Inhibition of CK2 Diminishes Fibrotic Scar Formation and Improves Outcomes After Ischemic Stroke via Reducing BRD4 Phosphorylation.

Fibrotic scars play important roles in tissue reconstruction and functional recovery in the late stage of nervous system injury. However, the mechanisms underlying fibrotic scar formation and regulation remain unclear. Casein kinase II (CK2) is a protein kinase that regulates a variety of cellular functions through the phosphorylation of proteins, including bromodomain-containing protein 4 (BRD4). CK2 and BRD4 participate in fibrosis formation in a variety of tissues. However, whether CK2 affects fibrotic scar formation remains unclear, as do the mechanisms of signal regulation after cerebral ischemic injury. In this study, we assessed whether CK2 could modulate fibrotic scar formation after cerebral ischemic injury through BRD4. Primary meningeal fibroblasts were isolated from neonatal rats and treated with transforming growth factor-ß1 (TGF-ß1), SB431542 (a TGF-ß1 receptor kinase inhibitor) or TBB (a highly potent CK2 inhibitor). Adult SD rats were intraperitoneally injected with TBB to inhibit CK2 after MCAO/R. We found that CK2 expression was increased in vitro in the TGF-ß1-induced fibrosis model and in vivo in the MCAO/R injury model. The TGF-ß1 receptor kinase inhibitor SB431542 decreased CK2 expression in fibroblasts. The CK2 inhibitor TBB reduced the increases in proliferation, migration and activation of fibroblasts caused by TGF-ß1 in vitro, and it inhibited fibrotic scar formation, ameliorated histopathological damage, protected Nissl bodies, decreased infarct volume and alleviated neurological deficits after MCAO/R injury in vivo. Furthermore, CK2 inhibition decreased BRD4 phosphorylation both in vitro and in vivo. The findings of the present study suggested that CK2 may control BRD4 phosphorylation to regulate fibrotic scar formation, to affecting outcomes after ischemic stroke.

M2a macrophages regulate fibrosis and affect the outcome after stroke via PU.1/mTOR pathway in fibroblasts.

The moderate formation of the fibrotic scar plays an important role in functional recovery after stroke. M2a macrophages have been identified as an important source of early fibrosis after cerebral ischemia. However, the underlying mechanisms by which macrophages interact with fibroblasts in this context remain largely unknown. Therefore, our study aimed to further investigate the potential mechanisms underlying the effects of macrophages on fibroblasts following ischemic stroke. In vitro and in vivo, recombinant rat interleukin 4 (IL4) was used to induce macrophages to polarize into M2a macrophages. In vitro, primary Sprague-Dawley newborn rat meningeal-derived fibroblasts were treated with PU.1 knockdown, the PU.1 inhibitor DB1976 or the mTOR inhibitor rapamycin, which were then co-cultured with M2a macrophage conditioned medium (MCM). In vivo, Sprague-Dawley adult rats were infected with negative control adenoviruses or PU.1-shRNA adenoviruses. Ten days after infection, an injury model of middle cerebral artery occlusion/reperfusion (MCAO/R) was constructed. Subsequently, IL4 was injected intracerebroventricularly to induce M2a macrophages polarization. In vitro, M2a MCM upregulated PU.1 expression and promoted the differentiation, proliferation, migration and extracellular matrix generation of fibroblasts, which could be reversed by treatment with the PU.1 inhibitor DB1976 or PU.1 knockdown. In vivo, PU.1 expression in fibroblasts was increased within ischemic core following MCAO/R, and this upregulation was further enhanced by exposure to IL4. Treatment with IL4 promoted fibrosis, increased angiogenesis, reduced apoptosis and infarct volume, as well as mitigated neurological deficits after MCAO/R, and these effects could be reversed by PU.1 knockdown. Furthermore, both in vivo and in vitro studies showed that IL4 treatment increased the levels of phosphorylated Akt and mTOR proteins, which were markedly decreased by PU.1 knockdown. Additionally, the use of an mTOR inhibitor rapamycin obviously suppressed the migration and differentiation of fibroblasts, and Col1 synthesis. In conclusion, our findings suggest for the first time that M2a macrophages, at least in part, regulate fibrosis and affect the outcome after cerebral ischemic stroke via the PU.1/mTOR signaling pathway in fibroblasts.

Low vitamin B12 levels may predict the risk of ischemic stroke: A cross-sectional study.

BACKGROUND: To examine serum vitamin B12 concentrations in relation to the risk of ischemic stroke among hospitalized patients in the Department of Neurology. METHODS: We performed a cross-sectional study involving 2,212 inpatients discharged from the Department of Neurology of the First Affiliated Hospital of Chongqing Medical University, from January 2020 to January 2022. The results of laboratory assays such as serum vitamin B12, homocysteine, and folate levels were measured. Logistic regression analysis was used to investigate the association between serum vitamin B12 concentrations and ischemic stroke, with adjustment for a number of relevant demographic and lifestyle factors and comorbidities. RESULTS: A total of 961 (43.4%) patients had an ischemic stroke. In the fully adjusted model, logistic regression analysis suggested a positive association between serum vitamin B12 levels<150 pg/mL (aOR: 1.42; 95% CI 1.02-1.97; p = 0.035), serum vitamin B12 150-300 pg/mL (aOR: 1.37; 95% CI 1.11-1.68; p = 0.003) and the prevalence of ischemic stroke. Furthermore, an inverse association was observed between serum vitamin B12 levels ≥ 900 pg/mL (aOR: 0.38; 95% CI: 0.19-0.77; p =0.007) and the prevalence of ischemic stroke. Moreover, the cut-off value of vitamin B12 concentration was 316.4 pg/mL and the discrimination power of the score evaluated by AUC-ROC was 0.71 (95%CI 0.68-0.73, p<0.001) in the vitamin B12 and ischemic stroke. CONCLUSION: Findings suggest that low vitamin B12 levels may predict the risk of ischemic stroke, early and timely supplementation of vitamin B12 can improve the short-term prognosis of ischemic stroke patients.

New insights into Sirt1: potential therapeutic targets for the treatment of cerebral ischemic stroke.

Ischemic stroke is one of the main causes of mortality and disability worldwide. However, the majority of patients are currently unable to benefit from intravenous thrombolysis or intravascular mechanical thrombectomy due to the limited treatment windows and serious complications. Silent mating type information regulation 2 homolog 1 (Sirt1), a nicotine adenine dinucleotide-dependent enzyme, has emerged as a potential therapeutic target for ischemic stroke due to its ability to maintain brain homeostasis and possess neuroprotective properties in a variety of pathological conditions for the central nervous system. Animal and clinical studies have shown that activation of Sirt1 can lessen neurological deficits and reduce the infarcted volume, offering promise for the treatment of ischemic stroke. In this review, we summarized the direct evidence and related mechanisms of Sirt1 providing neuroprotection against cerebral ischemic stroke. Firstly, we introduced the protein structure, catalytic mechanism and specific location of Sirt1 in the central nervous system. Secondly, we list the activators and inhibitors of Sirt1, which are primarily divided into three categories: natural, synthetic and physiological. Finally, we reviewed the neuroprotective effects of Sirt1 in ischemic stroke and discussed the specific mechanisms, including reducing neurological deficits by inhibiting various programmed cell death such as pyroptosis, necroptosis, ferroptosis, and cuproptosis in the acute phase, as well as enhancing neurological repair by promoting angiogenesis and neurogenesis in the later stage. Our review aims to contribute to a deeper understanding of the critical role of Sirt1 in cerebral ischemic stroke and to offer novel therapeutic strategies for this condition.

Vitamin B12 supplementation improves cognitive function in middle aged and elderly patients with cognitive impairment / La administración de suplementos de vitamina B12 mejora la capacidad cognitiva en pacientes de mediana edad y ancianos con deterioro cognitivo

Objectives: to determine the effects of vitamin B12 supplementation on neuropsychological function and disease progression in middle aged and elderly patients with cognitive impairment. Methods: this was a prospective case-control study. From May 2020 to May 2021, 307 participants clinically diagnosed with cognitive impairment in the Department of Neurology of the First Affiliated Hospital of Chongqing Medical University were enrolled. A total of 115 patients were included in this study. Meanwhile, 115 participants with cognitive impairment were randomly assigned in equal proportions to two groups: vitamin B12 treatment group (n = 58, vitamin B12 500 mg/d intramuscularly for seven days, followed by cobamamide 0.25 mg/d and methylcobalamin 0.50 mg/d) and the control group (n = 57). Demographic characteristics and blood biochemical variables were obtained from all participants. Cognitive performance was measured using the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Cognitive performance was measured at baseline and after six months. Results: the vitamin B12 supplementation treatment patients who presented with cognitive impairment showed significant improvement, especially in attention, calculation (p < 0.01) and visual-constructional ability (p < 0.05), in their neuropsychological function compared to their matched group. Conclusion: vitamin B12 supplementation may improve frontal function in patients with cognitive decline. Vitamin B12 levels should be investigated in all patients with cognitive impairment.(AU)

Objetivos: determinar los efectos de la suplementación con vitamina B12 en la función neuropsicológica y la progresión de la enfermedad enpacientes de mediana edad y adultos mayores con deterioro cognitivo.Métodos: se realizó un estudio prospectivo de casos y controles; se estudiaron 307 participantes, desde mayo de 2020 a mayo de 2021,diagnosticados clínicamente con deterioro cognitivo en el Departamento de Neurología, el Primer Hospital Anexado a la Universidad Médica deChongqing. En el estudio se incluyeron un total de 115 pacientes con deterioro cognitivo que fueron asignados aleatoriamente en proporcionesiguales a dos grupos: un grupo de tratamiento con vitamina B12 (n = 58, vitamina B12 500 mg/d intramuscular durante 7 días, seguido decobamamida 0,25 mg/d y metilcobalamina 0,50 mg/d) y un grupo de control (n = 57). Se obtuvieron las características demográficas y lasvariables bioquímicas sanguíneas de todos los participantes. El rendimiento cognitivo se midió mediante el miniexamen del estado mental (MMSE)y la evaluación cognitiva de Montreal (Moca) al inicio del estudio y a los 6 meses.Resultados: los pacientes con deterioro cognitivo que recibieron tratamiento de suplementación con vitamina B12 mostraron una mejorasignificativa, especialmente en la atención, el cálculo (p < 0,01) y la capacidad visuoespacial (p < 0,05), en su función neuropsicológica encomparación con el grupo control.Conclusión: la suplementación con vitamina B12 puede mejorar la función frontal en pacientes con deterioro cognitivo. Los pacientes condeterioro cognitivo deben conocer sus propios niveles de vitamina B12.(AU)

Vitamin B12 supplementation improves cognitive function in middle aged and elderly patients with cognitive impairment.

Introduction: Objectives: to determine the effects of vitamin B12 supplementation on neuropsychological function and disease progression in middle aged and elderly patients with cognitive impairment. Methods: this was a prospective case-control study. From May 2020 to May 2021, 307 participants clinically diagnosed with cognitive impairment in the Department of Neurology of the First Affiliated Hospital of Chongqing Medical University were enrolled. A total of 115 patients were included in this study. Meanwhile, 115 participants with cognitive impairment were randomly assigned in equal proportions to two groups: vitamin B12 treatment group (n = 58, vitamin B12 500 mg/d intramuscularly for seven days, followed by cobamamide 0.25 mg/d and methylcobalamin 0.50 mg/d) and the control group (n = 57). Demographic characteristics and blood biochemical variables were obtained from all participants. Cognitive performance was measured using the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Cognitive performance was measured at baseline and after six months. Results: the vitamin B12 supplementation treatment patients who presented with cognitive impairment showed significant improvement, especially in attention, calculation (p < 0.01) and visual-constructional ability (p < 0.05), in their neuropsychological function compared to their matched group. Conclusion: vitamin B12 supplementation may improve frontal function in patients with cognitive decline. Vitamin B12 levels should be investigated in all patients with cognitive impairment.

Introducción: Objetivos: determinar los efectos de la suplementación con vitamina B12 en la función neuropsicológica y la progresión de la enfermedad en pacientes de mediana edad y adultos mayores con deterioro cognitivo. Métodos: se realizó un estudio prospectivo de casos y controles; se estudiaron 307 participantes, desde mayo de 2020 a mayo de 2021, diagnosticados clínicamente con deterioro cognitivo en el Departamento de Neurología, el Primer Hospital Anexado a la Universidad Médica de Chongqing. En el estudio se incluyeron un total de 115 pacientes con deterioro cognitivo que fueron asignados aleatoriamente en proporciones iguales a dos grupos: un grupo de tratamiento con vitamina B12 (n = 58, vitamina B12 500 mg/d intramuscular durante 7 días, seguido de cobamamida 0,25 mg/d y metilcobalamina 0,50 mg/d) y un grupo de control (n = 57). Se obtuvieron las características demográficas y las variables bioquímicas sanguíneas de todos los participantes. El rendimiento cognitivo se midió mediante el miniexamen del estado mental (MMSE) y la evaluación cognitiva de Montreal (Moca) al inicio del estudio y a los 6 meses. Resultados: los pacientes con deterioro cognitivo que recibieron tratamiento de suplementación con vitamina B12 mostraron una mejora significativa, especialmente en la atención, el cálculo (p < 0,01) y la capacidad visuoespacial (p < 0,05), en su función neuropsicológica en comparación con el grupo control. Conclusión: la suplementación con vitamina B12 puede mejorar la función frontal en pacientes con deterioro cognitivo. Los pacientes con deterioro cognitivo deben conocer sus propios niveles de vitamina B12.

Sirt1 Overexpression Inhibits Fibrous Scar Formation and Improves Functional Recovery After Cerebral Ischemic Injury Through the Deacetylation of 14-3-3ζ.

Cerebral ischemic stroke is one of the leading causes of human death. The fibrous scar is one of major factors influencing repair in central nervous system (CNS) injury. Silencing information regulator 2-related enzyme 1 (Sirt1) can regulate peripheral tissue and organ fibrosis. However, it is unclear how the fibrous scar forms and is regulated and it is unknown whether and how Sirt1 regulates the formation of the fibrous scar after cerebral ischemic stroke. Therefore, in the present study, we examined the effects of Sirt1 on the formation of the fibrotic scar after middle cerebral artery occlusion/reperfusion (MCAO/R) injury in vivo and on the transforming growth factor ß1 (TGF-ß1)-induced meningeal fibroblast fibrotic response in vitro, and we explored the molecular mechanisms underlying the Sirt1-regulated fibrosis process in vitro. We found that MCAO/R injury induced fibrotic scar formation in the ischemic area, which was accompanied by the downregulation of Sirt1 expression. The overexpression of Sirt1 reduced the infarct volume, improved Nissl body structure and reduced neurons injury, attenuated formation of fibrotic scar, upregulated growth associated protein43 (GAP43) and synaptophysin (SYP) expression, and promoted neurological function recovery. Similarly, Sirt1 expression was also downregulated in the TGF-ß1-induced fibrosis model. Sirt1 overexpression inhibited fibroblast migration, proliferation, transdifferentiation into myofibroblasts, and secretion of extracellular matrix(ECM) by regulating the deacetylation of lysine at K49 and K120 sites of 14-3-3ζ in vitro. Therefore, we believe that Sirt1 could regulate fibrous scar formation and improve neurological function after cerebral ischemic stroke through regulating deacetylation of 14-3-3ζ.

M2 macrophages mediate fibrotic scar formation in the early stages after cerebral ischemia in rats.

In the central nervous system, the formation of fibrotic scar after injury inhibits axon regeneration and promotes repair. However, the mechanism underlying fibrotic scar formation and regulation remains poorly understood. M2 macrophages regulate fibrotic scar formation after injury to the heart, lung, kidney, and central nervous system. However, it remains to be clarified whether and how M2 macrophages regulate fibrotic scar formation after cerebral ischemia injury. In this study, we found that, in a rat model of cerebral ischemia induced by middle cerebral artery occlusion/reperfusion, fibrosis and macrophage infiltration were apparent in the ischemic core in the early stage of injury (within 14 days of injury). The number of infiltrated macrophages was positively correlated with fibronectin expression. Depletion of circulating monocyte-derived macrophages attenuated fibrotic scar formation. Interleukin 4 (IL4) expression was strongly enhanced in the ischemic cerebral tissues, and IL4-induced M2 macrophage polarization promoted fibrotic scar formation in the ischemic core. In addition, macrophage-conditioned medium directly promoted fibroblast proliferation and the production of extracellular matrix proteins in vitro. Further pharmacological and genetic analyses showed that sonic hedgehog secreted by M2 macrophages promoted fibrogenesis in vitro and in vivo, and that this process was mediated by secretion of the key fibrosis-associated regulatory proteins transforming growth factor beta 1 and matrix metalloproteinase 9. Furthermore, IL4-afforded functional restoration on angiogenesis, cell apoptosis, and infarct volume in the ischemic core of cerebral ischemia rats were markedly impaired by treatment with an sonic hedgehog signaling inhibitor, paralleling the extent of fibrosis. Taken together, our findings show that IL4/sonic hedgehog/transforming growth factor beta 1 signaling targeting macrophages regulates the formation of fibrotic scar and is a potential therapeutic target for ischemic stroke.

Vitamin B12, Folate, Homocysteine, Inflammatory Mediators (Interleukin-6, Tumor Necrosis Factor-α and C-Reactive Protein) Levels in Adolescents with Anxiety or Depressive Symptoms.

Purpose: To evaluate the prevalence of abnormal vitamin B12, folate, total homocysteine (tHcy), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) levels, to analyze the relationship between these parameters and the severity of anxiety or depressive symptoms, and to explore the possible factors associated with abnormal levels of these parameters in adolescents with anxiety or depressive symptoms. Methods: Adolescent (aged 12-18 years) outpatients with anxiety or depressive symptoms were recruited. The patient health questionnaire-9 and generalized anxiety disorder scale-7 were used to measure the severity of depression and anxiety. Serum vitamin B12, folate, tHcy, IL-6, TNF-α, and CRP levels were determined. Results: 128 subjects were recruited. The prevalence of vitamin B12 and folate deficiency, tHcy, TNF-α, IL-6, and CRP elevation was 8.6%, 10.2%, 25.8%, 14.8%, 21.9%, and 10.2%, respectively, in adolescents with anxiety or depressive symptoms. Lower vitamin B12 levels were correlated with a higher risk of severe anxiety and depressive symptoms. The severity of some symptoms of anxiety or depression were weakly correlated with vitamin B12, folate, tHcy, IL-6, and CRP levels. Vitamin B12, folate, and tHcy levels were not associated with inflammatory mediators. Vitamin B12 deficiency was associated with older age and higher tHcy levels. Folate deficiency was associated with elevated tHcy. Elevated tHcy was associated with lower vitamin B12 and folate levels. IL-6 elevation was associated with elevated CRP and TNF-α. CRP elevation was associated with older age, higher BMI, and current drinking. Conclusion: Lower vitamin B12 levels were correlated with a higher risk of severe anxiety or depressive symptoms. Weak correlations were observed between the severity of some symptoms of anxiety or depression and vitamin B12, folate, tHcy, IL-6, and CRP levels. Vitamin B12, folate, and tHcy levels were related to each other. IL-6 elevation was associated with elevated CRP and TNF-α. CRP elevation was associated with older age, higher BMI, and current drinking.

Sirt1 regulates microglial activation and inflammation following oxygen-glucose deprivation/reoxygenation injury by targeting the Shh/Gli-1 signaling pathway.

BACKGROUND: Cerebral ischemic injury leads to over-activation of microglia, which release pro-inflammatory factors that deteriorate neurological function during the acute phase of stroke. Thus, inhibiting microglial over-activation is crucial for reducing ischemic injury. Sirtuin 1 (Sirt1) has been shown to play a critical role in stroke, neurodegenerative diseases and aging. However, the effect of Sirt1 on the regulation of microglial activation following cerebral ischemic injury, as well as the underlying mechanism, remain unknown. Therefore, the purpose of the present study is to mainly investigate the effect of Sirt1 on oxygen-glucose deprivation/reoxygenation (OGD/R)-treated N9 microglia following treatment with the Sirt1 agonists resveratrol and SRT1720 and the Sirt1 antagonist sirtinol. METHODS: Cell viability, Apoptosis, activation and inflammatory responses of microglia, expressions and activity of Shh signaling pathway proteins were detected by Cell Counting Kit 8, Flow Cytometry, immunocytochemistry, ELISA, and Western blotting, respectively. RESULTS: The results demonstrated that treatment with resveratrol or SRT1720 could inhibit the activation of microglia and inflammation during OGD/R. Moreover, these treatments also led to the translocation of the GLI family zinc finger-1 (Gli-1) protein from the cytoplasm to the nucleus and upregulated the expression of Sonic hedgehog (Shh), Patched homolog-1 (Ptc-1), smoothened frizzled class receptor and Gli-1. By contrast, the inhibition of Sirt1 using sirtinol had the opposite effect. CONCLUSION: These findings suggested that Sirt1 may regulate microglial activation and inflammation by targeting the Shh/Gli-1 signaling pathway following OGD/R injury. Schematic representation of Sirt1 regulating the microglial activation and inflammation following oxygen-glucose deprivation/reoxygenation injury via mediation of Shh/Gli-1 signaling pathway.

Resveratrol Inhibits Activation of Microglia after Stroke through Triggering Translocation of Smo to Primary Cilia.

Activated microglia act as a double-edged sword for stroke. In the acute phase of stroke, activated microglia might deteriorate neurological function. Therefore, it is of great clinical transforming potential to explore drugs or methods that can inhibit abnormal activation of microglia in the acute phase of stroke to improve neurological function after stroke. Resveratrol has a potential effect of regulating microglial activation and anti-inflammation. However, the molecular mechanism of resveratrol-inhibiting microglial activation has not been fully clarified. Smoothened (Smo) belongs to the Hedgehog (Hh) signaling pathway. Smo activation is the critical step that transmits the Hh signal across the primary cilia to the cytoplasm. Moreover, activated Smo can improve neurological function via regulating oxidative stress, inflammation, apoptosis, neurogenesis, oligodendrogenesis, axonal remodeling, and so on. More studies have indicated that resveratrol can activate Smo. However, it is currently unknown whether resveratrol inhibits microglial activation via Smo. Therefore, in this study, N9 microglia in vitro and mice in vivo were used to investigate whether resveratrol inhibited microglial activation after oxygen-glucose deprivation/reoxygenation (OGD/R) or middle cerebral artery occlusion/reperfusion (MCAO/R) injury and improved functional outcome via triggering translocation of Smo in primary cilia. We definitively found that microglia had primary cilia; resveratrol partially inhibited activation and inflammation of microglia, improved functional outcome after OGD/R and MCAO/R injury, and triggered translocation of Smo to primary cilia. On the contrary, Smo antagonist cyclopamine canceled the above effects of resveratrol. The study suggested that Smo receptor might be a therapeutic target of resveratrol for contributing to inhibit microglial activation in the acute phase of stroke.

The role of mesenchymal stem cell transplantation for ischemic stroke and recent research developments.

Ischemic stroke is a common cerebrovascular disease that seriously affects human health. However, most patients do not practice self-care and cannot rely on the current clinical treatment for guaranteed functional recovery. Stem cell transplantation is an emerging treatment studied in various central nervous system diseases. More importantly, animal studies show that transplantation of mesenchymal stem cells (MSCs) can alleviate neurological deficits and bring hope to patients suffering from ischemic stroke. This paper reviews the biological characteristics of MSCs and discusses the mechanism and progression of MSC transplantation to provide new therapeutic directions for ischemic stroke.

Inhibition of BRD4 decreases fibrous scarring after ischemic stroke in rats by inhibiting the phosphorylation of Smad2/3.

AIMS: Fibrous scarring may play a much more important role in preventing secondary expansion of tissue damage and hindering repair and regeneration than glial scarring after central nervous system (CNS) injury. However, relatively little is known about how fibrous scars form and how fibrous scar formation is regulated after CNS injury. Bromodomain-containing protein 4 (BRD4) is involved in fibrosis in many tissues, and transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signaling is one of the critical pathways of fibrosis. However, it is unclear whether and how BRD4 affects fibrous scar formation after ischemicbraininjury. In the present study, whether BRD4 can regulate the formation of fibrous scars after ischemic stroke via TGF-ß1/Smad2/3 signaling was assessed. MATERIALS AND METHODS: Primary meningeal fibroblasts isolated from neonatal SD rats were treated with TGF-ß1, SB431542 (a TGF-ß1 receptor inhibitor) and JQ1 (a small-molecule BET inhibitor that can also inhibit BRD4). BRD4 was knocked down in adult Sprague-Dawley (SD) rats by using adenovirus before middle cerebral artery occlusion/reperfusion (MCAO/R) injury. The proliferation and migration of meningeal fibroblasts in vitro were evaluated with the Cell Counting Kit-8 (CCK-8) assay and scratch test, respectively. Neurological function was assessed with Longa scores, modified Bederson Scores and modified neurological severity scores (mNSSs). The infarct volume was assessed with TTC staining. The protein expression of synaptophysin (SY), BRD4, Smad2/3, p-Smad2/3, α-smooth muscle actin (α-SMA), collagen-1 (COL1) and fibronectin (FN) in vivo and in vitro was examined with immunocytochemistry, immunofluorescence, and Western blotting. KEY FINDINGS: BRD4 expression was upregulated in a TGF-ß1-induced meningeal fibroblast fibrosis model and was downregulated by the TGF-ß1 receptor inhibitor SB431542 in vitro. JQ1, a small-molecule BET inhibitor, inhibited BRD4 and decreased TGF-ß1-induced meningeal fibroblast proliferation, migration and activation. Furthermore, MCAO/R injury induced fibrosis and upregulated BRD4 expression in the cerebral infarct center. BRD4 knockdown by adenovirus inhibited fibrous scarring, promoted synaptic survival, decreased the infarct volume, and improved neurological function after MCAO/R injury. Moreover, inhibition of BRD4, either by JQ1 in vitro or adenovirus in vivo, decreased the phosphorylation of Smad2/3. CONCLUSIONS: This study is the first to indicate that inhibition of BRD4 delays fibrous scarring after ischemic stroke through mechanisms involving the phosphorylation of Smad2/3.

Potential mechanisms and therapeutic targets of mesenchymal stem cell transplantation for ischemic stroke.

Ischemic stroke is one of the major causes of death and disability in the world. Currently, most patients cannot choose intravenous thrombolysis or intravascular mechanical thrombectomy because of narrow therapeutic windows and severe complications. Stem cell transplantation is an emerging treatment and has been studied in various central nervous system diseases. Animal and clinical studies showed that transplantation of mesenchymal stem cells (MSCs) could alleviate neurological deficits and bring hope for ischemic stroke treatment. This article reviewed biological characteristics, safety, feasibility and efficacy of MSCs therapy, potential therapeutic targets of MSCs, and production process of Good Manufacturing Practices-grade MSCs, to explore the potential therapeutic targets of MSCs in the process of production and use and provide new therapeutic directions for ischemic stroke.

Resveratrol pretreatment protects neurons from oxygen-glucose deprivation/reoxygenation and ischemic injury through inhibiting ferroptosis.

Ferroptosis, a newly discovered iron-dependent cell death, is involved in brain ischemia-reperfusion injury. Iron scavengers or ferroptosis inhibitors could reduce infarct volume and improve neurological function in mice. Resveratrol has neuroprotective and neurorestorative effects. However, it is unclear whether resveratrol can play a neuroprotective role via inhibiting ferroptosis. Our study showed that resveratrol pretreatment had a similar effect with ferrostatin­1, which inhibited neuronal ferroptosis-related changes, such as iron overload, damages of oxidation-reduction system, and destruction of mitochondrial structure, after oxygen-glucose deprivation/reoxygenation (OGD/R) and application of ferroptosis inducers. In addition, middle cerebral artery occlusion/reperfusion (MCAO/R) injury in vivo also induced ferroptosis, and resveratrol pretreatment could inhibit ferroptosis and reduce degenerative neurons, cerebral ischemic damage and infarction volume. Our results are the first to indicate that resveratrol pretreatment might inhibit ferroptosis induced by OGD/R and ferroptosis inducers in neurons, and MCAO/R in rats.

Effect of blood pressure on early neurological deterioration of acute ischemic stroke patients with intravenous rt-PA thrombolysis may be mediated through oxidative stress induced blood-brain barrier disruption and AQP4 upregulation.

OBJECTIVE: To study association between blood pressure (BP) and development of early neurological deterioration (END) in acute ischemic stroke patients with intravenous rt-PA thrombolysis and its possible mechanism. METHODS: We prospectively collected data of acute ischemic stroke patients with intravenous rt-PA thrombolysis from March 2015 to June 2019. The collected data include general, clinical data and laboratory test. Moreover, serum levels or activity of malondialdehyde (MDA), superoxide dismutase (SOD), matrix metalloproteinase-9 (MMP-9), occludin(OCLN), ZO-1 and aquaporin 4(AQP-4) were determined. RESULTS: A total of 357 acute ischemic stroke patients received intravenous rt-PA thrombolysis and 16 cases were eventually excluded. Finally, 341 patients were eligible in this study and 65 patients (19.06%) experienced END. Mean systolic blood pressure (SBP) within 24 h, serum levels or activity of MDA, SOD, MMP-9, ZO-1, OCLN, AQP-4 at 24 h after thrombolysis were the independent predictors for END in the total and hypertension population using multivariate logistic regression analysis, and mean SBP within 24 h was the best predictor for END. Receiver operating characteristic (ROC) analysis found that cutoff mean SBP for END was 148.16 mmHg, and sensitivity was 85.6%. The best target SBP level is 140ཞ149 mmHg. Further, spearman correlation tests indicated that BP levels were directly proportional to serum levels or activity of MDA, MMP-9, ZO-1, OCLN, AQP-4 at 24 h after thrombolysis and neurological deterioration severity. CONCLUSIONS: END frequently occurs after thrombolysis, and the best predictor of END is SBP which may be mediated through oxidative stress induced blood-brain barrier disruption and AQP4 upregulation.

Resveratrol Treatment in Different Time-Attenuated Neuronal Apoptosis After Oxygen and Glucose Deprivation/Reoxygenation via Enhancing the Activation of Nrf-2 Signaling Pathway In Vitro.

Recent studies have indicated that resveratrol has protective effects against cerebral ischemia/reperfusion injury. However, the best therapeutic time for resveratrol treatment after acute ischemic stroke remains unknown. We aim to investigate whether resveratrol, administrated at different times after neuronal oxygen and glucose deprivation/reoxygenation (OGD/R) reduced neuronal injury in vitro. There were six experimental groups: normal, model, resveratrol pretreatment, resveratrol post-treatment, resveratrol OGD-treatment, and resveratrol whole-processing group. We found that resveratrol in a concentration-dependent manner decreased the activity of lactate dehydrogenase (LDH) and increased the activity of superoxide dismutase (SOD). Moreover, resveratrol, administrated at different times, increased neuronal viability, reduced neuronal apoptosis, upregulated the protein expressions of Nuclear factor erythroid 2-related factor 2 (Nrf-2), NAD(P)H: quinone oxidoreductase 1 (NQO-1), heme oxygenase 1 (HO-1), and Bcl-2, downregulated the protein expression of Caspase-3, and promoted Nrf-2 to transfer into the nuclei from the cytoplasm. The most effective treatment group was the whole-processing treatment group. These results suggest that resveratrol treatment at different times increased neuronal viability and inhibited neuronal apoptosis in vitro, at least in part, via enhancing the activation of the Nrf-2 signaling pathway.

Primary cilia mediate sonic hedgehog signaling to regulate neuronal-like differentiation of bone mesenchymal stem cells for resveratrol induction in vitro.

Mesenchymal stem cells (MSCs) can differentiate into neuronal-like cell types under specific conditions. The classical antioxidant inducers such as ß-mercaptoethanol (BME), butylated hydroxyanisol (BHA), and dimethylsulfoxide (DMSO) are limited in clinical because of toxicity. Resveratrol, a safer, natural antioxidant, can stimulate osteoblastic differentiation of MSCs. However, its effect of inducing MSCs to differentiate into neuronal-like cells is less well studied, and its differentiated mechanisms are not well understood. Sonic hedgehog (Shh) signaling, mediated by the primary cilia, is crucial for embryonic development and tissue differentiation, but relatively little is known about the role of Shh signaling and primary cilia in neuronal-like differentiation of MSCs. Here we show that primary cilia, harboring patched 1 (Ptc1), are present in growth-arrested MSCs and that smoothened (Smo) and Gli1 are present in cytoplasm of MSCs, which are important components of the Shh signaling pathway. After resveratrol induction, MSCs acquire neuronal-like cell morphologies and phenotypes, Smo translocates to the primary cilia, Gli1 enters the nucleus, and expressions of Smo and Gli1 proteins increase, which can be inhibited by cyclopamine, a Smo antagonist. Meanwhile, Smo agonist (SAG) attains similar effects compared with the resveratrol group. These data indicate that resveratrol can induce MSCs to differentiate into neuronal-like cells and activate Shh signaling pathway in the primary cilia. Moreover, the primary cilia and Shh signaling are essential for resveratrol inducing neuronal-like differentiation of MSCs. Our finding is important for understanding the neuronal-like differentiation mechanism of MSCs for resveratrol and promoting its clinical therapeutic utility.

Resveratrol pretreatment attenuates cerebral ischemic injury by upregulating expression of transcription factor Nrf2 and HO-1 in rats.

Oxidative stress damage plays a vital role in cerebral ischemia/reperfusion (I/R) pathogenesis. The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway can be activated by pharmacological and dietary means to attenuate cellular oxidative stress. Resveratrol, a plant-derived polyphenolic compound, has antioxidant property. Recent studies have demonstrated that resveratrol has protective effects against cerebral I/R injury. However, little is known about its mechanism. Hence, this study identified the neuroprotective effect of resveratrol pretreatment and elucidate the Nrf2/ARE signaling mechanism after focal cerebral I/R injury in rats. Adult male Sprague-Dawley rats were randomly assigned to sham-operated group, ischemia/reperfusion physiological saline-treated group, and ischemia/reperfusion resveratrol-pretreatmented (15 and 30 mg/kg) groups. Rats were pretreatmented with resveratrol or physiological saline of corresponding volume administered intraperitoneally for 7 days before surgery and 30 min before middle cerebral artery occlusion. At 24 h after reperfusion, neurological score, infarct volume, and brain water content were assessed. Oxidative stress was evaluated by malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity. Pathological changes of brain tissue were observed by HE staining. RT-PCR and Western blot analysed the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). TUNEL staining detected apoptotic cells. The protein expression of Caspase-3 were studied by immunohistochemistry. Resveratrol pretreatment significantly ameliorated neurological scores, reduced infarct volume and brain water content, decreased MDA levels, restored the SOD activity, upregulated the protein and mRNA expression of Nrf2 and HO-1, downregulated the protein expression of caspase-3. TUNEL-positive cells significantly decreased compared with the physiological saline-treated group. HE staining also showed that resveratrol significantly improved neuronal injury. These results showed that resveratrol pretreatment had neuroprotective effects on cerebral I/R injury. This neuroprotective effect is likely exerted by upregulated expression of transcription factor Nrf2 and HO-1 to ameliorate oxidative damage, decreased the protein expression of caspase-3. Our finding is important for understanding the neuroprotective mechanism of resveratrol and promoting its clinical therapeutic utility.