| Sepsis is a clinical disease caused by infection,which mainly characterized by tissue damage and systemic inflammation.It can lead to circulatory failure and multiple organ dysfunction,is one of the most common causes of death in patients in intensive care units.Muscle atrophy(the loss of muscle mass)is the main pathophysiological feature of septic myopathy,which seriously affects the function of respiratory system and peripheral motor system.Studies have shown that the increased level of protein degradation is the main cause of muscle atrophy in sepsis.Altuough autophagy lysosomal system is one of the pathways of protein degradation,the role of autophagy in septic skeletal muscle system remains a huge controversy.In this study,the sepsis model of SD rats and the inflammation model of L6 myotube cells were established to detect the expression of autophagy related proteins in tibialis anterior muscle and L6 myotube cells,and to observe the role of autophagy in septic muscular atrophy.The expression of autophagy related protein in tibialis anterior muscle and L6 myotube cells was detected by Western blot after treated by exogenous neuregulin-1(NRG-1),which was used to explore the effect of NRG-1 on autophagy.Finally,the possible molecular mechanism of NRG-1 regulating skeletal muscle autophagy in sepsis was explored.This study explored the effect of NRG-1 on septic skeletal muscle autophagy and its potential mechanism,and will provide new ideas and methods for the clinical treatment of septic muscular atrophy.Objective(1)To observe the level of autophagy in sepsis model of SD rats and the inflammation model of L6 myotube cells,and to explore the role of autophagy in sepsis muscle atrophy;(2)To explore the possible molecular mechanism of NRG-1 in regulating skeletal muscle autophagy;(3)To determine the effect of NRG-1 on skeletal muscle atrophy in sepsis through regulating autophagy by Akt/m TOR signaling pathway.Methods(1)L6 myoblasts were cultured and differentiated into myotubes using 2% fetal bovine serum.Sepsis model of SD rats was established by caecal ligation and punture.After 24 hours,septic serum was extracted from the inferior vena cava by centrifugation.L6 myotubes were treated with filtered septic serum for 72 hours to establish the cell inflammation model.The expression of differentiation marker molecules and inflammatory factors in L6 myoblasts were detected by RT-PCR.The morphological changes of cells were observed under microscope.(2)L6 myotubes were divided into 7 groups: control group(Con),sepsis group(Sep),neuregulin-1 5n M group(NRG-1 5n M),neuregulin-1 10 n M group(NRG-1 10 n M),neuregulin-1 15 n M group(NRG-1 15 n M),neuregulin-1 20 n M group(NRG-1 20 n M),and neuregulin-1 25 n M group(NRG-1 25 n M).The expression of p-Akt and Akt protein was detected by Western blot,and the optimal concentration of NRG-1 was determined.(3)L6 myotubes were divided into four groups: control group(Con),sepsis group(Sep),neuregulin-1 group(SN),inhibitor LY294002 group(SNLY).The optimal concentration of NRG-1 was added to the medium in the SN group,and the optimal concentration of NRG-1and inhibitor LY294002 were added to the medium in the SNLY group.CCK8 assay was used to detect the cell viability of each group.The expression of NRG-1 was detected by immunofluorescence confocal technique.The expressions of autophagy related proteins ULK-1,p-Beclin-1,Beclin-1,LC3 B,p62 and Akt /m TOR pathway were detected by Western blot.(4)Healthy adult male SPF clean grade(SD)rats(240-280 g)were randomly divided into three groups: Sham operation group(Sham)(n=3),sepsis group(Sep)(n=7)and neuregulin-1group(NRG-1)(n=7).In the sham operation group,only laparotomy was performed without cecal ligation and puncture.The sepsis group was ligated and punctured at middle of cecum of SD rats.The neuregulin-1 group was immediately injected with NRG-1 through tail vein once12 hours after operation.72 h after caecal ligation and puncture,the tibialis anterior muscle tissues of rats were extracted.The changes of body weight and survival of rats within 72 hours after operation were recorded.HE staining and microscope observation were performed to measure the diameter of tibialis anterior muscle.The expression of LC3 B in the tibialis anterior muscle was detected by immunohistochemistry.The protein expressions of NRG-1,autophagy related proteins ULK-1,p-Beclin-1,Beclin-1,LC3 B,p62 and Akt /m TOR pathway related proteins were detected by Western blot.Results(1)After 6 days of differentiation,L6 myoblasts turned into myotubes,in which the expression of Myo D gene was significantly decreased,and the expression of MRF4,Myo G,and Desmin genes were significantly increased.After differentiation,the viability of myotube cells,treated with septic serum,was decreased gradually.After 72 h,the relative expressions of IL-6 and TNF-α m RNA in L6 myotubes were increased significantly.(2)The expression of p-Akt was decreased in Sep group compared with Con group.Adding 10 n M NRG-1,the level of phosphorylation of Akt protein maximized.Therefor the optimal concentration of NRG-1 was 10 n M.(3)Compared with Con group,the expression of autophagy marker proteins ULK-1,p-Beclin-1,Beclin-1 and LC3 B II/I in myotubes in Sep group were increased,while the expression of p62 was decreased.Compared with Sep group,the autophagy level in SN group decreased,however the level of autophagy in SNLY group was higher than that in SN group.The expression of p-Akt and p-m TOR in Sep group was decreased than that in Con group and SN group.When the inhibitor LY294002 was added,expression of p-Akt and p-m TOR was decreased again.(4)After cecal ligation and puncture,the survival rate of Sep group and NRG-1 group was lower than that in Sham group,among that NRG-1 group was higher than Sep group.At the time of 48h、60h and 72 h after CLP,the weight of Sep group was decreased significantly,while NRG-1 group was significantly higher than that of Sep group at 60 h and 72 h after CLP.Compared with Sham group,the fiber diameter of anterior tibial muscle in Sep group and NRG-1 group was significantly decreased,however the diameter of fibers in NRG-1 group was higher than Sep group significantly.(5)Compared with Sham group,the expression levels of autophagy related marker proteins ULK-1,p-Beclin-1,Beclin-1 and LC3 B II/I ratio in tibial anterior muscle in Sep group were increased,while the expression of p62 and NRG-1 were significantly decreased.The protein levels of ULK-1,p-Beclin-1,Beclin-1 and LC3 B II/I in SN group were decreased compared with Sep group,while the protein levels of p62 and NRG-1 were increased.The expression of LC3 B protein detected by Immunohistochemical indicated that it was increased in the tibialis anterior muscle of Sep group compared with Sham group and NRG-1 group.(6)The expression of p-Akt and p-m TOR pathway-related proteins in Sep group was decreased compared with the Sham group and NRG-1 group.Conclusion(1)In the acute stage of sepsis,L6 myotubes and SD rats were induced muscle atrophy,and the level of autophagy in skeletal muscle was significantly increased.(2)NRG-1 can regulate skeletal muscle autophagy through Akt/m TOR signaling pathway.(3)NRG-1 alleviates sepsis-induced muscle atrophy by inhibiting the level of autophagy in skeletal muscle. |
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