The Role Of Autophagy In Sepsis-induced Neuromuscular Dysfunction Associated With Qualitative Changes To Acetylcholine Receptors

ObjectiveSepsis-induced neuromuscular dysfunction is a heavy burden for patients during respiratory failure as well as after discharge.Autophagy is one of the innate immune defense mechanisms against microbial challenges and has been proven to be a protective factor in heart,lung,liver,kidney,and immune system.However,no evidence showed the significance of autophagy in sepsis-induced neuromuscular dysfunction.Therefore,the aim of this study was to investigate the kinetics of autophagy in the septic skeletal muscle and to use pharmacologic agents to modulate autophagy to study its functional significance.Methods170 healthy adult male Sprague--Dawley(SD)rats were purchased.Experiment was divided into 3 parts,to explore the autophagy related proteins(LC3 II,p62),heterogenized receptors(α7-nAChR,γ-nAChR),and neuromuscular function(compound muscle action potential,CMAP)within 24 h of Cecal ligation and puncture(CLP)model;regulating autophagy by Rapamycin(Rapa)or trimethyladenine(3-MA)to study its significance on neuromuscular function and heterogenized receptors in sepsis acute phase;regulating autophagy by Rapamycin(Rapa)or trimethyladenine(3-MA)to study its significance on neuromuscular function and survival rates in sepsis chronic phase.The sepsis model wasestablished using cecal ligation and puncture(CLP),and the Sham model was established by Sham operations(laparotomy and suture).Biochemical,histological,functional and survival analyses were performed for each group at corresponding time points after modeling.Histological analysis of the lungs,spleen,small intestine was performed by HE staining;serum CRP was analyzed by ELISA;the interleukin-6(IL-6)of serum and muscle tissue was measured by cytometric bead array(CBA);blood and tibial anterior muscle samples were taken for bacterial culture and bacterial load was counted;using electromyography to measure the tibialis anterior muscle compound muscle action potential(CMAP),and to calculate the amplitude,latency period,duration,and nerve conduction velocity(NCV);Western blot was used to detect the expressions of LC3 II,P62,NRG-1,α7-nAChR and γ-nAChR in tibialis anterior muscle;the expression of α7-nAChR and γ-nAChR in tibialis anterior muscle was determined by immunofluorescence double-labeling;the survival curves of the Sham group and sepsis group were described by survival analysis method.ResultsIn the first part,the CMAP amplitude of tibial anterior muscle in CLP-24 h group was more than 20% lower than that of Sham group;tibial anterior muscle LC3 II,CLP groups were significantly higher than that of Sham group(P < 0.05);tibial anterior muscle p62,CLP-4h group was significantly lower than Sham group(P < 0.05),and CLP-16 h and CLP-24 h groups were significantly higher than Sham group(P < 0.05);The nrg-1,CLP-8h group was significantly higher than the Sham group(P < 0.05),and the CLP-24 h group was significantly lower than the Sham group(P <0.05);tibialis anterior muscle α7-nAChR and γ-nAChR,CLP-16 h and CLP-24 h groups were significantly higher than that of Sham group(P <0.05),and showed a continuous increasing trend.In the second part,serumbacterial load was significantly higher in the CLP group than in the Sham group(P < 0.05),and significantly lower in the CLP-Rapa group than in the CLP-DMSO group and CLP-3MA group(P < 0.05);Compared with the CLP-DMSO group,the latency,amplitude and nerve conduction velocity of the CLP-Rapa group were improved(P < 0.05),while the CLP-3MA group was significantly worse(P < 0.05);Compared with the CLPDMSO group,nrg-1 was significantly increased in the CLP-Rapa group,and α7-nAChR and γ-nAChR were significantly decreased(P < 0.05).In the third part,compared with the CLP-DMSO group,the 7-day survival rate was significantly increased in the CLP-Rapa group and significantly decreased in the CLP-3MA group(P < 0.05);compared with the CLPDMSO group,the latency,duration,amplitude and nerve conduction velocity of the CLP-Rapa group were improved in the tibial anterior muscle CMAP group(P < 0.05).ConclusionWithin 24 h of CLP modeling,autophagy decompensated,NRG-1expression decreased,and α7-nAChR and γ-nAChR increased continuously,accompanied by skeletal muscle dysfunction.After CLP modeling,rapamycin can enhance autophagy,and increase the expression of NRG-1through non-inflammatory pathways,reduce the expression of heterogeneous receptors,and improve neuromuscular function.The regulation of autophagy has an effect on the muscle function in the acute phase(24h)as well as in the chronic phase(7d)of sepsis,and can significantly increase the 7-day survival rate.