舒芬太尼对高糖孵育下H9c2细胞缺氧复氧损伤的影响

Effect of sufentanil on anoxia/reoxygenation-induced injury to H9c2 cells with high-glucese incubation

目的评价舒芬太尼对高糖孵育下H9c2细胞缺氧复氧损伤的影响。方法取H9c2细胞在含10％胎牛血清低糖DMEM／F12完全培养基中培养、传代，细胞接种于96孔（100出／孔）和6孔（2ml／孔）细胞培养板，接种密度10^5／ml。采用随机数字表法，将细胞随机分为5组，每组12孔，正常糖对照组（NC组）用低糖培养基孵育细胞48h；高糖对照组（HC组）用高糖（25mmol／L）培养基孵育细胞48h；高糖＋缺氧复氧组（HAR组）用高糖培养基孵育细胞48h后，行缺氧3h复氧3h；高糖＋舒芬太尼＋缺氧复氧组（HSAR组）用高糖培养基孵育细胞48h后加入舒芬太尼（终浓度为10μmol／L），15min后行缺氧复氧处理；高糖＋纳洛酮＋缺氧复氧组（HNAR组）用高糖培养基孵育细胞48h后加入纳洛酮（终浓度为10μmol／L），10min后加入舒芬太尼（终浓度为10^-9mol／L），15min后行缺氧复氧处理。各组处理后收集细胞，用MTT法测定细胞活力；收集细胞培养液测定乳酸脱氢酶（LDH）漏出量；超声破碎法采集细胞匀浆液，采用黄嘌呤氧化酶法测定细胞SOD活性。结果与NC组比较，其余各组细胞活力和SOD活性降低，LDH漏出量增多（P〈0．01）。与HC组比较，HAR组和HNAR组细胞活力和SOD活性降低，LDH漏出量增多（P〈0．01）。与HAR组比较，HSAR组细胞活力和SOD活性升高，LDH漏出量减少（P〈0．01），HNAR组上述指标差异无统计学意义（P〉0．05）。与HSAR组比较，HNAR组细胞活力和SOD活性降低，LDH漏出量增多（P〈0．01）。结论舒芬太尼可减轻高糖孵育下H9c2细胞缺氧复氧损伤，其机制可能与激活阿片受体有关。

Objective To investigate the effect of sufentanil on anoxia/rexogenation （A/R）-induced injury to H9c2 cells incubated in high-glucose culture medium. Methods The H9c2 ceils were cultured in low-glucose DMEM/F12 culture medium supplemented with 10% fetal bovine serum. The cells were seeded in 96-well or 6- well plates and randomly divided into 5 groups （ n = 12 wells each） ： normal glucose control group （group NC）, high-glucose control group （group HC）, high-glucose ＋ A/R group （group HA/R）, high-glucose ＋ sufentanil ＋ A/R group （group HSA/R）, high glucose ＋ naloxone ＋ A/R group （group HNA/R）. The cells were exposed to 95 % N2-5% CO2 in an incubator at 37 ℃ for 3 h followed by 3 h reoxygenation. In group NC, the ceils were incubated in low-glucose culture medium for 48 h. In group HC, the cells were incubated in high-glucose culture naedium for 48 h. In group HA/R, the cells were incubated in high-glucose culture medium for 48 h before anoxia. In group HSA/R, after the cells were incubated in high-glucose culture medium for 48 h, sufentanil was added to the culture medium with the final concentration of 10-9 mol/L at 15 rain before anoxia. In group HNA/R, after the cells were incubated in high-glucose culture medium for 48 h, naloxone was added to the culture medium with the final concentration of 10^-6 tool/L, 10 rain later sufentanil was added with the final concentration of 10-9 mol/L at 15 rain before anoxia. The cell viability was measured by MTT assay. Tile amount of lactic dehydrogenase （LDH） released in the supernatant and superoxide dismutase （SOD） activity were measured. Results The cell viability and SOD activity were significantly lower, while the amount of LDH released was signifcantly higher in the other groups than in group NC, in groups HA/R and HNA/R than in group HC, and in group HNA/R than in group HSA/R （P 〈 0.01）. The cell viability and SOD activity were significantly higher, while the amount of LDH released was significantly lower in group HSA/R than in group HA/R （P 〈 0.01 ）. There was no significant difference in the parameters mentioned above between group HNA/R and group HA/R （ P 〉 0.05）. Conclusion Sufentanil can attenuate A/R-induced injury to H9c2 cells with high-glucose incubation, and the mechanism may be related to the activation of opioid receptors.