Delayed administration of nafamostat mesylate inhibits thrombin-mediated blood-spinal cord barrier breakdown during acute spinal cord injury in rats.

BACKGROUND: Nafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action. METHODS: Time to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood-spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence. RESULTS: The optimal administration time window of nafamostat was 2-12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2-12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site. CONCLUSIONS: Our study provides preclinical information of nafamostat about the administration time window of 2-12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.

Bone Marrow-Derived SH-SY5Y Neuroblastoma Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus Display Unique Infection Phenotypes and Growth Properties.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an important oncogenic virus previously shown to be neurotropic, but studies on neuronal cell infection and pathogenesis are still very limited. Here, we characterized the effects of KSHV infection on neuronal SH-SY5Y cells by the recombinant virus rKSHV.219, which expresses both green fluorescent protein (GFP) and red fluorescent protein (RFP) to reflect the latent and lytic phases of infection. We demonstrated that infected cells have a higher growth rate and that KSHV infection can be sustained. Interestingly, the infected cells can transition spontaneously back and forth between lytic and latent phases of infection, producing progeny viruses but without any adverse effects on cell growth. In addition, transcriptome analysis of viral and cellular genes in latent and lytic cells showed that unlike other infected cell lines, the latently infected cells expressed both latent and most, but not all, of the lytic genes required for infectious virion production. The viral genes uniquely expressed by the lytic cells were mainly involved in the early steps of virus binding. Some of the cellular genes that were deregulated in both latently and lytically infected cells are involved in cell adhesion, cell signal pathways, and tumorigenesis. The downregulated cellular CCDN1, PAX5, and NFASC and upregulated CTGF, BMP4, YAP1, LEF1, and HLA-DRB1 genes were found to be associated with cell adhesion molecules (CAMs), hippo signaling, and cancer. These deregulated genes may be involved in creating an environment that is unique in neuronal cells to sustain cell growth upon KSHV infection and not observed in other infected cell types. IMPORTANCE Our study has provided evidence that neuronal SH-SY5Y cells displayed unique cellular responses upon KSHV infection. Unlike other infected cells, this neuronal cell line displayed a higher growth rate upon infection and can spontaneously transition back and forth between latent and lytic phases of infection. Unlike other latently infected cells, a number of lytic genes were also expressed in the latent phase of infection in addition to the established latent viral genes. They may play a role in deregulating a number of host genes that are involved in cell signaling and tumorigenesis in order to sustain the infection and growth advantages for the cells. Our study has provided novel insights into KSHV infection of neuronal cells and a potential new model for further studies to explore the underlying mechanism in viral and host interactions for neuronal cells and the association of KSHV with neuronal diseases.

Signatures of altered long noncoding RNAs and messenger RNAs expression in the early acute phase of spinal cord injury.

Spinal cord injury (SCI) is a highly severe disease and it can lead to the destruction of the motor and sensory function resulting in temporary or permanent disability. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt that play a critical role in central nervous system (CNS) injury. However, the exact roles of lncRNAs and messenger RNAs (mRNAs) in the early acute phase of SCI remain to be elucidated. We examined the expression of mRNAs and lncRNAs in a rat model at 2 days after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. Subsequently, a comprehensive bioinformatics analysis was also performed to clarify the interaction between DE mRNAs. A total of 3,193 DE lncRNAs and 4,308 DE mRNAs were identified between the injured group and control group. Classification, length distribution, and chromosomal distribution of the dysregulated lncRNAs were also performed. The gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to identify the critical biological processes and pathways. A protein-protein interaction (PPI) network indicated that IL6, TOP2A, CDK1, POLE, CCNB1, TNF, CCNA2, CDC20, ITGAM, and MYC were the top 10 core genes. The subnetworks from the PPI network were identified to further elucidate the most significant functional modules of the DE mRNAs. These data may provide novel insights into the molecular mechanism of the early acute phase of SCI. The identification of lncRNAs and mRNAs may offer potential diagnostic and therapeutic targets for SCI.

MicroRNA-29a regulates neural stem cell neuronal differentiation by targeting PTEN.

Neural stem cells (NSCs) are self-renewing, pluripotent, and undifferentiated cells which have benefits as candidates for central nervous system (CNS) injury. However, the transplanted NSCs usually maintain their undifferentiated characteristics, or differentiated potentially along the glial lineage after transplantation. MicroRNAs (miRNAs) are small, non-coding RNAs that play key roles in cell differentiation. However, it is unknown whether miR-29a could play a role in the process of NSC's differentiation. Primary NSCs were derived from rat embryonic cortex. Lentiviral vector-mediated miR-29a (LV-miR-29a) and negative control (LV-null) were infected into NSCs. Quantitative real-time PCR was used to detect expression of miR-29a and PTEN. Immunocytochemistry was used to stain neurons, astrocytes, and oligodendrocytes. Dual luciferase assay to study the interaction between miR-29a and PTEN. The current study showed that the expression of miR-29a was upregulated during NSC differentiation, while the expression of PTEN was downregulated during NSC differentiation. After infection with LV-miR-29a, MAP-2-positive neurons significantly increased, and GFAP-positive astrocytes significantly decreased. Furthermore, we demonstrated that PTEN is a molecular target of miR-29a. miR-29a promote the neuronal differentiation and decrease the astrocytes differentiation of NSCs via targeting PTEN. This may give insight into a novel mechanism of NSC differentiation and provide a promising therapeutic target.

Identification and Verification of Candidate Genes Regulating Neural Stem Cells Behavior Under Hypoxia.

BACKGROUND/AIMS: Neural stem cells (NSCs) reside in a hypoxic environment, and hypoxia plays an important role in their development and differentiation. This study aimed to explore the underlying mechanisms by which hypoxia affects NSC behavior. METHODS: In the current study, we downloaded the gene expression dataset GSE68572 and identified the differentially expressed genes (DEGs) by analyzing high-throughput gene expression in hypoxic and normoxic NSCs. Subsequently, we analyzed these data using a combined bioinformatics approach and predicted the microRNAs (miRNAs) targeting the key gene using miRNA databases. Quantitative real-time PCR (qRT-PCR) was used to validate the expression of the top five DEGs. RESULTS: In total, 1347 genes were identified as DEGs. We identified the predominant gene ontology categories and Kyoto Encyclopedia of Genes and Genomes pathways that were significantly over-represented in the hypoxic NSCs. A protein-protein interaction network he identification of miRNAs and their putative targets may offer new diagnostic and therapeutic strategies for liver cancer the top 10 core genes. Vascular endothelial growth factor A (VEGFA) had the highest degree and may be the key gene concerning NSC behavior under hypoxia. Further validation of the top five DEGs by qRT-PCR demonstrated that four DEGs were significantly higher and one DEG was significantly lower in the hypoxic group than in the control group. Seven miRNAs were predicted and proved to target VEGFA. CONCLUSION: This preliminary study can prompt the understanding of the molecular mechanisms by which hypoxia has an impact on NSC behavior and can help to optimize stem cell therapies for central nervous system injuries and diseases.

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.

Lysine-specific demethylase 1 cooperates with BRAF-histone deacetylase complex 80 to enhance HIV-1 Tat-mediated transactivation.

Despite the notable success of combination antiretroviral therapy, how to eradicate latent HIV-1 from reservoirs poses a challenge. The Tat protein plays an indispensable role in HIV reactivation and histone demethylase LSD1 promotes Tat-mediated long terminal repeats (LTR) activation. However, the role of LSD1 in remodeling chromatin and the role of its component BHC80 in activation of latent HIV-1 in T cells are unknown. Our findings indicate that LSD1 could decrease the level of histone H3 lysine 4 trimethylation (H3K4me3) at the HIV-1 promoter by recruiting histone lysine demethylase 5A (KDM5A) and preventing histone methyltransferase Set1A and WD-40 repeat protein 5 (WDR5) from binding to LTR. Moreover, BHC80 is necessary for LSD1-triggered LTR activation and assists LSD1 in activating LTR by binding to nucleotides 305-631 of LTR. In activated J-Lat-A2 cells, BHC80 expression was elevated and its isoform BHC80-6 promoted the association of BHC80 with LSD1. These results suggest that the LSD1-BHC80 complex enhances HIV-1 transcription by a decrease of H3K4me3 level at the viral promoter. Therefore, it might be used as a new drug target to reactivate latent HIV-1.

Gene expression analysis at multiple time-points identifies key genes for nerve regeneration.

INTRODUCTION: The purpose of this study was to provide a comprehensive understanding of gene expression during Wallerian degeneration and axon regeneration after peripheral nerve injury. METHODS: A microarray was used to detect gene expression in the distal nerve 0, 3, 7, and 14 days after sciatic nerve crush. Bioinformatic analysis was used to predict function of the differentially expressed mRNAs. Microarray results and the key pathways were validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Differentially expressed mRNAs at different time-points (3, 7, and 14 days) after injury were identified and compared with a control group (0 day). Nine general trends of changes in gene expression were identified. Key signal pathways and 9 biological processes closely associated with nerve regeneration were identified and verified. CONCLUSIONS: Differentially expressed genes and biological processes and pathways associated with axonal regeneration may elucidate the molecular-biological mechanisms underlying peripheral nerve regeneration. Muscle Nerve 55: 373-383, 2017.

The roles of microRNAs in spinal cord injury.

BACKGROUND AND PURPOSE: Spinal cord injury (SCI) involves serious damage that can result in abnormal or absent motor and sensory functions and a disruption of autonomic function, and a series of pathological reactions occur after the injury. As a type of small non-coding RNA, microRNAs (miRNAs) have been verified to inhibit gene expression via post-transcriptional regulation. This review mainly focuses on recent advances regarding the roles of miRNAs following SCI. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were adopted. The studies regarding the roles of miRNAs following SCI were identified through PubMed, Embase and Web of Science. We summarise the changes in expression levels of miRNAs and discuss the roles of miRNAs after SCI. RESULTS: A total of 77 empirical studies meeting the inclusion criteria were identified. Existing studies showed that miRNAs were temporally altered and had effects on apoptosis, inflammation, angiogenesis, astrogliosis, oligodendrocyte development, axonal regeneration and remyelination after SCI. The alteration of miRNAs and the regulative action of pathological reactions can also provide opportunities for potential therapeutic interventions. "miRNA replacement therapy" aims to transfer miRNAs into diseased cells via delivery techniques and improve targeting effectiveness in cells, and this novel therapeutic tool provides a promising technique to promote the repair of SCI and reduces functional deficits. CONCLUSIONS: This review is helpful for understanding the underlying mechanisms of SCI and the potential clinical value of miRNAs. miRNAs have the potential to be attractive tools and targets for novel diagnostic and therapeutic approaches of SCI.

The 57th amino acid conveys the differential subcellular localization of human immunodeficiency virus-1 Tat derived from subtype B and C.

The multifunctional transactivator Tat protein is an essentially regulatory protein for HIV-1 replication and it plays a role in pathogenesis of HIV-1 infection. At present, numerous experimental studies about HIV-1 Tat focus on subtype B, very few has been under study of subtype C-Tat. In view of the amino acid variation of the clade-specific Tat proteins, we hypothesized that the amino acid difference contributed to differential function of Tat proteins. In the present study, we documented that subtype B NL4-3 Tat and subtype C isolate HIV1084i Tat from pediatric patient in Zambia exhibited distinct nuclear localization by over-expressing fusion protein Tat-EGFP. Interestingly, 1084i Tat showed uniform nuclear distribution, whereas NL4-3 Tat primarily localized in nucleolus. The 57th amino acid, highly conserved between B-Tat (arginine) and C-Tat (serine), is located in the basic domain of Tat, and played an important role in this subcellular localization. Meanwhile, we found that substitution of arginine to serine at the site 57 decreases Tat transactivation of the HIV-1 LTR promoter.

ERK2 small interfering RNAs prevent epidural fibrosis via the efficient inhibition of collagen expression and inflammation in laminectomy rats.

Laminectomy is a widely accepted treatment for lumbar disorders. Epidural Fibrosis (EF) is a common post-laminectomy or post-discectomy complication, which is thought to cause recurrent pain. RNA interference (RNAi) is a process by which double-stranded RNA triggers the destruction of mRNAs sharing the same sequence. Previously, extra-cellular signal-regulated kinase (ERK) 2 plays crucial roles in suppressing the collagen expression. To investigate the effects of lentiviral ERK2 siRNA on the prevention of post-laminectomy EF formation in a rat model, a controlled double-blinded study was conducted in 75 healthy adult Wistar rats that underwent laminectomy. They were divided randomly into 3 groups according to the treatment method: (1) control group; (2) ERK scrRNA group; (3) ERK siRNA group. All rats were euthanized humanely 4 weeks post-laminectomy. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions were performed. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions all suggested better results in ERK siRNA group than other two groups. None of the rats expired and no obvious adverse effects were observed. Local delivery of a lentiviral siRNA targeting ERK2 can prevent epidural scar adhesion in post-laminectomy rat via inhibiting collagen expression and inflammation.

Endoscopic ultrasound-guided fine-needle aspiration in the diagnostic value of focal liver lesions: A systematic analysis of 88 cases (with video).

BACKGROUND AND AIMS: Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) is an important diagnostic tool for suspected parenchymal lesions in the gastrointestinal tract and adjacent organs. Our study aimed to evaluate the safety and effectiveness of EUS-FNA in focal liver lesions (FLLs). METHOD: Data from 88 patients diagnosed with FLLs by imaging who underwent EUS-FNA from 1 January 2017 to 31 August 2022 were reviewed in our retrospective study at the Second Affiliated Hospital of Soochow University and Ruijin Hospital of the School of Medicine of Shanghai Jiao Tong University. The EUS-FNA biopsy results were compared with the final diagnosis to evaluate diagnostic value. The relevant factors were analysed to determine their influence on EUS-FNA biopsy results. RESULTS: The 88 patients analysed in this study resulted in a final diagnosis of 86 malignant and two benign cases. The overall diagnostic accuracy of EUS-FNA in FLLs was 93.18 % (82/88; 95 % Confidence Interval [CI], 87.9-98.5), with a sensitivity, specificity, positive predictive value, and negative predictive value of 93.02 % (80/86; 95 %CI, 87.6-98.4), 100 % (2/2; 95 %CI, 100-100), 100 % (80/80; 95 %CI, 100-100), and 25 % (2/8; 95 %CI, -5-55.0), respectively. The parameters related to lesion and procedure were not significantly different between these two groups (p > 0.05). The number of puncture needles in the groups showed a statistically significant difference between multiple and single punctures (p = 0.001). CONCLUSION: Our data revealed that EUS-FNA is a safe and reliable diagnostic method for FLLs that shows high accuracy.

New strategy to treat spinal cord injury: Nafamostat mesilate suppressed NLRP3-mediated pyroptosis during acute phase.

Spinal cord injury (SCI) is a devastating condition for which effective clinical treatment is currently lacking. During the acute phase of SCI, myriad pathological changes give rise to subsequent secondary injury. The results of our previous studies indicated that treating rats post-SCI with nafamostat mesilate (NM) protected the blood-spinal cord barrier (BSCB) and exerted an antiapoptotic effect. However, the optimal dosage for mice with SCI and the underlying mechanisms potentially contributing to recovery, especially during the acute phase of SCI, have not been determined. In this study, we first determined the optimal dosage of NM for mice post-SCI (5 mg/kg/day). Subsequently, our RNA-seq findings revealed that NM has the potential to inhibit pyroptosis after SCI. These findings were further substantiated by subsequent Western blot (WB) and Immunofluorescence (IF) analyses in vivo. These results indicate that NM can alleviate NLRP3 (NOD-like receptor thermal protein domain associated protein 3)-mediated pyroptosis by modulating the NF-κB signaling pathway and reducing the protein expression levels of NIMA-related kinase 7 (NEK7) and cathepsin B (CTSB). In vitro experimental results supported our in vivo findings, revealing the effectiveness of NM in suppressing pyroptosis induced by adenosine triphosphate (ATP) and lipopolysaccharide (LPS) in BV2 cells. These results underscore the potential of NM to regulate NLRP3-mediated pyroptosis following SCI. Notably, compared with other synthetic compounds, NM exhibits greater versatility, suggesting that it is a promising clinical treatment option for SCI.

Celastrol inhibits oligodendrocyte and neuron ferroptosis to promote spinal cord injury recovery.

BACKGROUND: Spinal cord injury (SCI) is a traumatic injury to the central nervous system and can cause lipid peroxidation in the spinal cord. Ferroptosis, an iron-dependent programmed cell death, plays a key role in the pathophysiology progression of SCI. Celastrol, a widely used antioxidant drug, has potential therapeutic value for nervous system. PURPOSE: To investigate whether celastrol can be a reliable candidate for ferroptosis inhibitor and the molecular mechanism of celastrol in repairing SCI by inhibiting ferroptosis. METHODS: First, a rat SCI model was constructed, and the recovery of motor function was observed after treatment with celastrol. The regulatory effect of celastrol on ferroptosis pathway Nrf2-xCT-GPX4 was detected by Western blot and immunofluorescence. Finally, the ferroptosis model of neurons and oligodendrocytes was constructed in vitro to further verify the mechanism of inhibiting ferroptosis by celastrol. RESULTS: Our results demonstrated that celastrol promoted the recovery of spinal cord tissue and motor function in SCI rats. Further in vitro and in vivo studies showed that celastrol significantly inhibited ferroptosis in neurons and oligodendrocytes and reduced the accumulation of ROS. Finally, we found that celastrol could inhibit ferroptosis by up-regulating the Nrf2-xCT-GPX4 axis to repair SCI. CONCLUSION: Celastrol effectively inhibits ferroptosis after SCI by upregulating the Nrf2-xCT-GPX4 axis, reducing the production of lipid ROS, protecting the survival of neurons and oligodendrocytes, and improving the functional recovery.

Nicotinamide Riboside Promotes the Proliferation of Endogenous Neural Stem Cells to Repair Spinal Cord Injury.

Activation of endogenous neural stem cells (NSC) is one of the most potential measures for neural repair after spinal cord injury. However, methods for regulating neural stem cell behavior are still limited. Here, we investigated the effects of nicotinamide riboside promoting the proliferation of endogenous neural stem cells to repair spinal cord injury. Nicotinamide riboside promotes the proliferation of endogenous neural stem cells and regulates their differentiation into neurons. In addition, nicotinamide riboside significantly restored lower limb motor dysfunction caused by spinal cord injury. Nicotinamide riboside plays its role in promoting the proliferation of neural stem cells by activating the Wnt signaling pathway through the LGR5 gene. Knockdown of the LGR5 gene by lentivirus eliminates the effect of nicotinamide riboside on the proliferation of endogenous neural stem cells. In addition, administration of Wnt pathway inhibitors also eliminated the proliferative effect of nicotinamide riboside. Collectively, these findings demonstrate that nicotinamide promotes the proliferation of neural stem cells by targeting the LGR5 gene to activate the Wnt pathway, which provides a new way to repair spinal cord injury.

ROS-mediated lysosomal membrane permeabilization and autophagy inhibition regulate bleomycin-induced cellular senescence.

Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects.Abbreviations: AT2 cells: type II alveolar epithelial cells; ATG7: autophagy related 7; bEnd.3: mouse brain microvascular endothelial cells; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCL2: C-C motif chemokine ligand 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; FTH1: ferritin heavy polypeptide 1; γ-H2AX: phosphorylated H2A.X variant histone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cells; HT22: hippocampal neuronal cell lines; Il: interleukin; LAMP: lysosomal-associated membrane protein; LMP: lysosome membrane permeabilization; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NCOA4: nuclear receptor coactivator 4; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; RPS6KB/S6K: ribosomal protein S6 kinase; SA-GLB1/ß-gal: senescence-associated galactosidase, beta 1; SAHF: senescence-associated heterochromatic foci; SASP: senescence-associated secretory phenotype; SEC62: SEC62 homolog, preprotein translocation; SEP: superecliptic pHluorin; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.

Targeted Repair of Spinal Cord Injury Based on miRNA-124-3p-Loaded Mesoporous Silica Camouflaged by Stem Cell Membrane Modified with Rabies Virus Glycoprotein.

Spinal cord injury (SCI) has no effective treatment modalities. It faces a significant global therapeutical challenge, given its features of poor axon regeneration, progressive local inflammation, and inefficient systemic drug delivery due to the blood-spinal cord barrier (BSCB). To address these challenges, a new nano complex that achieves targeted drug delivery to the damaged spinal cord is proposed, which contains a mesoporous silica nanoparticle core loaded with microRNA and a cloaking layer of human umbilical cord mesenchymal stem cell membrane modified with rabies virus glycoprotein (RVG). The nano complex more readily crosses the damaged BSCB with its exosome-resembling properties, including appropriate size and a low-immunogenic cell membrane disguise and accumulates in the injury center because of RVG, where it releases abundant microRNAs to elicit axon sprouting and rehabilitate the inflammatory microenvironment. Culturing with nano complexes promotes axonal growth in neurons and M2 polarization in microglia. Furthermore, it showed that SCI mice treated with this nano complex by tail vein injection display significant improvement in axon regrowth, microenvironment regulation, and functional restoration. The efficacy and biocompatibility of the targeted delivery of microRNA by nano complexes demonstrate their immense potential as a noninvasive treatment for SCI.

Exploration of the effect and potential mechanism of quercetin in repairing spinal cord injury based on network pharmacology and in vivo experimental verification.

Spinal cord injury (SCI) is a highly complex neurological disease, but there is no effective repair method. Quercetin is a flavonol drug and has a variety of biological activities, such as scavenging oxygen free radicals in the body to resist oxidation, inhibiting inflammation, and so on. In this study, quercetin was firstly demonstrated to reduce tissue damage, promote neuron survival and repair motor function after SCI in rats through in vivo experiments. Then, 293 potential targets of quercetin repair for SCI were predicted by network pharmacology. GO analysis revealed that the biological processes of potential targets focused mainly on signal transduction, negative regulation of the apoptotic process, protein phosphorylation, drug response, and so on. Similarly, KEGG analysis suggested that these potential targets were involved in cell growth regulation, differentiation, apoptosis, and a few metabolic pathways. PPI network analysis predicted that the key genes were EP300, CREBBP, SRC, HSP90AA1, TP53, PIK3R1, EGFR, ESR1, and CBL. Further, the molecular docking showed that quercetin binds well with these proteins. Finally, RT-qPCR and Western blotting experiments verified that quercetin downregulated the expression levels of PIK3R1 and EGFR. It is suggested that quercetin can repair SCI in rats through PI3K-AKT signaling pathway and EGFR/MAPK pathway, which may provide a new theoretical basis for the repair of spinal cord injury.

Spastin is required for human immunodeficiency virus-1 efficient replication through cooperation with the endosomal sorting complex required for transport (ESCRT) protein.

Human immunodeficiency virus-1 (HIV-1) encodes simply 15 proteins and thus depends on multiple host cellular factors for virus reproduction. Spastin, a microtubule severing protein, is an identified HIV-1 dependency factor, but the mechanism regulating HIV-1 is unclear. Here, the study showed that knockdown of spastin inhibited the production of the intracellular HIV-1 Gag protein and new virions through enhancing Gag lysosomal degradation. Further investigation showed that increased sodium tolerance 1 (IST1), the subunit of endosomal sorting complex required for transport (ESCRT), could interact with the MIT domain of spastin to regulate the intracellular Gag production. In summary, spastin is required for HIV-1 replication, while spastin-IST1 interaction facilitates virus production by regulating HIV-1 Gag intracellular trafficking and degradation. Spastin may serve as new target for HIV-1 prophylactic and therapy.

CREB1 Facilitates GABAergic Neural Differentiation of Human Mesenchymal Stem Cells through BRN2 for Pain Alleviation and Locomotion Recovery after Spinal Cord Injury.

The transplantation of GABAergic neuron cells has been reported to alleviate nerve pain and improve motor function after spinal cord injury (SCI). However, human mesenchymal stem cell (hMSC) differentiation into GABAergic neuron cells in a sufficient quantity remains to be accomplished. From a database screening, cAMP-responsive element-binding protein 1 (CREB1) was chosen as a potential modulator due to its critical role in the protein-protein interaction of genes related to GABAergic neural differentiation. Here, CREB1 was overexpressed in transfected hMSCs, where CREB1 could induce differentiation into GABAergic neuron cells with an upregulation of Map2 and GAD1 by 2- and 3.4-fold, respectively. Additionally, GABAergic neural differentiation was enhanced, while Notch signaling was inhibited, and BRN2 transcriptional activation played an important role in neuronal maturation. Moreover, transfected hMSCs injected into immunocompromised mice caused by CsA exhibited the neuronal markers Tuj1 and Map2 via the intraspinal route, suggesting an improvement in survival and neural differentiation. Significantly, improvement in both BMS scores (6.2 ± 1.30 vs. 4 ± 0) and thermal hyperalgesia latency (7.74 ± 2.36 s vs. 4.52 ± 0.39 s) was seen compared with the SCI naïve treatment at 4 weeks post-transplantation. Our study demonstrates that CREB1 is crucial in generating induced GABAergic neuron cells (iGNs) originating from hMSCs. Transplanting iGNs to injured spinal cord provides a promising strategy for alleviating neuropathic pain and locomotion recovery after SCI.