RIPK1-mediated NLRP3 activation via MAPK signaling pathway in the Pathogenesis of Trigeminal Neuralgia

Abstract Background: Trigeminal neuralgia is a typical kind of neuropathic pain featured with severe and recurrent pain attacks along the areas innervated by trigeminal nerve. Although mechanical injuries caused by vessels compression is highly related to the pathogenesis, the underlying molecular mechanism remains unclear. Methods: We constructed chronic constriction to infraorbital nerve (CCI) on Sprague Dawley rats, after which spontaneous pain and mechanical allodynia related behaviors were measured. Transcriptome of trigeminal ganglion (TG) affected by CCI were analyzed to select the related signaling pathway and genes. We examined the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and domain-like receptor protein 3 (NLRP3) expression in the trigeminal ganglion by western blotting and RT-PCR; studied the location of RIPK1 and NLRP3 expression by performing immunofluorescence. Necrostatin-1, the specific antagonist of RIPK1, was administered to identify the effect of RIPK1 inhibition on NLRP3 expression. Results: We identified mechanical allodynia within the whisker pad area after CCI. RNA-sequencing indicated that NLRP3 levels in the trigeminal ganglion were higher in CCI groups compared to the control group and MAPK signaling pathway was up-regulated after CCI. We also identified an increase in overall reactivity of RIPK1 in trigeminal ganglia after the surgery. A significant proportion of RIPK1+ cells in the TG of CCI rats colocalized with the neuron cells. RT-PCR result shows the expression of NLRP3 also increased following RIPK1 activation. The results from immunofluorescence staining revealed that NLRP3 was upregulated in TG neuron cells. Also, when RIPK1 is inhibited by Nec-1, NLRP3 expression in neurons from CCI rats also decreased. Nec-1 can alleviate the demyelination of TG and mechanical allodynia after CCI. Compared to control group, RIPK1 expression is down-regulated after Nec-1 injection, followed by a decreasing in NLRP3 expression. Conclusions: This study demonstrates RIPK1 in TG neurons is activated when nerves are mechanically injuried, leading to the over-expression of NLRP3 inflammasome by activating MAPK signaling pathway. The subsequent cascade of inflammatory feedback and pain signal transmission led to the occurrence of trigeminal neuralgia.