低氧对大鼠CYP2E1活性和蛋白表达的影响

Effect of Hypoxia on CYP2E1 Activity and Protein Expression of Rats

目的探讨模拟高原低氧对大鼠肝脏中CYP2E1活性和蛋白表达的影响。方法将12只健康雄性Wistar大鼠随机分成对照组(1500m)和低氧组(4100 m)。低氧组置于高原模拟氧舱,3d后解剖,取大鼠肝脏、提取肝微粒体,用于CPY2E1的分析。采用Thermo AcclaimTM120C18柱(3 mm×150 mm,3μm),流动相:甲醇-水(V∶V)60∶40,流速:1.0ml·min-1,柱温:25℃,检测波长:280nm,进样体积:20μl,建立测定氯唑沙宗浓度的HPLC方法,Western blot方法检测大鼠肝脏中CYP2E1的蛋白表达。结果活性测定结果显示,对照组氯唑沙宗在孵育体系中的平均反应速度为0.1384±0.0148μg·min-1,其酶动力学参数:最大反应速度(Vmax)为2.06μg·min-1,米氏常数(Km)为116.38±3.46μg·ml-1。低氧组氯唑沙宗平均反应速度为0.0822±0.0071μg·min-1,Vmax为2.60μg·min-1,Km为142.88±5.66μg·ml-1。Western blot结果显示,对照组CYP2E1的蛋白表达量是低氧组的9.88倍。结论与对照相比,低氧组大鼠的CYP2E1活性降低,蛋白表达量降低。

Objective To investigate the effect of CYP2 E1 activity and protein expressions of livers of rats at plateau hypoxia.Methods Twelve healthy male Wistar rats were randomly divided into the control group（1500 m）and hypoxia group（4100 m）.The hypoxia group was placed in a plateau-simulating oxygen chamber.Livers of the rats were anatomized after three days to extract liver microsomes for analysis of CPY2 E1.Liqiud chromatography was performed on Thermo AcclaimTM120 C18（3 mm×150 mm,3μm）.The mobile phase was as a mixed system that consisted of methanol/water（V∶V,60∶40）with gradient elution at a flow-rate of 1.0 ml·min-1.The column temperature was set at 25 ℃.The wavelength was set at 280 nm.The liver microsomes were incubated with chlorzoxazone in vitro and chlorzoxazone as a probe drug.The concentration of the probe drug was determined by the established HPLC method.Westen blot was used to detect CYP2 E1 protein expression in rats.Results In the control group（1500 m）,the average rate of reaction was 0.1384±0.0148μg·min-1,Vmaxwas 2.06μg·min-1 and Km was 116.38±3.46μg·ml-1.The mean velocity of the hypoxia group（4100 m）was 0.0822±0.0071μg·min-1,Vmaxwas 2.60μg·min-1,and Kmwas 142.88±5.66μg·ml-1.The protein expression level of CYP2 E1 in the control group（1500 m）was 9.88 times that of the hypoxia group（4100 m）.Conclusion The activity and protein expression of CYP2 E1 decrease in the hypoxia group（4100 m）compared with the control group.