| BackgroundA well-controlled immune response is beneficial to maintaining central nervous system (CNS) homeostasis. When dysregulated and exaggerated, neuroinflammation can lead to significant tissue damage of CNS. A growing number of studies indicate that neuroinflammation has been highly involved in pathologic processes of many CNS disorders including stroke, traumatic brain injury and neurodegenerative disease. Thus, researching the regulatory factors that modulate neuroinflammation may be beneficial for therapeutic strategy, as well as for a better understanding on the immunopathology of inflammation related CNS diseases.Danger-associated molecular patterns (DAMPs), known as alarmins, which signal tissue and cell damage are critical for the induction of innate and adaptive immune response, resulting in the production of sterile inflammation. High mobility group box 1 (HMGB1) has been known as one of the crucial members of DAMPs. It normally locates in the nucleus. Once pathogens or tissue injury occurred, HMGB1 can be either passively released from injured tissue cells or actively secreted by immune cells to extracellular milieu. Subsequently, HMGB1 binds to pattern recognition receptors on immune cells and triggers the intracellular signal cascades, resulting in a robust inflammatory response. In CNS, the release of HMGB1 has been found in a variety of disorders such as stroke, traumatic brain injury, Alzheimer’s disease (AD), Parkinson’s disease (PD) and multiple sclerosis (MS). The extracellular HMGB1 binds to receptors for advanced glycation endproduct, toll-like receptor (TLR)-2, TLR-4 or macrophage antigen complex 1 (Mac1), on microglia or infiltrated macrophages. The binding of HMGB1 to its receptors then recruits myeloid differentiation factor 88 (MyD88) to activate mitogen activated protein kinase (MAPK); subsequently, it induces nuclear factor-κB (NF-κB) to start the transcription of inflammatory cytokines, which leads to brain cell damage. The activated microglia and injured neurons, in turn, cause further HMGB1 release to trigger an autocrine signaling and contribute to severe inflammatory neuronal and vascular injury. Thus, a vicious cycle is reinforced to aggravate disease outcome.Intensive studies on the proinflammatory role of HMGB1 have been emerged, however, negative regulation signaling involved in HMGB1-mediated inflammatory pathway remains unclear. Regulatory RNase 1 (Regnase-1), also known as Zc3h12a and monocyte chemotactic protein-1 (MCP-1) induced protein-1 (MCPIP1), is a novel CCCH-type zinc finger motif-containing protein which has endonuclease activity. The purified Regnase-1 can specifically decay a set of cytokine-encoding mRNAs such as interleukin (IL)-6, interferon-y, IL-1β, IL-12β and IL-2 by recognizing the stem-loop structure in the 3’-untranslational terminal region of these mRNAs. Stimulation by MCP-1, lipopolysaccharides (LPS) and IL-1β can induce a rapid and potent transcription of Regnase-1 through NF-κB or MAPK. In CNS, Regnase-1 has been reported to participate in electroacupuncture-induced ischemic stroke tolerance and minocycline-mediated neuroprotection against ischemic brain injury. Regnase-1 also involves in LPS preconditioning-induced ischemic stroke tolerance by regulating the expression of proinflammatory cytokines. More importantly, suppression of Regnase-1 by microRNA (miR)-9 enhances inflammatory response in microglia. These findings collectively suggest that Regnase-1 can be induced by inflammatory milieu and functions as a regulatory factor to ameliorate neuroinflammatory injury in CNS. Given that MAPK and NF-κB pathways are shared processes of HMGB1-induced inflammation and the production of Regnase-1, we hypothesize that Regnase-1 can be induced by HMGB1 to elicit a negative feedback mechanism which limits the HMGB1-mediated inflammation and neuronal injury.In this study, we designed series of experiments to testify this hypothesis and found that purified recombinant HMGB1 could induce the expression of Regnase-1 in microglia in vitro and in vivo. Furthermore, knockdown of Regnase-1 in microglia enhanced transcription of IL-1β, IL-6 and exacerbated HMGB1-mediated inflammatory injury to neurons.Purpose1. To confirm the expression of inflammatory cytokines induced by HMGB1 in BV-2 cells.2. To confirm Regnase-1 expression induced by HMGB1 in BV2 cells.3. To confirm Regnase-1 expression induced by HMGB1 in rat brain and whether Regnase-1 co-localized with microglia in the brain of rat treated with HMGB1.4. To identify Regnase-1 whether negative regulates inflammatory cytokines released from BV2 cells and reduces the neurotoxicity of BV2 cells under HMGB1 treatment.Method1. Establishing the expression of inflammatory cytokines in BV2 cells after HMGB1 treatment.Microglia cell line BV2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and grown at 37℃ in a humidified environment (5% CO2,95% air). When BV2 cells got to about 80% confluence, different stimulus times (1 to 24 h) and doses (100 to 1,000 ng/ml) of pure recombinant HMGB1 were added to medium for cell stimulus experiments. After treatment, RNA from BV2 cells was obtained to test the expression of inflammatory cytokines in BV2 cells.2. Establishing the expression of Regnase-1 in BV2 cells after treatment of HMGB1.Microglia cell line BV2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and grown at 37℃ in a humidified environment (5% CO2,95% air). When BV2 cells got to about 80% confluence, different stimulus times (1-24 h) and doses (100-1,000 ng/ml) of pure recombinant HMGB1 were added to medium for cell stimulus experiments. After treatment, RNA from BV2 cells was obtained to test the expression of Regnase-1 in BV2 cells.3. Establishing the expression of Regnase-1 in vivo after HMGB1 treatment.The recombinant HMGB1 (8 μg/kg) was injected into the right lateral ventricle of rats. Rats in the control group were received same volume of vehicle saline. After 24 h, animals were anesthetized and transcardially perfused with phosphate buffer solution (PBS).(1) Western blot were performed to detect the expression of Regnase-1 in the brains of two groups after treatment of HMGB1.(2) The brains of two groups were then isolated and fixed with cold 4% paraformaldehyde. Serial brain sections (6μ m thick) were collected for immunohistochemistry to observe the expression of Regnase-1 in the brains of two groups. Immunofluorescence was used to observe the co-localization of Regnase-1 and microglia after treatment of HMGB1.4. Detection of inflammatory factors released from BV2 cells with or without Regnase-1 silencing after HMGB1 treatmeant.BV2 cells were randomly divided into two groups:cells transfected with Regnase-1 siRNA (siZc3h12a) and cells transfected with scrambled sequence (siControl). Then, transfected BV2 cells were treated with HMGB1 (1μg/ml) for 1h after Regnase-1 silencing. Experiments were performed in triplicate and repeated three times independently. QPCR was performed to observe gene expression of inflammatory factors including IL-6, IL-1β and tumor necrosis factor-a (TNF-a). Western blot and enzyme linked immunosorbent assay (ELISA) analysis were performed to observe protein expression secretion and of IL-1β.5. Detection of the cytotoxicity of medium collected from HMGB1-stimulated BV2 cells with or without HMGB1 silencing.BV2 cells were randomly divided into two groups:cells transfected with Regnase-1 siRNA (si-Zc3h12a) and cells transfected with scrambled sequence (si-Control). BV2 cells of two groups treated with 1 μg/ml HMGB1. One hour after treatment of HMGB1, the cell medium (conditioned media, CM) of two groups was collected and respectively added to SH-SY5Y cells for 8h,12h and 24h. Experiments were performed in triplicate and repeated three times independently. SH-SY5Y cell viability of two groups was measured by Cell Counting Kit-8 (CCK-8) assay and observed by optical microscope. The cell death rate was measured by Annexin V-FITC/PI followed by detection with flow cytometry.6. Statistical analysisStatistical software SPSS 20.0 analysis was used to analyze experiment data. Comparison between two groups was conducted by Student’t test. More comparison used single factor analysis of variance between groups; used Turkey multiple comparison when variance was equal; used Dunnett T3 when variance was unequal. Two-way ANOVA was used for two factors analysis, used Turkey multiple comparison when variance was equal; used Dunnett T3 when variance was unequal. All the experimental data were expressed by the mean±standard deviation (X±S). P<0.05 was considered significant.Results1. HMGB1 did increase mRNA expression of IL-1β, IL-6 and TNF-α in a dose-dependent manner (100 to 1,000 ng/ml) at 24 h. Time course studies showed that 1,000 ng/ml HMGB1 significantly up-regulated mRNA expression levels of IL-1β and IL-6, and peaked at 4 h, while the increase of TNF-α reached the peak at 24 h and presented a biphasic pattern.2. A significant increase of Regnase-1 protein expression was observed after 1,000 ng/ml HMGB1 treatment for 24 h. mRNA level of Regnase-1 increased in a dose-dependent manner following HMGB1 treatment in BV2 cells for 24 h. Meanwhile, compared with the control group,1,000 ng/ml HMGB1 treatment for 4, 12 and 24 h also increased Regnase-1 mRNA expression in BV2 cells3. Regnase-1 protein level in rats from HMGB1-treated group was much higher than vehicle administration. Consistently, immunohistochemistry and immunofluorescence assays showed more intensive staining of Regnase-1 in HMGB1 treated group rats than vehicle control. Immunofluorescence analysis showed the co-localization of Regnase-1 and Ibal (the microglia marker), indicating a specific response of microglia to HMGB1 in vivo.4. Upon HMGB1 treatment, the expressions of all three cytokines were much enhanced; IL-1β and IL-6, two putative substrates of Regnase-1, were further increased in Regnase-1-silenced BV2 cells. Interestingly, TNF-a was not increased, but decreased slightly with statistical significance upon Regnase-1 silencing, suggesting that TNF-a may not be the direct target of Regnase-1. Western blot and ELISA analysis showed that Regnase-1 knockdown promoted IL-10 protein expression and secretion after HMGB1 treatment in BV2 cells5.CM from HMGB1-treated BV2 cells with or without Regnase-1 knockdown was collected to incubate SH-SY5Y cells. Incubation of Regnase-1 knockdown microglia CM with SH-SY5Y cells for 12 h, but not 8 and 24 h led to lower cell viability than control silencing. This was further supported by the morphological changes. Annexin V-FITC/PI staining assay was conducted at 12 h after CM treatment and showed that knockdown of Regnase-1 greatly enhanced the cytotoxicity of CM to SH-SY5Y cells.Conclusions1. HMGB1 could increase the expression of Regnase-1 in microglia and the findings will provide experimental basis for further research in pathology of inflammation related CNS diseases.2. Silencing Regnase-1 in BV2 cells under HMGB1 stimulation condition increased the mRNA level of IL-1β and IL-6. It suggests that Regnase-1 plays a negative regulated role in HMGB1-mediated inflammation.3. The CM from BV2 cells with Regnase-1 silencing under HMGB1 stimulation condition brought higher neuronal toxicity. It indicates that Regnase-1 play a significant role in regulating the neurotoxicity of microglia under HMGB1-mediated inflammation and has potential protective effects in neuroprotection. |
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