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.

Inhibition of periostin gene expression via RNA interference suppressed the proliferation, apoptosis and invasion in U2OS cells.

BACKGROUND: Periostin originally designated osteoblast-specific factor 2 (OSF-2) is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line (U2OS), such as proliferation, apoptosis, invasion and the associated signal pathway. METHODS: A human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups: cells transfected with phosphate buffered saline as control group (the U2OS group), cells transfected with pGCsi as negative control group (the NC group) and cells transfected with periostin/pGCsi as experimental group (the pGCsi-periostin group). Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT) quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry. RESULTS: The transfection efficiency of periostin/pGCsi to U2OS cells was about 70% - 80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% (F = 564.71, P < 0.001) and 58% (F = 341.51, P < 0.001), respectively. Meantime, the earlier apoptosis value increased by 417% (F = 28.69, P < 0.001). The percentage of S phase pGCsi-periostin cells decreased by 21% (F = 47.00, P < 0.001), however, that of G0 - G1 phase cells increased by 12% (F = 14.50, P < 0.001). The capability of migration and invasion reduced by 41% (F = 17.79, P < 0.001) and 72% (F = 197.08, P < 0.001), respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-ß and vascular endothelial growth factor decreased by 17% (F = 73.99, P < 0.001) and 47% (F = 30.25, P < 0.001), respectively. A tendency of lower focal adhesion kinase (FAK) was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21% (F = 16.81, P < 0.001). CONCLUSIONS: RNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.