The Study On Mild Hypothermia Ameliorating Hypercatabolism In Septic Rats Via Hypothalamic AMPK-Autophagy-POMC Pathway

Among critically ill patients,hypercatabolism,which occurred early and rapidly,was characterized for the first time and more pronounced among patients with multiorgan failure.The body in a short period of time will appear loss of appetite,skeletal muscle protein,gluconeogenesis,insulin resistance and other metabolic disorders,showing a high metabolic state.This hypercatabolism is hard to explain by peripheral organs,such as liver,skeletal muscle and intestine.It is more like regulated by hypothalamic energy regulation center.Hypothalamus is the regulatory centre of temperature,metabolism and food intake.In previous studies,we found that the arcuate nucleus(ARC)of hypothalamus exerted a critical role in the regulation of acute skeletal muscle wasting during sepsis.Two major populations of neural in the ARC,AgRP and POMC neurons,play a pivotal role in calorie intake and energy expenditure.The former neuron co-expresses orexigenic peptides NPY and AgRP,which promote food intake and decrease metabolism by acting as an inverse agonist of the type-4 melanocortin receptor(MC4-R).Whereas the other neuron co-expresses the anorexigenic POMC and CARTPT/CART.POMC could be pyrolysed into a-mela-nocytestimulating hormone(a-MSH),binding to MC3-R and MC4-R to derive anorectic effects.HypothalamicAMPK activity was closely involved in the induction of autophagy and the regulation of energy balance.Intracerebroventricular(i.c.v.)administration of AICAR could increase food intake and body weight.However,little is known about the exact mechanisms of the central role.Among potential approaches,Mild hypothermia(MH)could be an efficacious one.Prompt MH significantly increased the the survival duration of septic rats maintained at 34 ? throughout the experiment compared with those maintained at 38?.The research reported by Chisholm K showed sepsis was associated with brain dysfunction and influenced cortical oxygenation and mitochondrial function,and that MH could be protective.Induced MH could reversed low oxygen consumption and ameliorate mitochondrial function in skeletal muscle.Whereas,the potential role of MH on alleviating hypermetabolic state and muscle wasting remains uncertain.In this thesis,we firstly analyzed the correlation between sepsis-induced hypercatabolism,neuropeptide and AMPK-induced autophagy.Secondly,We knockouted the hypothalamus POMC with a lentiviral vector containing shRNA via third ventricle injection.Then,rats were injected with AICAR into third ventricle and promptly intraperitoneally injected with LPS to clear the effect of AMPK-autophagy-POMC pathway on hypercatabolism in septic rats.Finally,mild hypothermia was performed to observe whether it could ameliorate hypercatabolism during sepsis and was associated with hypothalamus AMPK-autophagy-POMC pathway.This thesis is divided into three parts.Part I:establishing of septic hypercatabolism model and analyzing the correlation between hypercatabolism and hypothalamic AMPK-induced autophagy and neuropeptideObjective:establishing a stable and repeatable septic hypercatabolism model and analyzing the correlation between sepsis-induced hypercatabolism and hypothalamic AMPK-induced autophagy and neuropeptide.Methods:Adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide(LPS)(5 mg/kg)or saline.Twenty-four hours after injection,blood,hypothalamus,skeletal muscle were obtained.Hypothalamic AMPK-induced autophagy markers(p-AMPK,AMPK,p-ULK1,ULK1 and LC3)and neuropeptides expression were detected.Blood glucose,serum insulin,leptin and muscle wasting mRNA expression of muscle ring finger 1(MuRF-1)and muscle atrophy F-box(MAFbx)was measured as the hypercatabolism markers.Results:LPS injection led to significant decreased food intake,loss of body weight and EDL-BW ratio.LPS injection also led to significant decreased of blood glucose and the high levels of serum insulin and leptin.And the expression of two muscle atrophic gene,MuRF-1 and MAFbx mRNA were also significant increased.This result indicated that LPS induced sepsis would lead to the hypercatabolism.The ratio of p-AMPK/AMPK,p-ULK1/ULK1 and LC3 ?/? were significant decreased.This result indicated that sepsis could decrease the level of hypothalamic AMPK-autophagy.The anorexigenic POMC and CART were significant increased and orexigenic peptides NPY and AgRP were significant decreased.With the analysis of the correlation between sepsis-induced hypercatabolism and neuropeptide,we found that hypercatabolism was mainly related with POMC expression.Conclusion:Sepsis induced by LPS led to the hypercatabolism,which may be associated with the increased hypothalamic POMC expression and AMPK-autophagy pathway.Part ?:Hypothalamic AMPK-induced autophagy ameliorated hypercatabolism in septic rats by regulating POMC expressionObjective:To investigate the exact mechanism of hypothalamic AMPK-autophagy-POMC on hypercatabolism in septic rats.Methods:Firstly,We knockouted the hypothalamus POMC with a lentiviral vector containing shRNA(1*109,5ul)via third ventricle injection.Two weeks later,adult male Sprague-Dawley rats received intraperitoneal LPS injection or vehicle.Twenty-four hours after injection,hypothalamus,blood and skeletal muscle were obtained.Then,third ventricle cannulated rats received central infusion of AMPK activator(AICAR,6mg/ml,5ul)or vehicle and intraperitoneal LPS injection.Twenty-four hours after injection,hypothalamus,blood and skeletal muscle were obtained.Hypothalamic AMPK-induced autophagy markers and neuropeptides expression were detected.Blood glucose,serum insulin,leptin and corticosterone were also detected.Muscle wasting was measured by the mRNA and protein expression of muscle ring finger 1(MuRF-1)and muscle atrophy F-box(MAFbx).Results:Sepsis would decrease the level of hypothalamic AMPK-autophagy accompany with the alterations of POMC expression and hypercatabolism.POMC knockdown in the arcuate nucleus of the hypothalamus with a lentiviral vector containing shRNA could significantly ameliorate hypercatabolism,whereas hypothalamic other neuropeptides and AMPK-induced autophagy pathway was barely affected.Moreover,Central activation of AMPK-induced autophagy pathway via third ventricle injection of AICAR could efficiently ameliorate hypercatabolism as well as attenuate the elevated POMC expression rather than other neuropeptides.Conclusion:Hypothalamic AMPK-autophagy pathway as a regulatory pathway for POMC expression was essential for hypercatabolism during sepsis.And hypothalamic autophagy activation could attenuate the POMC expression to ameliorate hypercatabolism.Part ?:Mild hypothermia ameliorated hypercatabolism in septic rats associated with hypothalamic AMPK-autophagy-POMC pathwayObjective:To investigate the best sustained and beginning time of mild hypothermia in septic rats and the protective effect on hypercatabolism via hypothalamic AMPK-autophagy-POMC pathway.Methods:Adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide(LPS)and instantly induced at 33 ? for 0h,1h,3h,6h and 9h to assess the survival rate during sepsis and determine the best time for mild hypothermia.Secondly,after 0h,3h,6h,9h and 12h of LPS injection,rats received the best time of mild hypothermia to assess the survival rate of sepsis and determine the best beginning time for mild hypothermia.Thirdly,rats were intraperitoneally injected with LPS or saline and received mild hypothermia.Meanwhile,the control and sepsis groups were simultaneously placed on the thermal mattress to maintain the a normal temperature in control group whatever the changes induced by anesthesia.Twenty-four hours after injection,hypothalamus,blood and skeletal muscle were obtained.Hypothalamic AMPK-induced autophagy,inflammatory markers and neuropeptides expression were detected.Blood glucose,serum insulin,leptin and corticosterone were also detected.Muscle wasting was measured by the mRNA expression of two muscle atrophic genes,muscle ring finger 1(MuRF-1)and muscle atrophy F-box(MAFbx),as well as 3-methylhistidine(3-MH)and tyrosine release.Results:after LPS injection,the instantly mild hypothermia for 3h significantly increased the survival rate during sepsis.LPS administration significantly decreased hypothalamic AMPK-induced autophagy together with hypercatabolism.Also,increased hypothalamic POMC,CART and NPY mRNA and decreased AgRP mRNA were observed.The instantly mild hypothermia for 3h significantly increased hypothalamic AMPK-induced autophagy and ameliorated LPS-induced hypercatabolism and the elevated of POMC.Conclusion:The instantly mild hypothermia for 3h could alleviate hypercatabolism by LPS injection,which was associated with reversing the level of hypothalamic AMPK-induced autophagy and the alteration of POMC.These results suggested that mild hypothermia could be a potential treatment concept and a novel mechanism in management of hypercatabolism in critically ill patients.