IRF7 overexpression alleviates CFA-induced inflammatory pain by inhibiting nuclear factor-κB activation and pro-inflammatory cytokines expression in rats.

BACKGROUND: It is known that nerve signals arising from sites of inflammation lead to persistent changes in the spinal cord and contribute to the amplification and persistence of pain. Nevertheless, the underlying mechanisms have not yet been completely elucidated. We identified differentially expressed genes in the lumbar (L4-L6) segment of the spinal cord from complete Freund's adjuvant (CFA) rats compared to control animals via high throughput sequencing. Based on differential gene expression analysis, we selected interferon regulatory factor 7 (IRF7) for follow-up experiments to explore its antinociceptive potential. METHODS: An animal model of inflammatory pain was induced by intraplantar injection of CFA. We evaluated the effects of adeno-associated viral (AAV)-mediated overexpression of IRF7 in the spinal cord on pain-related behavior after CFA injection. Moreover, the activation of the nuclear factor-κB (NF-κB) and the expression of inflammatory cytokines were investigated to understand the underlying mechanisms related to the contribution of IRF7 to inflammatory pain. RESULTS: CFA intraplantar injection caused a significant decrease in the level of spinal IRF7, which is mainly expressed in the dorsal horn neurons and astrocytes. Moreover, IRF7 overexpression significantly attenuated pain-related behaviors, as well as the activity of NF-κB/p65 and the production of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the spinal cord of CFA rats. CONCLUSIONS: Our data indicated that spinal IRF7 plays an important role in the regulation of inflammatory pain. Thus, IRF7 overexpression at the spinal cord level might represent a potential target for the treatment of inflammatory pain.

Inhibition of Spinal Interleukin-33 Attenuates Peripheral Inflammation and Hyperalgesia in Experimental Arthritis.

Accumulating evidence indicates that the continuous and intense nociceptive from inflamed tissue may increase the excitability of spinal dorsal horn neurons, which can signal back and modulate peripheral inflammation. Previous studies have demonstrated that spinal interleukin (IL)-33 contributes to the hyperexcitability of spinal dorsal horn neurons. This study was undertaken to investigate whether spinal IL-33 can also influence a peripheral inflammatory response in a rat model of arthritis. Lentivirus-delivered short hairpin RNA targeting IL-33 (LV-shIL-33) was constructed for gene silencing. Rats with adjuvant-induced arthritis (AIA) were injected intrathecally with LV-shIL-33 3 days before the complete Freund's adjuvant (CFA) injection. During an observation period of 21 days, pain-related behavior and inflammation were assessed. In addition, the expression of spinal proinflammatory cytokines and the activation of spinal extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways were evaluated on 9 days after CFA treatment. The existence of tissue injury or inflammation in rats with AIA resulted in the upregulation of spinal IL-33, which is predominantly expressed in neurons, astrocytes, and oligodendrocytes. Intrathecal administration of LV-shIL-33 significantly alleviated hyperalgesia, paw swelling, and joint destruction, and attenuated the expression of proinflammatory cytokines [IL-6, IL-1ß, and tumor necrosis factor-α (TNF-α)], as well as the activation of ERK and NF-κB/p65 in the spinal cord. Our data suggest that spinal IL-33 contributes to the development of both peripheral inflammation and hyperalgesia. Thus, interference with IL-33 at the spinal level might represent a novel therapeutic target for painful inflammatory disorders.

Interleukin-33 modulates lipopolysaccharide-mediated inflammatory response in rat primary astrocytes.

Astrocytes have a crucial role in the modulation of the neuroinflammatory response. However, the underlying mechanisms have yet to be fully defined. Interleukin-33 (IL-33) is constitutively expressed in astrocytes, which has been found to orchestrate inflammatory responses in a large variety of immune-mediated and inflammatory diseases of the nervous system. Thus, the purpose of this study was to elucidate the potential effect of IL-33 in the regulation of inflammatory response in primary cultured astrocytes. We investigated the role of IL-33 in the regulation of inflammatory responses in the lipopolysaccharide-stimulated astrocytes. This study utilized lentiviral short hairpin RNA vectors to target IL-33 (LV-shIL-33) for gene silencing. After lipopolysaccharide stimulation, the expression levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α), as well as the activation of nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) signaling pathways, were evaluated to elucidate the mechanisms related to the contributions of IL-33 to the inflammatory response in astrocytes. We found that the expression IL-33 has increased in rat primary cultured astrocytes after lipopolysaccharide stimulation. Administration of LV-shIL-33 knocked down the expression of IL-33 and markedly reduced the overexpression of spinal IL-1ß, IL-6, and TNF-α, and attenuated the activation of ERK and NF-κB/p65. This study shows that IL-33 participates in regulating inflammatory responses in primary cultured astrocytes, which might provide additional targets for controlling inflammatory responses following neurological diseases. See Video abstract, http://links.lww.com/WNR/A627.

IL-33/ST2 signaling contributes to radicular pain by modulating MAPK and NF-κB activation and inflammatory mediator expression in the spinal cord in rat models of noncompressive lumber disk herniation.

BACKGROUND: Immune and inflammatory responses occurring in the spinal cord play a pivotal role in the progression of radicular pain caused by intervertebral disk herniation. Interleukin-33 (IL-33) orchestrates inflammatory responses in a wide range of inflammatory and autoimmune disorders of the nervous system. Thus, the purpose of this study is to investigate the expression of IL-33 and its receptor ST2 in the dorsal spinal cord and to elucidate whether the inhibition of spinal IL-33 expression significantly attenuates pain-related behaviors in rat models of noncompressive lumbar disc herniation. METHODS: Lentiviral vectors encoding short hairpin RNAs that target IL-33 (LV-shIL-33) were constructed for gene silencing. Rat models of noncompressive lumber disk herniation were established, and the spines of rats were injected with LV-shIL-33 (5 or 10 µl) on the first day after the operation. Mechanical thresholds were evaluated during an observation period of 21 days. Moreover, the expression levels of spinal tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and cyclooxygenase 2 (COX-2) and the activation of the mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) pathways were evaluated to gain insight into the mechanisms related to the contribution of IL-33/ST2 signaling to radicular pain. RESULTS: The application of nucleus pulposus (NP) to the dorsal root ganglion (DRG) induced an increase in IL-33 and ST2 expression in the spinal cord, mainly in the dorsal horn neurons, astrocytes, and oligodendrocytes. Spinally delivered LV-shIL-33 knocked down the expression of IL-33 and markedly attenuated mechanical allodynia. In addition, spinal administration of LV-shIL-33 reduced the overexpression of spinal IL-1ß, TNF-α, and COX-2 and attenuated the activation of C-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and NF-κB/p65 but not p38. CONCLUSIONS: This study indicates that spinal IL-33/ST2 signaling plays an important role in the development and progression of radicular pain in rat models of noncompressive lumber disk herniation. Thus, the inhibition of spinal IL-33 expression may provide a potential treatment to manage radicular pain caused by intervertebral disk herniation.

The overexpression of epithelial cell adhesion molecule (EpCAM) in glioma.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in various neoplasms as a tumor-associated antigen and absent in natural brain. However, little is known about EpCAM's expression in gliomas. To investigate the expression of EpCAM in gliomas and understand the correlation of EpCAM expression with malignancy, proliferation, angiogenesis, and prognosis, we studied the expression of EpCAM in 98 glioma samples by immunohistochemistry and by western blotting (N = 12). Correlative analysis of EpCAM overexpression with microvessel density (MVD), Ki-67 expression, age, and gender were made. Survival data was analyzed with Kaplan-Meier method and Cox Proportional Hazard Model. Immunohistochemistry results showed EpCAM was widely expressed in glioma (90.8 %). The overexpression rate of WHO grade IV gliomas was significantly higher EpCAM overexpression correlated significantly with Ki-67 expression and MVD. Western blot analysis also revealed a stepwise increase in EpCAM expression from WHO II to IV glioma. The overall survival of WHO III and IV glioma patients with EpCAM overexpression was obviously lower than that without EpCAM overexpression. EpCAM overexpression was an independent prognostic factor for overall survival in glioma patients. This study firstly shows that EpCAM overexpression correlates significantly with malignancy (WHO grades), proliferation (Ki67), angiogenesis (MVD), and prognosis in gliomas. EpCAM may participate in tumorgenesis of gliomas.

Activation of spinal NF-κB/p65 contributes to peripheral inflammation and hyperalgesia in rat adjuvant-induced arthritis.

OBJECTIVE: It is known that noxious stimuli from inflamed tissue may increase the excitability of spinal dorsal horn neurons (a process known as central sensitization), which can signal back and contribute to peripheral inflammation. However, the underlying mechanisms have yet to be fully defined. A number of recent studies have indicated that spinal NF-κB/p65 is involved in central sensitization, as well as pain-related behavior. Thus, the aim of this study was to determine whether NF-κB/p65 can facilitate a peripheral inflammatory response in rat adjuvant-induced arthritis (AIA). METHODS: Lentiviral vectors encoding short hairpin RNAs that target NF-κB/p65 (LV-shNF-κB/p65) were constructed for gene silencing. The spines of rats with AIA were injected with LV-shNF-κB/p65 on day 3 or day 10 after treatment with Freund's complete adjuvant (CFA). During an observation period of 20 days, pain-related behavior, paw swelling, and joint histopathologic changes were evaluated. Moreover, the expression levels of spinal tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), and cyclooxygenase 2 (COX-2) were assessed on day 14 after CFA treatment. RESULTS: The presence of peripheral inflammation in rats with AIA induced an increase in NF-κB/p65 expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Delivery of LV-shNF-κB/p65 to the spinal cord knocked down the expression of NF-κB/p65 and significantly attenuated hyperalgesia, paw edema, and joint destruction. In addition, spinal delivery of LV-shNF-κB/p65 reduced the overexpression of spinal TNFα, IL-1ß, and COX-2. CONCLUSION: These findings indicate that spinal NF-κB/p65 plays an important role in the initiation and development of both peripheral inflammation and hyperalgesia. Thus, inhibition of spinal NF-κB/p65 expression may provide a potential treatment to manage painful inflammatory disorders.

WW domain containing E3 ubiquitin protein ligase 1 (WWP1) negatively regulates TLR4-mediated TNF-α and IL-6 production by proteasomal degradation of TNF receptor associated factor 6 (TRAF6).

BACKGROUND: Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation. METHODOLOGY/RESULTS: Knocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6. CONCLUSIONS/SIGNIFICANCE: We identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.

Small interfering RNA-mediated knockdown of NF-κBp65 attenuates neuropathic pain following peripheral nerve injury in rats.

Recent reports show that the nuclear factor-κB (NF-κB) can control numerous genes encoding inflammatory and nociceptive mediators and play an important role in the development of central pain sensitization. The aim of the present study is to assess the role of NF-κB signal pathway and its downstream pro-inflammatory cytokines in the modulation of neuropathic pain, by using small interfering RNAs (siRNAs) technique, which has been shown to result in potent, long-lasting post-transcriptional silencing of specific genes. We developed a highly efficient method of lentivirus-mediated delivery of short-hairpin RNA (shRNA) targeting NF-κBp65 for gene silencing. This method successfully transduced LV-shNF-κBp65 into cultured spinal cord neurons in vitro and spinal cord cells in vivo, inhibited the expression of NF-κBp65 and pro-inflammatory factors (TNF-α, IL-1ß and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia for more than 4weeks in chronic constriction injury (CCI) model of rats. Taken together, our results suggest that siRNA against NF-κBp65 is a potential strategy for analgesia. Furthermore, the lentiviral vector derived shRNA approach shows a great promise for the management of neuropathic pain and the study of functional NF-κBp65 gene expression.

[Effect of electroacupuncture on the expression of spinal glial fibrillary acidic protein, tumor necrosis factor-alpha and interleukin-1beta in chronic neuropathic pain rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of spinal glial fibrillary acidic protein (GFAP), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in chronic constriction injury (CCI) rats. METHODS: Seventy-two SD rats were randomized into sham operation (sham), CCI (model) and EA groups (n = 24/group). The mechanical and thermal pain thresholds were measured by using Von Frey filaments and radiant-heat irridiation separately. The immunoactivity of GFAP of spinal dorsal horn (L4-L5) was assessed by immunohistochemistry, and the expression of spinal TNF-alpha mRNA and IL-1beta mRNA was detected by real time-PCR. RESULTS: Compared with pre-CCI and sham group, both mechanical and thermal pain thresholds decreased considerably in rats with CCI (P < 0.05), and in comparison with model group, those of EA group increased markedly (P < 0.05). Compared with sham group, GFAP immunoactivity (mainly in the lamina I-II of the spinal dorsal horn), TNF-alpha mRNA and IL-1beta mRNA expression in the ipsilateral spinal cord on the CCI side in model group increased considerably (P < 0.05), while compared with model group, the expression of GFAP, TNF-alpha mRNA and IL-1beta mRNA in EA group was down-regulated remarkably (P < 0.05). CONCLUSION: EA can effectively suppress CCI-induced up-regulation of expression of spinal GFAP, TNF-alpha mRNA and IL-1beta mRNA, which may contribute to its effect in reducing mechanical allodynia and thermal hyperalgesia in neuropathic pain rats.