Therapeutic Interrupting C/EBPβ-Clec7a Cross-talk-mediated NLRP3 Inflammasome-dependent Pyroptosis for Relieving Neuropathic Pain

Neuropathic pain (NP) is a complex chronic pain due to the nervous system damage or diseases.During NP development and progression, the neuroinflammation has been observed along the pain pathways from the spinal cord to the thalamus and the parietal cortex. It may be caused by the activation of glial cells, especially microglia, with production of cytokines and other inflammatory mediators within the central nervous system (CNS), especially in spinal cord. In this study, we used high-throughput RNA-seq technology to detect the global gene expression in spinal cord of rats in sham group and CCI model group(an animal model of neuropathic pain), and the differentially expressed genes between diseases and control group were obtained.Significant differentially expressed genes (DEGs) of the Sham vs. CCI groups were identified using the criteria of a fold change>1.5 and P value <0.05. Finally, we focused on C/EBPβ-Clec7a Cross-talk-mediated NLRP3 Inflammasome-dependent Pyroptosis pathway and further studied its role in neuropathic pain. SC tissues (L4-L6) collected from the rats in the Sham and CCI groups (n=4 per group) on postoperative day 10 were immersed in TRIzol (Invitrogen, CA, USA) and immediately frozen in liquid nitrogen for the detection.Sequencing libraries were generated using NEBNextUltra RNA Library Prep Set for Illumina (NEB, USA.). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500/2000 platform. Gene expression profiling was analyzed using R (http://www.rproject.org/, version 3.1.1) with Bioconductor packages (http://www.bioconductor.org/). Raw intensities were normalized using Robust multi-array average (RMA method).

神经病理性疼痛（Neuropathic Pain, NP）是一类因神经系统损伤或疾病引发的复杂慢性疼痛。在神经病理性疼痛的发生与进展过程中，研究人员可在从脊髓到丘脑、顶叶皮层的痛觉通路上观测到神经炎症反应。该病症的发病机制可能与胶质细胞（尤其是小胶质细胞）的激活相关，激活后的胶质细胞会在中枢神经系统（Central Nervous System, CNS）尤其是脊髓内产生细胞因子及其他炎症介质。本研究采用高通量RNA测序（RNA-seq）技术，对假手术组与慢性压迫性损伤（Chronic Constriction Injury, CCI）模型组（神经病理性疼痛动物模型）大鼠的脊髓组织进行全基因表达水平检测，进而获取疾病组与对照组间的差异表达基因。本研究以差异倍数>1.5且P值<0.05作为筛选标准，鉴定出假手术组与CCI模型组间的显著差异表达基因（Differentially Expressed Genes, DEGs）。最终，本研究聚焦于C/EBPβ-Clec7a串扰介导的NLRP3炎性小体依赖性细胞焦亡通路，并深入探究其在神经病理性疼痛中的作用。本研究于术后第10天，采集假手术组与CCI模型组大鼠（每组n=4）的脊髓（spinal cord, SC）L4-L6节段组织，将其置于TRIzol试剂（Invitrogen，美国加利福尼亚州）中，并立即放入液氮中冻存以待后续检测。测序文库的构建采用适配Illumina平台的NEBNextUltra RNA文库制备试剂盒（NEB，美国）。带有索引标签的样本聚类操作在cBot集群生成系统上完成，所用试剂为TruSeq SR Cluster Kit v3-cBot-HS（Illumina）。集群生成完成后，将文库制备物在Illumina Hiseq 2500/2000测序平台上进行测序。基因表达谱的数据分析采用R软件（版本3.1.1，http://www.rproject.org/）及Bioconductor数据包（http://www.bioconductor.org/）完成。原始信号强度采用稳健多阵列平均法（Robust multi-array average, RMA）进行归一化处理。