group (simvastatin 1.8mg / kg), Huoxue group (0.135g / kg) and Huoxuejiedu capsule Coptis capsules, each 0.135g / kg). Continuous gavage 7d, 1h after the last administration, abdominal aorta blood preparation containing serum. HUVECs were prepared by collagenase digestion. HUVECs were treated with ox-LDL (100μg / L) and corresponding drug-containing serum for 24 hours. The morphology of the cells was observed under an inverted microscope 24 hours later. The effect of lactate dehydrogenase (LDH) leakage rate on cell membrane damage was observed. The activity of superoxide dismutase (SOD) in cells was detected by colorimetric assay. Cell viability was detected by thiobarbituric acid assay Malondialdehyde (MDA) was detected by flow cytometry. Annexin V double staining of fluorescein isothiocyanate / propidium iodide was used to determine the apoptosis rate of HUVEC. Results: Compared with the blank control group, the activity of HUVEC and the activity of SOD decreased after 24 hours ox-LDL stimulation, and the content of MDA in the cells increased. The proportion of apoptosis in early and late stage of HUVEC increased (P <0.01, P <0.05). Compared with ox-LDL alone, the drug-containing serums of Huoxue, Huoxue Jiedu and Simvastatin all significantly reduced the injury of HUVEC induced by ox-LDL and inhibited the early apoptosis of HUVEC (P <0.01, P <0.05) But there was no obvious inhibitory effect on the leakage of LDH. The serum containing traditional Chinese medicine and simvastatin could obviously reduce the content of MDA in HUVEC and increase the activity of SOD (P <0.01, P <0.05) influences. There was no significant difference in the serum levels of blood activating drugs and blood activating detoxification drugs. Conclusion: The compatibility of traditional Chinese medicine with promoting blood circulation and activating blood circulation to detoxification has some protective effects on oxidative damage and early apoptosis of HUVEC induced by ox-LDL.