Unraveling proteomic signatures and neuroinflammatory networks in a CCI rat model of early sciatica: insights for neuropathic pain mechanisms

Neuropathic pain (NP) is a complex and prevalent chronic pain condition that affects millions of individuals worldwide. Previous studies have shown that prior exercise protects against NP caused by nerve injury. However, the underlying mechanisms of this protective effect remain to be uncovered. Therefore, the purpose of this study is to investigate how prior exercise affects protein expression in NP model rats and thus gain comprehensive insights into the molecular mechanisms involved. To achieve this objective, 6-week-old male Sprague–Dawley rats were randomly assigned into three groups, named as chronic constriction injury (CCI) of the sciatic nerve, CCI with prior 6-week swimming training (CCI_Ex), and sham operated (Sham). The CCI_Ex group underwent 6 weeks of swimming training before CCI surgery, while the CCI and sham groups had no intervention. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were used as the main observation indicators to evaluate the behavioral changes associated with pain. Tissues from the spinal dorsal horn of the rats in the three groups were collected at 4 weeks after operation. LC–MS/MS proteomic analysis based on the label-free approach was used to detect protein profiles, and volcano plots, Venn diagrams, and clustering heatmaps were used to identify differentially expressed proteins (DEPs). Gene Ontology (GO) annotations, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and protein–protein interaction networks were employed to explore the biological importance of DEPs. At 14, 21, and 28 days following CCI, CCI rats with prior exercise showed a significant increase in the MWT and TWL of the injured lateral hind paw compared with those without exercise. A total of 122 proteins with significant changes in abundance were detected after CCI surgery, and 55 proteins were detected in the comparison between the CCI_Ex and CCI groups. GO and KEGG enrichment analysis revealed that oxygen transport capacity and the complement and coagulation cascades may be the critical mechanism by which prior exercise protects against NP. Serpina1, DHX9, and Alb are the key proteins in this process and warrant further attention, as confirmed by the results of Western blot analysis. In conclusion, this study provides new evidence that active physical activity can accelerate the relief of hyperalgesia after NP. Proteomic analyses revealed the potential target proteins and pathways for this process, offering valuable data resources and new insights into the pathogenesis and therapeutic targets of NP.

Objective To investigate the expression of matrix metalloproteinase-9 ( MMP-9 ) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the sciatic nerve after chronic constriction injury.Methods Ninety male SD rats were divided into control group and constriction group randomly.The chronic sciatic nerve constriction injury model was made by Mackinnon method in the constriction group.Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry staining techniques were applied to detect the expression of MMP-9 and TIMP-1 in the sciatic nerve.Results After chronic constriction injury of the sciatic nerves,demyelination occurred in the nerve fibers at the 2nd week ; at the 4th week,there was obvious hyperplasia of connective tissue; after 12 weeks,fibrous separations were increased in the nerves progressively,and fibrosis occurred in the constricted nerve.During the early phase of the constriction,the expression of MMP-9 and TIMP-1 was increased,MMP-9 mRNA reached the peak at the 2nd week by 0.0485,and TIMP-1 at the 10th week by O.1592,MMP-9/TIMP-1 was increased.As time prolonged,the expression of MMP-9 was significantly reduced while TIMP-I was elevated continuously; MMP-9/TIMP-1 was decreased evidently.Conclusion Chronic constriction injury causes the peripheral nerve fibrosis,which is related to the abnormal changes of MMP-9/TIMP-1. Key words: Peripheral nerve; Chronic constriction injury; Matrix metalloproteinase-9; Tissue inhibitor of matrix metalloproteinase-1; Fibrosis

This study analyzed protein sequencing from rat samples, categorized into a control group (Chronic Constriction Injury, CCI) and test (treated with dracorhodin perchlorate, DP), The purpose of this study was to identify key target proteins of DP in the treatment of sciatica and elucidate its underlying mechanisms. By comparing the differentially expressed proteins (DEPs) of the two groups at 1, 3, and 7 days post - treatment, it was revealed that DP consistently and significantly downregulated the expression of the Interleukin−6 (IL-6) and the cytokine-Induced neutrophil chemoattractant−3 (CINC-3), while significantly upregulating the expression of the neurotrophic factor ciliary neurotrophic factor (CNTF). Functional enrichment analysis indicated that these DEPs are primarily involved in inflammatory signaling pathways, nerve repair, and the chemotaxis of neutrophils. Validation via Western blot and ELISA demonstrated that the expression levels of IL-6 and CINC-3 were significantly reduced in the DP group, whereas the expression level of CNTF was significantly elevated, thereby confirming the significant therapeutic effect of DP on sciatica. Furthermore, experiments using a zebrafish inflammation model further confirmed that DP could significantly inhibit the recruitment and migration of neutrophils. In summary, this study not only uncovered potential target proteins for DP in the treatment of sciatica but also provided crucial evidence for its mechanism of action, suggesting that DP may effectively alleviate sciatica by downregulating IL-6 and CINC-3, inhibiting neutrophil migration, and upregulating CNTF to promote neurotrophic effects.

To investigate whether mammalian target of rapamycin (mTOR) signaling pathway is involved in peripheral nerve injury-induced hyperalgesia through activation of spinal dorsal astrocytes in rats. Methods: A total of 30 male Sprague-Dawley (SD) rats were randomly divided into 6 groups (n=5): the 1 day group (D1 group), the 4 days group (D4 group), the 7 days group (D7 group), the 14 days group (D14 group), the normal group and the sham group. The sciatic nerve chronic constriction injury (CCI) model was established in the D1, D4, D7 and D14 group. The normal group received no treatment while the sham group was only exposed the sciatic nerve. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at the 1st, 4th, 7th, and 14th day after CCI in the different groups. Lumbar spinal cord were harvested on the 1st, 4th, 7th and 14th day in the D1, D4, D7, D14 group correspondingly, which were harvested on the 14th day in the normal group and the sham group. Distribution of mTOR in rat spinal cord was assessed by immunohistochemistry. The expressions of mTOR mRNA and protein in the spinal cord in different groups were determined by real-time PCR and Western blotting, respectively. Another 30 male intrathecal catheterized SD rats were randomly divided into 6 groups (n=5): a blank group, a CCI group, a CCI+early rapamycin (RAPA) group, a CCI+early dimethylsulfoxide (DMSO) group, a CCI+ later RAPA group, and a CCI+later DMSO group. The blank group didn't received any treatment; The CCI group was carried out the treatment of CCI model in the left hind limbs. 10 μL of 1% RAPA was given to the CCI+early RAPA group intrathecally at 4 hours after CCI for 3 days; the CCI+later RAPA group were treated with the same dose of RAPA on the 7th days after CCI for 3 days; the CCI+early DMSO group and the CCI+later DMSO group were injected with the same volume of 4% DMSO at the corresponding time as controls. The PWTL and PWMT were measured before and after intrathecal catheterization, and every other day after CCI. The lumbar spinal cords were selected and the expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn were examined by immunohistochemistry in the 14th day after CCI. Results: The immunohistochemistry positive particles of mTOR were widely distributed in the cytoplasm of the normal spinal neurons. Compared with the base line, the PWMT in the D14 group on the 1st, 4th, 7th and 14th day after CCI were significantly lower, and the PWTL on the 4th, 7th and 14th day after CCI were also significantly lower (P<0.05 or P<0.01). The expressions of mTOR mRNA and protein in the CCI groups (D1, D4, D7 and D14 group) were significantly increased than those in the normal group (P<0.05 or P<0.01). Compared with the CCI+early DMSO group, the PWMT and PWTL in the CCI+early RAPA group were obviously increased on 4th, 6th, 8th, 10th, 12th or 14th day after CCI (P<0.05 or P<0.01); compared with the CCI+later DMSO group, the PWMT and PWTL in the CCI+later RAPA group were also significantly increased at the 8th, 10th or 14th day after CCI (P<0.01 or P<0.05). The GFAP immunohistochemistry positive area and absorbance value in the dorsal horn of the lumbar spinal cord in the CCI rats were decreased in the CCI+early RAPA group compared with the CCI+early DMSO group (P<0.05 or P<0.01), and which were also decreased in the CCI+later RAPA group compared with the CCI+later DMSO group (P<0.05 or P<0.01). Conclusion: mTOR signaling pathway may be involved in hyperalgesia induced by peripheral nerve injury via spinal astrocyte activation in the dorsal horn of the spinal cord.

Vein wrapping facilitates basic fibroblast growth factor-induced heme oxygenase-1 expression following chronic nerve constriction injury.

Objective To evaluate the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in cognitive dysfunction caused by chronic pain in rats.Methods The experiment was performed in 2 parts.In experiment Ⅰ,24 pathogen-free male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 4 groups (n =6 each) using a random number table:sham operation group (group S),m-AIP injected before sham operation group (group M-S),chronic sciatic nerve injury group (group N-C),and m-AIP injected before chronic constriction injury (CCI) group (group M-C).The sciatic nerve was only exposed but not ligated.Chronic pain was induced by CCI in N-C and M-C groups.The animals were anesthetized with intraperitoneal 1％ pentobarbital sodium.The sciatic nerve was exposed and 4 ligatures were placed on the sciatic nerve at 1 mm intervals.Normal saline 20 μ1 and m-AIP 20 μ/ were injected intrathecally at 15 min before sham operation in S and M-S groups,respectively,and at 15 min before CCI in N-C and M-C groups,respectively.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before CCI and on 4,7,10,14,17,21 and 28 days after CCI.Step-through latency (STL) was measured before CCI and on 7,14,21 and 28 days after CCI.In experiment Ⅱ,18 pathogen-free male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 3 groups (n =6 each) using a random number table:m-AIP injected after sham operation group (group C-N),control after CCI group (group C-N) and m-AIP injected after CCI group (group C-M).Group S-M received intrathecal injection of m-AIP 20 μl at 7 days after sham operation.Normal saline 20 μl and m-AIP 20 μ/ were injected intrathecally at 7 days after CCI in C-N and C-M groups,respectively.MWT,TWL and STL were measured before administration and at 2,4 and 8 h after administration.Results In experiment Ⅰ,compared with group S,MWT was significantly decreased at each time point after CCI,TWL was shortened at each time point after CCI and STL was shortened on 7,14 and 21 days after CCI in N-C group,and MWT was significandy decreased at each time point,TWL was shortened at each time point,and STL was shortened on 14 and 21 days after CCI in group M-C.Compared with group N-C,MWT was significantly increased on 4,7 and 10 days after CCI,TWL was prolonged on 4 and 7 days after CCI,and STL was prolonged on 7 days after CCI in group M-C.In experiment Ⅱ,compared with group S-M,MWT was significantly decreased,and TWL and STL were shortened at each time point after administration in C-N group,and TWL at 8 h after administration and STL at each time point after administration were shortened,MWT was decreased at 8 h after administration,and no significant change was found in MWT and TWL at 2 and 4 h after administration in group C-M Compared with group C-N,MWT was significantly increased,and TWL was prolonged at 2 and 4 h after administration,and no significant change was found in STL at each time point after administration in group C-M.Conclusion CaMK Ⅱ is involved in the development of cognitive dysfunction caused by chronic pain in rats. Key words: Calcium-calmodulin-dependent protein kinase type 2; Neuralgia; Cognition disorders

Objective To investigate the effect of hyperbaric oxygen on the expression of fractalkine (FKN) in the nerve tissues of rats with neuropathic pain (NP). Methods Thirty-two male Sprague-Dawley rats, weighing 250-280 g, aged 10-12 weeks, were divided into 4 groups (n= 8 each) using the random number table: control group (group C), sham operation group (group S), group NP, and hyperbaric oxygen group (group H). NP was induced by chronic constriction injury (CCI) in anesthetized rats. The left sciatic nerve was exposed, and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread. Group H received hyperbaric oxygen therapy once a day for 5 consecutive days starting from day 1 after CCI. The rats were placed into the hyperbaric oxygen chamber, which was pressurised to 2 atmosphere absolute at a rate of 10 kPa/min, and maintained at this level for 60 min. The pressure was then decreased to the normal pressure at a rate of 10 kPa/min. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before CCI, and 3, 5, 7 and 14 days after CCI. After measurement of pain threshold at 3 and 7 days after CCI, 4 rats were selected and sacrificed. The sciatic nerve and lumbar segment of the spinal cord were removed for determination of the expression of FKN by Western blot. Results Compared with group C, the MWT was significantly decreased, and TWL was shortened at each time point after CCI, the expression of FKN in the sciatic nerve at 3 days after CCI, and in the sciatic nerve and spinal cord at 3 and 7 days after CCI was up-regulated in group NP (P 0.05). Compared with group NP, the MWT was significantly increased, and TWL was prolonged at each time point after CCI, and the expression of FKN in the sciatic nerve at 3 days after CCI, and in the sciatic nerve and spinal cord at 3 and 7 days after CCI was down-regulated in group H (P<0.05). Conclusion The mechanism by which hyperbaric oxygen mitigates NP is related to inhibition of over-expression of FKN in the nerve tissues of rats. Key words: Hyperbaric oxygenation; Neuralgia; Chemotactic factors

Editor's evaluation: Deep proteome profiling reveals signatures of age and sex differences in paw skin and sciatic nerve of naïve mice

Although efficacious clinical results have been reported after vein wrapping for the treatment of recurrent compressive neuropathy, the mechanism of nerve protection remains uncertain. Eight-week-old male Wistar rats (n = 90) were randomly divided into three groups: sham procedure, chronic constriction injury, and chronic constriction injury plus vein wrapping. Mechanical withdrawal thresholds and walking patterns were measured with use of von Frey filaments and the CatWalk system, respectively. We investigated L4-L5 dorsal root ganglia immunohistochemically at fourteen days postsurgery and sciatic nerves histologically at fourteen days and again five months postsurgery. Concentrations of several sciatic neurotrophic factors in the ligated sciatic nerves were quantified with use of ELISA (enzyme-linked immunosorbent assay). In behavioral tests, the rats in which the chronic constriction injury had been followed by vein wrapping displayed significantly greater pain responses than the sham group, and the group with untreated chronic constriction injury showed greater pain responses than the vein-wrapping group (both p &lt; 0.05). Immunoreactive markers of inflammation and nerve damage, calcitonin gene-related peptide (CGRP) and activating transcription factor-3 (ATF3), were upregulated in dorsal root ganglion neurons in the constriction-injury and vein-wrapping groups compared with those in the sham group, with greater upregulation in the constriction-injury group than in the vein-wrapping group (both p &lt; 0.01). Histologic observation showed marked nerve degeneration and scar tissue formation around the sciatic nerve in the constriction-injury group, but these effects were prevented to some extent in the vein-wrapping group. Vascular endothelial growth factor (VEGF) levels at one and three days postsurgery and hepatocyte growth factor (HGF) levels at three, seven, fourteen, and twenty-eight days postsurgery were significantly higher in the vein-wrapping group than in the other groups (p &lt; 0.05). Vein wrapping decreased pain-associated behavior and nerve damage caused by chronic constriction injury. VEGF and HGF produced in response to vein grafts may play a mechanistic role. These findings may lead to development of new therapies employing growth factors, with or without other materials, that simulate vein wrapping.

Objective To evaluate the role of extracellular signal-related kinase (ERK) 1/2 signal transduction pathway at the supraspinal level in maintenance of neuropathic pain in mice.Methods Sixty-four Kunming mice,aged 2 months,weighing 18-20 g,were randomly divided into 4 groups (n =16 each):sham operation group (group S),chronic constrictive injury (CCI) group; CCI + U0126 (MEK inhibitor) group; CCI + dimethyl sulfoxide (DMSO) group.Neuropathic pain was induced by CCI.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1mmintervals with 4-0 silk thread in CCI,CCI + U0126 and CCI +DMSO groups.On 5 days after CCI,5 μg U0126 (in 5 μl of 5％ DMSO) and 5％ DMSO 5 μl were injected into the lateral cerebral ventricle over 10 s in CCI + U0126 and CCI + DMSO groups,respectively,and the time of.needle retaining was 20 s.Paw withdrawal threshold to mechanical stimulation with yon Frey filament (MWT) and paw withdrawal latency to thermal stimulation (TWL) were measured before operation (baseline),before intracerebroventricular injection (T1),and at 30 min and 2,6,12 and 24h after intracerebroventricular injection (T2-6).Resuits Compared with group S,MWT was significantly decreased and TWL was shortened at T1-6 in CCI and CCI +DMSO groups,and at T1 in CCI + U0126 group (P ＜ 0.05),while no significant change in MWT and TWL was found at T2-6 in group CCI + U0126 (P ＞ 0.05).Compared with group CCI,MWT was significantly increased and TWL was prolonged at T2-6 in group CCI + U0126 (P ＜ 0.05),while no significant change in MWT and TWL was found in group CCI + DMSO (P ＞ 0.05).Conclusion ERK1/2 signaling transduction pathway at the supraspinal level is involved in maintenance of neuropathic pain in mice. Key words: Neuralgia; Extracellular signal-regulated MAP kinases

Objective It has been shown that the AMPK signaling pathway helps maintain the structure of nerve cells and prevent damage caused by hypoxia by preserving the energy available in these cells. This signaling pathway is activated by various drugs, including metformin. Methods In this study, thirty male rats were randomly divided into healthy rats, CCI (chronic constriction injury), CCI + metformin, CCI + dorsomorphin, and CCI + metformin + dorsomorphin groups. Behavioral tests were performed on the third, seventh, and fourteenth days after the induction of CCI. On the last day, the inflammatory cytokines and pathology were investigated after dissecting the spinal cords and sciatic nerves of the animals, respectively. Results The results of this study indicated that metformin could improve performance in behavioral tests and reduce the levels of inflammatory cytokines. Histological analyses further revealed that metformin could decrease inflammation and necrosis in sciatic nerve tissue. Notably, this effect was observed despite dorsomorphin injection, which prevented the therapeutic effects of metformin in the fifth group. Conclusion The results of this study indicated that stimulation of the AMPK signaling pathway could effectively preserve the structure of the sciatic nerve, reduce inflammation, and improve responses to behavioral tests.

BackgroundNeuropathic pain is caused by damage to the nervous system, resulting in aberrant pain, which is associated with gene expression changes in the sensory pathway. However, the molecular mechanisms are not fully understood.MethodsWistar rats were employed for the establishment of the chronic constriction injury (CCI) models. Using the Illumina HiSeq 4000 platform, we examined differentially expressed genes (DEGs) in the rat dorsal horn by RNA sequencing (RNA-seq) between CCI and control groups. Then, enrichment analyses were performed for these DEGs using Gene Ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Hierarchical Cluster, and protein-protein interaction (PPI) network.ResultsA total of 63 DEGs were found significantly changed with 56 upregulated (e.g., Cxcl13, C1qc, Fcgr3a) and 7 downregulated (e.g., Dusp1) at 14 days after CCI. Quantitative reverse-transcribed PCR (qRT-PCR) verified changes in 13 randomly selected DEGs. GO and KEGG biological pathway analyses showed that the upregulated DEGs were mostly enriched in immune response-related biological processes, as well as 14 immune- and inflammation-related pathways. The downregulated DEGs were enriched in inactivation of mitogen-activated protein kinase (MAPK) activity. PPI network analysis showed that Cd68, C1qc, C1qa, Laptm5, and Fcgr3a were crucial nodes with high connectivity degrees. Most of these genes which have previously been linked to immune and inflammation-related pathways have not been reported in neuropathic pain (e.g., Laptm5, Fcgr3a).ConclusionsOur results revealed that immune and defense pathways may contribute to the generation of neuropathic pain after CCI. These mRNAs may represent new therapeutic targets for the treatment of neuropathic pain.

Glial NF-kappa B inhibition alters neuropeptide expression after sciatic nerve injury in mice

Neuropathic pain (NP) is one of the most common forms of chronic pain, yet current treatment options are limited in effectiveness. Peripheral nerve injury activates spinal microglia, altering their inflammatory response and phagocytic functions, which contributes to the progression of NP. Most current research on NP focuses on microglial inflammation, with relatively little attention to their phagocytic function. Early growth response factor 2 (EGR2) has been shown to regulate microglial phagocytosis, but its specific role in NP remains unclear. This study aims to investigate how EGR2 modulates microglial phagocytosis and its involvement in NP, with the goal of identifying potential therapeutic targets. Adult male Sprague-Dawley (SD) rats were used to establish a chronic constriction injury (CCI) model of the sciatic nerve. Pain behaviors were assessed on days 1, 3, 7, 10, and 14 post-surgery to confirm successful model induction. The temporal and spatial expression of EGR2 in the spinal cord was examined using real-time quantitative PCR (RT-qPCR), Western blotting, and immunofluorescence staining. Adeno-associated virus (AAV) was used to overexpress EGR2 in the spinal cord, and behavioral assessments were performed to evaluate the effects of EGR2 modulation of NP. CCI and lipopolysaccharide (LPS) models were established in animals and microglial cell lines, respectively, and changes in phagocytic activity were measured using RT-qPCR and fluorescent latex bead uptake assays. After confirming the involvement of microglial phagocytosis in NP, AAV was used to overexpress EGR2 in both in vivo and in vitro models, and phagocytic activity was further evaluated. Finally, eukaryotic transcriptome sequencing was conducted to screen differentially expressed mRNAs, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to identify potential downstream effectors of EGR2. The CCI model successfully induced NP. Following CCI, EGR2 expression in the spinal cord was upregulated in parallel with NP development. Overexpression of EGR2 via spinal AAV injection enhanced microglial phagocytic activity and increased pain hypersensitivity in rats. Both animal and cellular models showed that CCI or LPS stimulation enhanced microglial phagocytosis, which was further amplified by EGR2 overexpression. Transcriptomic analysis of spinal cord tissues from CCI rats overexpressing EGR2 revealed upregulation of numerous genes associated with microglial phagocytosis and pain regulation. Among them, Lag3 emerged as a potential downstream target of EGR2. EGR2 contributes to the maintenance of NP by enhancing microglial phagocytosis in the spinal dorsal horn.

The aim of the present study was to identify potential key candidate genes and mechanisms associated with rheumatoid arthritis (RA). Gene expression data from GSE55235, GSE55457 and GSE1919 datasets were downloaded from the Gene Expression Omnibus database. These datasets comprised 78 tissue samples collectively, including 25 healthy synovial membrane samples and 28 RA synovial membrane samples, whilst the 25 osteoarthritis (OA) samples were not included in the analysis. The differentially expressed genes (DEGs) between the two types of samples were identified with the Linear Models for Microarray Analysis package in R. Gene Ontology (GO) functional term and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analyses were also performed. In addition, Protein-Protein Interaction (PPI) network and module analyses were visualized using Cytoscape, and subsequent hub gene identification as well as GO and KEGG enrichment analyses of the modules was performed. Finally, reverse transcription-quantitative PCR (RT-qPCR) was used to validate the expression of the DEGs identified by GO and KEGG analysis in vitro. The analysis identified 491 DEGs, including 289 upregulated and 202 downregulated genes, which were mainly enriched in the following pathways: ‘Cytokine-cytokine receptor interaction’, ‘Rheumatoid arthritis’, ‘Chemokine signaling pathway’, ‘Intestinal immune network for IgA production’ and ‘Primary immunodeficiency’. The top 10 hub genes identified from the PPI network were IL-6, protein tyrosine phosphatase receptor type C, VEGFA, CD86, EGFR, C-X-C chemokine receptor type 4, matrix metalloproteinase 9, CC-chemokine receptor type (CCR)7, CCR5 and selectin L. KEGG signaling pathway enrichment analysis of the top two modules identified from the PPI network revealed that the genes in Module 1 were mainly enriched in the ‘Cytokine-cytokine receptor interaction’ and ‘Chemokine signaling pathway’, whereas analysis of Module 2 revealed that the genes were mainly enriched in ‘Primary immunodeficiency’ and ‘Cytokine-cytokine receptor interaction’. Finally, the results of the RT-qPCR and western blot analysis demonstrated that the expression levels of inflammation and NF-κB signaling pathway-related mRNAs were significantly upregulated following lipopolysaccharide stimulation. In conclusion, the findings of the present study identified key genes and signaling pathways associated with RA, which may improve the current understanding of the molecular mechanisms underlying its development and progression. The identified hub genes may also be used as potential targets for RA diagnosis and treatment.