Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1.

BACKGROUND: Macrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood. METHODS: We investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice. RESULTS: Ficolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice. CONCLUSION: Our work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

Inhibition of ADORA3 promotes microglial phagocytosis and alleviates chronic ischemic white matter injury.

BACKGROUND: Adenosine A3 receptor (ADORA3) belongs to the adenosine receptor families and the role of ADORA3 in vascular dementia (VaD) is largely unexplored. The present study sought to determine the therapeutic role of ADORA3 antagonist in a mouse model of VaD. METHODS: The GSE122063 dataset was selected to screen the differential expression genes and pathways between VaD patients and controls. A mouse model of bilateral carotid artery stenosis (BCAS) was established. The cognitive functions were examined by the novel object recognition test, Y maze test, and fear of conditioning test. The white matter injury (WMI) was examined by 9.4 T MRI, western blot, and immunofluorescence staining. The mechanisms of ADORA3-regulated phagocytosis by microglia were examined using qPCR, western blot, dual immunofluorescence staining, and flow cytometry. RESULTS: The expression of ADORA3 was elevated in brain tissues of VaD patients and ADORA3 was indicated as a key gene for VaD in the GSE122063. In BCAS mice, the expression of ADORA3 was predominantly elevated in microglia in the corpus callosum. ADORA3 antagonist promotes microglial phagocytosis to myelin debris by facilitating cAMP/PKA/p-CREB pathway and thereby ameliorates WMI and cognitive impairment in BCAS mice. The therapeutic effect of ADORA3 antagonist was partially reversed by the inhibition of the cAMP/PKA pathway. CONCLUSIONS: ADORA3 antagonist alleviates chronic ischemic WMI by modulating myelin clearance of microglia, which may be a potential therapeutic target for the treatment of VaD.

AZD1390, an ataxia telangiectasia mutated inhibitor, attenuates microglia-mediated neuroinflammation and ischemic brain injury.

AIMS: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown. METHODS: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay. RESULTS: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway. CONCLUSION: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke.

γ-Glutamylcysteine ameliorates blood-brain barrier permeability and neutrophil extracellular traps formation after ischemic stroke by modulating Wnt/ß-catenin signalling in mice.

During the inflammatory response after stroke, the blood-brain barrier (BBB) is significantly disrupted, compromising its integrity. This disruption allows many peripheral neutrophils to infiltrate the injury site in the brain and release neutrophil extracellular traps (NETs), which further increase BBB permeability. In this study, we aimed to investigate the protective effects of γ-Glutamylcysteine (γ-GC), an immediate precursor of GSH, against BBB breakdown and NET formation after ischemic stroke. Our data indicated that γ-GC treatment effectively attenuated BBB damage, decreased neutrophil infiltration, and suppressed the release of NETs, ultimately leading to the amelioration of ischemic injury. Transcriptomic data and subsequent validation studies revealed that mechanistically, γ-GC exerts its effect by activating the Wnt/ß-catenin pathway after ischemic stroke. This research suggests that γ-GC may hold promise as a therapeutic agent for alleviating brain injury following an ischemic stroke.

Neuroprotective effects of Aucubin against cerebral ischemia-reperfusion injury.

AIMS: To study the role of Aucubin (AU) in cerebral ischemia-reperfusion injury and investigate the potential mechanisms. METHODS: For the in vitro experiment, primary microglia were cultured and stimulated by Lipopolysaccharides (LPS) and treated with AU. Male C57/BL6J mice were used and middle cerebral artery occlusion (MCAO) model was performed to induce cerebral ischemia-reperfusion injury. For the short-term effects, mice administrated with AU (40 mg/kg) for 3 days after MCAO were evaluated for the infarct volume and neurological deficits. The neuroinflammatory factors and microglia activation were determined by Real-time PCR, western blot and immunofluorescence staining. For the long-term effects, MCAO mice were injected daily with AU (5 mg/kg or 10 mg/kg) for 28 days. Behavior tests were used to assess the neurological deficits of MCAO mice, and white matter integrity was determined by myelin basic protein (MBP) staining and black-gold staining. RESULTS: AU suppressed LPS-induced activation of microglia and pro-inflammatory cytokines release, and downregulated the NF-κB and MAPK pathways in primary microglia. In addition, AU attenuated ischemic injury and inhibited the neuro-inflammatory response in MCAO mice. Moreover, AU induced prolonged improvements in sensorimotor function and memory function following MCAO, and preserved white matter integrity in the long-term experiments. CONCLUSIONS: AU protected against ischemic injury, which might be correlated with the downregulation of NF-κB and MAPK signaling pathways. Furthermore, AU alleviated cognitive impairment after stroke and restored white matter integrity. Our data indicated that AU might be a potential compound for the treatment of stroke and post-stroke cognitive impairment.

Echinatin protects from ischemic brain injury by attenuating NLRP3-related neuroinflammation.

BACKGROUND: Microglia-mediated neuroinflammation is the major contributor to the secondary brain injury of ischemic stroke. NLRP3 is one of the major components of ischemia-induced microglial activation. Echinatin, a chalcone found in licorice, was reported to have the activity of anti-inflammation and antioxidant. However, the relative study of echinatin in microglia or ischemic stroke is still unclear. METHODS: We intravenously injected echinatin or vehicle into adult ischemic male C57/BL6J mice induced by 60-min transient middle cerebral artery occlusion (tMCAO). The intraperitoneal injection was performed 4.5 h after reperfusion and then daily for 2 more days. Infarct size, blood brain barrier (BBB) leakage, neurobehavioral tests, and microglial-mediated inflammatory reaction were examined to assess the outcomes of echinatin treatment. LPS and LPS/ATP stimulation on primary microglia were used to explore the underlying anti-inflammatory mechanism of echinatin. RESULTS: Echinatin treatment efficiently decreased the infarct size, alleviated blood brain barrier (BBB) damage, suppressed microglial activation, reduced the production of inflammatory factors (e.g., IL-1ß, IL-6, IL-18, TNF-α, iNOS, COX2), and relieved post-stroke neurological defects in tMCAO mice. Mechanistically, we found that echinatin could suppress the NLRP3 assembly and reduce the production of inflammatory mediators independently of NF-κB and monoamine oxidase (MAO). CONCLUSION: Based on our study, we have identified echinatin as a promising therapeutic strategy for the treatment of ischemic stroke.

Effect of femtosecond laser process parameters on the thermal denaturation degree of skin tissue.

The influence of femtosecond laser parameters on the degree of thermal denaturation was studied experimentally. The relationship between the degree of thermal denaturation and the characteristic parameters of skin microstructure and the secondary structure of skin tissue proteins in characterizing the degree of thermal damage was analyzed. The results showed the interaction of laser power, laser power, and scanning speed had a significant effect on the degree of thermal denaturation; greater degrees of thermal denaturation were associated with larger second-order moments of the texture angle of the skin microtissue and smaller entropy values and contrast, indicating a greater degree of thermal damage; and higher peak temperature, the lower peak intensity of Raman spectra, decrease in the percentage area of α-helix fitted curves and increase in the percentage area of ß-sheet and ß-turn fitted curves indicate that the protein is denatured to a large extent that means thermal damage is large.

SRGN amplifies microglia-mediated neuroinflammation and exacerbates ischemic brain injury.

BACKGROUND: Microglia is the major contributor of post-stroke neuroinflammation cascade and the crucial cellular target for the treatment of ischemic stroke. Currently, the endogenous mechanism underlying microglial activation following ischemic stroke remains elusive. Serglycin (SRGN) is a proteoglycan expressed in immune cells. Up to now, the role of SRGN on microglial activation and ischemic stroke is largely unexplored. METHODS: Srgn knockout (KO), Cd44-KO and wild-type (WT) mice were subjected to middle cerebral artery occlusion (MCAO) to mimic ischemic stroke. Exogenous SRGN supplementation was achieved by stereotactic injection of recombinant mouse SRGN (rSRGN). Cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Neurological functions were evaluated by the modified neurological severity score (mNSS) and grip strength. Microglial activation was detected by Iba1 immunostaining, morphological analysis and cytokines' production. Neuronal death was examined by MAP2 immunostaining and FJB staining. RESULTS: The expression of SRGN and its receptor CD44 was significantly elevated in the ischemic mouse brains, especially in microglia. In addition, lipopolysaccharide (LPS) induced SRGN upregulation in microglia in vitro. rSRGN worsened ischemic brain injury in mice and amplified post-stroke neuroinflammation, while gene knockout of Srgn exerted reverse impacts. rSRGN promoted microglial proinflammatory activation both in vivo and in vitro, whereas Srgn-deficiency alleviated microglia-mediated inflammatory response. Moreover, the genetic deletion of Cd44 partially rescued rSRGN-induced excessed neuroinflammation and ischemic brain injury in mice. Mechanistically, SRGN boosted the activation of NF-κB signal, and increased glycolysis in microglia. CONCLUSION: SRGN acts as a novel therapeutic target in microglia-boosted proinflammatory response following ischemic stroke.

Linarin ameliorates ischemia-reperfusion injury by the inhibition of endoplasmic reticulum stress targeting AKR1B1.

Due to various factors, there is still a lack of effective neuroprotective agents for ischemic stroke in clinical practice. Neuroinflammation and neuronal apoptosis mediated by endoplasmic reticulum stress are some of the important pathological mechanisms in ischemic stroke. Linarin has been reported to have anti-inflammation, antioxidant, and anti-apoptotic effects in myocardial ischemia, osteoarthritis, and kidney disease. Whether it exerts neuroprotective functions in ischemic stroke has not been investigated. The results showed that linarin could reduce the infarct volume in cerebral ischemia animal models, improve the neurological function scores and suppress the expression of inflammatory factors mediating the NF-κB. Meanwhile, it could protect the neurons from OGD/R-induced-apoptosis, which was related to the PERK-eIF2α pathway. Our results suggested linarin could inhibit neuronal inflammation and apoptosis induced by endoplasmic reticulum stress. Furthermore, the neuroprotective effect of linarin may be related to the inhibition of AKR1B1. Our study offers new insight into protecting against ischemia-reperfusion injury by linarin treatment in stroke.

MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination.

Chronic cerebral hypoperfusion is one of the pathophysiological mechanisms contributing to cognitive decline by causing white matter injury. Microglia phagocytosing myelin debris in a timely manner can promote remyelination and contribute to the repair of white matter. However, milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a microglial phagocytosis-related protein, has not been well studied in hypoperfusion-related cognitive dysfunction. We found that the expression of MFG-E8 was significantly decreased in the brain of mice after bilateral carotid artery stenosis (BCAS). MFG-E8 knockout mice demonstrated more severe BCAS-induced cognitive impairments in the behavioral tests. In addition, we discovered that the deletion of MFG-E8 aggravated white matter damage and the destruction of myelin microstructure through fluorescent staining and electron microscopy. Meanwhile, MFG-E8 overexpression by AAV improved white matter injury and increased the number of mature oligodendrocytes after BCAS. Moreover, in vitro and in vivo experiments showed that MFG-E8 could enhance the phagocytic function of microglia via the αVß3/αVß5/Rac1 pathway and IGF-1 production to promote the differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes. Interestingly, we found that MFG-E8 was mainly derived from astrocytes, not microglia. Our findings suggest that MFG-E8 is a potential therapeutic target for cognitive impairments following cerebral hypoperfusion.

GM-CSF Promotes the Development of Dysfunctional Vascular Networks in Moyamoya Disease.

Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease with the development of a network of abnormal vessels. Immune inflammation is associated with the occurrence and development of MMD. However, the mechanisms underlying the formation of the abnormal vascular network remain unclear. Twenty-eight patients with MMD, 26 ischemic stroke patients, and 26 unrelated healthy volunteers were enrolled in this study The data showed that the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) were higher in MMD patients than in healthy controls (P <0.01), and GM-CSF was mainly from Th1 and Th17 cells in MMD. We found that increased GM-CSF drove monocytes to secrete a series of cytokines associated with angiogenesis, inflammation, and chemotaxis. In summary, our findings demonstrate for the first time the important involvement of GM-CSF in MMD and that GM-CSF is an important factor in the formation of abnormal vascular networks in MMD.

Growth arrest specific protein 6 alleviated white matter injury after experimental ischemic stroke.

Ischemic white matter injury leads to long-term neurological deficits and lacks effective medication. Growth arrest specific protein 6 (Gas6) clears myelin debris, which is hypothesized to promote white matter integrity in experimental stroke models. By the middle cerebral artery occlusion (MCAO) stroke model, we observed that Gas6 reduced infarcted volume and behavior deficits 4 weeks after MCAO. Compared with control mice, Gas6-treatment mice represented higher FA values in the ipsilateral external capsules by MRI DTI scan. The SMI32/MBP ratio of the ipsilateral cortex and striatum was profoundly alleviated by Gas6 administration. Gas6-treatment group manifested thicker myelin sheaths than the control group by electron microscopy. We observed that Gas6 mainly promoted OPC maturation, which was closely related to microglia. Mechanically, Gas6 accelerated microglia-mediated myelin debris clearance and cholesterol transport protein expression (abca1, abcg1, apoc1, apoe) in vivo and in vitro, accordingly less myelin debris and lipid deposited in Gas6 treated stroke mice. HX531 (RXR inhibitor) administration mitigated the functions of Gas6 in speeding up debris clearance and cholesterol transport protein expression. Generally, we concluded that Gas6 cleared myelin debris and promoted cholesterol transportation protein expression through activating RXR, which could be one critical mechanism contributing to white matter repair after stroke.

Acute ischemia induces spatially and transcriptionally distinct microglial subclusters.

BACKGROUND: Damage in the ischemic core and penumbra after stroke affects patient prognosis. Microglia immediately respond to ischemic insult and initiate immune inflammation, playing an important role in the cellular injury after stroke. However, the microglial heterogeneity and the mechanisms involved remain unclear. METHODS: We first performed single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics (ST) on middle cerebral artery occlusion (MCAO) mice from three time points to determine stroke-associated microglial subclusters and their spatial distributions. Furthermore, the expression of microglial subcluster-specific marker genes and the localization of different microglial subclusters were verified on MCAO mice through RNAscope and immunofluorescence. Gene set variation analysis (GSVA) was performed to reveal functional characteristics of microglia sub-clusters. Additionally, ingenuity pathway analysis (IPA) was used to explore upstream regulators of microglial subclusters, which was confirmed by immunofluorescence, RT-qPCR, shRNA-mediated knockdown, and targeted metabolomics. Finally, the infarct size, neurological deficits, and neuronal apoptosis were evaluated in MCAO mice after manipulation of specific microglial subcluster. RESULTS: We discovered stroke-associated microglial subclusters in the brains of MCAO mice. We also identified novel marker genes of these microglial subclusters and defined these cells as ischemic core-associated (ICAM) and ischemic penumbra-associated (IPAM) microglia, according to their spatial distribution. ICAM, induced by damage-associated molecular patterns, are probably fueled by glycolysis, and exhibit increased pro-inflammatory cytokines and chemokines production. BACH1 is a key transcription factor driving ICAM generation. In contrast, glucocorticoids, which are enriched in the penumbra, likely trigger IPAM formation, which are presumably powered by the citrate cycle and oxidative phosphorylation and are characterized by moderate pro-inflammatory responses, inflammation-alleviating metabolic features, and myelinotrophic properties. CONCLUSIONS: ICAM could induce excessive neuroinflammation, aggravating brain injury, whereas IPAM probably exhibit neuroprotective features, which could be essential for the homeostasis and survival of cells in the penumbra. Our findings provide a biological basis for targeting specific microglial subclusters as a potential therapeutic strategy for ischemic stroke.

Infiltrating myeloid cell-derived properdin markedly promotes microglia-mediated neuroinflammation after ischemic stroke.

BACKGROUND: Emerging evidence has shown that myeloid cells that infiltrate into the peri-infarct region may influence the progression of ischemic stroke by interacting with microglia. Properdin, which is typically secreted by immune cells such as neutrophils, monocytes, and T cells, has been found to possess damage-associated molecular patterns (DAMPs) properties and can perform functions unrelated to the complement pathway. However, the role of properdin in modulating microglia-mediated post-stroke neuroinflammation remains unclear. METHODS: Global and conditional (myeloid-specific) properdin-knockout mice were subjected to transient middle cerebral artery occlusion (tMCAO). Histopathological and behavioral tests were performed to assess ischemic brain injury in mice. Single-cell RNA sequencing and immunofluorescence staining were applied to explore the source and the expression level of properdin. The transcriptomic profile of properdin-activated primary microglia was depicted by transcriptome sequencing. Lentivirus was used for macrophage-inducible C-type lectin (Mincle) silencing in microglia. Conditioned medium from primary microglia was administered to primary cortex neurons to determine the neurotoxicity of microglia. A series of cellular and molecular biological techniques were used to evaluate the proinflammatory response, neuronal death, protein-protein interactions, and related signaling pathways, etc. RESULTS: The level of properdin was significantly increased, and brain-infiltrating neutrophils and macrophages were the main sources of properdin in the ischemic brain. Global and conditional myeloid knockout of properdin attenuated microglial overactivation and inflammatory responses at the acute stage of tMCAO in mice. Accordingly, treatment with recombinant properdin enhanced the production of proinflammatory cytokines and augmented microglia-potentiated neuronal death in primary culture. Mechanistically, recombinant properdin served as a novel ligand that activated Mincle receptors on microglia and downstream pathways to drive primary microglia-induced inflammatory responses. Intriguingly, properdin can directly bind to the microglial Mincle receptor to exert the above effects, while Mincle knockdown limits properdin-mediated microglial inflammation. CONCLUSION: Properdin is a new medium by which infiltrating peripheral myeloid cells communicate with microglia, further activate microglia, and exacerbate brain injury in the ischemic brain, suggesting that targeted disruption of the interaction between properdin and Mincle on microglia or inhibition of their downstream signaling may improve the prognosis of ischemic stroke.

A newly-synthesized compound CP-07 alleviates microglia-mediated neuroinflammation and ischemic brain injury via inhibiting STAT3 phosphorylation.

Background and Objectives: Overactivated glial cells, especially microglia, are core components in the progression of pathologic neuroinflammation, and the application of anti-inflammatory reagents has been regarded as a potential therapy in the management of infarction/reperfusion (I/R) brain injury. This research aims to clarify the anti-inflammatory efect of a novel lipophilic compound N-(2-[4-tert-butylphenyl]-2-[pyrrolidine-1-yl]ethyl)-7-methyl-4-oxo-4H-chromene-2-carboxamide (named CP-07 in this study) in LPS-stimulated BV2 cell line and primary mouse microglia, and its therapeutic effect on I/R brain injury. Method: Cell Counting Kit-8 assay was used to determine the maximal nontoxic dose of CP-07. The mRNA levels of representative proinflammatory cytokines were determined by quantitative real-time polymerase chain reaction both in vitro and in vivo. TTC staining was performed to calculate infarct volumes while behavioral tests were used to assess the neurological deficits at 24 h after middle cerebral artery occlusion (MCAO). Flow cytometry analysis and immunofluorescence staining were performed to calculate the percentage of pro-inflammatory microglia in vivo.A selective JAK2/STAT3 pathway inhibitor, AG490 was used to block STAT3 phosphorylation before the CP-07 anti-inflammation tests in vitro. Results: CP-07 could effectively suppress the mRNA levels of IL-6, IL-1ß, iNOS and TNF-α induced by lipopolysaccharide (LPS) in vitro, and markedly block the evaluation of the fluorescence intensity of Iba-1 in primary mouse microglia. In middle cerebral arteryocclusion models, intraperitoneal injection with 1 mg/kg CP-07 significantly reduced cerebral infarct volumes at 24 h after surgery compared with vehicle treatment group, and promoted the recovery of neurological functions in MCAO mice. Further studies validated that CP-07 administration reduced the percentage of CD86 positive microglia after I/R injury, and the expression level of p-STAT3 was also markedly reduced in both microglial cells and the penumbra tissues. Blocking STAT3 phosphorylation with AG490 could completely eliminate the anti-inflammatory effects of CP-07, at least in vitro. Conclusion: We showed that a newly synthesized compound, CP-07, could effectively reduce the inflammatory responses in LPS-stimulated BV2 cells and primary mouse microglia, and overproduction of cytokines in middle cerebral artery occlusion mouse models by inhibiting STAT3 phosphorylation, leading to a neuroprotective effect on I/R brain injury.

Research on the effect of process parameters on the performance of femtosecond laser-bonded skin microstructure.

In this paper, the effect of the femtosecond laser process parameters on the texture characteristics of the microstructure was analyzed with the response surface method. The correspondence between the temperature of skin during laser bonding and microscopic tissue texture characteristics parameters was explored. The results show that the three process parameters of laser power, scanning speed, and scanning times and the interaction between the parameters have different patterns of influence on the four texture characteristics parameters of skin microstructure angular second-order moments, entropy, contrast, and relevance. Angular second-order moments and relevance of skin microstructure textures increase with increasing temperature, while entropy values and contrast decrease. It provides another way to evaluate the performance of femtosecond laser-bonded skin with microstructure.

QHRD106 ameliorates ischemic stroke injury as a long-acting tissue kallikrein preparation.

Ischemic stroke is the second leading cause of death worldwide, and there are limited effective treatment strategies. QHRD106, a polyethyleneglycol (PEG)-modified long-acting tissue kallikrein preparation, has not been reported previously. In this study, we aimed to investigate the therapeutic effect of QHRD106 in ischemic stroke and its possible mechanism. We found that QHRD106 treatment alleviated brain injury after stroke via bradykinin (BK) receptor B2 (B2R) instead of BK receptor B1 (B1R). Mechanistically, QHRD106 reduced high-mobility group box 1 (HMGB1)-induced apoptosis and inflammation after ischemic stroke in vivo and in vitro. Moreover, we confirmed that QHRD106 reduced the level of acetylated HMGB1 and reduced the binding between heat shock protein 90 alpha family class A member 1 (HSP90AA1) and HMGB1, thus inhibiting the translocation and release of HMGB1. In summary, these findings indicate that QHRD106 treatment has therapeutic potential for cerebral ischemic stroke.

Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization.

Microglia-mediated neuroinflammation is involved in various neurological diseases, including ischemic stroke, but the endogenous mechanisms preventing unstrained inflammation is still unclear. The anti-inflammatory role of transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) in macrophages and microglia has previously been identified. However, the endogenous mechanisms that how NR4A1 restricts unstrained inflammation remain elusive. Here, we observed that NR4A1 is up-regulated in the cytoplasm of activated microglia and localizes to processing bodies (P-bodies). In addition, we found that cytoplasmic NR4A1 functions as an RNA-binding protein (RBP) that directly binds and destabilizes Tnf mRNA in an N6-methyladenosine (m6A)-dependent manner. Remarkably, conditional microglial deletion of Nr4a1 elevates Tnf expression and worsens outcomes in a mouse model of ischemic stroke, in which case NR4A1 expression is significantly induced in the cytoplasm of microglia. Thus, our study illustrates a novel mechanism that NR4A1 posttranscriptionally regulates Tnf expression in microglia and determines stroke outcomes.

Pharmacological Upregulation of Microglial Lipid Droplet Alleviates Neuroinflammation and Acute Ischemic Brain Injury.

Lipid droplets (LDs) were reported to play an important role in the modulation of inflammation and various cellular processes among multiple cell types. However, LDs accumulation, its function and mechanisms of its formation during ischemic stroke remained poorly-identified. In this study, we observed increased LDs accumulation in microglia at the acute stage of ischemic stroke by immunofluorescence and flow cytometry. Transcriptomic analysis indicated that microglia accumulated with LDs were associated with inflammation and phagocytosis. Both inflammatory activation and phagocytosis of tissue debris in microglia could contribute to LDs formation. Moreover, through specific LDs depletion and overload experiments by pharmacological approaches, we proposed that LDs was critical for the maintenance of anti-inflammatory properties of microglia. Furthermore, Atglistatin, a specific adipose triglyceride lipase (ATGL) inhibitor, was shown to prevent proinflammatory cytokines production in primary microglia through decreased LDs lipolysis. After Atglistatin treatment, middle cerebral artery occlusion (MCAO) mice showed decreased infarct volume and improved neurobehavioral performance at the acute stage of stroke. Our findings provided a biological basis for microglial LDs regulation as a potential therapeutic strategy for acute ischemic stroke and uncovered the neuroprotective role of Atglistatin in the treatment of MCAO mice.

Compound from Magnolia officinalis Ameliorates White Matter Injury by Promoting Oligodendrocyte Maturation in Chronic Cerebral Ischemia Models.

Chronic cerebral hypoperfusion leads to white matter injury (WMI), which subsequently causes neurodegeneration and even cognitive impairment. However, due to the lack of treatment specifically for WMI, novel recognized and effective therapeutic strategies are urgently needed. In this study, we found that honokiol and magnolol, two compounds derived from Magnolia officinalis, significantly facilitated the differentiation of primary oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes, with a more prominent effect of the former compound. Moreover, our results demonstrated that honokiol treatment improved myelin injury, induced mature oligodendrocyte protein expression, attenuated cognitive decline, promoted oligodendrocyte regeneration, and inhibited astrocytic activation in the bilateral carotid artery stenosis model. Mechanistically, honokiol increased the phosphorylation of serine/threonine kinase (Akt) and mammalian target of rapamycin (mTOR) by activating cannabinoid receptor 1 during OPC differentiation. Collectively, our study indicates that honokiol might serve as a potential treatment for WMI in chronic cerebral ischemia.