Activation Of MTOR Pathway Is Involved In Lipid Disorder Under Condition Of Hypoxic Environment

Objective: Mechanical ventilation is one of the most frequently used method of supportive therapy for acute and chronic respiratory failure,during the process of mechanial ventilation,a wide variety ofconditions leading to decrease progressively of diaphragmatic contractility and diaghragmatic atrophy,a condition initially termed ventilator-induced diaphragmatic dysfunction(VIDD)which showed difficult weaning.Studies have shown that abnormal lipid metabolism induced mitochondrial damage induced by mechanical ventilation had importance implications for VIDD,however,the celluar basis for this phenomenon is poorly understood.Recently several of evidence have showed that activation of mammalian target of rapamycin(m TOR)is involved in the progression of lipid metabolism of atherosclerosis,but its role in the development of VIDD is unclear.The hypoxic environment induced by mechanical ventilation,which is caused by the decrease of oxygen supply,may be one of several stimulation factors of membrane lipid deposition,which leads to the increase of oxidative stress and diaphragmatic dysfunction.Hence,It is speculated that the disorder of lipid metabolism in the process of mechanical ventilation through the activation of m TOR signaling pathway,which leads to the aggravation of lipid deposition in diaphragm and accelerates the development of diaphragmatic dysfunction in early stage of mechanical ventilation.The purpose of this study was to establish C2C12 myoblasts hypoxia model,observe the molecular mechanisms of activation of m TOR signaling pathway on cell lipid which is exposed to low oxygen environment.To the extend,it is revealed that the initiate and regulation mechanism of muscle atrophy under the condition of mechanical ventilation,which can provide a theoretical basis forclinical prevention and treatment of VIDD and suggest promising avenues future research in this area.Methods: C2C12 myoblasts were cultured with DMEM high glucose medium containing 10% fetal bovine serum.Then,which were cultured with DMEM high glucose medium containing 2% horse serum in which C2C12 myoblasts can be actived to initiate proliferation and differentiation to myotubes through microscope to observe the changes in cell morphology,and divided into four experimental groups: 1)control group: C2C12 myoblasts which possessed myotubes were cultured in carbon dioxide incubator with 5% CO2 at 37℃。 2)hypoxia group: C2C12 myoblasts which possessed myotubes were cultured in incubator with 5% CO2,1% O,balance 94% at 37℃ for 12 hours,24 hours respectively.3)rapamycin group: C2C12 myoblasts which possessed myotubes were cultured in the medium with a final concentration of 10ng/ml rapamycin for 12 hours,24 hours respectively.4)rapamycin plus hypoxia group: C2C12 myoblasts which possessed myotubes were cultured in the medium with a final concentration of 10ng/ml rapamycin and with 5% CO2,1% O,balance 94% at 37 for ℃12 hours,24 hours respectively.Intracellular lipid deposition was evaluated by OR staining at different time points.Then,the cells were collected,and the protein expressions of molecules relation to p-m TOR,p-4EBP1,p-S6K1,SCAP,SREBP-2,and LDLR were examined by Western Blot and the m RNA expressions of molecules relation to m TOR,4EBP1,S6K1,SCAP,SREBP-2,LDLR by real-time polymerase chain reaction(PCR)at different time points.Results:(1)C2C12 myoblasts with DMEM high glucose medium containing 2% horse serum cells can be differentiated into myotubes for 7 days or so.(2)The results of OR staining showed that there were intracellular lipid deposition in each group,and rapamycin could reduce the intracellular lipid deposition.(3)Under the condition of hypoxia,the protein and m RNA expression involved in m TOR signaling pathway and Lipid metabolism pathway increased accompanied the add of intracellularlipid deposition.(4)With prolonged hypoxia time,the protein and m RNA expression involved in m TOR signaling pathway and Lipid metabolism pathway generally increased and Rapamycin can reduce intracellular lipid deposition.Conclusion:(1)Abnormal lipid metabolism may be a stimulus to the activation of m TOR signaling pathway(2)The activation of m TOR signaling pathway aggravates the deposition of lipid in cells,which may be a target for regulating intracellular lipid metabolism...