BackgroundNeuroinflammation contributes to delayed (secondary) neurodegeneration following traumatic brain injury (TBI). Tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling may promote post-TBI neuroinflammation, thereby exacerbating secondary injury.MethodsThis study investigated the pathogenic functions of TRAF6 signaling following TBI in vivo and in vitro. A rat TBI model was established by air pressure contusion while lipopolysaccharide (LPS) exposure was used to induce inflammatory-like responses in cultured astrocytes. Model rats were examined for cell-specific expression of TRAF6, NF-κB, phosphorylated (p)-NF-κB, MAPKs (ERK, JNK, p38), p-MAPKs, chemokines (CCL2 and CXCL1), and chemokine receptors (CCR2 and CXCR2) by immunofluorescence, RT-qPCR, western blotting, and ELISA, for apoptosis by TUNEL staining, and spatial cognition by Morris water maze testing. These measurements were compared between TBI model rats receiving intracerebral injections of TRAF6-targeted RNAi vector (AAV9-TRAF6-RNAi), empty vector, MAPK/NF-κB inhibitors, or vehicle. Primary astrocytes were stimulated with LPS following TRAF6 siRNA or control transfection, and NF-κB, MAPKs, chemokine, and chemokine receptor expression levels evaluated by western blotting and ELISA. ResultsTRAF6 was expressed mainly in astrocytes and neurons of injured cortex, peaking 3 days post-TBI. Knockdown by AAV9-TRAF6-RNAi improved spatial learning and memory, decreased TUNEL-positive cell number in injured cortex, and downregulated expression levels of p-NF-κB, p-JNK, p-ERK, p-p38, CCL2, CCR2, CXCL1, and CXCR2 post-TBI. Inhibitors of NF-κB, ERK, JNK, and p38 significantly suppressed CCL2, CCR2, CXCL1, and CXCR2 expression following TBI. Furthermore, TRAF6-siRNA inhibited LPS-induced NF-κB, ERK, JNK, p38, CCL2, and CXCL1 upregulation in cultured astrocytes.ConclusionsTargeting TRAF6-MAPK/NF-κB-chemokine signaling pathways may provide a novel therapeutic approach for reducing post-TBI neuroinflammation and concomitant secondary injury.
Background: Neuroinflammation contributes to delayed (secondary) neurodegeneration following traumatic brain injury (TBI). Tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling may promote post-TBI neuroinflammation, thereby exacerbating secondary injury. Methods: This study investigated the pathogenic functions of TRAF6 signaling following TBI in vivo and in vitro. A rat TBI model was established by air pressure contusion while lipopolysaccharide (LPS) exposure was used to induce inflammatory-like responses in cultured astrocytes. Model rats were examined for cell-specific expression of TRAF6, NF-κB, phosphorylated (p)-NF-kB, MAPKs (ERK, JNK, p38), p-MAPKs, chemokines (CCL2 and CXCL1), and chemokine receptors (CCR2 and CXCR2) by immunofluorescence, RT-qPCR, western blotting, and ELISA, for apoptosis by TUNEL staining, and spatial cognition by Morris water maze testing. These measurements were compared between TBI model rats receiving intracerebral injections of TRAF6-targeted RNAi vector (AAV9-TRAF6-RNAi), empty vector, MAPK/NF-kB inhibitors, or vehicle. Primary astrocytes were stimulated with LPS following TRAF6 siRNA or control transfection, and NF-κB, MAPKs, chemokine, and chemokine receptor expression levels evaluated by western blotting and ELISA. Results: TRAF6 was expressed mainly in astrocytes and neurons of injured cortex, peaking 3 days post-TBI. Knockdown by AAV9-TRAF6-RNAi improved spatial learning and memory, decreased TUNEL-positive cell number in injured cortex, and downregulated expression levels of p-NF-κB, p-JNK, p-ERK, p-p38, CCL2, CCR2, CXCL1, and CXCR2 post-TBI. Inhibitors of NF-κB, ERK, JNK, and p38 significantly suppressed CCL2, CCR2, CXCL1, and CXCR2 expression following TBI. Furthermore, TRAF6-siRNA inhibited LPS-induced NF-κB, ERK, JNK, p38, CCL2, and CXCL1 upregulation in cultured astrocytes. Conclusions: Targeting TRAF6-MAPK/NF-κB-chemokine signaling pathways may provide a novel therapeutic approach for reducing post-TBI neuroinflammation and concomitant secondary injury.
Background It is well known that hyperbaric oxygen (HBO) therapy achieves neuroprotective effects by suppressing or relieving neuroinflammatory responses. However, its underlying therapeutic mechanisms are not yet fully elucidated. Based on our previous studies, we further investigated whether HBO therapy exerts neuroprotective effects in vivo by regulating the NF-κB/ MAPKs-CXCL1 inflammatory pathway. Methods A rat model of traumatic brain injury (TBI) was established by controlled cortical impact (CCI). The cellular distribution of CXCL1 and CXCR2 was observed by double immunofluorescence labeling. The neurological function of TBI rats was assessed by modified neurological severity scores and Morris water maze methods. TUNEL staining was performed to observe apoptosis of neuronal cells in the injured cortical area. The changes in neural function, neuronal apoptosis, and expression of CXCL1, CXCR2, NF-κB, and MAPKs (ERK and JNK) were observed in TBI rats treated with CXCR2 antagonist, ERK, JNK, and NF-κB inhibitor or HBO therapy. Results The expression of CXCL1 and CXCR2 increased after TBI, and cell localization analysis revealed that CXCL1 was mainly expressed in astrocytes, while CXCR2 was mainly expressed in neurons. Increased apoptosis of cortical neurons in the injury area was also found after TBI. Reduced neuronal apoptosis with improved neurological function was observed after application of a CXCR2 antagonist. The expression of p-ERK, p-JNK and p-NF-κB increased after TBI, and application of ERK, JNK and NF-κB inhibitors decreased expression of CXCL1 and CXCR2 in rats. We further found that HBO therapy down-regulated the expression of p-ERK, p-JNK, p-NF-κB, CXCL1, and CXCR2, and reduced neuronal apoptosis, improved the neurological function of TBI rats, and ultimately alleviated the secondary injury. Conclusions CXCL1- CXCR2 mediates the interaction of activated astrocytes and neurons, exacerbating secondary injury after TBI. HBO therapy exerts neuroprotective effects by regulating the NF-κB/ MAPKs (JNK and ERK)- CXCL1 inflammatory pathway to control neuroinflammation after TBI, which provides the theoretical and experimental basis for the clinical application of HBO therapy in the treatment of TBI.
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