Ubiquitin ligase Triad1 promotes neurite outgrowth by inhibiting MDM2-mediated ubiquitination of the neuroprotective factor pleiotrophin.

Spinal cord injury (SCI) is the most severe result of spine injury, but no effective therapy exists to treat SCI. We have previously shown that the E3 ubiquitin ligase Two RING fingers and DRIL 1 (Triad1) promotes neurite outgrowth after SCI. However, the mechanism by which Triad1 affects neuron growth and the potential involvement of its ubiquitination activity is unclear. Neuroprotective cytokine pleiotrophin (PTN) can promote microglia proliferation and neurotrophic factor secretion to achieve neuroprotection. We find using immunostaining and behavioral assays in rats that the expression of Triad1 and the PTN was peaked at 1 day after SCI and Triad1 improved motor function and histomorphological injury after SCI. We show using flow cytometry and astrocyte/neuronal coculture assays that Triad1 overexpression promoted PTN protein levels, neurotrophic growth factor (NGF) expression, brain-derived neurotrophic factor (BDNF) expression, astrocyte and neuronal viability, and neurite outgrowth but suppressed astrocyte apoptosis, while shRNA-mediated knockdown of Triad1 and PTN had the opposite effects. Ubiquitin ligase murine double mutant 2 (MDM2) has previously been demonstrated to participate in the process of neurite outgrowth and mediate ubiquitination of p53. Furthermore, we demonstrate overexpression of MDM2 downregulated PTN protein levels, NGF expression and BDNF expression in astrocytes, and inhibited neurite outgrowth of neurons. In addition, MDM2 facilitated PTN ubiquitination, which was reversed by Triad1. Finally, we show simultaneous sh-PTN and MDM2 overexpression attenuated the neurite outgrowth-promoting effect of Triad1 overexpression. In conclusion, we propose Triad1 promotes astrocyte-dependent neurite outgrowth to accelerate recovery after SCI by inhibiting MDM2-mediated PTN ubiquitination.

Effect of Different Accumulative Temperate Zones in Heilongjiang on Glycine Soja Metabolites as Analyzed by Non-Target Metabolomics.

To study the effect of growth temperature on the nutritional components and metabolites of the wild soybean (Glycine soja), we analyzed the nutritional components and metabolic gases of the wild soybean in six accumulated temperature regions of the Heilongjiang Province, China, by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). A total of 430 metabolites, including organic acids, organic oxides, and lipids, were identified and analyzed using multivariate statistical analysis, orthogonal partial least squares discriminant analysis, principal component analysis, and cluster analysis. Eighty-seven metabolites significantly differed in the sixth accumulated temperature region compared with the other five accumulated temperature regions. The 40 metabolites (such as threonine (Thr) and lysine (Lys)) were found to be elevated in soybeans from the sixth accumulated temperature zone compared with the other five accumulated temperature zones. Through analyzing the metabolic pathways of these metabolites, amino acid metabolism had the greatest influence on wild soybean quality. The results of the amino acid analysis were consistent with those of the GC-TOF-MS and showed that amino acids in wild soybeans from the sixth accumulated temperature zone significantly differed from those of the other zones. Threonine and lysine were the main substances driving these differences. The growth temperature affected the type and concentrations of metabolites in wild soybeans, and the GC-TOF-MS analysis of the effect of growth temperature on wild soybean metabolites was shown to be feasible.

Upregulation of P2Y12 inhibits chondrocyte apoptosis in lumbar osteoarthritis through the PI3K/AKT signaling pathway.

Lumbar facet osteoarthritis (FJOA) is a major cause of severe lower back pain and disability worldwide. However, the mechanism underlying cartilage degeneration in FJOA remains unclear. The purpose of this study was to investigate the regulation and mechanism of P2Y12 on chondrocyte apoptosis in FJOA. The experimental rats were randomly divided into non-operation (n = 20) and operation groups (n = 20). In the operation group, Sodium iodoacetate (MIA, Sigma, 200 mg/mL) was injected into the right L4/5 facet process using a blunt nanoneedle 26 (WPI, Sarasota, FL, USA) under the control of an injection pump. The final injection volume was 5µL and the injection rate was 2µL/min. The facet joint was removed four weeks after surgery. After the operation, samples were stored at -80 °C until further use, whereby the right facet joints in each group were tested. Hematoxylin and eosin (HE) and iron-red solid green staining were used to observe the degeneration of articular chondrocytes in rats. Immunohistochemistry and western blotting were used to observe the expressions of P2Y12, Matrix metalloproteinase 13 (MMP13), Collagen II (COL2), and other cartilage degeneration and apoptosis-related genes. Co-localization of P2Y12-cleaved caspase-3 in the apoptosis model was detected by dual-standard immunofluorescence staining. Apoptosis was also detected by flow cytometry and TUNEL assay.P2Y12 is highly expressed in OA cartilage tissue, and inhibits IL-1ß -induced chondrocyte apoptosis through PI3K/AKT signaling pathway, thus playing a certain protective role on cartilage.

Knockdown of TRAF6 inhibits chondrocytes apoptosis and inflammation by suppressing the NF-κB pathway in lumbar facet joint osteoarthritis.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a regulator of NF-κB signaling, has been discovered recently to be probably related to osteoarthritis, while the function of TRAF6 in lumbar facet joint osteoarthritis(FJOA)still remains unknown. The aim of this study was to probe the specific function of TRAF6 in chondrocytes and its connection with the pathophysiology of FJOA. We found upregulation of TRAF6 in FJOA cartilage by western blot analysis. In vitro, we stimulated immortalized human chondrocytes by LPS to establish the cells apoptosis model. Western blot analysis demonstrated that levels of TRAF6 and cleaved caspase-3/8 in the chondrocyte injury model increased significantly. Knockdown of TRAF6 suppressed the expression of matrix metallopeptidase-13 (MMP-13) and interleukin-6 (IL-6) induced by LPS, and alleviated cell apoptosis. Meanwhile, western blot and immunofluorescent staining demonstrated that IκBα degradation and p65 nuclear transportation were also inhibited, revealing that knockdown of TRAF6 suppressed activation of the NF-κB pathway in LPS-induced chondrocytes apoptosis model. Collectively, our findings suggest that TRAF6 plays a crucial role in FJOA development by regulating NF-κB signaling pathway. Knockdown of TRAF6 may supply a potential therapeutic strategy for FJOA.

Wnt16 protects chondrocytes from lumbar facet joint osteoarthritis through the Wnt/ß-catenin pathway in low back pain patients.

PURPOSE: Low back pain (LBP) is a long-lasting and chronic symptom without any exact cause. This study attempts to propose a new staging system based on the original grading system combined with pathological results and clinical symptoms to better clarify the dynamic evolution of LBP related to cartilage degeneration during facet joint osteoarthritis (FJOA). To explore a potential target for diagnosis, treatment, and drug intervention of facet joint osteoarthritis related LBP via protecting chondrocytes. MATERIALS AND METHODS: All the facet joints were divided into 4 groups according to our new degenerative staging system based on Weishaupt grade, CT and MRI. Collect the facet joint samples from patients whom suffered lumbar fusion surgery for lumbar disc herniation. Molecular biology experiments were used to explore the effect of Wnt16 on the degeneration of facet joints. Micro-CT examination and pain stimulation test checked the biological function of Wnt16 in rats. RESULTS: Wnt16 was significantly increased and more aggregated in the facet joint chondrocytes in the Phase III and Phase IV, which is consistent with the pathological findings of cartilage degeneration (OARSI). We found that Wnt16 participated in the regulation of FJOA via Wnt/ß-catenin pathway in vitro, which was inhibited by specific inhibitor DKK1. The rats, rich expressed Wnt16, showed higher paw withdrawal thresholds and prolonged paw withdrawal latency to FJOA related LBP. Micro-CT examination for the lumbar spine of rats showed Wnt16 protected the chondrocytes from FJOA. CONCLUSIONS: This study defined a new staging system for LBP related cartilage degeneration of facet joint based on the original grading system combined with pathological results and clinical symptoms. Wnt16 is expected to be a potential target for treatment of FJOA via protecting chondrocytes.

Novel Role of HAX-1 in Neurons Protection After Spinal Cord Injury Involvement of IRE-1.

Spinal cord injury (SCI) is one of the diseases with high probability of causing disability in human beings, and there is no reliable treatment at present. Neuronal apoptosis is a vital component of secondary injury and plays a critical role in the development of neurological dysfunction after spinal cord injury. In this study, we found that the expression and distribution of HAX-1 in neurons increased 1 day after SCI. PC12 cells overexpressing HAX-1 showed decreased apoptosis and PC12 cells are more likely to undergo apoptosis after down-regulating HAX-1, which was confirmed via TUNEL experiments. We found GRP94 showed the same trend as HAX-1 in expression and interacted with HAX-1 and IRE-1 in both spinal cord tissue and PC12 cells, and this interaction seems to be enhanced after SCI. When the expression of HAX-1 was up-regulated, GRP94 also increased, but IRE-1 did not change at all. Further studies showed that overexpression of HAX-1 decreased the expression of pIRE-1, rather than IRE-1, and downstream proteins of the IRE signaling pathway (Caspase12, pJNK and CHOP) were significantly reduced, and vice versa. In animals treated with HAX-1 expressing adenovirus there are more neuronal cells remaining in the damaged spinal cord tissue, and hindlimb motor function of rats was significantly improved. So, we speculate that HAX-1 might play a role in protecting neurons from apoptosis after SCI by regulating the IRE-1 signaling pathway via promoting the dissociation of GRP94 from IRE-1. This may provide a theoretical basis and a potential therapeutic target for clinical improvement of neural function recovery after SCI.

Chest computed tomography findings of coronavirus disease 2019 (COVID-19) pneumonia.

OBJECTIVE: To retrospectively analyze the chest computed tomography (CT) features in patients with coronavirus disease 2019 (COVID-19) pneumonia. METHODS: From January 9, 2020, to February 26, 2020, totally 56 laboratory-confirmed patients with COVID-19 underwent chest CT. For 40 patients, follow-up CT scans were obtained. The CT images were evaluated for the number, type and distribution of the opacity, and the affected lung lobes. Furthermore, the initial CT scan and the follow-up CT scans were compared. RESULTS: Forty patients (83.6%) had two or more opacities in the lung. Eighteen (32.7%) patients had only ground-glass opacities; twenty-nine patients (52.7%) had ground-glass and consolidative opacities; and eight patients (14.5%) had only consolidation. A total of 43 patients (78.2%) showed two or more lobes involved. The opacities tended to be both in peripheral and central (30/55, 54.5%) or purely peripheral distribution (25/55, 45.5%). Fifty patients (90.9%) had the lower lobe involved. The first follow-up CT scans showed that twelve patients (30%) had improvement, 26 (65%) patients had mild-moderate progression, and two patients (5%) had severe progression with "white lungs." The second follow-up CT showed that 22 patients (71%) showed improvement compared with the first follow-up CT, four patients (12.9%) had aggravated progression, and five patients (16.1%) showed unchanged radiographic appearance. CONCLUSIONS: The common CT features of COVID-19 pneumonia are multiple lung opacities, multiple types of the opacity (ground-glass, ground-glass and consolidation, and consolidation alone), and multiple lobes especially the lower lobe involved. Follow-up CT could demonstrate the rapid progression of COVID-19 pneumonia (either in aggravation or absorption). KEY POINTS: • The predominant CT features of COVID-19 pneumonia are multiple ground-glass opacities with or without consolidation and, with both lungs, multiple lobes and especially the lower lobe affected. • CT plays a crucial role in early diagnosis and assessment of COVID-19 pneumonia progression. • CT findings of COVID-19 pneumonia may not be consistent with the clinical symptoms or the initial RT-PCR test results.

Triad1 regulates the expression and distribution of EHD1 contributing to the neurite outgrowth of neurons after spinal cord injury.

Traumatic spinal cord injury is a common and severe complication after an accident. As we all know that neurite outgrowth of neurons is difficult after a spinal cord injury. Endosome system is associated with cargoes transportation and contributes in promoting the neuronal capability for neurite outgrowth. EH domain-containing protein 1 (EHD1) transports proteins through the endosome system, especially in the recycling endosomes and regulating the neurite outgrowth. In mammalian cells, the involvement of the ubiquitin-proteasome system in endosomal sorting has been well established. Two RING fingers and a DRIL (double RING finger-linked) 1 (Triad1) plays an important role in membrane trafficking and its mutant results in the wrong accumulation of receptors in endosomes and plasma membrane. In this current study, we reasonably integrated the results of the above research and investigated the regulating function of Triad1 to EHD1 following the spinal cord injury. We characterized the upregulated expression and distribution of Triad1 and EHD1 in the neurons after SCI and declared the interaction between Triad1 with EHD1 both in vitro and in vivo. Triad1 regulated the interaction between itself and the full-length or EH domain of EHD1, which influenced the neurite outgrowth of PC12 cells. Our data delineate a novel interaction between Triad1 and EHD1 that may contribute to the regulation of neurite outgrowth for neurons after the spinal cord injury.

Up-regulation of TRAF2 inhibits chondrocytes apoptosis in lumbar facet joint osteoarthritis.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) has been demonstrated that it plays a significant role in cell death receptor signal transduction. The purpose of this study was to investigate the expression of TRAF2 and its possible role in FJOA. We observed an up-regulation of TRAF2 in FJOA by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) compared to normal tissues. In vitro, we used TNF-α to stimulate Human SW1353 chondrosarcoma cells to establish the chondrocytes injury model. Western blot analysis revealed significant expression of TRAF2 and cleaved caspase-3/8 in SW1353 cells. Co-localization of TRAF2/cleaved caspase-3/8 was detected in the cells injury model by double-labeling immunofluorescent staining. We demonstrated a possible anti-apoptotic effect of TRAF2 in chondrocyte apoptosis in FJOA by knockdown of its expression with siRNA. Moreover, TRAF2 knockdown was demonstrated to enhance TNF-α-induced apoptosis by flow cytometry assay. In conclusion, our results show that the up-regulation of TRAF2 may play an important role in the inhibition of chondrocyte apoptosis of FJOA.

E3 Ubiquitin Ligase c-cbl Inhibits Microglia Activation After Chronic Constriction Injury.

E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1ß and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.

Altered Wnt and NF-κB Signaling in Facet Joint Osteoarthritis: Insights from RNA Deep Sequencing.

Facet joint osteoarthritis is common lumbar osteoarthritis characterized by facet joint cartilage degeneration. However, the molecular basis of facet joint osteoarthritis remains largely undetermined. In the current study, we collected facet joint tissue samples from 10 control patients and 48 patients with facet joint osteoarthritis (20 patients with moderate degeneration and 28 with severe degeneration). The control patients underwent internal fixation of the lumbar spine due to vertebral fracture. RNA deep sequencing was performed, and Bioinformatic tools were applied. Among top 30 enriched signaling pathways, we focused on two inflammation-related signaling pathways, Wnt and NF-κB signaling pathways. Subsequently, using the quantitative RT-PCR analysis, we confirmed that in Wnt signaling pathway, the mRNA levels of Dickkopf WNT Signaling Pathway Inhibitor 2 (DKK2), Sex-determining Region Y-box 17 (SOX17), MYC, Cyclin D1, Calcium/Calmodulin Dependent Protein Kinase II Alpha (CAMK2A), and Wnt Family Member 11 and 5 were increased in facet joint osteoarthritis, while the mRNA levels of WNT Inhibitory Factor 1, Casein Kinase 1 Alpha 1, Transcription Factor 7/Lymphoid Enhancer Binding Factor 1 (TCF7/LEF1), and VANGL Planar Cell Polarity Protein 2 were decreased. In NF-κB signaling pathway, the mRNA levels of C-C Motif Chemokine Ligand 4 (CCL4) and C-C Motif Chemokine Ligand 4 Like 2 (CCL4L2) were increased, while the mRNA levels of BCL2 Related Protein A1 were decreased. These results suggest that Wnt and NF-κB signaling may be altered in the process of facet joint cartilage degeneration. The present study will expand our understanding of the molecular bases underlying facet joint osteoarthritis.

PDK1 promotes the inflammatory progress of fibroblast-like synoviocytes by phosphorylating RSK2.

This study investigated the role of PDK1 in inflammatory response which is initiated by TNF-α and analyzed the association between PDK1 and RSK2. TNF-α were added into MH7A cells to induce inflammation condition. Through overexpressing or suppressing PDK1 in MH7A cells, the role of PDK1 in cell invasiveness and inflammatory factors was determined. Levels of MMPs protein and inflammatory cytokines were assessed with PDK1 siRNA and TNF-α treatment. Inhibition of RSK2 was used to investigate the function of RSK2 on PDK1-induced inflammation. The phosphorylation of RSK2 was detected when PDK1 was inhibited. Luciferase reporter assay was performed to detect the transcriptional activity of NF-κB. We found highly expressed PDK1 could promote cell invasion and secretion of IL-1ß and IL-6 in MH7A cells. Inhibition of RSK2 reduced the PDK1-induced cell invasion and cytokines secretion in MH7A cells. In response to TNF-α, PDK1 could phosphorylate RSK2 and activated RSK2, then promoting the activation of NF-κB. This may be a possible therapeutic option of rheumatoid arthritis.

Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation.

Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, ßIII-tubulin and GFAP were the most highest in the DPSCs + bFGF + NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF + NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.

The Expression of IGFBP6 after Spinal Cord Injury: Implications for Neuronal Apoptosis.

IGFBP6, a member of the insulin-like growth factor-binding proteins family that contains six high affinity IGFBPs, modulates insulin-like growth factor (IGF) activity and also showed an independent effect of IGF, such as growth inhibition and apoptosis. However, the role of IGFBP6 in spinal cord injury (SCI) remains largely elusive. In this study, we have performed an acute SCI model in adult rats and investigated the dynamic changes of IGFBP6 expression in the spinal cord. Our results showed that IGFBP6 was upregulated significantly after SCI, which was paralleled with the levels of apoptotic proteins p53 and active caspase-3. Immunofluorescent labeling showed that IGFBP6 was co-localizated with active caspase-3 and p53 in neurons. To further investigate the function of IGFBP6, an apoptosis model was established in primary neuronal cells. When IGFBP6 was knocked down by specific short interfering RNA (siRNA), the protein levels of active caspase-3 and Bax as well as the number of apoptotic primary neurons were significantly decreased in our study. Taken together, our findings suggest that the change of IGFBP6 protein expression plays a key role in neuronal apoptosis after SCI.

Chitosan scaffolds induce human dental pulp stem cells to neural differentiation: potential roles for spinal cord injury therapy.

Cell-based transplantation strategies hold great potential for spinal cord injury (SCI) repair. Chitosan scaffolds have therapeutic benefits for spinal cord regeneration. Human dental pulp stem cells (DPSCs) are abundant available stem cells with low immunological incompatibility and can be considered for cell replacement therapy. The purpose of this study is to investigate the role of chitosan scaffolds in the neural differentiation of DPSCs in vitro and to assess the supportive effects of chitosan scaffolds in an animal model of SCI. DPSCs were incubated with chitosan scaffolds. Cell viability and the secretion of neurotrophic factors were analyzed. DPSCs incubated with chitosan scaffolds were treated with neural differentiation medium for 14 days and then neural genes and protein markers were analyzed by Western blot and reverse transcription plus the polymerase chain reaction. Our study revealed a higher cell viability and neural differentiation in the DPSC/chitosan-scaffold group. Compared with the control group, the levels of BDNF, GDNF, b-NGF, and NT-3 were significantly increased in the DPSC/chitosan-scaffold group. The Wnt/ß-catenin signaling pathway played a key role in the neural differentiation of DPSCs combined with chitosan scaffolds. Transplantation of DPSCs together with chitosan scaffolds into an SCI rat model resulted in the marked recovery of hind limb locomotor functions. Thus, chitosan scaffolds were non-cytotoxic and provided a conducive and favorable microenvironment for the survival and neural differentiation of DPSCs. Transplantation of DPSCs might therefore be a suitable candidate for treating SCI and other neuronal degenerative diseases.

Upregulation of PSMB4 is Associated with the Necroptosis after Spinal Cord Injury.

Spinal cord injury (SCI) is one of the most common and severe complications in spine injury. It is difficult to prevent cell necroptosis and promote the survival of residual neurons after SCI. Proteasome beta-4 subunit (PSMB4) is the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo, and our previous study showed that PSMB4 is significantly associated with neuronal apoptosis in neuroinflammation. However, PSMB4 function in the necroptosis after SCI is unkown. RIP3, a key regulatory factor of necroptosis, correlates with the induction of necroptosis in various types of cells and signaling pathway. Upregulation of the RIP3 expression may play a role as a novel molecular mechanism in secondary neural tissue damage following SCI. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of PSMB4 during the pathological process of SCI. We found PSMB4 expression was significantly up-regulated 3 days after injury by western blot and immunohistochemical staining. Double immunofluorescent staining indicated obvious changes of PSMB4 expression occurred in neurons. Significant up-regulation of PSMB4 expression was observed in Rip3 positive neurons at 3 days after SCI, which indicated that PSMB4 might play a vital role in the regulation of Rip3. Overexpress and knockdown PSMB4 could intervene the RIP3 and Mixed lineage kinase domain-like protein (MLKL) pathway in Tumor necrosis factor-α (TNF-α) induced necroptosis cell model. Based on our experimental data, we boldly conclude that PSMB4 is associated with RIP3 involved necroptosis after SCI.

[Experimental Comparison Research between Two Kinds of Modified Poly(lactic acid)Material In Vitro].

This study aims to compare two kinds of modified poly(lactic acid)(PLA)materials:PLA-chitosan(PLA-CTS)and PLA-poly(glycolic acid)(PLA-PGA).PLA-CTS and PLA-PGA scaffolds were prepared and observed under electron microscope.The scaffold porosity was calculated and the pH of the degradation solution was measured.Then rat olfactory ensheathing cells(OECs)were cultivated,and mixed cultured respectively with two scaffolds as two groups.The proliferation,adhesion rate and growth condition of the OECs were observed and compared between the two groups.Results showed that both the prepared PLA-CTS and PLA-PGA scaffolds were threedimensional porous structure and the porosity of PLA-CTS was 91%,while that of PLA-PGA was 87%.The pH of degradation solution decreased gradually,of which PLA-PGA fell faster than PLA-CTS.After added to the two scaffolds,most OECs could grow well,and there were no significant differences between the two groups on MTT test and nuclei number determined by fluorescent microscope.However,the cell adhesion rate of PLA-CTS group was significantly higher than that of PLA-PGA.It can be concluded that compared with PLA-PGA,PLA-CTS might be a better choice as OECs scaffold.

Expression of Peroxiredoxin 1 After Traumatic Spinal Cord Injury in Rats.

Reactive astrogliosis and microgliosis after spinal cord injury (SCI) contribute to glial scar formation that impedes axonal regeneration. The mechanisms underlying reactive astrocyte and microglia proliferation upon injury remain partially understood. Peroxiredoxin 1 (PRDX1) is an antioxidant participating in cell proliferation, differentiation, and apoptosis. However, PRDX1 functions in SCI-induced astrocyte and microglia proliferation are unknown. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of PRDX1 during the pathological process of SCI. We found the palpable expression increase of PRDX1 after SCI by western blot and immunohistochemistry staining. Double immunofluorescence staining showed that PRDX1 expression mainly increased in astrocytes and microglia. In addition, PRDX1/proliferating cell nuclear antigen (PCNA) colocalized in astrocytes and microglia. Furthermore, PCNA expression also elevated after SCI, as well as was positively correlated with PRDX1 expression. In vitro, PRDX1 expression in primary rat spinal cord astrocytes and microglia changed in a concentration- and time-dependent manner according to LPS treatment. In addition, PRDX1 knockdown in astrocytes and microglia resulted in the decrease of PCNA expression after LPS stimulation, showing that PRDX1 promoted astrocyte and microglia proliferation after inflammation. Our results suggested that PRDX1 might play a crucial role in astrocyte and microglia proliferation after SCI.

VIRMA promotes neuron apoptosis via inducing m6A methylation of STK10 in spinal cord injury animal models.

BACKGROUND: Spinal cord injury (SCI) occurs as a devastating neuropathic disease. The role of serine-threonine kinase 10 (STK10) in the development of SCI remains unclear. OBJECTIVE: This study aimed to investigate the action of m6A methylation on STK10 in the apoptosis of spinal cord neurons in the pathogenesis of SCI and the possible underlying mechanisms. METHODS: Rat model of SCI was established and subsequently evaluated for motor function, pathological conditions, and apoptosis of spinal cord neurons. And the effects of overexpression of STK10 on neuronal cells in animal models of spinal cord injury and glyoxylate deprivation (OGD) cell models were evaluated. m6A2Target database and SRAMP database were used to predict the m6A methylation sites of STK10. The methylation kits were used to detect overall m6A methylation. Finally, the interaction between STK10 and vir like m6A methyltransferase associated (VIRMA) was explored in animal and cellular models. RESULTS: STK10 is markedly decreased in spinal cord injury models and overexpression of STK10 inhibits neuronal apoptosis. VIRMA can induce m6A methylation of STK10. VIRMA is over-expressed in spinal cord injury models and negatively regulates the expression of STK10. m6A methylation and apoptosis of neuronal cells are reduced by the knockdown of VIRMA and STK10 shRNA have shown the opposite effects. CONCLUSIONS: VIRMA promotes neuronal apoptosis in spinal cord injury by regulating STK10 m6A methylation.

Contribution of ApoB-100/SORT1-Mediated Immune Microenvironment in Regulating Oxidative Stress, Inflammation, and Ferroptosis After Spinal Cord Injury.

This study aims to explore the impacts of ApoB-100/SORT1-mediated immune microenvironment during acute spinal cord injury (SCI), and to investigate the potential mechanism. CB57BL/6 mice underwent moderate thoracic contusion injury to establish the SCI animal model, and received ApoB-100 lentivirus injection to interfere ApoB-100 level. Functional recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) score and footprint analysis. Transmission electron microscopy was applied to observe the ultrastructure of the injured spinal cord tissue. Hematoxylin-eosin (HE) staining and Perls staining were conducted to assess histological changes and iron deposition. Biochemical factor and cytokines were detected using their commercial kits. M1/M2 macrophage markers were detected by immunofluorescence assay in vivo and by flow cytometry in vitro. HT22 neurons were simulated by lipopolysaccharide (LPS), followed by incubation with polarized macrophage medium to simulate the immune microenvironment of injured spinal cord in vitro. The local immune microenvironment is changed in SCI mice, accompanied with the occurrence of oxidative stress and the elevation of both M1 and M2 macrophages. Knockdown of ApoB-100 ameliorates oxidative stress and lipid disorder, and inhibits inflammation and ferroptosis in SCI mice. Importantly, knockdown of ApoB-100 can partly restrict M1 macrophages but does not change M2 macrophage proportion in SCI mice. Further, M1 macrophages are observed to attenuate the inflammatory response, oxidative stress, and ferroptosis levels of LPS-induced HT22 cells, which is further strengthened by SORT1 knockdown. Blockage of ApoB-100/SORT1-mediated immune microenvironment plays a protective role against SCI via inhibiting oxidative stress, inflammation, lipid disorders, and ferroptosis, providing novel insights of the targeted therapy of SCI.