大黄素和黄芩苷联用对急性胰腺炎模型大鼠Akt/Nrf2通路的影响研究

Effects of Emodin Combined with Baicalin on Akt/Nrf2 Pathway in Acute Pancreatitis Model Rats

目的:研究大黄素和黄芩苷联用对急性胰腺炎(AP)模型大鼠的改善作用和机制。方法:将70只大鼠随机分为假手术组、模型组、大黄素组(250 mg/kg)、黄芩苷组(250 mg/kg)、奥曲肽组(阳性对照,10μg/kg)、N-乙酰半胱氨酸组(阳性对照,180 mg/kg)和大黄素+黄芩苷组(125 mg/kg大黄素+125 mg/kg黄芩苷),每组10只。除假手术组外,其余各组大鼠均复制AP模型,造模成功后静脉注射给予相应药物。在给药后3、6、12、24 h,测定各组大鼠血清中淀粉酶、脂肪酶水平;在给药24 h后,检测大鼠血清中超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)、髓过氧化物酶(MPO)和胰腺组织中半胱氨酸天冬氨酸酶3(Caspase-3)水平,并采用Western blot法检测各组大鼠胰腺组织中蛋白激酶B(Akt)磷酸化和核因子E2相关因子2(Nrf2)蛋白表达水平。另取40只大鼠随机分为假手术组、模型组、大黄素+黄芩苷组(125 mg/kg大黄素+125 mg/kg黄芩苷)和大黄素+黄芩苷+LY294002(Akt特异性抑制剂)组(125 mg/kg大黄素+125 mg/kg黄芩苷+100 mg/kg LY294002),每组10只,进行机制验证实验。同法造模、给药,24 h后检测大鼠胰腺组织中Akt磷酸化和Nrf2蛋白表达水平。结果:与假手术组比较,从给药后3 h开始,模型组大鼠血清中淀粉酶、脂肪酶水平即显著升高(P<0.01);给药后24 h,大鼠血清中MDA、MPO和胰腺组织中Caspase-3水平均显著升高(P<0.01),血清中SOD、GSH和胰腺组织中Akt磷酸化及Nrf2蛋白表达水平均显著降低(P<0.01)。与模型组比较,从给药后6 h开始,各给药组大鼠血清中淀粉酶和脂肪酶水平均显著降低(P<0.01);给药24 h后,各组大鼠血清中MDA、MPO和胰腺组织中Caspase-3水平均显著降低(P<0.01),血清中SOD、GSH和胰腺组织中Akt磷酸化及Nrf2蛋白表达水平均显著升高(P<0.01)。机制验证实验中,与大黄素+黄芩苷组比较,大黄素+黄芩苷+LY294002组大鼠胰腺组织中Akt磷酸化及Nrf2蛋白表达水平均显著降低(P<0.01)。结论:大黄素和黄芩苷联用对AP模型大鼠具有一定的改善作用,其机制可能与促进Akt磷酸化以上调Nrf2蛋白表达,并促进其下游抗氧化酶的表达,从而降低体内氧化应激水平有关。

OBJECTIVE：To study the effects of emodin combined with baicalin on acute pancreatitis（AP）model rats and its possible mechanism. METHODS：A total of 70 rats were randomly divided into sham operation group,model group,emodin group（250 mg/kg）,baicalin group（250 mg/kg）,octreotide group（positive control,10 μg/kg）,N-acetyl cysteine group（positive control,180 mg/kg）,emodin ＋ baicalin group（125 mg/kg emodin ＋ 125 mg/kg baicalin）,with 10 rats in each group. Except for sham operation group,AP model was induced in each group. After modeling,relevant medicines were given intravenously. After 3,6,12,24 h of medication,the serum levels of amylase and lipase were determined. After 24 h medication,serum levels of SOD,MDA,GSH,MPO and level of Caspase-3 in pancreatic tissue were determined. The levels of Akt phosphorylation and expression of Nrf2 protein in pancreatic tissue of rats were determined by Western blot assay. Another 40 ratss were randomly divided into sham operatsion group,model group,emodin ＋ baicalin group（125 mg/kg emodin ＋ 125 mg/kg baicalin） and emodin ＋ baicalin ＋LY294002（Akt specific inhibitor）group（125 mg/kg emedin＋ 125 mg/kg baicalin＋ 100 mg/kg LY294002）,with 10 ratss in each group. Mechanism verification test was performed. The modeling and medication method of those groups were same to above groups,24 h later. The level of Akt phosphorylation and the expression of Nrf2 protein were determined in pancreatic tissue of rats.RESULTS：Compared with sham operation group,the serum levels of amylase,lipase of rats were significuntly increased in model group after 3 h of medication（P〈0.01）;levels of MDA,MPO in serum and Caspase-3 in pancreatic tissue were significantly increased in model group（P〈0.01）,while the levels of SOD,GSH in serum and the levels of Akt phosphorylation and the expression of Nrf2 protein were decreased significantly in pancreatic tissue of rats（P〈0.01）. Compared with model group,the serum levels of amylase and lipase were decreased significantly in administration groups after 6 h of medication（P〈0.01）. After24 h medication,the levels of MDA,MPO in serum and Caspase-3 in pancreatic tissue were decreased significantly（P〈0.01）,while the levels of SOD,GSH in serum and the levels of Akt phosphorylation and the expression of Nrf2 protein were increased significantly in pancreatic tissue of rats（P〈0.01）. In mechanism verification test,compared with emodin ＋ baicalin group,the levels of Akt phosphorylation and the expression of Nrf2 protein in pancreatic tissue were decreased significantly in emodin ＋baicalin ＋ LY294002 group（P〈0.01）. CONCLUSIONS：Emodin combined with baicalin shows certain improvement effect on AP rats,the mechanism is that the level of Akt phosphorylation is promoted so as to up-regulate the expression of Nrf2 protein,and the expression of antioxidant enzyme is promoted so as to reduce the level of oxidative stress in AP model rats.