Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 µM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 µM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Dysregulated MiR-3150a-3p Promotes Lumbar Intervertebral Disc Degeneration by Targeting Aggrecan.

BACKGROUND/AIMS: Low back pain has become one of the most common musculoskeletal diseases in the world. Studies have shown that intervertebral disc degeneration (IDD) is an important factor leading to low back pain, but the mechanisms underlying IDD remain largely unknown. Research over the past decade has suggested critical roles for microRNAs (miRNAs) in natural growth and disease progression. However, it remains poorly understood whether circular RNAs participate in IDD. METHODS: Clinical IDD samples were collected from 20 patients who underwent discectomy. Weighted gene co-expression network analysis was used to identify the co-expression miRNA network modules (highly co-expressed clusters of miRNAs) that were associated with IDD grade. RESULTS: miR-3150a-3p was the most significantly up-regulated miRNA in module "Blue." Notably, aggrecan (ACAN) was identified as a direct target gene of miR-3150a-3p and ACAN expression was regulated by miR-3150a-3p. Overexpression of miR-3150a-3p decreased ACAN expression in nucleus pulposus cells, whereas inhibition of miR-3150a-3p increased ACAN expression. In addition, ACAN expression was negatively correlated with IDD grade. CONCLUSION: Our study suggests that the reduction of ACAN expression induced by the upregulation of miR-3150a-3p might participate in the development of IDD.

New Insights into the Complement Receptor of the Ig Superfamily Obtained from Structural and Functional Studies on Two Mutants.

The extracellular region of the complement receptor of the Ig superfamily (CRIg) binds to certain C3 cleavage products (C3b, iC3b, C3c) and inhibits the alternative pathway (AP) of complement. In this study, we provide further insight into the CRIg protein and describe two CRIg mutants that lack multiple lysine residues as a means of facilitating chemical modifications of the protein. Structural analyses confirmed preservation of the native CRIg architecture in both mutants. In contrast to earlier reports suggesting that CRIg binds to C3b with an affinity of ∼1 µM, we found that wild-type CRIg binds to C3b and iC3b with affinities <100 nM, but to C3c with an affinity closer to 1 µM. We observed this same trend for both lysine substitution mutants, albeit with an apparent ∼2- to 3-fold loss of affinity when compared with wild-type CRIg. Using flow cytometry, we confirmed binding to C3 fragment-opsonized Staphylococcus aureus cells by each mutant, again with an ∼2- to 3-fold decrease when compared with wild-type. Whereas wild-type CRIg inhibits AP-driven lysis of rabbit erythrocytes with an IC50 of 1.6 µM, we observed an ∼3-fold reduction in inhibition for both mutants. Interestingly, we found that amine-reactive crosslinking of the CRIg mutant containing only a single lysine results in a significant improvement in inhibitory potency across all concentrations examined when compared with the unmodified mutant, but in a manner sensitive to the length of the crosslinker. Collectively, our findings provide new insights into the CRIg protein and suggest an approach for engineering increasingly potent CRIg-based inhibitors of the AP.

Genome Sequence of a Novel Soil Actinomycete, Protaetiibacter sp. Strain SSC-01.

The family Microbacteriaceae represents a diverse and important group of soil bacteria in the phylum Actinobacteria Here, we report the genome sequence of a soil Microbacteriaceae strain, Protaetiibacter sp. strain SSC-01, the second putative species of the genus. Iron acquisition and xylose metabolism are central pathways identified in the annotated genome.

Cytokine expressions of spinal cord injury treated by neurotropin and nafamostat mesylate.

BACKGROUND: Spinal cord injury (SCI) leads to severe physical disability and sensory dysfunction. Neurotropin (NTP) has been used clinically to alleviate neuropathic pain, while nafamostat mesylate (NM) used clinical on pancreatitis patients through inhibiting synthetic serine protease. Our previous studies showed that NTP and NM were able to repair SCI. However, the underlying mechanism has not been fully explored after treatment with these 2 different drugs. METHODS: The drugs NTP and NM were administered on a contusion SCI Wistar rat model. Cytokine array analysis was performed to describe the changes of 67 proteins after acute SCI. Hierarchical clustering and volcano plot analysis were conducted to clarify protein change profiles. The differently expressed proteins related to biological processes were analyzed by functional protein association networks, Gene Ontology and pathway analysis. Flow cytometric analysis was detected to reflect the activation of immune system after drug intervention, while withdrawal threshold and BBB score were detected to evaluated the mechanical allodynia and functional recovery after SCI. RESULTS: HGF, ß-NGF, and activin were the 3 most upregulated proteins, while the receptor for RAGE, IL-1α, and TNF-α were the 3 most downregulated proteins after NTP treatment. Adiponectin, decorin and CTACK were the 3 most upregulated proteins, while RAGE, IL-1α, and IL-1ß were the 3 most downregulated proteins in the NM group. Number of lymphocytes was decreased while BBB score was increased both in NTP and NM group. But only NTP could improve mechanical pain threshold after SCI. CONCLUSIONS: The PI3K-Akt, Jak-STAT signaling pathway and apoptosis might participate in SCI restoration by NTP, while the MAPK and NOD-like receptor signaling pathway may participated in repairing SCI with NM. We concluded that NTP regulated the microenvironment via a neuroprotective effect and inhibition of inflammation to repair SCI, while NM healed SCI through an anti-inflammatory effect. Both NTP and NM could down-regulate the activation of immune system and improve the functional recovery while only NTP could improve the pathological neuralgia after SCI. Elucidating the molecular mechanisms of these 2 clinical drugs indicates that they their expected to be effective clinical treatment for SCI.

A novel, minimally invasive technique to establish the animal model of spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a traumatic disease that is associated with high morbidity, disability, and mortality worldwide. The animal spinal cord contusion model is similar to clinical SCI; therefore, this model is often used to study the pathophysiological changes and treatment strategies for humans after SCI. The present study aimed to introduce a novel, minimally invasive technique to establish an SCI model, and to evaluate its advantages compared with conventional methods. METHODS: Incision length, blood loss, length of time, and model success rate during the operation were recorded. Postoperative hematuria, incision hematoma, scoliosis [detected by micro computed tomography (Micro-CT)] and mortality were analyzed to evaluate surgical complications. The visual observation of the tissue was used to compare the effect of laminectomy by 2 methods on the scar hyperplasia at the injured site. Basso-Beattie-Bresnahan (BBB) score and catwalk automated quantitative gait analysis were conducted to measure behavioral function recovery. To evaluate the nerve function recovery of rats postoperatively, somatosensory evoked potential (SEP) and motor evoked potential (MEP) were studied by electrophysiological analyses. RESULTS: The results of operation-related parameters of the two models (conventional surgery group vs. minimally invasive surgery group) were as follows: surgical incision length: 23.58±1.58 versus 12.67±1.50 mm (P<0.05), blood loss: 3.96±1.05 versus 1.34±0.87 mL (P<0.05), and total operative time: 12.67±1.78 versus 10.33±1.92 min (P<0.05). In addition, the success rate of the 2 models was 100%. Surgical complications (conventional surgery group vs. minimally invasive surgery group) were as follows: hematuria: 25% versus 8.3%, kyphosis: 25% versus 0%, incision hematoma: 30% versus 9%, and mortality: 25% versus 8.3%. Micro-CT indicated severe scoliosis in the conventional surgery group. Gross tissue results showed that the conventional surgery group had more severe fibrous scar hyperplasia. The results of the BBB scores, catwalk automated quantitative gait analysis, and electrophysiology showed that the difference between the two groups was statistically significant in terms of behavioral recovery and neuroelectrophysiology. CONCLUSIONS: The minimally invasive technique has the advantages of small incision and reduced tissue damage and surgical complications, and may be used as an alternative spinal cord contusion method.

Identification of four genes and biological characteristics associated with acute spinal cord injury in rats integrated bioinformatics analysis.

BACKGROUND: Spinal cord injury (SCI) is a serious condition that can cause physical disability and sensory dysfunction. Cytokines play an extremely important role in the acute phase of SCI. Clarifying the cytokine expression profile is of great importance. METHODS: Cytokine array analysis was used to explore the changes in 67 different proteins at 0 hours, 2 hours, 1 day, 3 days, and 7 days after acute SCI in rats. The differentially expressed cytokines in the various periods were analyzed and compared. The biological processes related to the differentially expressed proteins were examined using Gene Ontology (GO) analysis. RESULTS: Immediately after SCI (0 hours), only ciliary neurotrophic factor (CNTF) was slightly up-regulated, while 23 other proteins were down-regulated. At 2 hours after SCI, there were 3 upregulated and 21 downregulated proteins. At 1 day after SCI, there were 5 upregulated and 6 downregulated proteins. At 3 days after SCI, there were 6 upregulated and 4 downregulated proteins. At 7 days after SCI, there were 4 upregulated and 9 downregulated proteins. Erythropoietin (EPO) and Fms related tyrosine kinase 3 ligand (Flt-3L) were downregulated at all time points. CD48 was decreased at 2 hours to 7 days after SCI. Monocyte chemotactic protein-1 (MCP-1) was the only protein that was upregulated at 2 hours to 7 days. The GO and pathway analyses revealed that the cytokine-related pathways, cell death, and proliferation might play a key role during secondary SCI. CONCLUSIONS: This study identified 3 downregulated proteins during SCI, that being EPO, Flt-3L, and CD48. MCP-1 was the only upregulated protein, and its expression was upregulated till day 7 following SCI. These 4 identified genes may be potential therapeutic targets for the treatment of SCI.

Neurotropin exerts neuroprotective effects after spinal cord injury by inhibiting apoptosis and modulating cytokines.

BACKGROUND/OBJECTIVE: Spinal cord injury (SCI) severely and irreversibly damages the central nervous system. Neurotropin (NTP), a nonprotein extract obtained from inflamed rabbit skin inoculated with vaccinia virus, is a drug that has been used for more than sixty years to alleviate neuropathic pain. It also reportedly exerts a neuroprotective role in peripheral nerves and in response to various central nervous system diseases, such as brain injury and Alzheimer disease. However, whether NTP promotes SCI recovery remains unknown. This study evaluated NTP's effects after SCI and explored its underlying mechanisms in a rat contusion model of SCI. METHOD: NTP was intraperitoneally administered to adult female Wistar rats subjected to contusion-induced SCI. Functional recovery was evaluated with behavioural scores and electrophysiological examinations. Tissue recovery was assessed with magnetic resonance imaging as well as histological staining with haematoxylin and eosin and Luxol Fast Blue. Neuronal survival and gliosis were observed after NeuN and glial fibrillary acidic protein immunofluorescence. Levels of apoptosis were demonstrated with TdT-mediated dUTP nick-end labeling (TUNEL) staining, Caspase-3 and B-cell lymphoma-2 (Bcl-2) Western blot, and Annexin V/propidium iodide flow cytometry. A protein antibody chip analysis was performed to evaluate the expression levels of 67 rat cytokines. RESULTS: NTP treatment improved the hindlimb locomotor recovery of the injured animals as well as their electrophysiological outcomes after SCI. A dosage of 50 NTP units/kg was found to optimize the efficacy of NTP. Magnetic resonance imaging revealed that lesion sizes decreased after NTP treatment. The haematoxylin and eosin and Luxol Fast Blue staining showed significant increases in the amount of spared tissue. The NeuN and glial fibrillary acidic protein immunofluorescence revealed that NTP treatment increased neuronal survival and reduced gliosis in tissue samples obtained from the lesion's epicentre. That NTP inhibited apoptosis was confirmed by the decreased number of TUNEL-positive cells, level of Caspase-3 expression, and number of Annexin V/propidium iodide-positive cells, as well as the increased level of Bcl-2 expression. The protein array analysis identified 28 differentially expressed proteins in the NTP group, and the gene ontology (GO) analysis showed that the enriched differentially expressed proteins implicate janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathways. The expression levels of proinflammatory cytokines such as interleukin 6, thymus chemokine-1(TCK-1), and lipopolysaccharide-induced CXC chemokine (LIX) decreased after NTP treatment, whereas the levels of prorepair cytokine hepatocyte growth factor and adiponectin increased. CONCLUSION: Our research provides evidence that NTP can improve functional outcomes and alleviate secondary injury after SCI by inhibiting apoptosis and modulating cytokines. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The multicomponent NTP might have broad target spectra in SCI pathophysiology and halt the secondary injury cascade. As a safe drug that features sixty years of clinical use as an analgesic, translating this demonstrated efficacy of NTP to addressing SCI in human patients may potentially be accelerated.

SARS-CoV-2 Exacerbates COVID-19 Pathology Through Activation of the Complement and Kinin Systems.

Infection with SARS-CoV-2 triggers the simultaneous activation of innate inflammatory pathways including the complement system and the kallikrein-kinin system (KKS) generating in the process potent vasoactive peptides that contribute to severe acute respiratory syndrome (SARS) and multi-organ failure. The genome of SARS-CoV-2 encodes four major structural proteins - the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. However, the role of these proteins in either binding to or activation of the complement system and/or the KKS is still incompletely understood. In these studies, we used: solid phase ELISA, hemolytic assay and surface plasmon resonance (SPR) techniques to examine if recombinant proteins corresponding to S1, N, M and E: (a) bind to C1q, gC1qR, FXII and high molecular weight kininogen (HK), and (b) activate complement and/or the KKS. Our data show that the viral proteins: (a) bind C1q and activate the classical pathway of complement, (b) bind FXII and HK, and activate the KKS in normal human plasma to generate bradykinin and (c) bind to gC1qR, the receptor for the globular heads of C1q (gC1q) which in turn could serve as a platform for the activation of both the complement system and KKS. Collectively, our data indicate that the SARS-CoV-2 viral particle can independently activate major innate inflammatory pathways for maximal damage and efficiency. Therefore, if efficient therapeutic modalities for the treatment of COVID-19 are to be designed, a strategy that includes blockade of the four major structural proteins may provide the best option.

Circular RNA derived from TIMP2 functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR­185­5p and matrix metalloproteinase 2.

Intervertebral disc degeneration (IDD) is an important cause of lower back pain, although the underlying mechanisms remain poorly understood. The present study aimed to examine the role of a circular RNA derived from tissue inhibitor of metallopeptidases 2 (circ­TIMP2) in degenerative nucleus pulposus (NP) tissues, and to validate its function in cultured human NP cells. Overexpression of miR­185­5p in NP cells markedly inhibited the enhanced extracellular matrix (ECM) catabolism induced by tumor necrosis factor­α (TNF­α) and interleukin­1ß (IL­1ß) treatment. Bioinformatics analysis demonstrated that matrix metalloproteinase 2 (MMP2) was a potential target of miR­185­5p. MMP2 protein expression levels were increased following treatment with TNF­α and IL­1ß in NP cells compared with those in untreated cells, and this effect was attenuated by transfection with miR­185­5p. Compared with normal NP tissues, IDD samples exhibited higher circ­TIMP2 expression levels. In addition, overexpression of circ­TIMP2 promoted ECM catabolism and suppressed ECM anabolism. Furthermore, circ­TIMP2 sequestered miR­185­5p, which may potentially upregulate the target genes associated with ECM degradation. In conclusion, the results of the present study revealed that circ­TIMP2 promoted TNF­α­ and IL­1ß­induced NP cell imbalance between ECM anabolism and catabolism via miR­185­5p­MMP2 signaling. These findings provide a potential therapeutic option for the treatment of IDD.

Epidemiological profile of thoracolumbar fracture (TLF) over a period of 10 years in Tianjin, China.

CONTEXT/OBJECTIVE: The objective of the research was to illustrate the epidemiology profile of thoracolumbar fracture (TLF) in Tianjin Medical University General Hospital, China, from 2006-2015. DESIGN: Hospital-based retrospective study. SETTING: Tianjin Medical University General Hospital. METHODS: Medical records of inpatient patients with TLF from 1 January 2006 to December 2015 were collected. Detailed information on epidemiological characters were analyzed based on the medical records suffering from TLF from T11-L2 level, including incidence, age and sex, marital, occupation, etiology and fracture type, types of injuries. RESULTS: Totally 132 cases were identified. The incidence rate was 2.4 patient per million population at 2015. Male-to-female ratio was 1.4:1, with a mean age of 49.1 ± 17.7 years. The cases number in 46-60 group, totally 35 and accounting for 26.5%, was the largest. There is a significant differences of cases number between 2011-2015 group and 2006-2010 group. Retiree, taken up 48.5%, was the largest group among TLF patients. The most common injury level was T12 (34) accounting for 25.7%. Falls (57, 43.2%) (low falls and high falls) were the leading causes, followed by motor vehicle collisions (MVCs) (23, 17.4%).Compression is the only type of osteoporosis and took up 55.3%. CONCLUSIONS: The incidence ratio is increased annually in TMUGH. Male was more vulnerable than female based on different social character. The average age was older in 2011-2015, retiree accounted for the main proportion and compression took up the largest percentage, the mean age increased and osteoporosis takes more in recent years.

Ferroptosis inhibitor SRS 16-86 attenuates ferroptosis and promotes functional recovery in contusion spinal cord injury.

Cell death is a key issue in spinal cord secondary injury. Ferroptosis is recently discovered as an iron-dependent type of cell death that is distinct from other forms of cell death pathways such as apoptosis and necrosis. This research is aimed to investigate the role of ferroptosis in spinal cord injury (SCI) pathophysiology, and to explore the effectiveness of ferroptosis inhibitor on SCI. We examined the ferroptosis markers and the factors in a rat contusion SCI model. Seen from transmission electron microscopy (TEM) following SCI, mitochondria showed ferroptotic characteristic changes. Treatment with a ferroptosis inhibitor SRS 16-86 enhanced functional recovery after SCI through the upregulation of anti-ferroptosis factor GPX4, GSH and xCT, and the downregulation of the lipid peroxidation marker 4HNE. SRS 16-86 treatment alleviated astrogliosis and enhanced neuronal survival after SCI. The inflammatory cytokine levels (IL-1ß, TNF-α and ICAM-1) were decreased significantly post SRS 16-86 treatment after SCI. These findings suggest strong correlation between ferroptosis and the secondary injury of SCI. The effectiveness of ferroptosis inhibitor SRS-16-86 on SCI repair leads to the identification of a novel therapeutic target for SCI.

Deferoxamine promotes recovery of traumatic spinal cord injury by inhibiting ferroptosis.

Ferroptosis is an iron-dependent novel cell death pathway. Deferoxamine, a ferroptosis inhibitor, has been reported to promote spinal cord injury repair. It has yet to be clarified whether ferroptosis inhibition represents the mechanism of action of Deferoxamine on spinal cord injury recovery. A rat model of Deferoxamine at thoracic 10 segment was established using a modified Allen's method. Ninety 8-week-old female Wistar rats were used. Rats in the Deferoxamine group were intraperitoneally injected with 100 mg/kg Deferoxamine 30 minutes before injury. Simultaneously, the Sham and Deferoxamine groups served as controls. Drug administration was conducted for 7 consecutive days. The results were as follows: (1) Electron microscopy revealed shrunken mitochondria in the spinal cord injury group. (2) The Basso, Beattie and Bresnahan locomotor rating score showed that recovery of the hindlimb was remarkably better in the Deferoxamine group than in the spinal cord injury group. (3) The iron concentration was lower in the Deferoxamine group than in the spinal cord injury group after injury. (4) Western blot assay revealed that, compared with the spinal cord injury group, GPX4, xCT, and glutathione expression was markedly increased in the Deferoxamine group. (5) Real-time polymerase chain reaction revealed that, compared with the Deferoxamine group, mRNA levels of ferroptosis-related genes Acyl-CoA synthetase family member 2 (ACSF2) and iron-responsive element-binding protein 2 (IREB2) were up-regulated in the Deferoxamine group. (6) Deferoxamine increased survival of neurons and inhibited gliosis. These findings confirm that Deferoxamine can repair spinal cord injury by inhibiting ferroptosis. Targeting ferroptosis is therefore a promising therapeutic approach for spinal cord injury.

Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk.

Intervertebral disc degeneration (IDD) is an important factor leading to low back pain, but the underlying mechanisms remain poorly understood. Compared with normal nucleus pulposus (NP) tissues, the expression of circ-GRB10 was downregulated in IDD. Furthermore, overexpression of circ-GRB10 inhibited NP cell apoptosis. circ-GRB10 could sequester miR-328-5p, which could potentially lead to the upregulation of target genes related to cell proliferation via the ErbB pathway. In conclusion, the present study revealed that circ-GRB10/miR-328-5p/ERBB2 signaling pathway is involved in IDD development, suggesting that circ-GRB10 might be a novel therapeutic target for IDD.

Nafamostat mesilate attenuates inflammation and apoptosis and promotes locomotor recovery after spinal cord injury.

AIM: Spinal cord injury (SCI) leads to severe neural damage for which there is currently no effective treatment. Exploration of the neuroprotective effect among clinically approved drugs will speed up clinical translation of SCI. Nafamostat mesilate (NM) as a synthetic serine protease inhibitor has been used clinically in pancreatitis treatments. However, its effectiveness in SCI is unknown. The aim of this study was to confirm the efficacy of NM in ameliorating SCI. METHODS: Intraperitoneal administration of NM was performed on a contusion SCI model in Wistar rat. Hematoxylin and eosin staining (H&E staining) and Luxol fast blue (LFB) staining were used to observe the histological lesions. Apoptosis was examined by TUNEL staining, Annexin V-FITC/PI, caspase-3, and Bcl-2. Cytokines and neurotrophins were tested by Western blot. Locomotion recovery assessed by hindlimb BBB score and the inclined plane test. RESULTS: Nafamostat mesilate treatment significantly improved locomotion recovery as assessed by hindlimb BBB scores and the inclined plane test. H&E staining and LFB staining showed a significant increase in spared tissue in both gray matter and white matter. NM decreased the expression of the proinflammatory cytokines TNF-α and IL-6. In addition, apoptosis was also significantly decreased, as shown by TUNEL staining and Annexin V-FITC/PI and by Western blotting for caspase-3 and Bcl-2 expression. Due to the mechanism of action of NM as a serine protease inhibitor, the drug decreased thrombin expression in the damaged spinal cord. Furthermore, NM increased the expression of neurotrophins (NT-3, BDNF, and NGF). CONCLUSIONS: Upon NM treatment, the functional and histological outcomes were improved, and microenvironment upon SCI was modulated. As a clinically approved drug, NM holds promise for clinical use after spinal cord injury.

Gene expression profile identifies potential biomarkers for human intervertebral disc degeneration.

The present study aimed to reveal the potential genes associated with the pathogenesis of intervertebral disc degeneration (IDD) by analyzing microarray data using bioinformatics. Gene expression profiles of two regions of the intervertebral disc were compared between patients with IDD and controls. GSE70362 containing two groups of gene expression profiles, 16 nucleus pulposus (NP) samples from patients with IDD and 8 from controls, and 16 annulus fibrosus (AF) samples from patients with IDD and 8 from controls, was downloaded from the Gene Expression Omnibus database. A total of 93 and 114 differentially expressed genes (DEGs) were identified in NP and AF samples, respectively, using a limma software package for the R programming environment. Gene Ontology (GO) function enrichment analysis was performed to identify the associated biological functions of DEGs in IDD, which indicated that the DEGs may be involved in various processes, including cell adhesion, biological adhesion and extracellular matrix organization. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated that the identified DEGs were potentially involved in focal adhesion and the p53 signaling pathway. Further analysis revealed that there were 35 common DEGs observed between the two regions (NP and AF), which may be further regulated by 6 clusters of microRNAs (miRNAs) retrieved with WebGestalt. The genes in the DEG­miRNA regulatory network were annotated using GO function and KEGG pathway enrichment analysis, among which extracellular matrix organization was the most significant disrupted biological process and focal adhesion was the most significant dysregulated pathway. In addition, the result of protein­protein interaction network modules demonstrated the involvement of inflammatory cytokine interferon signaling in IDD. These findings may not only advance the understanding of the pathogenesis of IDD, but also identify novel potential biomarkers for this disease.

[Neuroprotective effects and mechanism of saikosaponin A on acute spinal cord injury in rats].

Objective: To investigate the effect of saikosaponin a (SSa) on the levels of immune inflammation in rats with acute spinal cord injury and its possible mechanism. Methods: Seventy-two Sprague Dawley rats (weighing, 220-250 g) were randomly divided into sham operation group (group A), spinal cord injury group (group B), and SSa treatment group (group C) respectively, 24 rats in each group. The spinal cord injury model was induced by using the Allen's method in groups B and C; the spinous process and vertebral plate at both sides were cut off by lamina excision to expose the spinal cord in group A. The rats were given intraperitoneal injection of 10 mg/kg SSa in group C and equal volume of normal saline in group B at immediate after injury. The spinal cord tissue was harvested from 18 rats of each group at 24 hours after operation to measure the levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by ELISA, to detect the expressions of nuclear factor κB (NF-κB) P65, NF-κB P-P65, and aquaporin 4 (AQP4) by Western blot and to observe the morphology of spinal cord by HE staining. The motor function of the lower limbs was evaluated by BBB score and tiltboard experiment in 6 rats at 1, 3, 7, 14, 21, and 28 days after injury. Results: The BBB score and tiltboard experiment maximum angle were significantly higher in group A than groups B and C at each time point ( P<0.05) and in group C than group B at 14, 21, and 28 days after operation ( P<0.05). ELISA test showed that the concentrations of TNF-α and IL-6 were significantly lower in group A than groups B and C, and in group C than group B ( P<0.05). Western blot results showed that the protein expression levels of NF-κB P65, NF-κB P-P65, and AQP4 were significantly lower in group A than groups B and C, and in group C than group B ( P<0.05). HE staining demonstrated normal neurons of the spinal cord and no obvious lesion in group A; neuronal cells were observed in the injured area of group B, with hemorrhage, neutrophil infiltration, and nerve cell edema in the injured area; the neuronal cells were visible in the spinal cord of group C, with microglia mild hyperplasia, and the pathological changes were improved when compared with group B. Conclusion: SSa has neuroprotective effects on acute spinal cord injury in rats by inhibiting NF-κB signaling pathway and AQP4 protein expression and reducing inflammation response and edema.

Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway.

Myelin formation during peripheral nervous system development, as well as myelin repair after injury and in disease, requires multiple intrinsic and extrinsic signals. Neurotrophin-4 (NT-4) is a member of the neurotrophin family, which regulates the development of neuronal networks by participating in the growth of neuronal processes, synaptic development and plasticity, neuronal survival, and differentiation. However, the intracellular signaling pathways by which NT-4 participates in myelination by Schwann cells remain elusive. In this study, we examined the effects of NT-4 on the expression of compact myelin proteins in cultured Schwann cells. Using real-time quantitative RT-PCR and western blotting, we found that NT-4 could significantly enhance the expression of myelin protein zero (MPZ) but not the expression of myelin basic protein or peripheral myelin protein 22. Further, knockdown of truncated TrkB with small interfering RNA could eliminate the effect of NT-4 on MPZ expression. Moreover, we demonstrated that the NT-4-enhanced MPZ expression depended on Akt and mTORC1 signaling. Taken together, these results suggest that NT-4 binds TrkB to enhance the expression of MPZ in Schwann cells, probably through the PI3K/Akt/mTORC1 signaling pathway, thus contributing to myelination.

Mechanisms underlying the promotion of functional recovery by deferoxamine after spinal cord injury in rats.

Deferoxamine, a clinically safe drug used for treating iron overload, also repairs spinal cord injury although the mechanism for this action remains unknown. Here, we determined whether deferoxamine was therapeutic in a rat model of spinal cord injury and explored potential mechanisms for this effect. Spinal cord injury was induced by impacting the spinal cord at the thoracic T10 vertebra level. One group of injured rats received deferoxamine, a second injured group received saline, and a third group was sham operated. Both 2 days and 2 weeks after spinal cord injury, total iron ion levels and protein expression levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß and the pro-apoptotic protein caspase-3 in the spinal cords of the injured deferoxamine-treated rats were significantly lower than those in the injured saline-treated group. The percentage of the area positive for glial fibrillary acidic protein immunoreactivity and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were also significantly decreased both 2 days and 2 weeks post injury, while the number of NeuN-positive cells and the percentage of the area positive for the oligodendrocyte marker CNPase were increased in the injured deferoxamine-treated rats. At 14-56 days post injury, hind limb motor function in the deferoxamine-treated rats was superior to that in the saline-treated rats. These results suggest that deferoxamine decreases total iron ion, tumor necrosis factor-α, interleukin-1ß, and caspase-3 expression levels after spinal cord injury and inhibits apoptosis and glial scar formation to promote motor function recovery.