Nrdp1-mediated Macrophage Phenotypic Regulation Promotes Functional Recovery in Mice with Mild Neurological Impairment after Intracerebral Hemorrhage.

Neuregulin receptor degradation protein 1 (Nrdp1) is a ring finger E3 ubiquitin ligase involved in some inflammation through ubiquitination, including macrophage polarization following cerebral hemorrhage. However, there is limited understanding regarding the mechanisms through which Nrdp1 modulates macrophage polarization and the potential impact of this modulation on neurological function. Using stereotactic injection and adenoviral transfection techniques, the corresponding animal models were constructed through injecting adenovirus, saline, or blood into the mouse striatum at different periods of time in this research. The alteration in the ratio of various M1/M2 phenotype-associated markers (e.g., CD86, CD206, IL-6, IL-10, etc.) was evaluated through immunohistochemistry, immunofluorescence, western blotting, and elisa assays. Additionally, neurological function scores and behavioral tests were utilized to evaluate changes in neurological function in mice after cerebral hemorrhage. Our results show that overexpression of Nrdp1 promotes the expression of a variety of M2 macrophage-associated markers and enhance transcriptional activity of arginase-1 (Arg1) protein through ubiquitination for early regulation M2 macrophage polarization. Additionally, Nrdp1 promotes hematoma absorption, increases IL-10 expression, inhibits inducible nitric oxide synthase (iNOS), IL-6, and TNF-α production, alleviates neurological impairment and brain edema, and accelerates functional recovery. These findings suggest that modulating macrophage polarization through Nrdp1 could be a therapeutic strategy for neurofunctional impairment in cerebral hemorrhage.

Hemoglobin enhances miRNA-144 expression and autophagic activation mediated inflammation of microglia via mTOR pathway.

Intracerebral hemorrhage promotes autophagic activation of microglia and enhances neuroinflammation. MiRNAs are key factors to autophagy, contributed to negatively and posttranscriptionally regulate gene expression and function. However, the specific miRNAs involved in the intracerebral hemorrhage mediated microglia autophagic activation are unidentified. In this experiment, microglia was treated with hemoglobin. And then, miRNA-144 expression, autophagic activation and inflammation of microglia were detected. In addition, the mTOR target of miRNA-144 and its regulation were identified. Our data demonstrated that hemoglobin promoted miRNA-144 expression and autophagic activation mediated inflammation. Additionally, miRNA-144 targeted mTOR by directly interacting with the 3' untranslated regions (UTRs), mutations of the binding sites abolish the miRNA-144 responsiveness. Overexpression of mTOR decreased autophagic activation and inflammation of microglia. Therefore, our results suggested that miRNA-144 contributed to hemoglobin mediated autophagic activation and inflammation of microglia via mTOR pathway. And miRNA based treatment provided novel therapeutical strategy for intracerebral hemorrhage.

MCP-1-mediated activation of microglia promotes white matter lesions and cognitive deficits by chronic cerebral hypoperfusion in mice.

Microglia activation played a vital role in the pathogenesis of white matter lesions (WMLs) by chronic cerebral hypoperfusion. In addition, hypoxia induced up-regulated expression of MCP-1, promotes the activation of microglia. However, the role of MCP-1-mediated microglia activation in chronic cerebral ischemia is still unknown. To explore that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in mice. The activation of microglia and the related signal pathway p38MAPK/PKC in white matter, and working memory of mice were observed. We found that stenosis of common carotid arteries could induce MCP-1-mediated activation of microglia through p38MAPK/PKC pathway and white matter lesions. Taken together, our findings represent a novel mechanism of MCP-1 involved in activation of microglia and provide a novel therapeutical strategy for chronic cerebral hypoperfusion.

C5a/C5aR Pathway Plays a Vital Role in Brain Inflammatory Injury via Initiating Fgl-2 in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a serious emergency with high mortality and morbidity. Up to date, a limited understanding of ICH pathogenesis is difficult to implement effective therapeutic strategy. Much evidence demonstrates that the complement cascade is activated after experimental ICH. However, the exact mechanism has not been well studied in ICH. In the current study, C57BL/6J mice were injected with autologous whole blood. C5a/C5aR levels, microglia infiltration, inflammatory cytokine, and fibrinogen-like protein 2 (Fgl-2) expression in the perihematomal region were analyzed following ICH. In addition, brain water content and neurological dysfunction were detected following ICH. Our data demonstrated that ICH induced complement activation, along with an increase of C5a/C5aR levels, microglia infiltration, and inflammatory cytokine levels. However, C5aR-/- mice exhibited significant attenuation of inflammatory reaction, accompanied by a remarkable reduction of Fgl-2, brain water content, and neurological dysfunction. Furthermore, inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 efficiently inhibited C5a-mediated Fgl-2 production following ICH. Taken together, these data suggest that C5a/C5aR plays a vital role in the ICH-induced inflammatory damage via Fgl-2, and ERK1/2 and p38 pathways also are involved in the pathogenesis of ICH. Therefore, inhibition of C5a/C5aR activation might enlarge our insights in ICH therapy.

Autophagy Promotes Microglia Activation Through Beclin-1-Atg5 Pathway in Intracerebral Hemorrhage.

Previous study demonstrates that intracerebral hemorrhage (ICH) promotes microglia activation and inflammation. However, the exact mechanism of microglia activation induced by ICH is not clear. In this experiment, microglia autophagy was examined using electron microscopy, conversion of light chain 3(LC3), and monodansylcadaverine (MDC) staining to detect autophagic vacuoles. We found that ICH induced microglia autophagy and activation. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (BECN1 and ATG5) decreased the microglia activation and inflammation in ICH. Moreover, autophagy inhibitors reduced brain damage in ICH. In conclusion, these data indicate that ICH contributes to microglia autophagic activation through BECN1 and ATG5 and provide the therapeutical strategy for ICH.

Programmed death (PD)-1 attenuates macrophage activation and brain inflammation via regulation of fibrinogen-like protein 2 (Fgl-2) after intracerebral hemorrhage in mice.

Neuroinflammation plays an important role in the recovery of brain injury in ICH. Macrophage is the major executor in the neuroinflammation and initiates neurological defects. Programmed death 1 (PD-1) delivers inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. PD-1 expression by macrophages plays a pathologic role in the innate inflammatory response. However, the exact role of PD-1 on inflammatory responses following ICH has not been well identified. In this experiment, PD-1 KO (PD-1 -/-) ICH mice and Wild-type (WT) ICH mice were caused by intracranial injection of type IV collagenase. The level of macrophage activation, inflammatory cytokines and fibrinogen-like protein 2 (Fgl-2) were detected using immunofluorescence staining and ELISA assays. In addition, brain edema and neurological scores of ICH mice were also measured. Our data demonstrated that ICH promoted PD-1 expression of macrophage and enhanced inflammatory cytokines and Fgl-2 concentrations. PD-1 -/- mice exhibited significantly higher expression of the inflammatory cytokines which initiate Fgl-2, than did their wild-type (WT) littermates. As a result, macrophage activation, cerebral edema and neurological deficit scores of PD-1 -/- mice were higher. In conclusion, our data demonstrate that PD-1 plays a vital role in brain inflammation via regulation of Fgl-2 after ICH, and that manipulation of PD-1 might be a promising therapeutical target in ICH.

Scavenger receptor SRA attenuates TLR4-induced microglia activation in intracerebral hemorrhage.

Scavenger receptor A (SRA) has been shown to participate in the pattern recognition of pathogen infection. However, its role in intracerebral hemorrhage has not been well defined. In this study, we detected SRA and TLR4 expression and inflammatory response of microglia treated with erythrocyte lysate in vitro, and observed the cerebral water content and neurological deficit of ICH mice in vivo. We found that SRA deficiency leads to greater sensitivity to erythrocyte lysate-induced inflammatory response. SRA down-regulated inflammatory response expression in microglia by suppressing TLR4-induced activation. Collectively, we have identified the molecular linkage between SRA and the TLR4 signaling pathways in ICH. And our results reveal that SRA has important clinical implications for TLR-targeted immunotherapeutical strategy in ICH.

MicroRNA367 negatively regulates the inflammatory response of microglia by targeting IRAK4 in intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) induces microglial activation and the release of inflammatory cytokines, leading to inflammation in the brain. IRAK4, an essential component of the MyD88-dependent pathway, activates subsets of divergent signaling pathways in inflammation. METHODS: In the experiment, microglia were stimulated with erythrocyte lysates, and then miR-367, IRAK4, NF-ĸB activation and downstream proinflammatory mediator production were analyzed. In addition, inflammation, brain edema, and neurological functions in ICH mice were also assessed. RESULTS: Here, we report that ICH downregulated miR-367 expression but upregulated IRAK4 expression in primary microglia. We also demonstrate that miR-367 suppressed IRAK4 expression by directly binding its 3'-untranslated region. MiR-367 inhibited NF-ĸB activation and downstream proinflammatory mediator production. Knocking down IRAK4 in microglia significantly decreased the IRAK4 expression and inhibited the NF-ĸB activation and the downstream production of proinflammatory mediators. In addition, our results indicate that miR-367 could inhibit expression of proinflammatory cytokines, reduce brain edema, and improve neurological functions in ICH mice. CONCLUSIONS: In conclusion, our study demonstrates that miR-367/IRAK4 pathway plays an important role in microglial activation and neuroinflammation in ICH. Our finding also suggests that miR-367 might represent a potential therapeutic target for ICH.

Recombinant adenovirus encoding NLRP3 RNAi attenuate inflammation and brain injury after intracerebral hemorrhage.

Numerous evidence have shown that microglia mediated inflammation plays a pivotal role in the development of brain injury after intracerebral hemorrhage (ICH). Therefore anti-inflammation therapy represents a potentially promising approach to ICH. Recently, NLRP3 inflammasome was discovered to facilitate the inflammatory response. However, the effect of NLRP3 inflammasome after ICH has not been fully studied. To explore the potential of NLRP3 inflammasome, we detected NLRP3 expression, inflammation, brain edema and neurological functions in vitro and in vivo. We found that ICH activated the NLRP3 inflammasome and inflammation. However, NLRP3 RNAi could attenuate inflammation and brain injury after ICH. Therefore, the findings suggested that recombinant adenovirus encoding NLRP3 RNAi might be valuable as a potential strategy for anti-inflammation therapy in ICH.

A novel nanoparticle containing neuritin peptide with grp170 induces a CTL response to inhibit tumor growth.

Malignant glioma is among the most challenging of all cancers to treat successfully. Despite recent advances in surgery, radiotherapy and chemotherapy, current treatment regimens have only a marginal impact on patient survival. In this study, we constructed a novel nanoparticle containing neuritin peptide with grp170. The nanoparticle could elicit a neuritin-specific cytotoxic T lymphocyte response to lyse glioma cells in vitro. In addition, the nanoparticle could inhibit tumor growth and improve the lifespan of tumor-bearing mice in vivo. Taken together, the results demonstrated that the nanoparticle can inhibit tumor growth and represents a promising therapy for glioma.

Adenovirus encoding Smad4 suppresses glioma cell proliferation and increases apoptosis through cell cycle arrest at G1 phase.

Mothers against decapentaplegic homologue 4 (Smad4) is associated with several human cancers. However, the exact mechanism of Smad4 in human glioma is still unknown. In this study, we constructed a recombinant adenovirus encoding Smad4 and transduced it into glioma cells. The results demonstrated that the overexpression of Smad4 not only suppressed glioma cell proliferation but also increased cell apoptosis by promoting cell cycle arrest at G1 phase. Furthermore, an adenovirus encoding Smad4 suppressed tumor formation in nude mice. These findings clearly demonstrate that Smad4 plays an important role in human glioma development by regulating cell proliferation. Moreover, Smad4 may represent a potential therapeutic target in glioma.

Hypoxia induces microglia autophagy and neural inflammation injury in focal cerebral ischemia model.

Much evidence demonstrated that autophagy played an important role in neural inflammation response after ischemia stroke. However, the specific effect of microglia autophagy in cerebral ischemia is still unknown. In the current study, we constructed focal cerebral ischemia model by permanent middle cerebral artery occlusion (pMCAO) in mice. We detected microglia autophagy and inflammation response in vivo, and observed infarct brain areas, edema formation, and neurological deficits of mice. We found that pMCAO induced microglia autophagy and inflammatory response. The suppression of autophagy using either pharmacologic inhibitor (3-MA) not only decreased the microglia autophagy and inflammatory response, but also significantly decreased infarct size, edema formation and neurological deficits in vivo. Taken together, these results suggested that cerebral ischemia induced microglia autophagy contributed to ischemic neural inflammation and injury. In addition, our findings also provided novel therapeutic strategy for ischemic stroke.

miR-203 protects microglia mediated brain injury by regulating inflammatory responses via feedback to MyD88 in ischemia.

Much evidence demonstrates that microglia mediated inflammatory responses play an important role in brain injury in ischemia. miRNA is the important factor in regulation of inflammation. However, the effect of miRNA in microglia mediated inflammatory responses has not been well studied. In the study, we demonstrate that miR-203 negatively regulates ischemia induced microglia activation by targeting MyD88, an important adapter protein involved in most Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) pathways. Through negative feedback, enforced expression of miR-203 or MyD88 siRNA silencing inhibits downstream NF-κß signaling and microglia activation, thereby alleviating neuronal injury. These findings reveal that miR-203 represents a novel target regulating neuroinflammation and brain injury, thus offering a new therapeutical strategy for cerebral hypoxic diseases.

MicroRNA-223 regulates inflammation and brain injury via feedback to NLRP3 inflammasome after intracerebral hemorrhage.

NLRP3 inflammasome, the multimeric protein complexes involved in the processing of IL-1ß through Caspase-1 cleavage, facilitates the inflammatory response. The control and activation of NLRP3 after intracerebral hemorrhage have not been fully studied. In the current study, we explore the specific microRNA which could regulate the NLRP3 inflammasome and inflammation after intracerebral hemorrhage. We detected the inverse relationship between the expression of miR-223 and NLRP3. We found that NLRP3 mRNA contains conserved miR-223 binding sites in its 3' UTR, and miR-223 could directly regulate NLRP3 expression through these 3' UTR sites. Our results indicate that miR-223 could downregulate NLRP3 to inhibit inflammation through caspase-1 and IL-1ß, reduce brain edema and improve neurological functions. Together, miR-223 may be a vital regulator of NLRP3 inflammasome activation. The results suggest that miR-223 represents a novel target reducing the inflammatory response, and offers a new therapeutical strategy following ICH.

Toll-like receptor-4-mediated autophagy contributes to microglial activation and inflammatory injury in mouse models of intracerebral haemorrhage.

AIMS: Much evidence demonstrates that Toll-like receptor-4 (TLR4)-mediated microglial activation is an important contributor to the inflammatory injury in intracerebral haemorrhage (ICH). However, the exact mechanism of TLR4-mediated microglial activation induced by ICH is not clear. In addition, microglial autophagy is involved other forms of nervous system injury. To explore the relationship between TLR4 and autophagy, we investigated the role of TLR4-mediated microglial autophagy and inflammation in ICH. METHODS: We detected TLR4 expression, autophagy and inflammation of microglia treated with lysed erythrocytes in vitro, and observed the cerebral water content and neurological deficit of ICH mice [TLR4-/- and wild type (WT)] in vivo. RESULTS: We found that lysed erythrocyte treated microglia (TLR4-/-) had reduced autophagy and inflammation compared with microglia (WT) in vitro. ICH mice (TLR4-/-) had reduced water content and neurological injury compared with ICH mice (WT). The autophagy inhibitor (3-methyladenine) decreased microglial activation and inflammatory injury due to lysed erythrocyte treatment, and improved the neurological function of ICH mice. CONCLUSIONS: Taken together, these data suggested that TLR4 induced autophagy contributed to the microglial activation and inflammatory injury and might provide novel therapeutic interventions for ICH.

Scavenger receptor SRA attenuates microglia activation and protects neuroinflammatory injury in intracerebral hemorrhage.

Microglia mediated neuroinflammation plays a crucial role in intracerebral hemorrhage (ICH). Therefore, the negative feedback immune mechanism to keep microglia homeostasis and inhibit the related inflammatory injury is important. Scavenger receptor A (SRA), a pattern recognition molecule, is a physiologic negative regulator of immune consequences. However, its role in microglia mediated immune response has not been well defined. In this study, we detected SRA expression and inflammatory response of microglia treated with erythrocyte lysate in vitro, and observed the cerebral water content and neurological deficit of ICH mice in vivo. We found that SRA was highly expressed in erythrocyte lysate treated microglia. Interestingly, genetic SRA ablation increased microglia activation and cytokine production, and sensitized mice to ICH induced neuron injury. In addition, we adoptive transferred microglia (WT) into ICH mice (SRA-/-), and found that the ICH-induced inflammation injury was effectively ameliorated. Therefore, the results demonstrated that SRA could attenuate microglia mediated inflammation injury in ICH. In addition, SRA mediated negative feedback mechanism in neuroimmune homeostasis might provide a novel therapeutical strategy for ICH. Scavenger receptor SRA restrains T cell activation and protects against concanavalin A-induced hepatic injury.

Regulatory T cells inhibit microglia activation and protect against inflammatory injury in intracerebral hemorrhage.

Numerous evidence demonstrate that microglia mediated inflammatory injury plays a critical role in intracerebral hemorrhage (ICH). Therefore, the way to inhibit the inflammatory response is greatly needed. Treg cells have been shown to play a critical role in immunologic self-tolerance as well as anti-tumor immune responses and transplantation. In the current study, we transfered Treg cells in the ICH model, and investigated the effect. The cytokines of microglia were measured by ELISA, JNK/ERK and NF-κB were measured by Western blot and EMSA (Electrophoretic Mobility Shift Assay), animal behavior was evaluated by animal behavioristics. We found that Treg cells could inhibit microglia mediated inflammatory response through NF-κB activation via the JNK/ERK pathway in vitro, and improve neurological function in vivo. Our findings suggest that Treg cells could suppress inflammatory injury and represent a novel cell-based therapeutical strategy in ICH.

Dendritic cells transduced with CPEB4 induced antitumor immune response.

Much evidence leads to the exploration of immunologic approaches for eliminating tumor cells. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is considered to be a novel therapeutical target for glioblastoma. In this study, we transduced DCs with CPEB4 to explore the immune response in vivo. We found that DCs transduced with recombinant adenovirus encoding CPEB4 could induce specific cytotoxic T lymphocytes (CTLs) to lyse glioma cells and augment the number of IFN-γ secreting T-cells in mice. In addition, the modified DCs could effectively protect mice from lethal challenges against glioma cells, reduce tumor growth and increase the mice life span. These results suggest that the DC transduced with CPEB4 may induce anti-tumor immunity against glioma cells and might be used as an efficient tumor vaccine in clinical applications.

Sinomenine inhibits microglia activation and attenuates brain injury in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) causes morbidity and mortality and commonly follows the reperfusion after an ischemic event. Microglial activation mediated cytokine and protease secretion contributes to brain injury in ICH. Previous studies have shown that sinomenine possesses potent immunoregulatory properties. However, little is known about its exact role in ICH. In the present study, to investigate the effect of sinomenine on microglial cells inflammation, we treated ICH-challenged BV2 microglial cells with sinomenine in vitro, and explored its neuroprotection role in intracerebral hemorrhage in vivo. Changes in inflammatory cytokines, such as TNF-α, IL-1ß and IL-6, reactive oxygen species (ROS) and NF-κB activation NF-κB were observed. In addition, the neurological deficit and cerebral water content of ICH mice were studied. The results demonstrated that sinomenine could inhibit the release of these cytokines and attenuate ROS production in a dose-dependent manner, and reduce NF-κB activation. Furthermore, sinomenine markedly inhibited cerebral water content and neurological deficit. In conclusion, our findings suggest that sinomenine played the protective effects through inhibition of microglial inflammation, and the findings also provided a novel therapy to treat ICH induced brain injury.

Microglial activation with reduction in autophagy limits white matter lesions and improves cognitive defects during cerebral hypoperfusion.

Microglial activation plays a vital role in the pathogenesis of white matter lesions (WMLs) during chronic cerebral hypo perfusion. Autophagy has been associated with both microglia survival and cell death. Yet, the role of autophagy during microglial activation in chronic cerebral ischemia is still unknown. We used a chronic cerebral hypoperfusion model by permanent stenosis of bilateral common carotid artery in mice to study microglial activation and autophagy. However, the autophagy inhibitor (3-methyladenine) could attenuate microglial autophagic activation, decrease white matter lesions, and improve working memory during chronic cerebral hypoperfusion in mice. In conclusion, chronic cerebral hypoperfusion that leads to microglial activation and autophagy induction exacerbates white matter lesions and cognitive deficits in mice. Our findings represent a potential novel target for chronic cerebral hypoperfusion therapy.