清活Ⅰ号中药复方对高糖培养微血管内皮细胞增殖的影响

Effect of a traditional Chinese medicinal preparation on microvascular endothelial cell proliferation in high-glucose culture

目的观察清活Ⅰ号中药复方水煎剂及其药物血清对高浓度葡萄糖培养的微血管内皮细胞增殖的影响。方法采用中药血清药理学研究方法制备药物血清。原代培养SD大鼠肺微血管内皮细胞,中药复方水煎剂实验细胞分为NG-1组(葡萄糖5.56 mmol/L)、HG-1组(葡萄糖25 mmol/L)、中药-1组(葡萄糖25 mmol/L+清活Ⅰ号水煎剂)和NAC-1组[葡萄糖25 mmol/L+10 mmol/L N-乙酰半胱氨酸(NAC)];药物血清实验细胞分为NG-2组(葡萄糖5.56 mmol/L)、HG-2组(葡萄糖25 mmol/L)、中药-2组(葡萄糖25 mmol/L+药物血清)和NAC-2组(葡萄糖25 mmol/L+10 mmol/L NAC)。培养24 h后,观察清活Ⅰ号水煎剂及其药物血清对25 mmol/L葡萄糖培养内皮细胞生长形态的影响,并采用锥虫蓝染色细胞计数观察细胞活力。结果NG-1、HG-1、中药-1、NAC-1组的平均细胞数分别为57 000、38 333、57 333和65 000个/孔,活细胞比率分别为96.5%、58.2%、94.0%和95.8%,各组间差异均有统计学意义(P值均<0.01)。NG-2、HG-2、中药-2、NAC-2组的平均细胞数分别为38 333、10000、37 083和55 000个/孔,活细胞比率分别为96.7%、41.7%、91.0%和94.7%,各组间差异均有统计学意义(P值均<0.01)。培养24 h后,HG-1组的内皮细胞增殖减少、活细胞减少;中药-1组的细胞生长情况和活细胞数恢复到NG-1组水平,与NAC-1组培养的细胞增殖情况相似;药物血清与中药水煎剂实验各组的细胞增殖情况基本一致。结论清活Ⅰ号水煎剂及其药物血清均能改善高浓度葡萄糖导致的血管内皮细胞损伤,其保护作用可能与其抗氧化作用有关。

Objective To observe the effect of Qinghuoyihao（QHYH）, a traditional Chinese medicinal preparation, on the proliferation of microvascular endothelial cells in high-glucose culture. Methods QHYH decoction was administered in SD rats to obtain the drug-containing serum. Pulmonary microvascular endothelial cells of SD rats were cultured for 24 h in the medium containing 5.56 mmol/L glucose, 25 mmol/L glucose, 25 mmol/L glucose＋ QHYH decoction, 25 mmol/L glucose ＋ QHYH serum, or 25 mmol/L glucose ＋ N-acetyl-L-cysteine （NAC）, respectively. The cell growth changes was observed with Trypan blue staining and MTT chromatometry. Results The average number of cells treated with glucose at the concentrations of 5.56 and 25 mmol/L, or with QHYH decoction or NAC in the presence of 25 mmol/L glucose was 57 000, 38 333, 57 333 and 65 000 per well, with viable cell percentage of 96.5 %, 58.2 %, 94.0 % and 95.8 %, respectively, showing significant differences in the cell viability between the groups（P〈 0.01）. The average number of cells treated with glucose at the concentrations of 5.56 and 25 mmol/L, QHYH serum ＋ 25 mmol/L glucose, or with NAC ＋ 25 mmol/L glucose was 38 333, 10 000, 37 083 and 55 000 per well, with viable cell percentages of 96.7%, 41.7%, 91.0% and 94, 7%, respectively, showing significant difference between the groups （all P〈 0. 01 ）. High glucose concentration （25 mmol/L） obviously inhibited the cell proliferation and resulted in reduced viable cells, but supplementation of QHYH decoction increased the number of viable cells similar to that in 5. 56 mmol/L glucose culture, with an effect comparable with that of NAC. Similar results were observed in the cells treated with QHYH serum. Conclusion QHYH both in the form of decoction and drug-containing serum can ameliorate rat microvascular endothelial cell damages due to high concentration glucose, the mechanism of which may involve the antioxidative effect of QHYH.