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.

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.].

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.

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.

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.

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.

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.

miR-766-3p Targeting BCL9L Suppressed Tumorigenesis, Epithelial-Mesenchymal Transition, and Metastasis Through the ß-Catenin Signaling Pathway in Osteosarcoma Cells.

Accumulating evidence has indicated that abnormal microRNAs (miRNAs) serve critical roles in carcinogenesis and development of osteosarcoma (OS). The purpose of the present study was to elucidate the relationship between miR-766-3p and development of osteosarcoma and explore the potential mechanism. In this study, we found that miR-766-3p was the most downregulated miRNA by analyzing GSE65071 from the GEO database. miR-766-3p was lowly expressed in OS tissue samples and cells, and high miR-766-3p expression repressed the malignant level of OS, including cell proliferation, EMT, migration, and invasion in vitro and in vivo. B-Cell Lymphoma 9-Like Protein (BCL9L) was negatively associated with miR-766-3p expression in OS cells and tissue samples and was validated as the downstream target by luciferase reporter assay and western blotting. Rescue experiment indicated that BCL9L could restore the influence of miR-766-3p on OS cells. In addition, the ß-Catenin/TCF-4 signal pathway was demonstrated to be related to the miR-766-3p/BCL9L axis. In summary, miR-766-3p, a negative regulator of BCL9L, plays the role of tumor metastasis suppressor via the ß-catenin signaling pathway in the progression of OS.

LINC00266-1/miR-548c-3p/SMAD2 feedback loop stimulates the development of osteosarcoma.

Osteosarcoma (OS) is one of the most common primary bone malignancies and accounts for 3.4% of pediatric tumors. Its 5-year survival is as low as about 20%. Differentially expressed lncRNAs in OS profiling were searched in the downloaded profile of GSE12865. As a result, LINC00266-1 was detected to be upregulated in both GSE12865 and OS tissues we collected. SMAD2 was the downstream target binding to promoter sites of LINC00266-1, displaying a positive regulatory interaction. Knockdown of LINC00266-1 suppressed the proliferative and metastatic abilities, and promoted the apoptosis in OS cells. Besides, knockdown of LINC00266-1 significantly alleviated the growth of OS in vivo. MiR-548c-3p was the sponge miRNA of LINC00266-1, which was able to reverse the regulatory effects of LINC00266-1 on OS cell phenotypes. Moreover, miR-548c-3p bound to the 3'-UTR of SMAD2 and thus downregulated SMAD2. Overexpression of SMAD2 partially reversed the regulatory effects of LINC00266-1 on OS cell phenotypes. Finally, we have identified that LINC00266-1/miR-548c-3p/SMAD2 feedback loop was responsible for stimulating the development of OS.

CircRNA LRP6 promotes the development of osteosarcoma via negatively regulating KLF2 and APC levels.

We aimed to investigate the biological functions of circLRP6 in the progression of osteosarcoma. CircLRP6 level in OS was detected by quantitative real-time polymerase chain reaction. Correlation between circLRP6 level with survival of OS patients was evaluated. Cell counting kit-8 and Transwell assay were conducted to detect proliferative, migratory and invasive capacities of OS cells. Cell cycle and apoptosis in OS cells influenced by circLRP6 were evaluated by flow cytometry. RNA immunoprecipitation was conducted to verify the binding relationship between circLRP6 with LSD1 and EZH2. Finally, the interaction between LSD1, EZH2 and promoter regions of KLF2, APC was clarified by chromatin immunoprecipitation. CircLRP6 level markedly increased in OS tissues. Besides, OS patients with high expression of circLRP6 showed shorter disease-free survival and over-all survival than those with low expression. CircLRP6 knockdown suppressed proliferative, migratory and invasive rates of OS cells. Moreover, circLRP6 knockdown induced apoptosis and arrested cell cycle in G0/G1 phase. The interaction between circLRP6 with LSD1 and EZH2 mediates their binding to the promoter regions of KLF2 and APC. Knockdown of circLRP6 weakened the binding abilities of LSD1, EZH2 to KLF2, APC. APC overexpression inhibited proliferation, induced apoptosis and arrested cell cycle. Moreover, the tumor-suppressor effect of downregulated circLRP6 on OS could be reversed by APC knockdown. Collectively, circLRP6 was highly expressed in OS and served as an oncogene by binding to LSD1 and EZH2 to inhibit expressions of KLF2 and APC.