A retrospective study at a single center examining risk factors associated with central nervous system involvement in individuals diagnosed with diffuse large B-cell lymphoma.

OBJECTIVE: The aim of this study is to identify risk factors contributing to central nervous system (CNS) invasion and to validate the suitability of the Central Nervous System International Prognostic Index (CNS-IPI) for individuals afflicted with diffuse large B-cell lymphoma (DLBCL). METHODS: Based on the presence or absence of CNS invasion, 365 patients were stratified into two groups: the CNS group and the non-CNS group. The clinical data of the patients were retrospectively analyzed using univariate and multivariate analysis, and the differences in survival curves were compared. The dependent variable in this study was the presence or absence of CNS invasion, while the independent variables included age, stage, extranodal involvement, renal/adrenal involvement, and others. Statistical methods included the chi-squared test and Fisher's exact test for intergroup comparison and binary logistic regression for multi-factor analysis. The related risk factors were modeled using the Cox proportional hazards model. The Kaplan-Meier method was used to generate survival curves, and the log-rank test was used to compare the differences between survival curves. The optimal cutoff value of beta-2 (ß2)-microglobulin was determined through the utilization of a receiver operating characteristic (ROC) curve. All P values were bidirectional, and P < 0.05 was considered statistically significant. Both SPSS 23.0 (IBM Inc., Armonk, NY, USA) and RStudio (R software version 4.0.2, R Project for Statistical Computing) software were used for data processing RESULTS: The five factors of the CNS-IPI were related to the prognosis of patients with CNS invasion. Bone involvement, albumin < 40 g/L, and P53 protein (+) were the risk factors for CNS invasion in patients with DLBCL. However, prognostic factors such as double strike, testicular involvement, breast involvement, uterine involvement, and bone marrow involvement did not apply to these patients. It was also discovered that elderly patients with DLBCL with reduced albumin levels were more susceptible to CNS invasion. Furthermore, extranodal involvement at multiple sites and elevated beta-2 (ß2) microglobulin were independent prognostic factors CONCLUSION: Patients older than 60 years with DLBCL and decreased albumin are at increased risk for CNS invasion. In addition to the five factors in the CNS-IPI, bone involvement, albumin levels < 40 g/L, and P53 protein expression are risk factors affecting the prognosis of CNS invasion in patients with DLBCL.

Activation of pyramidal neurons in the infralimbic cortex alleviates LPS-induced depressive-like behavior in mice.

The infralimbic (IL) cortex dysfunction has been implicated in major depressive disorder (MDD), yet the precise cellular and molecular mechanisms remain poorly understood. In this study, we investigated the role of layer V pyramidal neurons in a mouse model of MDD induced by repeated lipopolysaccharide (LPS) administration. Our results demonstrate that three days of systemic LPS administration induced depressive-like behavior and upregulated mRNA levels of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-ß (TGF-ß) in the IL cortex. Electrophysiological recordings revealed a significant decrease in the intrinsic excitability of layer V pyramidal neurons in the IL following systemic LPS exposure. Importantly, chemogenetic activation of IL pyramidal neurons ameliorated LPS-induced depressive-like behavior. Additionally, LPS administration significantly increased microglial activity in the IL, as evidenced by a greater number of Ionized calcium binding adaptor molecule-1 (IBA-1)-positive cells. Morphometric analysis further unveiled enlarged soma, decreased branch numbers, and shorter branch lengths of microglial cells in the IL cortex following LPS exposure. Moreover, the activation of pyramidal neurons by clozapine-N-oxide increased the microglia branch length but did not change branch number or cytosolic area. These results collectively suggest that targeted activation of pyramidal neurons in the IL cortex mitigates microglial response and ameliorates depressive-like behaviors induced by systemic LPS administration. Therefore, our findings offer potential therapeutic targets for the development of interventions aimed at alleviating depressive symptoms by modulating IL cortical circuitry and microglial activity.

Ion channels in cancer-induced bone pain: from molecular mechanisms to clinical applications.

Cancer-induced bone pain (CIBP) caused by bone metastasis is one of the most prevalent diseases, and current treatments rely primarily on opioids, which have significant side effects. However, recent developments in pharmaceutical science have identified several new mechanisms for CIBP, including the targeted modification of certain ion channels and receptors. Ion channels are transmembrane proteins, which are situated on biological cell membranes, which facilitate passive transport of inorganic ions across membranes. They are involved in various physiological processes, including transmission of pain signals in the nervous system. In recent years, there has been an increasing interest in the role of ion channels in chronic pain, including CIBP. Therefore, in this review, we summarize the current literature on ion channels, related receptors, and drugs and explore the mechanism of CIBP. Targeting ion channels and regulating their activity might be key to treating pain associated with bone cancer and offer new treatment avenues.

Circulating Tumor DNA Characteristics Based on Next Generation Sequencing and Its Correlation With Clinical Parameters in Patients With Lymphoma.

Background: Lymphoma is a heterogeneous group of tumors in terms of morphological subtypes, molecular alterations, and management. However, data on circulating tumor DNA (ctDNA) mutated genes are limited. The purpose of this study was to investigate the features of the ctDNA mutated genes, the prognosis, and the association between the ctDNA mutated genes and the clinical parameters in lymphoma. Methods: Differences in the ctDNA between the mutated genes and the prognosis of 59 patients with Hodgkin's lymphoma (HL) (10.2%), germinal center B-cell-like lymphoma (GCB) (28.8%), nongerminal center B-cell-like lymphoma (non-GCB) (50.8%), and marginal zone lymphoma (MZL) (10.2%) were analyzed by next generation sequencing (NGS) targeting 121 lymphoma-relevant genes. Results: Genetic alterations were identified in the ctDNA samples with a median of 6 variants per sample. The genetic variation of the ctDNA in the plasma was found to be significantly correlated with the clinical indices in lymphoma. The genetic heterogeneity of different lymphoma subtypes was clearly observed in the ctDNAs from HL, GCB, non-GCB, and MZL, confirming that distinct molecular mechanisms are involved in the pathogenesis of different lymphomas. Conclusion: Our findings suggest that NGS-based ctDNA mutation analysis reveals genetic heterogeneity across lymphoma subtypes, with potential implications for discovering therapeutic targets, exploring genomic evolution, and developing risk-adaptive therapies.

Systemic immune inflammation index, ratio of lymphocytes to monocytes, lactate dehydrogenase and prognosis of diffuse large B-cell lymphoma patients.

BACKGROUND: In malignant tumors, inflammation plays a vital role in the development, invasion, and metastasis of cancer cells. Diffuse large B-cell lymphoma (DLBCL), the most common malignant proliferative disease of the lymphatic system, is commonly associated with inflammation. The international prognostic index (IPI), which includes age, lactate dehydrogenase (LDH), number of extranodal lesions, Ann Arbor score, and Eastern Cooperative Oncology Group (ECOG) score, can evaluate the prognosis of DLBCL. However, its use in accurately identifying high-risk patients and guiding treatment is poor. Therefore, it is important to find novel immune markers in predicting the prognosis of DLBCL patients. AIM: To determine the association between the systemic immune inflammation index (SII), ratio of lymphocytes to monocytes (LMR), ratio of LMR to LDH (LMR/LDH), and prognosis of patients with DLBCL. METHODS: A total of 68 patients diagnosed with DLBCL, treated in our hospital between January 2016 and January 2020, were included. χ 2 test, Pearson's R correlation, Kaplan Meier curves, and Cox proportional risk regression analysis were used. The differences in the SII, LMR, and LMR/LDH among patients with different clinicopathological features were analyzed. The differences in progression-free survival time among patients with different SII, LMR, and LMR/LDH expressions and influencing factors affecting the prognosis of DLBCL patients, were also analyzed. RESULTS: The LMR and LMR/LDH in patients with Ann Arbor stage III-IV, ECOG score ≥ 2, and SII, IPI score 2-5 were significantly higher than those of patients with Ann Arbor stage I-II and ECOG score < 2 (P < 0.05). Patients with high SII, LMR, and LMR/LDH had progression-free survival times of 34 mo (95%CI: 32.52-38.50), 35 mo (95%CI: 33.42-36.58) and 35 mo (95%CI: 33.49-36.51), respectively, which were significantly lower than those with low SII, LMR, and LMR/LDH (P < 0.05); the SII, LMR, and LMR/LDH were positively correlated (P < 0.05). Cox proportional risk regression analysis showed that the SII, LMR, and LMR/LDH were influencing factors for the prognosis of DLBCL patients (hazard ratio = 1.143, 1.665, and 1.704, respectively; P < 0.05). CONCLUSION: The SII, LMR, and LMR/LDH are related to the clinicopathological features of DLCBL, and they also influence the prognosis of patients with the disease.

Epigallocatechin-3-gallate attenuates acute pancreatitis induced lung injury by targeting mitochondrial reactive oxygen species triggered NLRP3 inflammasome activation.

Green tea has been considered as a health-promoting beverage and is widely consumed worldwide. Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol derived from green tea leaves with potent antioxidative and chemopreventive activities, has been reported to offer protection against inflammation-driven tissue damage. Here, we evaluated the protective effects of EGCG against lung injury during acute pancreatitis (AP) and further revealed the detailed mechanism. The results showed that EGCG significantly attenuated l-arginine-induced AP and the consequent pulmonary damage in mice. Moreover, EGCG substantially attenuated oxidative stress and concurrently suppressed NOD-like receptor protein 3 (NLRP3) inflammasome activation in the lung. In vitro, EGCG considerably reduced the production of mitochondrial reactive oxygen species (mtROS) and oxidized mitochondrial DNA (ox-mtDNA) in alveolar macrophages (AMs) challenged with AP-conditioned plasma. Meanwhile, the amount of ox-mtDNA bound to NLRP3 decreased significantly by the treatment with EGCG, resulting in impaired NLRP3 inflammasome activation. In addition, the antagonism of NLRP3 signaling by EGCG was affected in the presence of the mtROS stimulant rotenone or scavenger Mito-TEMPO. Altogether, EGCG possesses potent activity to attenuate lung injury during AP progression by inhibiting NLRP3 inflammasome activation. As for the mechanism, the EGCG-conferred restriction of NLRP3 inflammasome activation probably arises from the elimination of mtROS as well as its oxidative product ox-mtDNA, which consequently enables the protection against AP-associated lung injury.

Thoracoscopic total laryngo-pharyngo-oesophagectomy for the pharyngoesophageal junction cancer: a single-center experience of multidisciplinary team.

PURPOSE: The aims of this study were to review the surgical experience and evaluate the feasibility of thoracoscopic total laryngo-pharyngo-oesophagectomy by multidisciplinary team in the patients with pharyngoesophageal junction cancer. METHODS: A total of 31 patients with pharyngoesophageal junction cancer who underwent thoracoscopic total laryngo-pharyngo-oesophagectomy with gastric pull-up reconstruction performed by a collaborative thoracic surgery and otolaryngology surgery team in our department from January 2009 to January 2019 were retrospectively analysed. Surgical experience, Postoperative morbidity, overall survival were evaluated. RESULTS: The median age was 62 years old. Among these patients, 20 had hypopharyngeal cancer, 11 had cervical oesophageal cancer. No patients died during the perioperative period, and the median operation time was 4 h 30 min. The mean hospital stay was 13 days. The rate of complications was 32.3%. There were two cases of anastomotic leakage, four cases of moderate pulmonary infection. The median follow-up period was 31 months. Four patients were lost to follow-up in the second and fourth years and were considered to have died at that time. The 3- and 5-year overall survival rates were 52.6% and 31.6%, respectively. CONCLUSION: As a salvage surgery, thoracoscopic total laryngo-pharyngo-oesophagectomy by multidisciplinary team can be performed with an acceptable level of perioperative morbidity and mortality, relatively good recovery, and acceptable survival outcome for patients with pharyngoesophageal junction cancer.

[Clinical Value of Serum Amyloid A and Misfolded Transthyretin for Relapsed/Refractory Diffuse Large B-Cell Lymphoma Patients].

OBJECTIVE: To evaluate the clinical value of serum amyloid A (SAA1/2) and misfolded transthyretin (TTR) for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) patients. METHODS: 30 R/R DLBCL patients were enrolled as observation group, 20 remission/stabilization DLBCL and 10 chronic lymphadenitis patients were enrolled as control group. SELDI technique, Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, the shotgun-LTQ-MS method, and bioinformatics technique were used to detected and analyzed SAA and TTR in R/R DLBCL patients. SPSS 21.0 software was used to analyze the relationship between the high expression of SAA, misfolded TTR in serum and the clinicopathological features, survival time of R/R DLBCL. patients Chi-square test was used to analyze clinical count data, Kaplan-Meier curve was used for survival analysis, and Log-Rank test was used to compare single-factor survival differences. RESULTS: The high expression of SAA and TTR (SAA+TTR+) was significantly associated with extranodal lesion, high level of LDH, and NCCN-IPI scores, and also correlated with non-GCB type. TTR+ was correlated with C-MYC in pathological tissue, while SAA+ was also associated with B-symptoms. The survival time of patients in SAA+, TTR+, and SAA+TTR+ group were shorter than that in control group. CONCLUSION: Both SAA and misfolded TTR are poor prognosis factors of R/R DLBCL patients.

miR-146a promoted breast cancer proliferation and invasion by regulating NM23-H1.

The study aimed to investigate the regulatory effect of miR-146a in proliferation, invasion and migration of breast cancer and its possible mechanism via NM23-H1. The expression levels of miR-146a in breast cancer with different pathological classification were significantly increased, while the expression levels of NM23-H1 were significantly decreased, which were closely correlated. Double luciferase reporter gene was used to verify the target regulatory relationship between miR-146 and NM23-H1 on a human breast cancer cell line. miR-146a was closely related to the proliferation and metastasis of breast cancer. miR-146a also promoted the growth of breast cancer in vivo via targeting NM23-H1. In conclusion, miR-146 can promote the proliferation and invasion of breast cancer by targeting NM23-H1.

[Effect of P53 Expression on Prognosis of Patients with Double Expressor Lymphoma].

OBJECTIVE: To investigate the effect of P53 expression on prognosis of patients with double expressor lymphoma(DEL) and the interaction between the expression of MYC, BCL2 and P53 in diffuse large B-cell lymphoma(DLBCL). METHODS: Eighty-eight patients with newly diagnosed DLBCL from 1st September 2012 to 31th May 2018 in Shanxi Dayi Hospital affiliated to Shanxi Medical University were selected. The expressions of MYC、BCL2、P53、CD10、BCL6、MUM and Ki-67 were tested by immunohistochemistry method. The overall survival of patients was analyzed by the Kaplan-Meier method and compared by the log-rank test. The prognostic effect of MYC, BCL2 and P53 expression was analyzed by univariate and multivariate analysis. RESULTS: Compared with patients without P53 expression, the patients with P53 expression had higher LDH level, higher NCCN-IPI scores, lower response to chemotherapy，poorer overall survival(OS) and a higher rate of death(P<0.05). In patients who had diffuse large B-cell lymphoma associated with MYC, BCL2 expression or MYC+/BCL2+ double expression， compared with the patients whom without P53 expression, P53 expression associated with a significant worse OS (P<0.05). The patients with concurrent MYC and P53 expression had a worse OS, compared with patients with either P53 or MYC expression(P<0.05). In patients with MYC+/P53+ co-expression, BCL2 expression did not correlate with poorer survival significantly(P>0.05). Among lymphoma patients with MYC+/P53+, MYC+/BCL2+ and BCL2+/P53+ co-expression, the patients with MYC+/P53+ co-expression had the worse OS (3 year OS rate：31.6%), followed by the subgroup of patients with MYC-/BCL2+/P53+(3 year OS rate：46.2%), patients with MYC+/BCL2+/P53- expression(3 year OS rate： 636%) showed a longer OS compared with the other two subgroups（P<0.05）. Multivariate analysis demonstrated that P53 expression and NCCN-IPI were independent prognostic factors in this patient cohort. CONCLUSION: P53 and MYC expressions have a synergistically negative prognostic effect in DLBCL patients. P53 expression augments the negative prognostic effect of MYC+/BCL2+ double expression. Patients with MYC+/P53+ co-expression have a worse prognosis in comparison with the patients with MYC+/BCL2+ double expression.

Increased CXCL13 and CXCR5 in Anterior Cingulate Cortex Contributes to Neuropathic Pain-Related Conditioned Place Aversion.

Pain consists of sensory-discriminative and emotional-affective components. The anterior cingulate cortex (ACC) is a critical brain area in mediating the affective pain. However, the molecular mechanisms involved remain largely unknown. Our recent study indicated that C-X-C motif chemokine 13 (CXCL13) and its sole receptor CXCR5 are involved in sensory sensitization in the spinal cord after spinal nerve ligation (SNL). Whether CXCL13/CXCR5 signaling in the ACC contributes to the pathogenesis of pain-related aversion remains unknown. Here, we showed that SNL increased the CXCL13 level and CXCR5 expression in the ACC after SNL. Knockdown of CXCR5 by microinjection of Cxcr5 shRNA into the ACC did not affect SNL-induced mechanical allodynia but effectively alleviated neuropathic pain-related place avoidance behavior. Furthermore, electrophysiological recording from layer II-III neurons in the ACC showed that SNL increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), decreased the EPSC paired-pulse ratio, and increased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio, indicating enhanced glutamatergic synaptic transmission. Finally, superfusion of CXCL13 onto ACC slices increased the frequency and amplitude of spontaneous EPSCs. Pre-injection of Cxcr5 shRNA into the ACC reduced the increase in glutamatergic synaptic transmission induced by SNL. Collectively, these results suggest that CXCL13/CXCR5 signaling in the ACC is involved in neuropathic pain-related aversion via synaptic potentiation.

Overexpression of miR-758 inhibited proliferation, migration, invasion, and promoted apoptosis of non-small cell lung cancer cells by negatively regulating HMGB.

Non-small cell lung cancer (NSCLC) is one of the most fatal types of cancer with significant mortality and morbidity worldwide. MicroRNAs (miRs) have been confirmed to have positive functions in NSCLC. In the present study, we try to explore the role of miR-758 in proliferation, migration, invasion, and apoptosis of NSCLC cells by regulating high-mobility group box (HMGB) 3 (HMGB3.) NSCLC and adjacent tissues were collected. Reverse transcription quantitative PCR (RT-qPCR) was employed to detect expression of miR-758 and HMGB3 in NSCLC and adjacent tissues, in BEAS-2B cells and NSCLC cell lines. The targetted relationship between miR-758 and HMGB3 was identified by dual luciferase reporter gene assay. The effects of miR-758 on proliferation, migration, invasion, cell cycle, and apoptosis of A549 cells. MiR-758 expression was lower in NSCLC tissues, which was opposite to HMGB3 expression. The results also demonstrated that miR-758 can target HMGB3. The cells transfected with miR-758 mimic had decreased HMGB3 expression, proliferation, migration, and invasion, with more arrested cells in G1 phase and increased apoptosis. Our results supported that the overexpression of miR-758 inhibits proliferation, migration, and invasion, and promotes apoptosis of NSCLC cells by negative regulating HMGB2. The present study may provide a novel target for NSCLC treatment.

Differential expressions of integrin-linked kinase, ß-parvin and cofilin 1 in high-fat diet induced prostate cancer progression in a transgenic mouse model.

High-fat diet induced obesity was associated with more aggressive prostate cancer. Recent research has demonstrated that integrin-linked kinase (ILK), ß-parvin and downstream cofilin 1 jointly affected cancer progression. Meanwhile, these proteins were also involved in energy metabolism. Therefore, the present study was conducted to investigate the potential function of ILK, ß-parvin and cofilin 1 in the high-fat diet-induced progression of prostate cancer. Transgenic mice with prostate cancer were employed, fed with different diets and sacrificed at 20 and 28 weeks. Tumor differentiation, extracapsular extension and metastasis were compared between the groups. Expression levels of ILK, ß-parvin and cofilin 1 in prostate were evaluated by immunohistochemical analysis and determined by an immunoreactivity score. Public databases were applied for analysis and validation. It was detected that high-fat diet feeding promoted cancer progression in transgenic mice with prostate cancer, with increased expressions of ß-parvin (P=0.038) and cofilin 1 (P=0.018). Higher expressions of ILK, ß-parvin and cofilin 1 were also associated with poorer cancer differentiation. Additionally, higher mRNA levels of CFL1 were correlated with a worse disease-free survival in patients of certain subgroups from The Cancer Genome Atlas database. Further studies were warranted in discussing the potential roles of ILK, ß-parvin and cofilin 1 in high-fat diet feeding induced progression of prostate cancer.

Evaluation of PSA-age volume score in predicting prostate cancer in Chinese population.

This study was performed to evaluate prostate-specific antigen-age volume (PSA-AV) scores in predicting prostate cancer (PCa) in a Chinese biopsy population. A total of 2355 men who underwent initial prostate biopsy from January 2006 to November 2015 in Huashan Hospital were recruited in the current study. The PSA-AV scores were calculated and assessed together with PSA and PSA density (PSAD) retrospectively. Among 2133 patients included in the analysis, 947 (44.4%) were diagnosed with PCa. The mean age, PSA, and positive rates of digital rectal examination result and transrectal ultrasound result were statistically higher in men diagnosed with PCa (all P < 0.05). The values of area under the receiver operating characteristic curves (AUCs) of PSAD and PSA-AV were 0.864 and 0.851, respectively, in predicting PCa in the entire population, both performed better than PSA (AUC = 0.805; P < 0.05). The superiority of PSAD and PSA-AV was more obvious in subgroup with PSA ranging from 2.0 ng ml-1 to 20.0 ng ml-1. A PSA-AV score of 400 had a sensitivity and specificity of 93.7% and 40.0%, respectively. In conclusion, the PSA-AV score performed equally with PSAD and was better than PSA in predicting PCa. This indicated that PSA-AV score could be a useful tool for predicting PCa in Chinese population.

Chemokine CXCL13 activates p38 MAPK in the trigeminal ganglion after infraorbital nerve injury.

Recent data demonstrated that chemokine CXCL13 mediates neuroinflammation and contributes to the maintenance of neuropathic pain after nerve injury in the spinal cord. Pro-nociceptive chemokines activate mitogen-activated protein kinases (MAPKs) which are potential signaling pathways contributing to the nociceptive behavior in inflammatory or neuropathic pain. However, whether activation of p38 and JNK MAPK signaling pathway in the trigeminal ganglion (TG) are involved in CXCL13 and its receptor CXCR5-mediated orofacial pain has not yet been clarified. Here, we show that the unilateral partial infraorbital nerve ligation (pIONL) induced a profound orofacial pain in wild-type (WT) mice. Western blot results showed that pIONL induced p38 but not JNK activation in the TG of WT mice. However, the orofacial pain induced by pIONL was alleviated in Cxcr5 -/- mice, and the activation of p38 was also abrogated in Cxcr5 -/- mice. Furthermore, intra-TG injection of CXCL13 evoked mechanical hypersensitivity and increased p-p38 expression in WT mice. But CXCL13 had no effect on pain behavior or p-p38 expression in Cxcr5 -/- mice. Finally, pretreatment with p38 inhibitor, SB203580, attenuated the pIONL-induced mechanical allodynia and decreased the mRNA expression of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the TG. Taken together, our data suggest that CXCL13 acts on CXCR5 to increase p38 activation and further contributes to the pathogenesis of orofacial neuropathic pain.

Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice.

DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under neuropathic pain condition remain largely unknown. Here we investigated how chemokine receptor CXCR3 is regulated by DNA methylation and how it contributes to neuropathic pain induced by spinal nerve ligation (SNL) in mice. SNL increased Cxcr3 mRNA and protein expression in the neurons of the spinal cord. Meanwhile, the CpG (5'-cytosine-phosphate-guanine-3') island in the Cxcr3 gene promoter region was demethylated, and the expression of DNA methyltransferase 3b (DNMT3b) was decreased. SNL also increased the binding of CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)/enhancer binding protein α (C/EBPα) with Cxcr3 promoter and decreased the binding of DNMT3b with Cxcr3 promoter in the spinal cord. C/EBPα expression was increased in spinal neurons after SNL, and inhibition of C/EBPα by intrathecal small interfering RNA attenuated SNL-induced pain hypersensitivity and reduced Cxcr3 expression. Furthermore, SNL-induced mechanical allodynia and heat hyperalgesia were markedly reduced in Cxcr3-/- mice. Spinal inhibition of Cxcr3 by shRNA or CXCR3 antagonist also attenuated established neuropathic pain. Moreover, CXCL10, the ligand of CXCR3, was increased in spinal neurons and astrocytes after SNL. Superfusing spinal cord slices with CXCL10 enhanced spontaneous EPSCs and potentiated NMDA-induced and AMPA-induced currents of lamina II neurons. Finally, intrathecal injection of CXCL10 induced CXCR3-dependent pain hypersensitivity in naive mice. Collectively, our results demonstrated that CXCR3, increased by DNA demethylation and the enhanced interaction with C/EBPα, can be activated by CXCL10 to facilitate excitatory synaptic transmission and contribute to the maintenance of neuropathic pain. SIGNIFICANCE STATEMENT: Peripheral nerve injury induces changes of gene expression in the spinal cord that may contribute to the pathogenesis of neuropathic pain. CXCR3 is a chemokine receptor. Whether it is involved in neuropathic pain and how it is regulated after nerve injury remain largely unknown. Our study demonstrates that spinal nerve ligation downregulates the expression of DNMT3b, which may cause demethylation of Cxcr3 gene promoter and facilitate the binding of CCAAT/enhancer binding protein α with Cxcr3 promoter and further increase CXCR3 expression in spinal neurons. The upregulated CXCR3 may contribute to neuropathic pain by facilitating central sensitization. Our study reveals an epigenetic mechanism underlying CXCR3 expression and also suggests that targeting the expression or activation of CXCR3 signaling may offer new therapeutics for neuropathic pain.

CXCL13/CXCR5 enhances sodium channel Nav1.8 current density via p38 MAP kinase in primary sensory neurons following inflammatory pain.

CXCL13 is a B lymphocyte chemoattractant and activates CXCR5 receptor in the immune system. Here we investigated whether CXCL13/CXCR5 mediates inflammatory pain in dorsal root ganglia (DRG) and the underlying mechanisms. Peripheral injection of complete Freund's Adjuvant (CFA) increased the expression of CXCL13 and CXCR5 in DRG neurons. In Cxcr5-/- mice, CFA-induced pain hypersensitivity were attenuated. Whole-cell patch-clamp recording showed that the excitability of dissociated DRG neurons was increased after CFA injection or CXCL13 incubation from wild-type (WT) mice, but not from Cxcr5-/- mice. Additionally, sodium channel Nav1.8 was co-expressed with CXCR5 in dissociated DRG neurons, and the increased neuronal excitability induced by CFA or CXCL13 was reduced by Nav1.8 blocker. Intrathecal injection of Nav1.8 blocker also attenuated intrathecal injection of CXCL13-induced pain hypersensitivity. Furthermore, CXCL13 increased Nav1.8 current density in DRG neurons, which was inhibited by p38 MAP kinase inhibitor. CFA and CXCL13 increased p38 phosphorylation in the DRG of WT mice but not Cxcr5-/- mice. Finally, intrathecal p38 inhibitor alleviated CXCL13-induced pain hypersensitivity. Taken together, these results demonstrated that CXCL13, upregulated by peripheral inflammation, acts on CXCR5 on DRG neurons and activates p38, which increases Nav1.8 current density and further contributes to the maintenance of inflammatory pain.

Chemokine receptor CXCR2 in dorsal root ganglion contributes to the maintenance of inflammatory pain.

Chemokines and their receptors have been demonstrated to be important contributors to the development and maintenance of chronic pain. Recent studies showed that chemokine (C-X-C motif) ligand 1 (CXCL1) and its major receptor CXCR2 are respectively expressed in astrocytes and neurons in the spinal cord and are involved in the maintenance of neuropathic pain and inflammatory pain via astrocytic-neuronal interaction. Here we investigated how CXCL1 and CXCR2 are regulated in the dorsal root ganglion (DRG) after peripheral injection of complete Freund's adjuvant (CFA) and its implication in inflammatory pain. CFA induced rapid increase of CXCL1 mRNA and protein in the DRG. Double immunostaining showed that CXCL1 was colocalized with calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200). Furthermore, CXCR2 mRNA was increased 3h after CFA injection and maintained for more than 3 days. CXCR2 protein was also increased and colocalized with CGRP, IB4, and NF200. Finally, perisciatic nerve injection of CXCR2 siRNA to specifically knock down CXCR2 in the DRG effectively attenuated CFA-induced mechanical allodynia and heat hyperalgesia, and the effect maintained for more than 5 days. Taken together, our results demonstrated that CXCL1 and CXCR2 may regulate the maintenance of inflammatory pain via an autocrine/paracrine way in DRG neurons.

CXCL13 drives spinal astrocyte activation and neuropathic pain via CXCR5.

Recent studies have implicated chemokines in microglial activation and pathogenesis of neuropathic pain. C-X-C motif chemokine 13 (CXCL13) is a B lymphocyte chemoattractant that activates CXCR5. Using the spinal nerve ligation (SNL) model of neuropathic pain, we found that CXCL13 was persistently upregulated in spinal cord neurons after SNL, resulting in spinal astrocyte activation via CXCR5 in mice. shRNA-mediated inhibition of CXCL13 in the spinal cord persistently attenuated SNL-induced neuropathic pain. Interestingly, CXCL13 expression was suppressed by miR-186-5p, a microRNA that colocalized with CXCL13 and was downregulated after SNL. Spinal overexpression of miR-186-5p decreased CXCL13 expression, alleviating neuropathic pain. Furthermore, SNL induced CXCR5 expression in spinal astrocytes, and neuropathic pain was abrogated in Cxcr5-/- mice. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. Intrathecal injection of CXCL13 was sufficient to induce pain hypersensitivity and astrocyte activation via CXCR5 and ERK. Finally, intrathecal injection of CXCL13-activated astrocytes induced mechanical allodynia in naive mice. Collectively, our findings reveal a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain. Thus, miR-186-5p and CXCL13/CXCR5-mediated astrocyte signaling may be suitable therapeutic targets for neuropathic pain.

Molecular hydrogen inhibits lipopolysaccharide-triggered NLRP3 inflammasome activation in macrophages by targeting the mitochondrial reactive oxygen species.

The NLRP3 inflammasome, an intracellular multi-protein complex controlling the maturation of cytokine interleukin-1ß, plays an important role in lipopolysaccharide (LPS)-induced inflammatory cascades. Recently, the production of mitochondrial reactive oxygen species (mtROS) in macrophages stimulated with LPS has been suggested to act as a trigger during the process of NLRP3 inflammasome activation that can be blocked by some mitochondria-targeted antioxidants. Known as a ROS scavenger, molecular hydrogen (H2) has been shown to possess therapeutic benefit on LPS-induced inflammatory damage in many animal experiments. Due to the unique molecular structure, H2 can easily target the mitochondria, suggesting that H2 is a potential antagonist of mtROS-dependent NLRP3 inflammasome activation. Here we have showed that, in mouse macrophages, H2 exhibited substantial inhibitory activity against LPS-initiated NLRP3 inflammasome activation by scavenging mtROS. Moreover, the elimination of mtROS by H2 resultantly inhibited mtROS-mediated NLRP3 deubiquitination, a non-transcriptional priming signal of NLRP3 in response to the stimulation of LPS. Additionally, the removal of mtROS by H2 reduced the generation of oxidized mitochondrial DNA and consequently decreased its binding to NLRP3, thereby inhibiting the NLRP3 inflammasome activation. Our findings have, for the first time, revealed the novel mechanism underlying the inhibitory effect of molecular hydrogen on LPS-caused NLRP3 inflammasome activation, highlighting the promising application of this new antioxidant in the treatment of LPS-associated inflammatory pathological damage.