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.

HAX1 is associated with neuronal apoptosis and astrocyte proliferation after spinal cord injury.

HS1-associated protein X-1 (HAX1) is a class of multifunctional protein, participated in various physiological processes such as cell apoptosis, proliferation and motility. However, the HAX1 expression and function in the spinal cord injury (SCI) pathological process have not been investigated. In our current research, the rat model of SCI was established, and then we explored the possible role of HAX1 after SCI. The results of western blot indicated that HAX1 was present in sham operated control group and significantly elevated at 3 days post SCI, then declined gradually. Immunohistochemical studies indicated HAX1 expression was enhanced significantly in white and gray matter at 3 days post SCI compared with sham operated group. Double immunofluorescence staining showed the proportion of cells, double-labeled HAX1 and neurons, astrocytes, increased significantly at 3 days post SCI. In addition, co-localization of HAX1/active caspase-3 and HAX1/PCNA was tested in cells. Furthermore, over-expression of HAX1 inhibited neuronal apoptosis in vitro, and in astrocytes HAX1 silencing could down-regulate PCNA expression post LPS treatment. Meanwhile, CCK8 assay showed that knockdown of HAX1 could inhibit the astrocyte proliferation. In summary, our data indicated that HAX1 might play significant roles in pathological process of neuronal apoptosis and astrocyte proliferation during SCI.

Vimentin Promotes Astrocyte Activation After Chronic Constriction Injury.

Vimentin, among the family of the intermediate filament, plays as the organizer of some critical proteins involved in migration, attachment, and cell signaling. In this study, the role of vimentin in chronic constriction injury (CCI) was investigated. Western blot revealed increased protein level of vimentin following CCI, peaking at 7 days. Double immunofluorescent staining showed that vimentin was mostly co-localized with astrocytes, not with neurons or microglia. In vitro, sensory neuronal injury stimulated astrocytes to produce more pro-inflammation cytokines, p-ERK (phosphorylated extracellular signal-regulated protein kinase), and vimentin. However, vimentin knockdown by siRNA (small interfering RNA) reversed the upregulation of p-ERK and vimentin expression and reduced the release of pro-inflammatory cytokines. Overall, stimulated astrocytes might release pro-inflammatory cytokines to promote the development of neuropathic pain via vimentin/ERK signaling.