| Objective: Spinal cord injury(SCI)is damage to the spinal cord tissue that may result in temporary or permanent changes in its neural function.This condition has severe consequences to patients,including muscle weakness,loss of sensation,and dysfunction of the bowel and bladder.With limited treatment strategies,SCI remains a severe medical problem.Glucagon-like peptide-1(GLP-1)secreted by intestinal L cells is shown to be an important incretin hormone in glucose homeostasis.Liraglutide,a long-acting GLP-1 analog,has been developed and explored to treat type 2 diabetes mellitus(T2DM),which also has been found to have high homosynaptic to natural GLP-1.Emerging evidence indicates that liraglutide,other than its basic function in decreasing glucose level,exhibits neurotrophic and neuroprotective effects in experimental models of multiple neurological diseases,such as Alzheimer’s disease,Parkinson’s disease.Macroautophagy,hereafter named as autophagy,is a homeostatic mechanism of cells by which the damaged organelles,toxic agents,unnecessary or dysfunctional components are targeted,disassembled and recycled.In addition,increasing evidence indicates that autophagy provides neuroprotective effects in plenty central nervous system.Based on these knowledges,the present study was designed to investigate the therapeutic potential of liraglutide and its effects on autophagy in spinal contusion injury,as well as the potential mechanisms.Our data may bring valuable insight and information to explore novel therapeutic target for SCI.Methods: Adult female Sprague–Dawley rats,220–240 g in weight,were selected obtained for the present study. The first part: This part aims to exam the neuroprotective effects of liraglutide and the level of autophagic responses.The animal model of SCI was induced through a modified weight-drop impactor in accordance with Allen’s method.A 10-g impactor,with diameter of 2 mm,was dropped from a 20 mm height,causing moderate spinal contusion injury with intact spinal dura.Animals were randomly divided into three groups: sham group,SCI group and SCI + liraglutide group.In liraglutide group,liraglutide(200μg/kg)was given immediately to the rodents via subcutaneous injection(s.c.)following SCI,and subsequently given once a day for the next two days.Animals in both the sham and SCI group were administrated with equal sterile PBS.To test the recovery of motor function after spinal contusion,the Basso,Beattie,and Bresnahan(BBB)scoring system was performed.HE staining was carried out to analysis the cavity size of spinal cord in each group.The number of ventral motor neuron was counted via Nissl staining.To identify and locate the autophagy markers in the damaged spinal tissue,we tested the expression of LC3 B,Beclin-1 and p62 via western blot and immunofluorescent staining.The second Part: This part aims to futher exam the potential signaling pathway of liraglutide in SCI.Animals were randomly divided into four groups: sham group,SCI group,SCI + liraglutide group and SCI + liraglutide + Compound C group.In liraglutide + Compound C group,the rodents were administrated with liraglutide(200μg/kg)subcutaneously and Compound C intraperitoneally(20mg/kg)immediately and once a day for the next two days after spinal contusion.To test the recovery of motor function after spinal contusion,the BBB scoring system was performed.HE staining was carried out to analysis the cavity size of spinal cord in each group.The number of ventral motor neuron was counted via Nissl staining.To identify and locate the autophagy markers in the damaged spinal tissue,we tested the expression of LC3 B,Beclin-1 and p62 via western blot and immunofluorescent staining.In addition,to investigate the potential mechanism,we detected the expression of AMPK,p-AMPK,FOXO3,and p-FOXO3 in spinal tissue via western blot to determine whether the AMPK-FOXO3 signaling pathway was activated in liraglutide-induced neuroprotection.Results: The first part: Our results showed that the rats treated with liraglutide scored significantly higher than those in the SCI group at 14,21,and 28 days post-operation.On the HE-stained segments,SCI group exhibited obvious damaged lesions of spinal cord tissue relative to that in the sham group.In comparison,the corresponding segments of liraglutide-treated group presented smaller tissue damages after contusion.With the comparison of the sham group,the rats in SCI group had significantly less motor neurons in the anterior horn.Notably,motor neurons were significantly preserved in liraglutidetreated rats compared with the rats in SCI group.Our results showed that the ratio of LC3BII/LC3B-I and the expression of Beclin-1 were higher in the SCI group compared with the sham group.Meanwhile,the application of liraglutide further enhanced the conversion of LC3B-I to LC3B-II,as well as the expression of Beclin-1.The expression of p62 was significantly suppressed following contusion compared with the sham group and liraglutide treatment further decreased the level of p62.The results of immunofluorescent staining were paralleled with WB. The second part: Our results futher confirmed that liraglutide facilitates motor function recovery after SCI;reduces the cavity of necrotic tissue and the loss of motor neurons;promotes autophagy and autophagic flux in neurons after spinal contusion injury.However,the beneficial effect of liraglutide on motor function recovery was attenuated by Compound C.The neuroprotective benefits of liraglutide,both in reducing necrotic area and preventing the loss of motor neurons,were blocked by Compound C.It is noteworthy that the combined application of liraglutide and Compound C remarkably neutralized the ratio of LC3B-II/LC3B-I and the expression of Beclin-1 compared with the liraglutidetreated group.Similar blocked effects were also detected in p62 via western blot.The results of immunofluorescent staining were also paralleled with WB.Compared with the sham group,the ratio of p-AMPK/AMPK,FOXO3,p-FOXO3 were notably aggrandized in injured spinal tissue of the SCI group.Meanwhile,liraglutide treatment significantly enhanced the up-regulation of the AMPK phosphorylation,as well as the expression of FOXO3,p-FOXO3.However,the application of Compound C markedly suppressed the ratio of p-AMPK/AMPK,and the expression of FOXO3 and p-FOXO3 compared with the liraglutide-treated group.Conclusion: The first part: Liraglutide exhibits neuroprotective effects after spinal contusion injury.Liraglutide facilitates motor function recovery;reduces the cavity of necrotic tissue and the loss of motor neurons;promotes autophagy and autophagic flux in neurons after spinal contusion injury. The second Part: Liraglutide treatment can benefit motor function recovery and decrease the destruction of spinal tissue and the loss of surviving neurons versus spinal contusion injury via enhancing autophagic responses through activating the AMPKFOXO3 signaling pathway. |
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