Detecting prokaryote-specific gene and other bacterial signatures in thrombi from patients with acute ischemic stroke.

BACKGROUND AND PURPOSE: Microbial infection has been associated with thrombogenesis. This study aimed to detect bacterium-specific genes and other signatures in thrombi from patients with acute ischemic stroke and to relate these signatures to clinical characteristics. METHODS: Blood samples were collected before thrombectomy procedures, and thrombus samples were obtained during the procedure. Identification and classification of bacteria in the samples were accomplished using 16 S rRNA gene sequencing. Bacterium-specific structures were observed with transmission electron microscopy. Bacterium-specific biomarkers were detected through immunohistochemical staining. RESULTS: 16 S rRNA gene was detected in 32.1% of the thrombus samples from 81 patients. Bacillus (0.04% vs. 0.00046%, p = 0.003), Parabacteroides (0.20% vs. 0.09%, p = 0.029), Prevotella (1.57% vs. 0.38%, p = 0.010), Streptococcus (1.53% vs. 0.29%, p = 0.001), Romboutsia (0.18% vs. 0.0070%, p = 0.029), Corynebacterium (1.61% vs. 1.26%, p = 0.026) and Roseburia (0.53% vs. 0.05%, p = 0.005) exhibited significantly higher abundance in thrombi compared to arterial blood. Bacteria-like structures were observed in 22 (27.1%), while whole bacteria-like structures were observed in 7 (8.6%) thrombi under transmission electron microscopy. Immunohistochemical staining detected bacterium-specific monocyte/macrophage markers in 51 (63.0%) out of 81 thrombi. Logistic regression analysis indicated that alcohol consumption was associated with a higher bacteria burden in thrombi (odds ratio = 3.19; 95% CI, 1.10-9.27; p = 0.033). CONCLUSION: Bacterial signatures usually found in the oral cavity and digestive tract were detected in thrombi from patients with ischemic stroke. This suggests a potential involvement of bacterial infection in the development of thrombosis. Long-term alcohol consumption may potentially enhance this possibility.

Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury.

BACKGROUND: Neuroinflammation is a vital pathophysiological process during ischemic stroke. Activated astrocytes play a major role in inflammation. Lipocalin-2 (LCN2), secreted by activated astrocytes, promotes neuroinflammation. Pyroptosis is a pro-inflammatory form of programmed cell death that has emerged as a new area of research in stroke. Nevertheless, the potential role of LCN2 in astrocyte pyroptosis remains unclear. METHODS: An ischemic stroke model was established by middle cerebral artery occlusion (MCAO) in vivo. In this study, in vitro, oxygen-glucose deprivation and reoxygenation (O/R) were applied to cultured astrocytes. 24p3R (the LCN2 receptor) was inhibited by astrocyte-specific adeno-associated virus (AAV-GFAP-24p3Ri). MCC950 and Nigericin sodium salt (Nig) were used to inhibit or promote the activation of NLRP3 inflammasome pharmacologically, respectively. Histological and biochemical analyses were performed to assess astrocyte and neuron death. Additionally, the neurological deficits of mice were evaluated. RESULTS: LCN2 expression was significantly induced in astrocytes 24 h after stroke onset in the mouse MCAO model. Lcn2 knockout (Lcn2-/-) mice exhibited reduced infarct volume and improved neurological and cognitive functions after MCAO. LCN2 and its receptor 24p3R were colocalized in astrocytes. Mechanistically, suppression of 24p3R by AAV-GFAP-24p3Ri alleviated pyroptosis-related pore formation and the secretion of pro-inflammatory cytokines via LCN2, which was then reversed by Nig-induced NLRP3 inflammasome activation. Astrocyte pyroptosis was exacerbated in Lcn2-/- mice by intracerebroventricular administration of recombinant LCN2 (rLCN2), while this aggravation was restricted by blocking 24p3R or inhibiting NLRP3 inflammasome activation with MCC950. CONCLUSION: LCN2/24p3R mediates astrocyte pyroptosis via NLRP3 inflammasome activation following cerebral ischemia/reperfusion injury.

Extracellular vesicles carrying proinflammatory factors may spread atherosclerosis to remote locations.

Most cells involved in atherosclerosis release extracellular vesicles (EVs), which can carry bioactive substances to downstream tissues via circulation. We hypothesized that EVs derived from atherosclerotic plaques could promote atherogenesis in remote locations, a mechanism that mimics the blood metastasis of cancer. Ldlr gene knockout (Ldlr KO) rats were fed on a high cholesterol diet and underwent partial carotid ligation to induce local atherosclerosis. EVs were separated from carotid artery tissues and downstream blood of carotid ligation by centrifugation. MiRNA sequencing and qPCR were then performed to detect miRNA differences in EVs from rats with and without induced carotid atherosclerosis. Biochemical analyses demonstrated that EVs derived from atherosclerosis could increase the expression of ICAM-1, VCAM-1, and E-selectin in endothelial cells in vitro. EVs derived from atherosclerosis contained a higher level of miR-23a-3p than those derived from controls. MiR-23a-3p could promote endothelial inflammation by targeting Dusp5 and maintaining ERK1/2 phosphorylation in vitro. Inhibiting EV release could attenuate atherogenesis and reduce macrophage infiltration in vivo. Intravenously administrating atherosclerotic plaque-derived EVs could induce intimal inflammation, arterial wall thickening and lumen narrowing in the carotids of Ldlr KO rats, while simultaneous injection of miR-23a-3p antagomir could reverse this reaction. The results suggested that EVs may transfer atherosclerosis to remote locations by carrying proinflammatory factors, particularly miR-23a-3p.

High-frequency repetitive transcranial magnetic stimulation (rTMS) protects against ischemic stroke by inhibiting M1 microglia polarization through let-7b-5p/HMGA2/NF-κB signaling pathway.

BACKGROUND: Microglia assume opposite phenotypes in response to ischemic brain injury, exerting neurotoxic and neuroprotective effects under different ischemic stages. Modulating M1/M2 polarization is a potential therapy for treating ischemic stroke. Repetitive transcranial magnetic stimulation (rTMS) held the capacity to regulate neuroinflammation and astrocytic polarization, but little is known about rTMS effects on microglia. Therefore, the present study aimed to examine the rTMS influence on microglia polarization and the underlying possible molecular mechanisms in ischemic stroke models. METHODS: Previously reported 10 Hz rTMS protocol that regulated astrocytic polarization was used to stimulate transient middle cerebral artery occlusion (MCAO) rats and oxygen and glucose deprivation/reoxygenation (OGD/R) injured BV2 cells. Specific expression levels of M1 marker iNOS and M2 marker CD206 were measured by western blotting and immunofluorescence. MicroRNA expression changes detected by high-throughput second-generation sequencing were validated by RT-PCR and fluorescence in situ hybridization (FISH) analysis. Dual-luciferase report assay and miRNA knock-down were applied to verify the possible mechanisms regulated by rTMS. Microglia culture medium (MCM) from different groups were collected to measure the TNF-α and IL-10 concentrations, and detect the influence on neuronal survival. Finally, TTC staining and modified Neurological Severity Score (mNSS) were used to determine the effects of MCM on ischemic stroke volume and neurological functions. RESULTS: The 10 Hz rTMS inhibited ischemia/reperfusion induced M1 microglia and significantly increased let-7b-5p level in microglia. HMGA2 was predicted and proved to be the target protein of let-7b-5p. HMGA2 and its downstream NF-κB signaling pathway were inhibited by rTMS. Microglia culture medium (MCM) collected from rTMS treated microglia contained lower TNF-α concentration but higher IL-10 concentration than no rTMS treated MCM, reducing ischemic volumes and neurological deficits of MCAO mice. However, knockdown of let-7b-5p by antagomir reversed rTMS effects on microglia phenotype and associated HMGA/NF-κB activation and neurological recovery. CONCLUSION: High-frequency rTMS could alleviate ischemic stroke injury through inhibiting M1 microglia polarization via regulating let-7b-5p/HMGA2/NF-κB signaling pathway in MCAO models.

Low-density lipoprotein receptor (LDLR) regulates NLRP3-mediated neuronal pyroptosis following cerebral ischemia/reperfusion injury.

BACKGROUND: Inflammatory response has been recognized as a pivotal pathophysiological process during cerebral ischemic stroke. NLRP3 inflammasome, involved in the regulation of inflammatory cascade, can simultaneously lead to GSDMD-executed pyroptosis in cerebral ischemia. Low-density lipoprotein receptor (LDLR), responsible for cholesterol uptake, was noted to exert potential anti-inflammatory bioactivities. Nevertheless, the role of LDLR in neuroinflammation mobilized by cerebral ischemia/reperfusion (I/R) has not been investigated. METHODS: Ischemic stroke mice model was accomplished by middle cerebral artery occlusion. Oxygen-glucose deprivation was employed after primary cortical neuron was extracted and cultured. A pharmacological inhibitor of NLRP3 (CY-09) was administered to suppress NLPR3 activation. Histological and biochemical analysis were performed to assess the neuronal death both in vitro and in vivo. In addition, neurological deficits and behavioral deterioration were evaluated in mice. RESULTS: The expression of LDLR was downregulated following cerebral I/R injury. Genetic knockout of Ldlr enhanced caspase-1-dependent cleavage of GSDMD and resulted in severe neuronal pyroptosis. LDLR deficiency contributed to excessive NLRP3-mediated maturation and release of IL-1ß and IL-18 under in vitro and in vivo ischemic conditions. These influences ultimately led to aggravated neurological deficits and long-term cognitive dysfunction. Blockade of NLRP3 substantially retarded neuronal pyroptosis in Ldlr-/- mice and cultured Ldlr-/- neuron after experimental stroke. CONCLUSIONS: These results demonstrated that LDLR modulates NLRP3-mediated neuronal pyroptosis and neuroinflammation following ischemic stroke. Our findings characterize a novel role for LDLR as a potential therapeutic target in neuroinflammatory responses to acute cerebral ischemic injury.

High-frequency repetitive transcranial magnetic stimulation improves functional recovery by inhibiting neurotoxic polarization of astrocytes in ischemic rats.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive treatment for ischemic stroke. Astrocytes regulation has been suggested as one mechanism for rTMS effectiveness. But how rTMS regulates astrocytes remains largely undetermined. There were neurotoxic and neuroprotective phenotypes of astrocytes (also denoted as classically and alternatively activated astrocytes or A1 and A2 astrocytes) pertaining to pro- or anti-inflammatory gene expression. Pro-inflammatory or neurotoxic polarized astrocytes were induced during cerebral ischemic stroke. The present study aimed to investigate the effects of rTMS on astrocytic polarization during cerebral ischemic/reperfusion injury. METHODS: Three rTMS protocols were applied to primary astrocytes under normal and oxygen-glucose deprivation/reoxygenation (OGD/R) conditions. Cell survival, proliferation, and phenotypic changes were assessed after 2-day treatment. Astrocytes culture medium (ACM) from control, OGD/R, and OGD/R + rTMS groups were mixed with neuronal medium to culture neurons for 48 h and 7 days, in order to explore the influence on neuronal survival and synaptic plasticity. In vivo, rats were subjected to middle cerebral artery occlusion (MCAO), and received posterior orbital intravenous injection of ACM collected from different groups at reperfusion, and at 3 days post reperfusion. The apoptosis in the ischemic penumbra, infarct volumes, and the modified Neurological Severity Score (mNSS) were evaluated at 1 week after reperfusion, and cognitive functions were evaluated using the Morris Water Maze (MWM) tests. Finally, the 10 Hz rTMS was directly applied to MCAO rats to verify the rTMS effects on astrocytic polarization. RESULTS: Among these three frequencies, the 10 Hz protocol exerted the greatest potential to modulate astrocytic polarization after OGD/R injury. Classically activated and A1 markers were significantly inhibited by rTMS treatment. In OGD/R model, the concentration of pro-inflammatory mediator TNF-α decreased from 57.7 to 23.0 Ñ€g/mL, while anti-inflammatory mediator IL-10 increased from 99.0 to 555.1 Ñ€g/mL in the ACM after rTMS treatment. The ACM collected from rTMS-treated astrocytes significantly alleviated neuronal apoptosis induced by OGD/R injury, and promoted neuronal plasticity. In MCAO rat model, the ACM collected from rTMS treatment decreased neuronal apoptosis and infarct volumes, and improved cognitive functions. The neurotoxic astrocytes were simultaneously inhibited after rTMS treatment. CONCLUSION: Inhibition of neurotoxic astrocytic polarization is a potential mechanism for the effectiveness of high-frequency rTMS in cerebral ischemic stroke.

Binding Differences of Two Homochiral [Ru(bpy)2dppz]2+ Complexes with poly(U)·poly(A)*poly(U) Triplex RNA.

The first investigation of chiral ruthenium(II) complexes Δ- and Λ-[Ru(bpy)2dppz]2+ and triplex RNA poly(U)·poly(A)*poly(U) was carried out, which showed that Δ enantiomer displayed significant ability in stabilizing model triplex RNA.

Metal-Phenolic Network with Pd Nanoparticle Nodes Synergizes Oxidase-Like and Photothermal Properties to Eradicate Oral Polymicrobial Biofilm-Associated Infections.

Designing an effective treatment strategy to combat oral diseases caused by complex polymicrobial biofilms remains a great challenge. Herein, a series of metal-phenolic network with Pd nanoparticle nodes using polyphenols as stabilizers and reducing agents is constructed. Among them, sulfonated lignin-Pd (SLS-Pd) with ultrafine size palladium nanoparticles and broadband near infrared absorption exhibit excellent oxidase-like activity and stable photothermal effect. In vitro experiments demonstrate that the superoxide radical generated by SLS-Pd oxidase-like activity exhibits selective antibacterial effects, while its photothermal effect induced hyperthermia exhibits potent antifungal properties. This difference is further elucidated by RNA-sequencing analysis and all-atom simulation. Moreover, the SLS-Pd-mediated synergistic antimicrobial system exhibits remarkable efficacy in combating various biofilms and polymicrobial biofilms. By establishing a root canal model and an oropharyngeal candidiasis model, the feasibility of the synergistic antimicrobial system in treating oral biofilm-related infections is further validated. This system provides a promising therapeutic approach for polymicrobial biofilm-associated infections in the oral cavity.

Supramolecular Photothermal Cascade Nano-Reactor Enables Photothermal Effect, Cascade Reaction, and In Situ Hydrogelation for Biofilm-Associated Tooth-Extraction Wound Healing.

Due to the emergence of drug resistance in bacteria and biofilm protection, achieving a satisfactory therapeutic effect for bacteria-infected open wounds with conventional measures is problematic. Here, a photothermal cascade nano-reactor (CPNC@GOx-Fe2+ ) is constructed through a supramolecular strategy through hydrogen bonding and coordination interactions between chitosan-modified palladium nano-cube (CPNC), glucose oxidase (GOx), and ferrous iron (Fe2+ ). CPNC@GOx-Fe2+ exhibits excellent photothermal effects and powers the GOx-assisted cascade reaction to generate hydroxyl radicals, enabling photothermal and chemodynamic combination therapy against bacteria and biofilms. Further proteomics, metabolomics, and all-atom simulation results indicate that the damage of the hydroxyl radical to the function and structure of the cell membrane and the thermal effect enhance the fluidity and inhomogeneity of the bacterial cell membrane, resulting in the synergistic antibacterial effect. In the biofilm-associated tooth extraction wound model, the hydroxyl radical generated from the cascade reaction process can initiate the radical polymerization process to form a hydrogel in situ for wound protection. In vivo experiments confirm that synergistic antibacterial and wound protection can accelerate the healing of infected tooth-extraction wounds without affecting the oral commensal microbiota. This study provides a way to propose a multifunctional supramolecular system for the treatment of open wound infection.

Aberrant oligodendroglial LDL receptor orchestrates demyelination in chronic cerebral ischemia.

Oligodendrocytes express low-density lipoprotein receptor (LDLR) to endocytose cholesterol for the maintenance of adulthood myelination. However, the potential role of LDLR in chronic cerebral ischemia-related demyelination remains unclear. We used bilateral carotid artery stenosis (BCAS) to induce sustained cerebral ischemia in mice. This hypoxic-ischemic injury caused a remarkable decrease in oligodendroglial LDLR, with impaired oligodendroglial differentiation and survival. Oligodendroglial cholesterol levels, however, remained unchanged. Mouse miR-344e-3p and the human homolog miR-410-3p, 2 miRNAs directly targeting Ldlr, were identified in experimental and clinical leukoaraiosis and were thus implicated in the LDLR reduction. Lentiviral delivery of LDLR ameliorated demyelination following chronic cerebral ischemia. By contrast, Ldlr-/- mice displayed inadequate myelination in the corpus callosum. Ldlr-/- oligodendrocyte progenitor cells (OPCs) exhibited reduced ability to differentiate and myelinate axons in vitro. Transplantation with Ldlr-/- OPCs could not rescue the BCAS-induced demyelination. Such LDLR-dependent myelin restoration might involve a physical interaction of the Asn-Pro-Val-Tyr (NPVY) motif with the phosphotyrosine binding domain of Shc, which subsequently activated the MEK/ERK pathway. Together, our findings demonstrate that the aberrant oligodendroglial LDLR in chronic cerebral ischemia impairs myelination through intracellular signal transduction. Preservation of oligodendroglial LDLR may provide a promising approach to treat ischemic demyelination.

Extracellular Vesicles as Messengers in Atherosclerosis.

Atherosclerosis is a major cause of cardiovascular diseases. Most cells involved in atherosclerosis can shed extracellular vesicles (EVs). Both atherogenic factors, such as hypoxia and oxidative stress, and atheroprotective factors, such as laminar blood flow, can influence the production of EV shedding. EVs can carry protein, DNA, mRNA, and noncoding RNA and act as mediators or messengers for cell-to-cell communications. EVs have been proven to promote or inhibit atherogenesis under particular circumstances. Therefore, EVs might be targeted for preventing or treating atherosclerotic diseases. The level of circulating EVs has been associated with the presence, progressiveness, or severity of atherosclerosis. Therefore, EVs may be utilized as indexes for diagnosing and grading atherosclerosis. Here, we reviewed the progress concerning the involvements of EVs in atherogenesis and atheroprotection. We also discussed the potential applications of EVs in managing atherosclerotic diseases.

Overexpression of BRCA1 in Neural Stem Cells Enhances Cell Survival and Functional Recovery after Transplantation into Experimental Ischemic Stroke.

Transplantation of neural stem cells (NSCs) is a promising therapy for ischemic stroke. However, the effectiveness of this approach is limited by grafted cell death. Breast cancer susceptibility protein 1 (BRCA1) could suppress apoptosis in neural progenitors and modulate oxidative stress in neurons. In this study, we found that BRCA1 was upregulated by oxygen-glucose deprivation/reoxygenation (OGD/R). Overexpression of BRCA1 in NSCs reduced cell apoptosis and oxidative stress after OGD/R insult. The molecule overexpression also stimulated cellular proliferation in OGD/R NSCs and increased the survival rate of grafted cells. Further, the transplantation of BRCA1-transfected NSCs into mice with ischemic stroke increased brain-derived neurotropic factor and nerve growth factor expression in the brain and elicited neurological function improvement. In addition, we found that RING finger domain and BRCT domain of BRCA1 could physically interact with p53 in NSCs. The cross talk between BRCA1 RING finger domain and p53 was responsible for p53 ubiquitination and degradation. Our findings indicate that modification with BRCA1 could enhance the efficacy of NSCs transplantation in ischemic stroke.

Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway.

Cellular oxidative stress plays a vital role in the pathological process of neural damage in cerebral ischemia/reperfusion (I/R). The breast cancer susceptibility protein 1 (BRCA1), a tumor suppressor, can modulate cellular antioxidant response and DNA repair. Yet the role of BRCA1 in cerebral I/R injury has not been explored. In this study, we observed that BRCA1 was mainly expressed in neurons and was up-regulated in response to I/R insult. Overexpression of BRCA1 attenuated reactive oxygen species production and lipid peroxidation. Enhanced BRCA1 expression promoted DNA double strand break repair through non-homologous end joining pathway. These effects consequently led to neuronal cell survival and neurological recovery. Mechanically, BRCA1 can interact with the nuclear factor (erythroid-derived 2)-like 2 (NRF2) through BRCA1 C-terminal (BRCT) domain. The cross-talk between BRCT and NRF2 activated the NRF2/Antioxidant Response Element signaling pathway and thus protected injured neurons during cerebral I/R. In conclusion, enhanced BRCA1 after cerebral I/R injury may attenuate or prevent neural damage from I/R via NRF2-mediated antioxidant pathway. The finding may provide a potential therapeutic target against ischemic stroke.

Effects of the fluorine substituent positions of the intercalating ligands on the binding behavior and third-strand stabilization of two Ru(II) complexes toward poly(U)•poly(A)*poly(U) triplex RNA.

Two new Ru(II) polypyridyl complexes containing fluorine substituents, [Ru(bpy)2(o-fpip)]2+ (Ru1, bpy=2,2'-bipyridine, o-fpip=2-(2-fluorophenyl)imidazo[4,5-f] [1,10]phenanthroline) and [Ru(bpy)2(p-fpip)]2+ (Ru2, p-fpip=2-(4-fluorophenyl)imidazo[4,5-f] [1,10]phenanthroline) have been synthesized as binders for poly(U)•poly(A)∗poly(U) triplex RNA. The binding of the two complexes with the triplex RNA has been investigated by spectroscopic methods and viscosity measurements. Analysis of the electronic absorption spectra indicates that the association of intercalating Ru2 with the triplex RNA is greater than that of Ru1, which is also supported by spectroscopic titrations and viscosity measurements. Thermal denaturation studies reflect that third-strand stabilization depend on the nature of the two complexes and Ru2 is more effective for stabilization of the triplex RNA. Circular dichroism spectra of the triplex RNA in the presence of metal complexes indicate that the binding-induced CD perturbation of the triplex structure is more obvious by Ru2. The main results obtained here suggest that the positions of fluorine substituent in the intercalating ligands have a significant effect on the two complexes stabilizing the third strand.

Interaction of octahedral ruthenium(II) polypyridyl complex [Ru(bpy)2(PIP)]2+ with poly(U)·poly(A)*poly(U) triplex: Increasing third-strand stabilization of the triplex without affecting the stability of the duplex.

Triple-helical RNA are of interest because of possible biological roles as well as the potential therapeutic uses of these structures, while the stability of triplexes is usually weaker than that of the Watson-Crick base pairing duplex strand due to the electrostatic repulsion between three polyanionic strands. Therefore, how to increase the stability of the specific sequences of triplexes are of importance. In this paper the binding of a Ru(II) complex, [Ru(bpy)2(PIP)]2+ (bpy=2.2'-bipyridine, PIP=2-phenyl-1H-imidazo[4,5-f]- [1,10]-phenanthroline), with poly(U)·poly(A)*poly(U) triplex has been investigated by spectrophotometry, spectrofluorometry, viscosimetry and circular dichroism. The results suggest that [Ru(bpy)2(PIP)]2+ as a metallointercalator can stabilize poly(U)·poly(A)*poly(U) triplex (where · denotes the Watson-Crick base pairing and * denotes the Hoogsteen base pairing),while it stabilizes third-strand with no obvious effect on the duplex of poly(U)·poly(A), reflecting the binding of this complex with the triplex is favored by the Hoogsteen paired poly(U) third strand to a great extent.