促红细胞生成素和还原型谷胱甘肽联用对肝缺血-再灌注损伤的保护作用

Protective effects of erythropoietin combined with reduced glutathione on hepatic ischemia-reperfusion injury

目的探讨促红细胞生成素(EPO)与还原型谷胱甘肽(GSH)联用最佳组方配比,及其对肝缺血-再灌注损伤的保护作用和机制。方法复制小鼠肝缺血-再灌注损伤模型,根据权重配方法设立不同配比组方6组,于缺血30 min-再灌注4 h后取血检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性为参考指标,确定组方的最佳理论配比。另取56只小鼠进行确证性实验,随机分为假手术组、模型组、EPO(2 000 U·kg^(-1))组、GSH(12.5 mg·kg^(-1))组、EPO(1 000 U·kg^(-1))+GSH(6.25 mg·kg^(-1))低剂量组、EPO(2 000 U·kg^(-1))+GSH(12.5 mg·kg^(-1))中剂量组、EPO(4 000 U·kg^(-1))+GSH(25 mg·kg^(-1))高剂量组,均于手术前1 h腹腔注射生理盐水或相应药物,检测缺血30 min-再灌注4 h后各组血清AST、ALT水平和肝组织丙二醛(MDA)、总超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-P_X)活性。结果组方实验结果显示EPO对肝缺血-再灌注的保护作用更明显,是主药,GSH是辅药,两者的理论优化组方为EPO 2 000 U·kg^(-1)+GSH 12.5 mg·kg^(-1)。确证性实验结果显示,与假手术组相比,模型组血清ALT、AST水平和肝脏MDA水平升高,肝脏SOD、GSH-P_X活性降低(P<0.01)。EPO组、EPO+GSH中剂量组和EPO+GSH高剂量组血清AST、ALT活性低于模型组(P<0.01),其中EPO组高于其他两组(P<0.01)。与模型组比较,各给药组MDA含量降低,SOD活性升高(P<0.05或P<0.01)。EPO+GSH中剂量组、EPO+GSH高剂量组GSH-P_X活性高于模型组(P<0.05)。结论 EPO和GSH对肝缺血-再灌注损伤有相加保护作用,理论优化组方为EPO 2 000 U·kg^(-1)+GSH 12.5mg·kg^(-1),其机制可能与抗氧化作用有关。

AIM To investigate the optimal combination ratio of erythropoietin （EPO） combined with reduced glutathione （GSH）, and their protective effects and mechanism on hepatic ischemia-reperfusion injury. METHODS Models of mice with hepatic ischemia- reperfusion were established. Six formulas with different combination ratios were designed according to the weighted modification method. After ischemia for 30 min and reperfusion for 4 h, the blood and hepatic tissue was collected. The results were analyzed for obtaining the theoretical optimal combination ratio, according to the decreased activity of alanine aminotransferase （ALT） and aspartate aminotransferase （AST）. Then in the further confirmation test, another 56 mice were randomized into sham group, model group, EPO （2 000 U·kg^-1） group, GSH （12.5 mg.kg^-1） group, low dose EPO （1 000 U·kg^-1） ＋ GSH （6.25 mg·kg^-1） group, middle dose EPO （2 000 U·kg^-1） ＋ GSH （12.5 mg·kg^-1） group, high dose EPO （4 000 U·kg^-1） ＋ GSH （25 mg·kg^-1） group. All of them were administered intraperitoneally with normal saline or appropriate drugs 1 hour before surgery. After ischemia for 30 min and reperfusion for 4 h, the blood serum levels of AST, ALT and the tissue concentrations of malondialdehyde （MDA）, superoxide dismutase （SOD）, glutathione synthetase （GSH-Px） were measured. RESULTS Formula experimental results showed that EPO was the main medicine, which had a more efficient protection for liver ischemia-reperfusion injury, and GSH was complementary medicine. The theoretical optimization combination was EPO 2 000 U·kg^-1 and GSH 12.5 mg·kg^-1. In the further confirmation test, compared with those in sham group, the serum levels of AST, ALT and the MDA concentration in the liver were increased, while the activities of SOD and GSH-Px in the liver decreased significantly in the model group （P 〈 0.01）. The serum activities of AST, ALT in EPO group, middle dose EPO ＋ GSH group and high dose EPO ＋ GSH group were lower than those in model group, and those in EPO group were higher than those in the other two groups （P 〈 0.01 ） . Compared with those in model group, MDA concentration decreased, and the activity of SOD was increased in each medicated group （P 〈 0.05 or P 〈 0.01）. The activities of GSH-Px in middle dose EPO ＋ GSH group and high dose EPO ＋ GSH group were higher than those in the model group （P 〈 0.05）. CONCLUSION Combination of EPO with GSH could produce additive protective effects on hepatic ischemia- reperfusion injury, and EPO 2 000 U·kg^-1 ＋ GSH 12.5 mg ·kg^-1 are the optimal combination dosage. The mechanism is partly related to the enhancement of antioxidation.