乌司他丁对糖尿病大鼠心肌细胞氧化应激损伤的影响：离体实验

Effect of ulinastatin on oxidative stress injury to myocardial cells in diabetic rats in vitro

目的评价乌司他丁对糖尿病大鼠心肌细胞氧化应激损伤的影响。方法H9c2细胞株，于DMEM培养基培养至对数生长期时，分别接种于96孔板（细胞密度1×10^4个／ml，200μl／孔）或6孔板（细胞密度1×10^5个／ml，2ml／孔）中，采用随机数字表法，将培养孔分为4组（n=18）：正常对照组（C组）、高糖组（HG组）、高糖＋氧化应激组（HG＋OS组）和乌司他丁＋高糖＋氧化应激组（U＋HG＋OS组）。HG组以高糖DMEM培养基（糖浓度25．0mmol／L）孵育细胞48h，HG＋OS组和U＋HG＋OS组以高糖DMEM培养基孵育24h时加入500Ixmol／LH2O2继续孵育24h；U＋HG＋OS组高糖DMEM培养基中加入400U／ml乌司他丁。收集细胞，测定细胞活力、细胞凋亡情况、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量，收集细胞培养液上清，测定乳酸脱氢酶（LDH）活性。结果与C组比较，其它3组细胞活力和SOD活性降低，细胞凋亡率、MDA含量及LDH活性升高（P〈0．05）；与HG组比较，HG＋OS组和U＋HG＋OS组细胞活力和SOD活性降低，细胞凋亡率、MDA含量及LDH活性升高（P〈0．05）；与HG＋OS组比较，U＋HG＋OS组细胞活力和SOD活性升高，细胞凋亡率、MDA含量及LDH活性降低（P〈0．05）。结论乌司他丁可减轻糖尿病大鼠心肌细胞氧化应激损伤，其机制与抑制细胞凋亡有关。

Objective To evaluate the effect of ulinastatin on oxidative stress injury to myocardial cells in diabetic rats in vitro. Methods The H9c2 ceils were cultured in DMEM culture medium and the cells at the logarithmic growth phase were seeded in 96-well plates （density 1×10^4 cells/ml, 200 μl/well） or in 6-well plates （density 1× 10^5 cells/ml, 2 ml/well）. The cells were randomly divided into 4 groups （n= 18 each） using a random number table： normal control group （group C）, high-glucose group （group HG）, high-glucose ＋ oxidative stress group （group HG＋OS）, ulinastatin ＋high-glucose＋oxidative stress group （group U＋HG＋OS）. The cells were cultured in high-glucose DMEM culture medium （25.0 mmol/L） for 48 h in group HG. After the ceils were cultured in high-glucose DMEM culture medium for 24 h, HzO2 with the final concentration of 500μmol/L was added to the high-glucose culture medium, and the ceils were continuously cultured for 24 h in HG＋OS and U＋HG＋OS groups. In group U＋HG＋OS, ulinastatin 400 U/ml was added to the high-glucose culture medium. The cells were collected for determination of cell viability, H9c2 apoptosis, activity of superoxide dismutase （SOD） and contents of malonadehyde （MDA）. Apoptosis rate was calculated. The cell culture supernatant was collected for detection of lactate dehydrogenase （LDH） activity. Results Compared with group C, the cell viability and SOD activity were significantly decreased, and the apoptosis rate, MDA content and LDH activity were increased in the other groups. Compared with HG group, the cell viability and SOD activity were significantly decreased, and the apoptosis rate, MDA content and LDH activity were increased in HG＋OS and U＋HG＋OS groups. Compared with group HG＋OS, the cell viability and SOD activity were significantly increased, and the apoptosis rate, MDA content and LDH activity were decreased in group U＋I-IG＋OS. Conclusion Ulinastatin can mitigate oxidative stress injury to myocardial cells in diabetic rats, and inhibited cell apoptosis is involved in the mechanism.