基于PI3K-Akt-eNOS通路探讨瘀毒清丸对铁过载导致的心肌损伤小鼠的保护作用

Exploration on the Protective Effect of Yuduqing Pills on Myocardial Injury Caused by Iron Overload Based on PI3K-Akt-eNOS Pathway

目的基于磷脂酰肌3激酶(PI3K)-蛋白激酶B(Akt)-内皮型一氧化氮合酶(e NOS)通路探讨瘀毒清丸对铁过载导致的心肌损伤的保护作用。方法将70只SPF级C57BL/6J小鼠随机分为空白对照组、模型组(右旋糖酐铁,91 mg/kg)、地拉罗司组(阳性对照,182 mg/kg),瘀毒清丸低、中、高剂量组(3.51、7.02、14.04 g/kg)和瘀毒清丸+PI3K抑制剂(LY294002)组,每组10只。连续给药12周。末次给药24 h后,摘除小鼠眼球取血,ELISA法检测血清肌钙蛋T(cTnT)、血清肌钙蛋I(cTnI)、肌酸激酶(CK)、乳酸脱氢酶(LDH)、一氧化氮(NO)、eNOS和血清中氧化应激的指标[超氧化物歧化酶(SOD)、丙二醛(MDA)]含量;Western blotting法检测小鼠心肌组织中P-Akt、P-eNOS、Akt、eNOS蛋白含量。结果1)瘀毒清丸低、中、高剂量组和地拉罗司组T波倒置以及ST段抬高得到改善。2)血清学检测方面,与空白对照组比较,模型组小鼠血清肌钙蛋白cTnT、cTnI、血清铁浓度、CK、LDH、MDA含量均显著升高,NO、e NOS、SOD的水平显著下降;与模型组比较,瘀毒清丸低、中、高剂量组和地拉罗司组小鼠血清肌钙蛋白cTnT、cTnI、血清铁浓度、CK、LDH、MDA的含量均显著下降,血清NO、eNOS、SOD的水平显著升高(均P<0.05或P<0.01)。3)蛋白表达水平方面,与空白对照组比较,模型组PAkt、P-eNOS蛋白的表达水平均显著下降(P<0.01);各药物组心肌组织P-Akt、P-eNOS蛋白的表达水平均显著上升(P<0.01或P<0.05)。结论瘀毒清丸通过PI3K-Akt-eNOS信号通路抑制氧化应激,改善心肌舒张功能,从而起到对铁过载导致的心肌损伤的保护作用。

Objective:Based on the pathway of PI3K protein kinase B(Akt)and endothelial nitric oxide synthetase(eNOS),the protective effect of Yuduqing Pills on myocardial injury caused by iron overload was explored.Methods:Seventy SPF-grade C57BL/6J mice were randomly divided into a blank control group,a model group(dextran iron,91 mg/kg),a deferasirox group(positive control,182 mg/kg),a stasis clearing pill low,medium and high dose group(3.51,7.02 and 14.04 g/kg)and a stasis clearing pill+PI3K inhibitor(LY294002)group,10 in each group.The drugs were administered continuously for 12 weeks.Electrocardiograms were collected using an animal ECG machine and recorded.After 24 h of the last administration,the blood was extracted from the eyes of mice to detect cardiac enzymes(c TnT,cTnI,CK,LDH)in the serum of each group using an enzyme marker;ELISA was used to detect the indexes of endothelial diastolic function(NO,e NOS)and oxidative stress(SOD,MDA)in the serum;immunohistochemistry was used to detect the level of eNOS in the myocardial tissue;Western blotting was used to detect the level of e NOS in the myocardial tissue of mice.P-Akt,P-eNOS,Akt,eNOS protein levels in mouse myocardial tissues were detected.Results:1)For ECG,the model group showed ST-segment elevation and T-wave inversion on ECG compared with the blank control group,indicating successful modeling.T-wave inversion as well as ST-segment elevation were improved in the low,medium and high dose groups of Stasis Clear Pill and Deferasirox group.2)Compared with the blank control group,the levels of serum troponin cTnT,cTnI,serum iron concentration,CK,LDH and MDA were significantly increased,and the levels of NO,eNOS and SOD were significantly decreased in the model group;compared with the model group,the levels of serum troponin cTnT,cTnI,serum iron concentration,CK,LDH and MDA were significantly increased in the low,medium andhigh dose groups of Ecotoxin Qing Pill and Dilaurus group.The levels of CK,LDH and MDA were significantly de-creased,and the levels of serum NO,e NOS and SOD were significantly increased(all P<0.05或P<0.01).3)Interms of protein expression levels,the expression levels of P-Akt and P-e NOS proteins were significantly de-creased in the model group compared with the blank control group(P<0.01);the expression levels of P-Akt andP-e NOS proteins in myocardial tissue were significantly increased in each drug group(P<0.01 or P<0.05).Conclusion:Yuduqing Pills can inhibit oxidative stress and improve myocardial diastolic function through PI3K-Akt-eNOS signaling pathway,thus it can protect the myocardial injury caused by iron overload.