Molecular Mechanisms Of Activation Of AMPK Inhibits Inflammatory Response During Hypoxia And Reoxygenation

INTRODUCTION:Cardiovascular disease,the main cause of death globally,was responsible for more than 17.3 million deaths in 2013,and that number is anticipated to reach more than 23.6 million by 2030.Ischemic heart disease is a condition where the heart muscle is weakened and the left ventricle is usually enlarged and dilated.This condition can result from coronary artery stenosis or occlusion or chronic myocardial ischemia.Among these deaths,those due to ischemic heart disease increased by an approximated 41.7% from 1990 to 2013.Therefore,the elucidation of key factors in the regulation of myocardial ischemia and the search for appropriate strategies for prevention and treatment of IHD are important issues that need to be resolved.AMP-activated protein kinase(AMP-activated protein kinase,AMPK)enzyme is one of the most important regulator of intracellular stress response,the activity is mainly affected by the level of intracellular energy regulation.AMPK activation increase the activity of ATP synthesis system while reducing the consumption of ATP.AMPK activation can modulate glucose and fatty acid metabolism via the phosphorylation of the downstream acetyl-Co A carboxylase(ACC)in order to protect the heart from ischemic damage caused by Ischemia/Re-perfusion(I/R).During I/R,excessive ROS can cause the mitochondrial permeability transition pore(m PTP)to open and activate harmful signaling.Mitochondrial dysfunction during I/R is mainly caused by the opening of the m PTP.Chronic energy deficiency stimulates AMPK in order to facilitate muscle mitochondrial biogenesis,but whether the activated AMPK can protect mitochondrial function during I/R remains uncertain.The c-Jun N-terminal protein kinase(JNK)is a mitogen activated protein kinase(MAPK)family member that modulates multiple cellular functions,including proliferation,differentiation,and apoptosis.Chronic AMPK activation decreased JNK activation via hydrogen peroxidein endothelial cells.The Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)is a nuclear transcription factor that can regulate gene expression,which is very important to apoptosis and inflammation signaling in many diseases,including ischemic pathology.Previous studies have shown that the NF-κB subunit p65 is associatedwith I/R injury in a liver model,due to increased inflammation.Moreover,the components of the mitogen-activated protein kinase(MAPK)signaling pathway,which include p38 MAPK,extracellular signal-regulated kinase(ERK)and JNK,also participate in inflammation and are considered to be upstream factors of NF-κB.All these three are very important regulatory factors.Does the AMPK signaling pathway protect mitochondrial function under ischemia-reperfusion condition? Does the AMPK signaling pathway regulate inflammatory responses through ROS-mediated JNK signaling pathways? All these questions are still to be clarified.Therefore,in this study,an in vitro hypoxia-reoxygenation model was constructed to simulate ischemia-reperfusion in vivo,and the regulation of the inflammatory response by the AMPK signaling pathway was explored.At he same time,related signaling mechanisms were analyzed.AIM: 1.To clarify the effect of AMPK on mitochondrial function during ischemia/reperfusion;2.To determine the regulation of AMPK in the inflammatory response during ischemia/reperfusion;3.To explore the specific molecular mechanisms among the above biological effects.METHODS and RESULTS: 1.In vitro hypoxia-reoxygenation(H/R)model was established using rat cardiac myoblasts.During experiments,the pharmacological method(AMPK inhibitor Compound C,JNK inhibitor JNK-IN-8)and the genetic method(the small interfering RNAs of AMPKα1/α2 and small interfering RNAs of JNK1/2)were used to demonstrate that there is no feedback Loop relationship between AMPK and JNK.That means the AMPK is an upstream regulatory molecule of JNK.2.The mitochondrial Oxygen Consumption Rate(OCR)was measured using the Seahorse XFe24 Cell Energy Metabolism Analyzer(Seahorse XFe24).The experimental results show that activated AMPK can maintain mitochondrial function,inhibit the reduction of oxygen consumption rate,and can inhibit the decrease of basal respiration,decrease of the maximum respiration,the decrease of spare capacity and the decrease of ATP production under hypoxia-reoxygenation stress conditions.3.The production of reactive oxygen species(ROS)was used ROS-ID total ROS detection kit to measure;at the same time,the opening of the mitochondrial permeability transition pore(m PTP)was measured using of flow cytometry.The experimental results show that the activated AMPK protein inhibits the production of reactive oxygen species(ROS)and inhibits the opening of the mitochondrial permeability transition pore(m PTP)during hypoxia/reoxygenation.4.Total RNA was isolated and then the m RNA levels of pro-inflammatory cytokines TNF-α and IL-6 were analyzed by Real-time Quantitative PCR Detecting System.The ELISA kit was used to determine the secretion of TNF-α and IL-6 in the culture medium.The experimental results show that the activated AMPK protein can inhibit the phosphorylation of p65 under hypoxia/reoxygenation stress condition,and it can also inhibit the m RNA and protein levels of pro-inflammatory cytokines TNF-α and IL-6.5.The apoptosis of H9c2 cells was measured by In Situ Cell Death Dection Kit(Roche)using TUNEL and DAPI staining.The experimental results show that the activated AMPK can inhibit apoptosis induced by Hypoxia/Reoxygenation.6.Metformin,which is commonly used to treat diabetes,is an AMPK agonist.It is used to pretreat H9c2 cells.The experimental results show that pretreatment of H9c2 myoblasts by metformin can reduce the phosphorylation of JNK and p65 during hypoxia/reoxygenation,reduce the level of mitochondrial permeability transition pore(m PTP)opening and reduce the reactive oxygen species(ROS)production.At the same time,it can also reduce the m RNA and proteins of proinflammatory cytokines(TNF-alpha and IL-6).CONCLUSIONS: AMPK activation modulates JNK-NF-κB signaling cascade during hypoxia and reoxygenation stress conditions.Cardiac AMPK activation plays a critical role in maintaining mitochondrial function and inhibiting the inflammatory response caused by ischemic insults.