Study On Molecular Mechanism Of Three Types Of Neuropathic Pain Based On RNA-seq

Neuropathic pain（NP）is most of the time a chronic disease caused by peripheral or central sensory nervous system injury.Unlike inflammatory pain,NP mostly does not alleviate with the remission of primary diseases（such as peripheral nerve injury,diabetes,etc.）.Because of the interaction between neurons and immune system,the plasticity of the sensory nervous system that causes the false adaptation is an important reason for NP.In clinical practice,more than half of NP patients have no response or poor response to analgesic drugs,and NP treatment has become a medical problem.Therefore,it is of great theoretical and clinical significance to deeply study the mechanism of NP.In this study,behavioral,RNA-seq,real-time fluorescent quantitative PCR（q RT-PCR）,immunofluorescence,Western blot and other technologies were used to explore the new molecular peripheral and central mechanisms of neuropathic pain induced by diabetes,sciatic nerve injury（CCI）and spinal nerve ligation（SNL）,combined with single cell sequencing（sc-RNA seq）,immunoprecipitation sequencing（Ch IP-seq）and other methods.The research contents and results include:1.The central and peripheral mechanisms of diabetes neuropathic pain（DNP）were explored.The experimental results are as follows.（1）The DNP model induced by diabetes in rats were successfully established.by means of high fat and high sugar+STZ.The weight of DNP rats on the 28th day was reduced,the blood sugar concentration was increased,and the threshold of mechanical pain was significantly reduced by means of behavioral and biochemical experiments.（2）Transcriptomics analysis showed that there were 110 and 106 up-regulated genes,119 and 110 down-regulated genes,respectively,enriched in 5 and 48 pathways,and 263and 219 molecular functions and biological processes at spinal cord horn and dorsal root ganglion of DNP.The important pathways antigen processing and presentation screened by GSEA suggest that they may affect pain at spinal dorsal horn and DRG through the immune role of dendritic cells.Further comprehensive analysis revealed that different pathways at spinal cord horn and dorsal root ganglion were activated in DNP,which at spinal cord horn were less than those at DRG.（3）Quantitative detection by q RT-PCRshowed that the expression of pain related candidate genes Fcrl2 and C1qtnf9 in spinal cord dorsal horn were decreased and the expression of Pot1b was increased,while the expression of pain related candidate gene Pcdh20 in DRG was increased.Analysis suggests that they affect the occurrence of NP by regulating immunity,inflammation,phagocytosis,NO,and the state of neurons,respectively.2.The central mechanisms of CCI-induced neuropathic pain were explored.The experimental results are as follows.（1）The CCI neuropathic model was successfully established by chronic sciatic nerve compression.The mechanical pain threshold values of CCI rats on the 8th and 28th days were significantly reduced through behavioral testing.（2）Transcriptomics analysis showed that CCI induced 131 and 215 up-regulated genes,173 and 221 down-regulated genes,15 and 14 enriched KEGG pathways,32 and34 molecular functions and biological processes in the spinal dorsal horn during the chronic and acute phases,respectively.The signal pathway of Staphylococcus aureus infection is an important pathway for CCI induced NP,which ultimately acts on neurons during the acute and chronic stages of pain.Further comprehensive analysis revealed that many of the same genes were activated during the acute and chronic stages of CCI,exercising some of the same functions,and some of the up-regulated genes in the acute（pre）stage of pain were down-regulated during the later（chronic）stage.（3）Quantitative detection of selected genes at the spinal dorsal horn and DRG by q RT-PCRshowed that the expression of genes Smc5 and Lgals5 at the spinal dorsal horn increased in the chronic phase,while the expression of Lmln decreased.The expression of genes Ceacam4 and Parvg increased in the acute phase,while the expression of Dnajb4and Klhdc9 decreased.These genes may affect pain by influencing cell replication and the number of neuronal cells,phagocytosis,cell division,and influencing phagocytosis,regulating protein folding and activity,cell cycle control,and cell matrix adhesion,respectively.（4）Western blot and immunofluorescence assay showed that the protein of Smc5 and Ceacam4 in spinal cord dorsal horn were upregulated induced by CCI.（5）Inhibition of protein function by injection of antibody neutralizing proteins Smc5and Ceacam4,behavioral methods detect that CCI induced pain threshold reduction is suppressed,further confirming the function of candidate genes.3.The central and peripheral mechanisms of SNL-induced neuropathic pain were explored.The experimental results are as follows.（1）The SNL neuropathic model was successfully established by L5 spinal nerve ligation.The mechanical pain threshold values of SNL rats on the 2th and 10th days were significantly reduced through behavioral testing.（2）Transcriptomics analysis showed that At DRG,there are 378,27,and 312 up-regulated genes in the early,late,and full stages,respectively,and 42,23,and 40 down-regulated genes.There are 58,28,and 63 up-regulated gene enriched KEGG pathways,respectively.There are 0,1,and 0 down-regulated gene enriched KEGG pathways,respectively.There are 45,12,and 55 up-regulated genes enriched GO molecular functions,and 771,156,and 803 biological processes,respectively,There are 15biological processes for early downregulation gene enrichment.（3）At the dorsal horn of the spinal cord,there are 143,820,and 1027 up-regulated genes in the early,late,and full stages,respectively,while there are 24,37,and 39down-regulated genes.There are 13,78,and 77 up-regulated gene enriched KEGG pathways,respectively.There are 0,5,and 0 down-regulated gene enriched KEGG pathways,respectively.There are 16,74,and 75 up-regulated gene enriched GO molecular functions,and 282,1385,and 1344 biological processes,respectively,The down regulated genes enriched GO molecular functions in 25,20,and 0,and biological processes in 26,9,and 3,respectively.（4）Studies shown that early NP at DRG have a large number of repair,immune,and proinflammatory reactions,while late NPs have chronic inflammatory or anti- inflammatory reactions.Early NP in the spinal cord tends to regulate cell cycle,while late NP is a process of repair,hematopoiesis,immunity,and metabolism.The repair and immune response at the DRG tend to lead to early pain,while the repair and immune response at the dorsal horn of the spinal cord tend to lead to late pain.The differential genes involved in DRG repair,immunity,and inflammation,as well as repair,cell cycle,immunity,hematopoiesis,and inflammation in the dorsal horn of the spinal cord,present certain correlations,but differ greatly.The differential genes involved in repair,immunity,and inflammation at DRG are mostly up-regulated in the early stage and down-regulated in the late stage;The differential genes involved in immunity,inflammation,cell cycle,hematopoiesis,and repair in the dorsal horn of the spinal cord are mostly down-regulated in the early stage and up-regulated in the late stage.（5）Four new pain related genes for DRG have been found,namely Cd8a,Bub1b,Ccna2,and Cdk1.Seven new pain related genes were found in the dorsal horn of the spinal cord,including B2m,Birc5,Cd3e,Csf2rb,Ddx58,Il2rb,and Casp8.（6）Using q PCR quantitative assay to detect the selected molecules,it was found that the expression of genes Cd8a,Cdk1,Ccna2,Bub1b,Cdkn1a,Btk,and Gngt2 at DRG increased in the early stage and decreased in the late stage;The expression of genes Csf2rb and Itgb2 in the dorsal horn of the spinal cord increased in the early stage and decreased in the late stage,while B2m,Cd3e,Ddx58,Il2rb,and Casp8 decreased in the early stage and increased in the late stage,while Birc5 increased in the early stage and increased in the late stage.（7）Western blot and immunofluorescence results further show that SNL induced the expression of important candidate genes Itgb2 and Csf2rb protein in the spinal dorsal horn and the expression of Cdk1 protein in DRG to increase.4.The study found that the NP induced by three models involved some of the same molecular mechanisms.The results are as follows.（1）In the spinal dorsal horn,DM and CCI activate many different genes,and chronic and acute CCI activate more genes,more KEGG pathways,and fewer GO functions than DM;In the early stage of SNL and DM,many different genes and completely different pathways were activated;Early SNL has completely different molecular functions and biological processes from DM,chronic and acute CCI.（2）In the dorsal horn of the spinal cord,the gene Tyrp1 is downregulated in CCI,SNL,and DNP;Interaction pathways between viral proteins and cytokines and cytokine receptors occur in CCI,late SNL,and DNP;Many same pathways are activated in both early and late SNL and DNP such as chemokine signaling pathway;SNL and CCI activate many same genes,both of which activate the pathway of staphylococcal infection.5.Predict the methods for applying the results of this study to NP molecular targets and drug therapy.The results are as follows.（1）Combined studies with affinity calculations have found that Cxcl10,Vdr,Pdk4,Angptl4,Usp18,and Crh,among the differentially expressed genes in DNP,have multiple compounds interacting with their proteins in the Binding DB database.（2）The staphylococcus aureus infection pathway in CCI can be C3,C4a（C4）,Fcgr3a,（FcγR）and Rat NP-3b（defensin,DEFA3,DEFA5）is used as a target genes for pain intervention.（3）Interaction between proteins corresponding to immune genes and MHC class I antibodies in SNLresults show that the peptide segments’FMSDDVFTV’of Il21r,’MPAQRRSPL’of Il1r1,’MRMATPLLM’of Cd74 at DRG,and’FLIARTWPA’of Mmp9 at the dorsal horn of the spinal cord,’RPRIQLSAL’of Cd244,and’MRMATPLLM’of Cd74 have the strongest affinity.The protein peptides’FLKELLFHI’and’SLLDIFVE M’corresponding to the differentially expressed gene Casp8 in the dorsal horn of the spinal cord have the strongest affinity with MHC I.The demethyl lovatinib quinolone with chemical formula C₂₀H₁₇Cl N₄O₅and the piloglucose group with chemical formula C₃₂H₃₆N₂O₄can interact with Casp8 protein.6.Explore the mechanism characteristics of NP participating molecules selected in this study.The results are as follows.（1）Combined with SNI single cell transcription analysis,it was found that the immune related gene Immune DRG involved in SNL induced NP at DRG was distributed in five ways:only in immune cells,only in non immune cells,only in immune and non immune cells,high（differential）expression in multiple cells,and almost no（differential）expression in all cells（type 1-5）;Immune DRG is divided into 9 types according to its homology,with the highest differential expression in specific types of cells.For example,the gene Apod is only highly（differentially）expressed in fibroblasts,or can be used as a biomarker of NP in fibroblasts;The nine homologous types of Immuno DRG have differential gene expression in all ten cells,while the genes with differential expression in Immuno DRG-1 type are mostly distributed in neurons,satellite cells,and Schwann cells.（2）Combined with Ch IP-seq（H3K4me1）analysis of SNL induced mouse NP at the dorsal horn of the spinal cord,it was found that the differential genes Ifngr1 and Lyn interacting with protein H3K4me1 may participate in NP through antigen antibody complex formation mediated by immunoglobulin Fc receptor,phagocytosis,and inflammatory reactions.In summary,studying the characteristics of NP involved molecules,their pathways and functions,as well as cellular heterogeneity may lead to new therapeutic methods targeting dysfunctional molecules,cells,and pathways of NP.