芦荟苷通过调控AMPK/NRF-2/GSH通路抑制异丙肾上腺素诱导的心肌肥厚

Aloin Inhibits Isoproterenol-Induced Myocardiac Hypertrophy by Regulating AMPK/NRF-2/GSH Pathway.

目的探讨芦荟苷(ALO)对小鼠心肌肥厚的保护作用及其分子机制。方法将30只C57小鼠随机分为5组:对照组、异丙肾上腺素(ISO)组、ALO干预组(低剂量组(ISO+low-dose ALO,I+L-A)、中剂量组(ISO+middle-dose ALO,I+M-A)、高剂量组(ISO+high-dose ALO,I+H-A)。ISO组:每日腹腔注射ISO;ALO干预组:在ALO灌胃的同时腹腔注射ISO;对照组:每日予以等量生理盐水腹腔注射及灌胃。以上各组每天处理1次,连续15 d后处死小鼠、称重,计算小鼠心脏体质量比(HW/BW);hematoxylin-eosin染色(HE)及Masson染色观察心脏形态和纤维化程度;实时荧光定量PCR(qRT-PCR)测定心肌细胞肥大相关基因心房利钠肽(ANP)、脑钠肽(BNP)和β-肌球蛋白重链(β-MHC)的mRNA表达;为检测组织中氧化还原情况,分光光度法测定抗氧化剂谷胱甘肽(GSH)及硫代巴比妥酸法测定脂质过氧化指标-丙二醛(MDA);Western Blot法测定心肌组织肥大标志物BNP、心肌损伤标志物肌酸激酶同工酶(CKMB)、抗氧化通路AMP依赖的蛋白激酶(AMPK)/磷酸化AMPK(p-AMPK)及转录因子E2相关因子2(NRF-2)的蛋白表达。结果与对照组比,ISO组HW/BW升高(P<0.05),HE及masson染色提示心肌细胞排列紊乱、细胞形状改变、纤维化增多,ANP、BNP和β-MHC的mRNA表达水平上调,MDA含量增加和GSH减少,p-AMPK/AMPK比值、CKMB、NRF-2蛋白表达减少;ALO干预后,小鼠HW/BW下降,心肌细胞变形及纤维化程度明显改善,肥厚相关基因表达明显减少,抗氧化通路AMPK/NRF-2/GSH的表达增加。结论ALO能抑制心肌肥厚的发生发展,其机制可能与增强AMPK/NRF-2/GSH通路的激活有关。

Objective To investigate the protective effect of aloin(ALO)on myocardial hypertrophy and its molecular mechanism in mice.Methods A total number of 30 C57 mice were randomly divided into 5 groups:Control group,Isoproterenol group(ISO),ALO intervention groups including:ISO+low-dose aloin(I+L-A)、ISO+middle-dose aloin(I+M-A)、ISO+high-dose aloin(I+A-H)).To investigate the protective effect of aloin(ALO)on myocardial hypertrophy in mice and its molecular mechanism.ISO group:daily intraperitoneal injection of ISO;ALO intervention group:Intraperitoneal injection of ISO at the same time of ALO intragastric administration;Control group:the same amount of normal saline intraperitoneal injection and gavage every day.Mice in each group were treated once a day.After 15 days,the mice were sacrificed and weighed to calculate the ratio of heart weight/body weight(HW/BW).Hematoxylin-eosin staining(HE)and Masson staining were performed to observe cardiac morphology and fibrosis degree.Real-time quantitative polymerase chain reaction(QRT-PCR)was used to determine the mRNA expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)andβ-myosin heavy chain(β-MHC)genes related to cardiac hypertrophy.Glutathione(GSH)was determined by spectrophotometry and malondialdehyde(MDA)was determined by thiobarbituric acid.Western blot was used to determine BNP(a marker of myocardial hypertrophy),CKMB(creatine kinase isoenzyme,a marker of myocardial injury),AMPK(adenosine monophosphate-activated protein kinase)/phosphorylated AMPK(P-AMPK)and nuclear factor Erythroid-2-related factor 2(NRF-2)protein expression.Results Compared with the control group,HW/BW in ISO group was increased(P<0.05),HE and Masson staining showed that myocardial cells were disordered with changes in cell shape and increase of fibrosis,the mRNA expression levels of ANP,BNP andβ-MHC were up-regulated,MDA content was significantly increased and GSH was decreased,and p-AMPK/AMPK ratio,CKMB and NRF-2 protein expression were decreased.After ALO intervention,HW/BW decreased,the degree of myocardial cell deformation and fibrosis was significantly improved,the expression of hypertrophy related genes was significantly decreased,and the expression of AMPK/NRF-2/GSH,the key factors in antioxidant pathway was increased.Conclusion ALO can inhibit the development of cardiac hypertrophy,and its mechanism may be related to the enhancement of the activation of AMPK/NRF-2/GSH pathway.