Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway.

BACKGROUND: The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported. METHODS: We constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin-eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway. RESULTS: CAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1ß, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1. CONCLUSION: CAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.

Blockade of the ADAM8-Fra-1 complex attenuates neuroinflammation by suppressing the Map3k4/MAPKs axis after spinal cord injury.

BACKGROUND: Mechanical spinal cord injury (SCI) is a deteriorative neurological disorder, causing secondary neuroinflammation and neuropathy. ADAM8 is thought to be an extracellular metalloproteinase, which regulates proteolysis and cell adherence, but whether its intracellular region is involved in regulating neuroinflammation in microglia after SCI is unclear. METHODS: Using animal tissue RNA-Seq and clinical blood sample examinations, we found that a specific up-regulation of ADAM8 in microglia was associated with inflammation after SCI. In vitro, microglia stimulated by HMGB1, the tail region of ADAM8, promoted microglial inflammation, migration and proliferation by directly interacting with ERKs and Fra-1 to promote activation, then further activated Map3k4/JNKs/p38. Using SCI mice, we used BK-1361, a specific inhibitor of ADAM8, to treat these mice. RESULTS: The results showed that administration of BK-1361 attenuated the level of neuroinflammation and reduced microglial activation and recruitment by inhibiting the ADAM8/Fra-1 axis. Furthermore, treatment with BK-1361 alleviated glial scar formation, and also preserved myelin and axonal structures. The locomotor recovery of SCI mice treated with BK-1361 was therefore better than those without treatment. CONCLUSIONS: Taken together, the results showed that ADAM8 was a critical molecule, which positively regulated neuroinflammatory development and secondary pathogenesis by promoting microglial activation and migration. Mechanically, ADAM8 formed a complex with ERK and Fra-1 to further activate the Map3k4/JNK/p38 axis in microglia. Inhibition of ADAM8 by treatment with BK-1361 decreased the levels of neuroinflammation, glial formation, and neurohistological loss, leading to favorable improvement in locomotor functional recovery in SCI mice.

Transplantation of A2 type astrocytes promotes neural repair and remyelination after spinal cord injury.

BACKGROUND: Limited progress in terms of an effective treatment for spinal cord injury (SCI) emphasizes the urgent need for novel therapies. As a vital central nervous system component, the resident astrocytes play crucial roles in regulating recovery after SCI. In this study, recovery after SCI was compared following the transplantation of either A1 or A2 astrocytes. A1 astrocytes are harmful as they upregulate the neurotoxic classical complement cascade genes. Conversely, A2 astrocytes are characterized as neuroprotective as they upregulate the production of many neurotrophic factors. METHODS: We used different supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4 to generate A1 and A2 astrocytes. We detected the influence of astrocytes on neurons by co-culturing A1 and A2 astrocytes with neurons. We transplanted astrocytes into the lesion site of the spinal cord and assessed lesion progression, neural restoration, glia formation and locomotor recovery. RESULTS: Astrocytes were polarized into A1 and A2 phenotypes following culture in the supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4, respectively. Furthermore, co-culturing A2 astrocytes with neurons significantly suppressed glutamate-induced neuronal apoptosis and promoted the degree of neuron arborization. Transplantation of these A2 astrocytes into the lesion site of the spinal cord of mice significantly improved motor function recovery, preserved spared supraspinal pathways, decreased glia scar deposition, and increased neurofilament formation at the site of injury compared to the transplantation of A1 astrocytes. Additionally, enhanced A2 astrocytes with potentially beneficial A2-like genes were also detected in the A2 group. Moreover, luxol fast blue staining and electron microscopy indicated increased preservation of myelin with organized structure after transplantation of A2 astrocytes than of A1 astrocytes. CONCLUSIONS: A2 astrocyte transplantation could be a promising potential therapy for SCI. Video abstract.

A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics.

BACKGROUND: A20 is an anti-inflammatory molecule in nucleus pulposus (NP) cells. The anti-inflammatory properties of A20 are mainly attributed to its ability to suppress the NF-κB pathway. However, A20 can protect cells from death independently of NF-κB regulation. This study aimed to investigate the effects of A20 on pyroptosis and apoptosis of NP cells induced by lipopolysaccharide (LPS). METHODS: NP cells induced by LPS were used as an in vitro model of the inflammatory environment of the intervertebral disc. Pyroptosis, apoptosis, and mitophagy marker proteins were detected. Then, NP cells were transfected with A20 overexpressed lentivirus or A20-siRNA. Annexin V FITC/PI, Western blotting, and immunofluorescence assays were used to detect the apoptosis, pyroptosis, and mitophagy of NP cells. Furthermore, the expressions of A20, related proteins, and related inflammatory cytokines were detected by western blotting, and ELISA. RESULTS: Apoptosis and pyroptosis of NP cells increased gradually treated with LPS for 12 h, 24 h, and 48 h. Differently, the level of mitophagy increased first and then decreased, and was the highest at LPS treatment for 12 h. Overexpression or knockdown of A20 in NP cells revealed that A20 attenuated the pyroptosis, apoptosis, and production of inflammatory cytokines of NP cells induced by LPS, while A20 sponsored mitophagy, reduced ROS production and collapse of mitochondrial membrane potential (ΔΨm). Moreover, A20 also promoted mitochondrial dynamic homeostasis and attenuated LPS-induced excessive mitochondrial fission. Excitingly, inhibition of mitophagy attenuated the effect of A20 on the negative regulation of pyroptosis of NP cells induced by LPS. Pyroptosis was accompanied by a large release of inflammatory cytokines. Inhibition of pyroptosis also significantly reduced apoptosis of NP cells. Finally, The mitochondria-targeted active peptide SS-31 inhibited LPS-induced pyroptosis and ROS production in NP cells. CONCLUSIONS: To sum up, A20 attenuates pyroptosis and apoptosis of NP cells via promoting mitophagy and stabilizing mitochondrial dynamics. Besides, A20 reduces LPS-induced NP cell apoptosis by inhibiting NLRP3 inflammasome-mediated pyroptosis. It provides theoretical support for the reduction of functional NP cell loss in the intervertebral disc through the gene-targeted intervention of A20.

Excess administration of miR-340-5p ameliorates spinal cord injury-induced neuroinflammation and apoptosis by modulating the P38-MAPK signaling pathway.

Spinal cord injury (SCI) is a destructive polyneuropathy that can result in loss of sensorimotor function and sphincter dysfunction, and even death in critical situations. MicroRNAs (miRs) are a series of non-coding RNA molecules that are involved in transcriptional regulation. Previous studies have demonstrated that modulation of multiple miRs is involved in neurological recovery after SCI. However, the functions of miR-340-5p in SCI remain uncertain. Therefore, we probed the therapeutic effect and mechanism of miR-340-5p in microglia in vitro and in vivo in SCI rats. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to examine the alterations in miR-340-5p and P38 levels in SCI rats. miR-340-5p targets in microglia were ascertained using luciferase reporter assays, immunofluorescence analyses, and western blotting. We also established an SCI model and administered miR-340-5p. The effects of miR-340-5p on the amelioration of inflammation, oxidative stress, and apoptosis following SCI were assessed using immunofluorescence, immunohistochemistry, and histological analyses. Finally, locomotor function recovery was determined using the Basso, Beattie, Bresnahan rating scale. In our study, the expression profiles and luciferase assay results clarified that P38 was a target of miR-340-5p, which was associated with activation of the P38-MAPK signaling pathway. Elevation of miR-340-5p decreased P38 expression, subsequently inhibiting the inflammatory reaction. SCI-induced secondary neuroinflammation was relieved under miR-340-5p treatment. Moreover, by controlling neuroinflammation, the increased levels of miR-340-5p might counter oxidative stress and reduce the degree of apoptosis. We also observed decreasing gliosis and glial scar formation and increasing neurotrophin expression at the chronic stage of SCI. Together, these potential effects of miR-340-5p treatment ultimately improved locomotor function recovery in SCI rats.

ACOD1, rather than itaconate, facilitates p62-mediated activation of Nrf2 in microglia post spinal cord contusion.

BACKGROUND: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI. METHODS: Adult male C57BL/6 ACOD1-/- mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay. RESULTS: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1. CONCLUSIONS: Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita. KEY POINTS: ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.

A Disintegrin and Metalloproteinase-8 Protects Against Erastin-Induced Neuronal Ferroptosis via Activating Nrf2/HO-1/FTH1 Signaling Pathway.

Ferroptosis is a type of iron-dependent programmed cell death caused by the imbalance between oxidants and antioxidants. A disintegrin and metalloproteinase-8 (ADAM8) is a metalloproteinase that mediates cell adhesion, cell migration, and proteolytic activity. However, the molecular mechanism of ADAM8 regulating ferroptosis after neural disorder is unclear, especially in the neuron. In the present study, we identified the protective role of ADAM8 in Erastin-induced ferroptosis in vitro of the HT22 cells. It was found that overexpression of ADAM8 resulted in upregulated expression of GPX4 and FTH1 along with the decreased reactive oxygen species (ROS) production and reduced neuronal death; however, knockdown of ADAM8 resulted in an opposite. Mechanically, using the Nrf2 activator NK-252 and inhibitor nrf2-IN-1, we dmonstrated that ADAM8 regulates Erastin-mediated neuronal ferroptosis via activating the Nrf2/HO-1/FTH1 signaling pathway. In conclusion, the current study suggested that ADAM8 inhibited Erastin-induced neuronal ferroptosis through activating the Nrf2/HO-1/FTH1 signaling pathway, playing a protective role in vitro of the HT22 cell line. ADAM8 may be a promising and feasible target for neuronal survival in diseases of neural disorder.

Corrigendum: miR-766-3p targeting BCL9L suppressed tumorigenesis, epithelial-mesenchymal transition, and metastasis through the ß-catenin signaling pathway in osteosarcoma cells.

[This corrects the article DOI: 10.3389/fcell.2020.594135.].

An update of interbody cages for spine fusion surgeries: from shape design to materials.

INTRODUCTION: Discectomy and interbody fusion are widely used in the treatment of intervertebral disc-related diseases. Among them, the interbody cage plays a significant role. However, the complications related to the interbody cage, such as nonunion or pseudoarthrosis, subsidence, loosening, and prolapse of the cage, cannot be ignored. By changing the design and material of the interbody fusion cage, a better fusion effect can be obtained, the incidence of appeal complications can be reduced, and the quality of life of patients after interbody fusion can be improved. AREAS COVERED: This study reviewed the research progress of cage design and material and discussed the methods of cage design and material to promote intervertebral fusion. EXPERT OPINION: Current treatment of cervical and lumbar degenerative disease requires interbody fusion to maintain decompression and to promote fusion and reduce the incidence of fusion failure through improvements in implant material, design, internal structure, and function. However, interbody fusion is not an optimal solution for treating vertebral instability.Abbreviations: ACDF, Anterior cervical discectomy and fusion; ALIF, anterior lumbar interbody fusion; Axi-aLIF, axial lumbar interbody fusion; BAK fusion cage, Bagby and Kuslich fusion cage; CADR, cervical artificial disc replacement; DBM, decalcified bone matrix; HA, hydroxyapatite; LLIF/XLIF, lateral or extreme lateral interbody fusion; MIS-TLIF, minimally invasive transforaminal lumbar interbody fusion; OLIF/ATP, oblique lumbar interbody fusion/anterior to psoas; PEEK, Poly-ether-ether-ketone; PLIF, posterior lumbar interbody fusion; ROI-C, Zero-profile Anchored Spacer; ROM, range of motion; SLM, selective melting forming; TLIF, transforaminal lumbar interbody fusion or.

Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury.

Background: Neuroinflammation following spinal cord injury (SCI) results in prolonged neurological damage and locomotor dysfunction. Polarization of microglia is vital to regulation of neuroinflammation, although the underlying mechanisms have not yet been elucidated. Endocannabinoid receptor subtype 2 (CB2R) is reported to ameliorate neurodegeneration via immunomodulation activities. However, the underlying machinery in the context of SCI remains unclear. Methods: A lipopolysaccharide-induced microglia inflammation model and a mouse model of SCI were employed to investigate the regulatory role of CB2R in the polarization of microglia in response to excess neuroinflammation. Markers of inflammation and autophagy were measured by Western blot analysis, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. Histological staining with hematoxylin and eosin, Nissl, and Luxol® fast blue was conducted using commercial kits. The locomotor function of the hindlimbs of the experimental mice was evaluated with the Basso Mouse Scale, Louisville Swim Scale, and footprint assay. Results: The results showed that CB2R promoted M2 differentiation, increased interleukin (IL)-10 expression, and inhibited M1 differentiation with decreased expression of IL-1ß and IL-6. CB2R activation also increased ubiquitination of the NLRP3 inflammasome and interacted with the autophagy-related proteins p62 and microtubule-associated proteins 1B light chain 3. Treatment with the CB2R activator JWH-133 reduced loss of myelin, apoptosis of neurons, and glial scarring, leading to improved functional recovery of the hindlimbs, while the CB2R antagonist AM630 produced opposite results. Conclusion: Taken together, these results suggested that CB2R activation attenuated neuroinflammation targeting microglial polarization by promoting NLRP3 clearance, thereby facilitating functional recovery post-SCI.

Deficiency of CD93 exacerbates inflammation-induced activation and migration of BV2 microglia by regulating the TAK1/NF-κB pathway.

The role of CD93 in inflammatory response has been reported in multiple previous studies. However, the underlying mechanism of CD93 in microglial activation and migration during neuroinflammation post spinal cord injury (SCI) remains elusive. In the current study, we performed western blot, qRT-PCR, immunofluorescence analyses Transwell assay, and ELISA to determine the expression change and in-depth molecular mechanism of CD93 in microglia post inflammatory initiation. We found that CD93 expression was increased in microglia after SCI in vivo or lipopolysaccharide (LPS) stimuli in vitro. Additionally, CD93 interacted with TAK1 to inhibit NF-κB activation, thus attenuating inflammation and migration of microglia after treatment with LPS. These findings indicate that CD93 might participate in microglia-induced neuroinflammation development post SCI, suggesting that CD93 is a promising target for neuroimmunological regulation.

Growth Differentiation Factor 15 Regulates Oxidative Stress-Dependent Ferroptosis Post Spinal Cord Injury by Stabilizing the p62-Keap1-Nrf2 Signaling Pathway.

Background: Spinal cord injury (SCI) is a severe traumatic disorder of the central nervous system (CNS) that causes irreversible damage to the nervous tissue. The consequent hemorrhage contributed by trauma induces neuronal ferroptosis post SCI, which is an important death mode to mediate neuronal loss. Growth differentiation factor 15 (GDF15) is a cytokine that regulates cell proliferation, differentiation, and death. However, the specific role of GDF15 in neuronal ferroptosis post SCI remains unknown. Materials and Methods: Neuronal ferroptosis in vitro was measured by detection of lipid peroxidation, glutathione, iron content, and reactive oxidative stress. In vivo, western blotting and immunofluorescence (IF) staining was utilized to measure ferroptosis post SCI. IF staining, TUNEL staining, hematoxylin-eosin staining, and Nissl staining were used to measure neurological damage. Finally, locomotor function recovery was analyzed using the Basso Mouse Scale and Louisville Swim Scale. Results: GDF15 was significantly increased in neuronal ferroptosis and silencing GDF15 aggravated ferroptosis both in vitro and in vivo. Besides, GDF15-mediated inhibition of neuronal ferroptosis is through p62-dependent Keap1-Nrf2 pathway. In SCI mice, knockdown of GDF15 significantly exacerbated neuronal death, interfered with axon regeneration and remyelination, aggravated ferroptosis-mediated neuroinflammation, and restrained locomotor recovery. Conclusion: GDF15 effectively alleviated neuronal ferroptosis post SCI via the p62-Keap1-Nrf2 signaling pathway and promoted locomotor recovery of SCI mice, which is suggested as a potential target on SCI pathogenesis and treatment.

Forsythoside B attenuates neuro-inflammation and neuronal apoptosis by inhibition of NF-κB and p38-MAPK signaling pathways through activating Nrf2 post spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a ruinous neurological pathology that results in locomotor and sensory impairment. Neuro-inflammation and secondary neuronal apoptosis contribute to SCI, with anti-inflammatory therapies the focus of many SCI studies. Forsythoside B (FTS•B), a phenylethanoid glycoside extracted from the leaves of Lamiophlomis rotata Kudo, has been shown previously to have anti-inflammatory properties. Nevertheless, the therapeutic effect of FTS•B on neuro-inflammation after SCI is unknown. METHODS: Neuro-inflammation was assessed by western blotting (WB), immunofluorescence (IF) staining, and enzyme-linked immunosorbent assay (ELISA) both in vitro and in vivo. Secondary neuronal apoptosis was simulated in a microglia-neuron co-culture model with the degree of apoptosis measured by WB, IF, and TUNEL staining. In vivo, FTS•B (10 mg/kg, 40 mg/kg) were intraperitoneally injected into SCI mice. Morphological changes following SCI were evaluated by Nissl, Hematoxylin-eosin, and Luxol Fast Blue staining. Basso Mouse Scale scores were used to evaluate locomotor function recovery. RESULTS: FTS•B markedly decreased the levels of iNOS, COX-2 and signature mediators of inflammation. Phosphorylated p38 and nuclear factor-kappa B (NF-κB) were markedly decreased by FTS•B. Additionally, FTS•B-induced inhibition of NF-κB and p38-MAPK signaling pathways was reversed by Nrf2 downregulation. Administration of FTS•B also significantly reduced apoptosis-related protein levels indicating that FTS•B ameliorated secondary neuronal apoptosis. FTS•B administration inhibited glial scar formation, decreased neuronal death, tissue deficiency, alleviated demyelination, and promoted locomotor recovery. CONCLUSION: FTS•B effectively attenuates neuro-inflammation and secondary neuronal apoptosis by inhibition of NF-κB and p38-MAPK signaling pathways through activating Nrf2 after SCI. This study demonstrates FTS•B to be a potential therapeutic for SCI.

FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion.

BACKGROUND: Traumatic spinal cord injury (SCI)-induced neuroinflammation results in secondary neurological destruction and functional disorder. Previous findings showed that microglial pyroptosis plays a crucial role in neuroinflammation. Thus, it is necessary to conduct a comprehensive investigation of the mechanisms associated with post-SCI microglial pyroptosis. The Fanconi Anemia Group C complementation group gene (FANCC) has been previously reported to have an anti-inflammation effect; however, whether it can regulate microglial pyroptosis remains unknown. Therefore, we probed the mechanism associated with FANCC-mediated microglial pyroptosis and neuroinflammation in vitro and in vivo in SCI mice. METHODS: Microglial pyroptosis was assessed by western blotting (WB) and immunofluorescence (IF), whereas microglial-induced neuroinflammation was evaluated by WB, Enzyme-linked immunosorbent assays and IF. Besides, flow cytometry, TdT-mediated dUTP Nick-End Labeling staining and WB were employed to examine the level of neuronal apoptosis. Morphological changes in neurons were assessed by hematoxylin-eosin and Luxol Fast Blue staining. Finally, locomotor function rehabilitation was analyzed using the Basso Mouse Scale and Louisville Swim Scale. RESULTS: Overexpression of FANCC suppressed microglial pyroptosis via inhibiting p38/NLRP3 expression, which in turn reduced neuronal apoptosis. By contrast, knockdown of FANCC increased the degree of neuronal apoptosis by aggravating microglial pyroptosis. Besides, increased glial scar formation, severe myelin sheath destruction and poor axon outgrowth were observed in the mice transfected with short hairpin RNA of FANCC post SCI, which caused reduced locomotor function recovery. CONCLUSIONS: Taken together, a previously unknown role of FANCC was identified in SCI, where its deficiency led to microglia pyroptosis, neuronal apoptosis and neurological damage. Mechanistically, FANCC mediated microglia pyroptosis and the inflammatory response via regulating the p38/NLRP3 pathway.

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury.

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown. Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations. Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro. Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.

Ruxolitinib attenuates secondary injury after traumatic spinal cord injury.

Excessive inflammation post-traumatic spinal cord injury (SCI) induces microglial activation, which leads to prolonged neurological dysfunction. However, the mechanism underlying microglial activation-induced neuroinflammation remains poorly understood. Ruxolitinib (RUX), a selective inhibitor of JAK1/2, was recently reported to inhibit inflammatory storms caused by SARS-CoV-2 in the lung. However, its role in disrupting inflammation post-SCI has not been confirmed. In this study, microglia were treated with RUX for 24 hours and then activated with interferon-γ for 6 hours. The results showed that interferon-γ-induced phosphorylation of JAK and STAT in microglia was inhibited, and the mRNA expression levels of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, interleukin-6, and cell proliferation marker Ki67 were reduced. In further in vivo experiments, a mouse model of spinal cord injury was treated intragastrically with RUX for 3 successive days, and the findings suggest that RUX can inhibit microglial proliferation by inhibiting the interferon-γ/JAK/STAT pathway. Moreover, microglia treated with RUX centripetally migrated toward injured foci, remaining limited and compacted within the glial scar, which resulted in axon preservation and less demyelination. Moreover, the protein expression levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were reduced. The neuromotor function of SCI mice also recovered. These findings suggest that RUX can inhibit neuroinflammation through inhibiting the interferon-γ/JAK/STAT pathway, thereby reducing secondary injury after SCI and producing neuroprotective effects.

Pharmacological Disruption of Phosphorylated Eukaryotic Initiation Factor-2α/Activating Transcription Factor 4/Indian Hedgehog Protects Intervertebral Disc Degeneration via Reducing the Reactive Oxygen Species and Apoptosis of Nucleus Pulposus Cells.

Excessive reactive oxygen species (ROS) and apoptosis in nucleus pulposus (NP) cells accelerate the process of intervertebral disc degeneration (IDD). Here, we integrated pathological samples and in vitro and in vivo framework to investigate the impact of phosphorylation of eukaryotic initiation factor-2α (eIF2α)/activating transcription factor 4 (ATF4)/Indian hedgehog (Ihh) signaling in the IDD. From the specimen analysis of the IDD patients, we found phosphorylated eIF2α (p-eIF2α), ATF4 and Ihh protein levels were positively related while the NP tissue went degenerative. In vitro, tumor necrosis factor (TNF)-α caused the NP cell degeneration and induced a cascade of upregulation of p-eIF2α, ATF4, and Ihh. Interestingly, ATF4 could enhance Ihh expression through binding its promoter region, and silencing of ATF4 decreased Ihh and protected the NP cells from degeneration. Moreover, ISRIB inhibited the p-eIF2α, which resulted in a suppression of ATF4/Ihh, and alleviated the TNF-α-induced ROS production and apoptosis of NP cells. On the contrary, further activating p-eIF2α aggravated the NP cell degeneration, with amplification of ATF4/Ihh and a higher level of ROS and apoptosis. Additionally, applying cyclopamine (CPE) to suppress Ihh was efficient to prevent NP cell apoptosis but did not decrease the ROS level. In an instability-induced IDD model in mice, ISRIB suppressed p-eIF2α/ATF4/Ihh and prevented IDD via protecting the anti-oxidative enzymes and decreased the NP cell apoptosis. CPE prevented NP cell apoptosis but did not affect anti-oxidative enzyme expression. Taken together, p-eIF2α/ATF4/Ihh signaling involves the ROS level and apoptosis in NP cells, the pharmacological disruption of which may provide promising methods in preventing IDD.

Disrupting RhoA activity by blocking Arhgef3 expression mitigates microglia-induced neuroinflammation post spinal cord contusion.

Excess inflammatory microglia activation deteriorates the pathological degree of spinal cord injury (SCI). We here employed microglia samples in vitro and murine model in vivo to trace the role of inhibition of Arhgef3 in inflammatory response post SCI. From the specimen analysis of lipopolysaccharide (LPS)-induced inflammatory microglia, we found that Arhgef3 expression was positively relative to microglia activation. In vitro, LPS caused the microglia inflammatory activation and induced upregulation of the Arhgef3 expression. Interestingly, presence of Arhgef3 could activate RhoA through promoting Rho GTPases, but silencing of Arhgef3 decreased RhoA activation and inhibited the microglia inflammation. Moreover, disruption of Arhgef3 inhibited the GTP-RhoA, resulted in a suppression of proinflammatory cytokines, and alleviated the LPS-elicited inflammatory genes expression. Moreover, artificially decreasing Arhgef3 expression remarkedly reduced ROS generation after LPS treatment. In vivo of a mouse mechanical contusion-induced SCI model, inhibition of Arhgef3 reduced the ratio of GTP-RhoA/Total-RhoA, and prevented SCI via mitigating the microglial inflammatory phenotype and decreased secondary neurological injury. Besides, inhibition of Arhgef3 prevented alleviated the degree of demyelination but did not affect neuronal regeneration. Meaningfully, absence of Arhgef3 improved mouse locomotor recovery post SCI. Taken together, Arhgef3 involves the microglial activation and inflammatory response following neural injury, and targeted disrupting of which may indicate a promising therapeutic direction in preventing SCI.

Silencing COX-2 blocks PDK1/TRAF4-induced AKT activation to inhibit fibrogenesis during skeletal muscle atrophy.

Skeletal muscle atrophy with high prevalence can induce weakness and fatigability and place huge burden on both health and quality of life. During skeletal muscle degeneration, excessive fibroblasts and extracellular matrix (ECM) accumulated to replace and impair the resident muscle fiber and led to loss of muscle mass. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in synthesis of prostaglandin, has been identified as a positive regulator in pathophysiological process like inflammation and oxidative stress. In our study, we found injured muscles of human subjects and mouse model overexpressed COX-2 compared to the non-damaged region and COX-2 was also upregulated in fibroblasts following TGF-ß stimulation. Then we detected the effect of selective COX-2 inhibitor celecoxib on fibrogenesis. Celecoxib mediated anti-fibrotic effect by inhibiting fibroblast differentiation, proliferation and migration as well as inactivating TGF-ß-dependent signaling pathway, non-canonical TGF-ß pathways and suppressing generation of reactive oxygen species (ROS) and oxidative stress. In vivo pharmacological inhibition of COX-2 by celecoxib decreased tissue fibrosis and increased skeletal muscle fiber preservation reflected by less ECM formation and myofibroblast accumulation with decreased p-ERK1/2, p-Smad2/3, TGF-ßR1, VEGF, NOX2 and NOX4 expression. Expression profiling further found that celecoxib could suppress PDK1 expression. The interaction between COX-2 and PDK1/AKT signaling remained unclear, here we found that COX-2 could bind to PDK1/AKT to form compound. Knockdown of COX-2 in fibroblasts by pharmacological inactivation or by siRNA restrained PDK1 expression and AKT phosphorylation induced by TGF-ß treatment. Besides, si-COX-2 prevented TGF-ß-induced K63-ubiquitination of AKT by blocking the interaction between AKT and E3 ubiquitin ligase TRAF4. In summary, we found blocking COX-2 inhibited fibrogenesis after muscle atrophy induced by injury and suppressed AKT signaling pathway by inhibiting upstream PDK1 expression and preventing the recruitment of TRAF4 to AKT, indicating that COX-2/PDK1/AKT signaling pathway promised to be target for treating muscle atrophy in the future.

Silencing TAK1 reduces MAPKs-MMP2/9 expression to reduce inflammation-driven neurohistological disruption post spinal cord injury.

Microglia activation post traumatic spinal cord injury (SCI) provokes accumulation of inflammatory metabolites, leading to increasing neurological disruption. Our previous studies demonstrated that blocking MAPKs pathway mitigated microglia inflammatory activation and prevented cords from neuroinflammation-induced secondary injury. Transforming growth factor-ß-activated kinase 1 (TAK1) is an upstream gate regulating activation of MAPKs signaling. To validate the therapeutic effect of TAK1 inhibition in neuroinflammation post SCI, in the current study, cultures of microglia BV2 line was undergone lipopolysaccharide (LPS) stimulation in the presence of TAK1 inhibitor 5Z-7-Oxozeaenol (ZO), LPS, or control. LPS triggered inflammatory level, cell migration, and matrix metalloproteinase (MMP) 2/9 production, which was reduced in ZO-treated cultures. TAK1 inhibition by ZO also decreased activation of MAPKs pathway, indicating that ZO-mediated alleviation of neuroinflammation is likely modulated via TAK1/MAPKs axis. In vivo, neuroinflammatory level and tissue destruction were assessed in adult male mice that were undergone SCI by mechanical trauma, and treated with ZO by intraperitoneal injection. Compared with SCI mice, ZO-treated mice exhibited less microglia pro-inflammatory activation and accumulation adjacent to injured core linked to reduced MMP2/9 expression, leading to minor tissue damage and better locomotor recovery. To sum up, the obtained data proved that in the early phase post SCI, TAK1 inhibition impedes microglia biological activities including activation, enzymatic synthesis, and migration via downregulation of MAPKs pathway, and the effects may be accurately characterized as potent anti-inflammation.