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.

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.

MYPT1SMKO Mice Function as a Novel Spontaneous Age- and Hypertension-Dependent Animal Model of CSVD.

Cerebral small vessel disease (CSVD) is the most common progressive vascular disease that causes vascular dementia. Aging and hypertension are major contributors to CSVD, but the pathophysiological mechanism remains unclear, mainly due to the lack of an ideal animal model. Our previous study revealed that vascular smooth muscle cell (VSMC)-specific myosin phosphatase target subunit 1 (MYPT1) knockout (MYPT1SMKO) leads to constant hypertension, prompting us to explore whether hypertensive MYPT1SMKO mice can be considered a novel CSVD animal model. Here, we found that MYPT1SMKO mice displayed age-dependent CSVD-like neurobehaviors, including decreased motion speed, anxiety, and cognitive decline. MYPT1SMKO mice exhibited remarkable white matter injury compared with control mice, as shown by the more prominent loss of myelin at 12 months of age. Additionally, MYPT1SMKO mice were found to exhibit CSVD-like small vessel impairment, including intravascular hyalinization, perivascular space enlargement, and microbleed and blood-brain barrier (BBB) disruption. Last, our results revealed that the brain of MYPT1SMKO mice was characterized by an exacerbated inflammatory microenvironment, which is similar to patients with CSVD. In light of the above structural and functional phenotypes that closely mimic the conditions of human CSVD, we suggest that MYPT1SMKO mice are a novel age- and hypertension-dependent animal model of CSVD.

Imperatorin inhibits mitogen-activated protein kinase and nuclear factor kappa-B signaling pathways and alleviates neuroinflammation in ischemic stroke.

AIMS: Microglia-mediated neuroinflammation plays an important role in the pathological process of ischemic stroke, and the effect of imperatorin on post-stroke neuroinflammation is not fully understood. METHODS: Primary microglia were treated with imperatorin for 2 h followed by LPS (100 ng/ml) for 24 h. The expression of inflammatory cytokines was detected by RT-PCR, ELISA, and Western blot. The activation of MAPK and NF-κB signaling pathways were analyzed by Western blot. The ischemic insult was determined using a transient middle cerebral artery occlusion (tMCAO) model in C57BL/6J mice. Behavior tests were used to assess the neurological deficits of MCAO mice. TTC staining was applied to measure infract volume. RESULTS: Imperatorin suppressed LPS-induced activation of microglia and pro-inflammatory cytokines release and attenuated ischemic injury in MCAO mice. The results of transcriptome sequencing and Western blot revealed that downregulation of MAPK and NF-κB pathways might contribute to the protective effects of imperatorin. CONCLUSIONS: Imperatorin downregulated MAPK and NF-κB signaling pathways and exerted anti-inflammatory effects in ischemic stroke, which indicated that imperatorin might be a potential compound for the treatment of stroke.

γ-Glutamylcysteine Alleviates Ischemic Stroke-Induced Neuronal Apoptosis by Inhibiting ROS-Mediated Endoplasmic Reticulum Stress.

Ischemic stroke is a severe and acute neurological disorder with limited therapeutic strategies currently available. Oxidative stress is one of the critical pathological factors in ischemia/reperfusion injury, and high levels of reactive oxygen species (ROS) may drive neuronal apoptosis. Rescuing neurons in the penumbra is a potential way to recover from ischemic stroke. Endogenous levels of the potent ROS quencher glutathione (GSH) decrease significantly after cerebral ischemia. Here, we aimed to investigate the neuroprotective effects of γ-glutamylcysteine (γ-GC), an immediate precursor of GSH, on neuronal apoptosis and brain injury during ischemic stroke. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic cerebral ischemia in mice, neuronal cell lines, and primary neurons. Our data indicated that exogenous γ-GC treatment mitigated oxidative stress, as indicated by upregulated GSH and decreased ROS levels. In addition, γ-GC attenuated ischemia/reperfusion-induced neuronal apoptosis and brain injury in vivo and in vitro. Furthermore, transcriptomics approaches and subsequent validation studies revealed that γ-GC attenuated penumbra neuronal apoptosis by inhibiting the activation of protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α) in the endoplasmic reticulum (ER) stress signaling pathway in OGD/R-treated cells and ischemic brain tissues. To the best of our knowledge, this study is the first to report that γ-GC attenuates ischemia-induced neuronal apoptosis by suppressing ROS-mediated ER stress. γ-GC may be a promising therapeutic agent for ischemic stroke.

Poncirin suppresses lipopolysaccharide (LPS)-induced microglial inflammation and ameliorates brain ischemic injury in experimental stroke in mice.

BACKGROUND: Based on accumulating evidence, excessive activation of microglia-mediated inflammatory responses plays an essential role in ischemic stroke. Poncirin (Pon) exerts anti-hyperalgesic, anti-osteoporotic and anti-tumor effects on various diseases. However, the roles of Pon in microglial activation and the underlying mechanism have not been elucidated. This study aimed to explore whether Pon inhibits lipopolysaccharide (LPS)-induced microglial neuroinflammation and protects against brain ischemic injury in experimental stroke in mice. METHODS: Primary microglia cells were prepared from the cerebral cortices of 1- to 2-day-old C57BL/6J mice. Murine BV2 cells and primary microglia were stimulated with LPS and the effects of a non-cytotoxic concentration of Pon on LPS-stimulated pro-inflammatory factors were measured using real-time PCR and enzyme-linked immunosorbent assays (ELISAs). Western blot analyses were used for mechanistic studies. In an in vivo study, 8-week-old male C57BL/6J mice were subjected to focal cerebral ischemia through middle cerebral artery occlusion (MCAO). Pon (30 mg/kg, i.p.) or the same volume of saline was administered after the MCAO model was established, and the infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. We also evaluated animal behaviours, the expression of pro-inflammatory cytokines and microglial activation in the ischemic hemisphere. RESULTS: Pon prevented the release of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha (TNF-α) in both BV2 cells and primary microglia stimulated with LPS. The inhibitory effects of Pon were associated with the regulation of the ERK1/2, JNK and nuclear factor kappa B (NF-κB) signaling pathways. In mice that underwent MCAO, Pon administration decreased the lesion size and improved neurological deficits. Furthermore, Pon attenuated the production of inflammatory cytokines mainly by restraining microglial activation after ischemic stroke. CONCLUSIONS: Based on the findings from the present study, Pon provides neuroprotection through its anti-inflammatory effects on microglia and it may be a useful treatment for ischemic stroke.

A SIRPα-Fc fusion protein enhances the antitumor effect of oncolytic adenovirus against ovarian cancer.

Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein-α (SIRPα)-IgG1 Fc fusion gene (termed SG635-SF) was constructed, which could block the CD47 'don't eat me' signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635-SF was found to specifically proliferate in hTERT-positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK-OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro, the antitumor effect of SG635-SF was significantly superior to that of SG635 in vivo. Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK-OV3 cells but not in those of CD47-negative HepG2 cells, indicating that the enhanced antitumor effect of SG635-SF was CD47-dependent. Collectively, these findings highlight a potent antitumor effect of SG635-SF in the treatment of CD47-positive cancers.

TMEM16A Inhibition Preserves Blood-Brain Barrier Integrity After Ischemic Stroke.

The inflammatory response plays a pivotal role in Blood-Brain Barrier (BBB) destruction following ischemic brain injury. Enhanced leukocyte adhesion to vascular endothelial cells is an essential event in the inflammatory process. TMEM16A, a newly discovered protein regulating calcium-activated chloride channels, is widely expressed in eukaryotes. Recent studies have suggested that upregulated expression of TMEM16A is associated with the occurrence and development of many diseases. However, the role of TMEM16A in regulating BBB integrity after ischemic stroke has not been fully investigated. In this study, we found that TMEM16A is mainly expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. Caccinh-A01, an TMEM16A inhibitor that reduced its upregulation, attenuated brain infarct size and neurological deficits after ischemic stroke. ICAM-1 and MPO expression and BBB permeability were decreased after TMEM16A inhibitor administration. In addition, TMEM16A silencing rescued oxygen-glucose deprivation/reoxygenation (OGD/R)-induced transendothelial permeability in vitro accompanied by decreased ICAM-1 expression and leukocyte adhesion. Furthermore, our mechanistic study showed that TMEM16A knockdown alleviated NF-κB activation and nuclear translocation, indicating that TMEM16A knockdown downregulated OGD/R-induced ICAM-1 expression in an NF-κB-dependent manner. Finally, NF-κB inhibitor treatment also alleviated OGD/ R-induced BBB permeability, confirming that activated NF-κB and increased ICAM-1 are essential factors involved in ischemia-induced BBB damage. Thus, our research provides a promising treatment strategy against BBB destruction after ischemic stroke, and TMEM16A may become a potential target for the treatment of ischemic stroke.

11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity.

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

Efficient synthesis of boron-containing α-acyloxyamide analogs via microwave irradiation.

In this report, a Passerini three-component reaction utilizing boron-containing carboxylic acids or aldehydes is discussed. The reaction was carried out in water and facilitated by the use of microwave irradiation. This methodology allowed for the efficient formation of a broad range of boron-containing α-acyloxyamides under mild conditions within a short time. Two series of boron-containing α-acyloxyamides were synthesized and subsequently screened for cytotoxicity using the MTT cell viability assay. Two potential lead compounds were found to have potent activity against the HepG2 cancer cell line, demonstrating the potential of this methodology for use in the development of novel pharmaceuticals.