The Role Of Neuregulin-1? On The Diaphragm Properties (contractility And AChE Activity) Of Rats With Sepsis And The Possible Mechanisms

Objective:To observe the effects of neuregulin-1? on the diaphragm property(contractility and AChE activity)of rats with sepsis and explore the possible mechanisms.Methods:First part: Cecal ligation and puncture(CLP)was used to establish a rat model of low-grade and midgrade sepsis.At 24 h after surgery,diaphragm contractile measurements,an assay of thiobarbituric acid reactive species(TBARS)and protein carbonyls(PC)contents,as well as the myeloperoxidase(MPO),superoxide dismutase(SOD),and catalase(CAT)activity,was conducted.AChE activity was measured by biochemical and histological detection.Second part: CLP was used to establish a rat model of midgrade sepsis,and NRG-1? was given to rats via tail vein injection 30 min before the surgery.At 24 h after surgery,inferior vena blood samples were obtained for determination of serum glutamic oxaloacetic transaminase(AST),lactate dehydrogenase(LDH),creatine kinase isoenzymes(CK)activity and myohemoglobin(Myo)contents.The diaphragm was removed for determination of the contractile tension,TBARS,PC content,MPO and AChE activity.Hematoxylin-eosin staining(HE)staining was performed to detect the diaphragm pathological situation.Third part: CLP was used and antibiotics were given postoperatively to establish a rat model of sepsis.NRG-1? was given every 12 h for 72 h after CLP.At 72 h after surgery,diaphragm contraction tension,HE staining for determination of diaphragmatic muscle cross-sectional area(cross sectional area,CSA),Western blot for diaphragmatic protein expression of PI3K/Akt pathway were conducted.In addition,L6 myoblasts were cultured and differentiated for subsequent experiments.Lipopolysaccharide(LPS)was used to stimulate cells to form sepsis model,while NRG-1? and PI3 K inhibitor LY294002 were co-incubated with differentiated L6 cells.Afterwards,Western blot for PI3K/Akt pathway related protein expression and toluidine blue staining for the atrophy situation of L6 cells were determined.Results:First part: At 24 h after CLP,diaphragm contractile force,AChE and CAT activity in the diaphragm decreased,while the contents of TBARS and PC,the activity of MPO and SOD,and the SOD/CAT ratio increased.The above changes were much more significant in the mid-grade septic group than in the low-grade septic group.The colour of the AChE activity staining at the NMJ gradually lightened from the sham surgery group to the mid-grade septic group.AChE activity was significantly negatively correlated with the levels of TBARS and PC.Second part: At 24 h after CLP,the level of serum AST,LDH,CK activity and Myo content increased.The contractile force and AChE activity of diaphragm decreased,while TBARS,PC contents and MPO activity increased.HE staining showed inflammatory infiltration of diaphragm.After NRG-1? treatment,the diaphragm contractile force and AChE activity increased,while serum AST,LDH,CK activity and Myo content?TBARS,PC content and MPO activity of the diaphragm decreased.Additionally,the degree of inflammatory infiltration of diaphragm was decreased.Third part: At 72 h after CLP,the contractile force,CSA and phosphorylated protein expression of PI3K/Akt pathway decreased in the diaphragm of septic rats.Contractile force and CSA increased,and PI3K/Akt pathway was activated in the diaphragm of rats treated with NRG-1?.In vitro,NRG-1? improved the decrease of PI3K/Akt pathway related phosphorylated protein expression and cell atrophy induced by LPS stimulation.PI3 K inhibitor LY294002 partially reversed the above effects of NRG-1?.Conclusion:During the early period of sepsis,two vital physiological properities(contractile force,AChE activity)of rat diaphragm changed,and oxidative stress may be responsible for these changes.NRG-1? treatment increased the contraction force and AChE activity of diaphragm during sepsis,which was related to its role of antioxidant stress.At 72 h after CLP,diaphragm dysfunction and atrophy occur,and NRG-1? treatment can mitigate the diaphragm dysfunction and atrophy,which is associated with the activation of PI3K/Akt pathway of diaphragm.