槲皮素通过PI3K/AKT/mTOR通路减轻脓毒症小鼠心肌损伤

目的基于磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路探究槲皮素减轻脓毒症小鼠心肌损伤的分子机制,为槲皮素治疗脓毒症提供理论依据。方法采用盲肠结扎穿孔法构建脓毒症小鼠模型,将造模成功的小鼠随机分为模型组、槲皮素组(200 mg/kg)、抑制剂组(PI3K/mTOR抑制剂NVP-BEZ235 60 mg/kg)和槲皮素+抑制剂组(槲皮素200 mg/kg+PI3K/mTOR抑制剂NVP-BEZ235 60 mg/kg),每组15只,另取15只小鼠作为假手术组。假手术组和模型组小鼠灌胃等体积生理盐水,其余各组灌胃相对应药物,给药体积10 m L/kg。给药24 h后,超声检测平均动脉压(MAP)、左心室收缩压(LVSP)、左心室等容舒张期压力下降最大速率(-dp/dtmax)和左心室等容舒张期压力上升最大速率(+dp/dtmax);全自动生化分析仪检测血清肌酸激酶同工酶(CK-MB)和心肌肌钙蛋白I(cTnI)水平;苏木素-伊红(HE)染色观察心肌组织病理学变化;酶联免疫吸附(ELISA)法检测心肌组织白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、丙二醛(MDA)和超氧化物歧化酶(SOD)含量;蛋白免疫印迹(Western blot)法检测心肌组织PI3K、AKT和mTOR蛋白磷酸化水平。结果心肌组织病理损伤:模型组小鼠心肌组织出现结构紊乱、心肌细胞变性、心肌纤维断裂、心肌横纹模糊、细胞间质水肿等病理损伤;槲皮素组较模型组减轻,抑制剂组较模型组加重;槲皮素+抑制剂组较槲皮素组加重。MAP、LVSP、-dp/dtmax、+dp/dtmax、SOD含量及PI3K、AKT、mTOR蛋白磷酸化水平:模型组较假手术组降低;槲皮素组较模型组升高,抑制剂组较模型组降低;槲皮素+抑制剂组较槲皮素组降低,较抑制剂组升高,差异均有统计学意义(P <0.05)。CK-MB、cTnI水平及TNF-α、IL-6、MDA含量:模型组较假手术组升高;槲皮素组较模型组降低,抑制剂组较模型组升高;槲皮素+抑制剂组较槲皮素组升高,较抑制剂组降低,差异均有统计学意义(P <0.05)。结论槲皮素可能通过上调PI3K/AKT/mTOR通路降低脓毒症小鼠心肌组织炎症和氧化应激反应,从而减轻心肌损伤。

Quercetin alleviates myocardial damage in septic mice by PI3K/AKT/mTOR pathway

Objective To explore the molecular mechanism of quercetin in alleviating myocardial damage in septic mice based on phosphatidylinositol-3-kinase( PI3 K)/protein kinase B( AKT)/mammalian target of rapamycin( mTOR) pathway,so as to provide theoretical basis for quercetin treatment of sepsis. Methods The septic mice models were established by cecal ligation and puncture method. The successful mice were randomly divided into model group,quercetin( 200 mg/kg)group,inhibitor group( PI3 K/mTOR inhibitor NVP-BEZ235 60 mg/kg) and quercetin + inhibitor group( quercetin 200 mg/kg + PI3 K/mTOR inhibitor NVP-BEZ235 60 mg/kg),with 15 mice in each group,another 15 mice were used as sham operation group. The mice in the sham operation group and model group were intragastrically given the same volume of normal saline,and the other groups were given corresponding drugs by gavage, the dosage volume was 10 m L/kg. At 24 hours after administration,the mean arterial pressure( MAP),left ventricular systolic pressure( LVSP),the maximum rate of left ventricular isovolumic diastolic pressure decline(-dp/dtmax) and the maximum rate of left ventricular isovolumic diastolic pressure rise( + dp/dtmax) were detected by ultrasound;the levels of serum creatine kinase isoenzyme( CK-MB) and troponin I( cTnI) were detected by automatic biochemical analyzer;HE staining was used to observe the pathological changes of myocardium;the contents of interleukin-6( IL-6),tumor necrosis factor-α( TNF-α),malondialdehyde( MDA)and superoxide dismutase( SOD) were detected by enzyme-linked immunosorbent assay( ELISA);the protein phosphorylation levels of PI3 K,AKT and mTOR were detected by Western blot. Results Compared with those in the sham operation group,the myocardial tissue of the model group showed structural disorder, myocardial cell degeneration, myocardial fiber rupture, myocardial transverse striation blur,intercellular edema and other pathological damage,the MAP,LVSP,-dp/dtmax,+ dp/dtmax,the content of SOD and protein phosphorylation levels of PI3 K, AKT and mTOR were significantly lower( P < 0. 05),and the levels of CK-MB and cTnI,the contents of TNF-α,IL-6 and MDA were significantly higher( P < 0. 05). Compared with those in the model group,the above pathological damage of myocardial tissue in quercetin group was reduced,the MAP,LVSP,-dp/dtmax, + dp/dtmax,the content of SOD and protein phosphorylation levels of PI3 K,AKT and mTOR were significantly higher( P < 0. 05),and the levels of CK-MB and cTnI,the contents of TNF-α,IL-6 and MDA were significantly lower( P < 0. 05);meanwhile,the above pathological damage of myocardial tissue in the inhibitor group was aggravated,the MAP,LVSP,-dp/dtmax,+ dp/dtmax,the content of SOD and protein phosphorylation levels of PI3 K,AKT and mTOR were significantly lower( P < 0. 05),and the levels of CK-MB and cTnI,the contents of TNF-α,IL-6 and MDA were significantly higher( P < 0. 05). Compared with those in quercetin group,the pathological damage of myocardium in quercetin + inhibitor group was more serious,the MAP,LVSP,-dp/dtmax,+ dp/dtmax,content of SOD and protein phosphorylation levels of PI3 K,AKT and mTOR were significantly lower( P < 0. 05),and the levels of CK-MB and cTnI,the contents of TNF-α,IL-6 and MDA were significantly higher( P < 0. 05). Compared with the inhibitor group,the MAP,LVSP,-dp/dtmax,+ dp/dtmax,content of SOD and protein phosphorylation levels of PI3 K,AKT and mTOR in quercetin +inhibitor group were significantly higher( P < 0. 05),and the levels of CK-MB and cTnI,the contents of TNF-α,IL-6 and MDA were significantly lower( P < 0. 05). Conclusions Quercetin may reduce myocardial inflammation and oxidative stress,and reduce myocardial damage in septic mice by up-regulating PI3 K/AKT/mTOR pathway.