The Changes Of Acetylcholinesterase Activity At The Neuromuscular Junction In The Diaphragm Of Rats With Sepsis And The Possible Mechanisms

Objective:To observe the changes of acetylcholinesterase(ACh E)activity at the neuromuscular junction in the diaphragm of rats during sepsis and investigate the possible mechanisms.Methods:First part:A rat model of acute sepsis was established by cecal ligation and puncture.After 24 h of surgery,the time-response curve of the antagonistic actions of 0.1 or 0.5μmol/L of neostigmine on rocuronium(10μmol/L)-depressed diaphragm twitch tension was measured.Meanwhile,the activity of ACh E at the neuromuscular junction was detected using a modified Karnovsky and Roots method.The m RNA levels of the primary transcript and the type T transcript of ACh E(ACh E_T)in the diaphragm were determined by real-time reverse transcription-polymerase chain reaction.Second part:N-acetylcystein and deferoxamine were given to rats after surgery for therapy when acute sepsis was surgically induced using the cecal ligation and puncture method.At 24 h after surgery,the diaphragm was removed for determination of the malondialdehyde(MDA)content and activities of ACh E,myeloperoxidase(MPO),superoxide dismutase(SOD)and catalase(CAT).SOD/CAT ratio was calculated.Third part:A rat model of chronic sepsis was established by cecal ligation and puncture followed by antibiotic treatment after surgery.On days 1,3,7 and 14 after surgery,the ACh E activity at the neuromuscular junction of diaphragm was detected using a modified Karnovsky and Roots method.Meanwhile,ACh E expression at the neuromuscular junction and the whole muscle fibers of the diaphragm was determined by immunohistofluorescence staining and Western blot analysis,respectively.Results:First part:The time-response curve of both two concentrations of neostigmine during sepsis showed upward shift.Meanwhile,the ACh E activity at the neuromuscular junction was decreased.The ACh E and ACh E_T m RNA expression levels in the diaphragm were reduced.Second part:During acute sepsis,the ACh E and CAT activities in the diaphragm were decreased while the MDA content,MPO activity and SOD/CAT ratio were increased.After antioxidant therapy,the ACh E and CAT activities were increased while the MDA content,MPO activity and SOD/CAT ratio were decreased.Third part:During chronic sepsis,the ACh E activity and expression at the neuromuscular junction of diaphragm began to decrease on day 1,reached their lowest levels on day 7,and then partially recovered on day 14.They were positively correlated.The ACh E protein level in the whole muscle fibers also decreased after surgery,with the lowest level observed on day 3.Conclusion:Sepsis strengthened the antagonistic actions of neostigmine on rocuronium-depressed twitch tension of the diaphragm by inhibiting the activity of ACh E at the neuromuscular junction.Oxidative stress might be responsible for the decreased activity of the ACh E in the diaphragm of rats induced with sepsis.The decreased ACh E activity also might be related to its reduced expression at the neuromuscular junction during sepsis.