Rameshrad Maryam, Maleki-Dizaji Nasrin, Soraya Hamid, Toutounchi Negisa Seyed, Barzegari Abolfazl, Garjani Alireza
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; Student Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Iran J Basic Med Sci. 2016 Dec;19(12):1308-1317. doi: 10.22038/ijbms.2016.7917.
TLR-4 activates a number of inflammatory signaling pathways. Also, AMPK could be involved in anti-inflammatory signaling. The aim of this study was to identify whether stimulation of AMPK could inhibit LPS-induced -4 gene expression in mice hearts.
Heart AMPK activity and/or -4 expression was stimulated in different mice groups, using respectively IP injection of A-769662 (10 mg/kg) and LPS (2 mg/kg) or a combination of both agents. Moreover, compound-C (20 mg/kg), as an AMPK antagonist, was intraperitoneally co-administrated with both A-769662 and LPS in another group to investigate the role of AMPK activity on -4 regulation. After 8 hr, in addition to peripheral neutrophil cell count, myocardial p-AMPK, p-ACC as well as MyD88 protein contents and -4 expression was assessed by Western blotting and real-time qRT-PCR, respectively. TNF-α and IL-6 expression levels were also determined by ELISA.
LPS induced heart -4 expression (<0.001) associating with an increase in the myocardial MyD88 protein content (<0.001), elevation of heart TNF-α (<0.01) and IL-6 (<0.05) concentrations, and rise in the peripheral neutrophil cell count (<0.001). Administration of A-769662 decreased LPS-induced -4 expression (<0.01) and alleviated peripheral neutrophil cell count (<0.01). The inhibitory effect of A-769662 on LPS-induced -4 expression was reversed by antagonizing AMPK with compound-C (<0.001) which reduced p-AMPK (<0.05) and p-ACC (<0.01) myocardial protein contents in the LPS+A-769662 group.
This study demonstrated that activation of AMPK, by A-769662 agent, could inhibit -4 expression and activity, suggesting a link between AMPK and Tlr-4 in heart tissue.
Toll样受体4（TLR-4）可激活多种炎症信号通路。此外，腺苷酸活化蛋白激酶（AMPK）可能参与抗炎信号传导。本研究旨在确定激活AMPK是否能抑制脂多糖（LPS）诱导的小鼠心脏中TLR-4基因表达。
分别通过腹腔注射A-769662（10 mg/kg）和LPS（2 mg/kg）或两者联合使用，刺激不同小鼠组的心脏AMPK活性和/或TLR-4表达。此外，在另一组中，将化合物C（20 mg/kg）作为AMPK拮抗剂与A-769662和LPS同时腹腔内给药，以研究AMPK活性对TLR-4调控的作用。8小时后，除了检测外周中性粒细胞计数外，分别通过蛋白质免疫印迹法和实时定量逆转录聚合酶链反应（qRT-PCR）评估心肌磷酸化AMPK（p-AMPK）、磷酸化乙酰辅酶A羧化酶（p-ACC）以及髓样分化因子88（MyD88）蛋白含量和TLR-4表达。还通过酶联免疫吸附测定（ELISA）法测定肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的表达水平。
LPS诱导心脏TLR-4表达（<0.001），同时伴有心肌MyD88蛋白含量增加（<0.001）、心脏TNF-α（<0.01）和IL-6（<0.05）浓度升高以及外周中性粒细胞计数增加（<0.001）。给予A-769662可降低LPS诱导的TLR-4表达（<0.01）并减轻外周中性粒细胞计数（<0.01）。在LPS+A-769662组中，用化合物C拮抗AMPK可逆转A-769662对LPS诱导的TLR-4表达的抑制作用（<0.001），化合物C降低了p-AMPK（<0.05）和p-ACC（<0.01）的心肌蛋白含量。
本研究表明，A-769662激活AMPK可抑制TLR-4表达和活性，提示在心脏组织中AMPK与Tlr-4之间存在联系。
