Inhibition of CCR2 attenuates neuroinflammation and neuronal apoptosis after subarachnoid hemorrhage through the PI3K/Akt pathway.

BACKGROUND: Neuroinflammation and neuronal apoptosis are closely associated with a poor prognosis in patients with subarachnoid hemorrhage (SAH). We investigated the role of C-C motif chemokine receptor 2 (CCR2) in SAH. METHODS: Pre-processed RNA-seq transcriptome datasets GSE167110 and GSE79416 from the Gene Expression Omnibus (GEO) database were screened for genes differentially expressed between mice with SAH and control mice, using bioinformatics analysis. The endovascular perforation model was performed to establish SAH. RS504393 (a CCR2 antagonist) and LY294002 (PI3K inhibitor) were administered to explore the mechanism of neuroinflammation after SAH. SAH grading, neurological scoring, brain water content and blood-brain barrier (BBB) permeability determination, enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence were performed. An in vitro model of SAH was induced in H22 cells by hemin treatment. The protective mechanism of CCR2 inhibition was studied by adding RS504393 and LY294002. Clinical cerebrospinal fluid (CST) samples were detected by ELISA. RESULTS: Expression of CCR2 was upregulated in both datasets and was identified as a hub gene. CCR2 expression was significantly upregulated in the cytoplasm of neurons after SAH, both in vitro and in vivo. RS significantly reduced the brain water content and blood-brain barrier permeability, alleviated neuroinflammation, and reduced neuronal apoptosis after SAH. Additionally, the protective effects of CCR2 inhibition were abolished by LY treatment. Finally, the levels of CCR2, inflammatory factors, and apoptotic factors were elevated in the CSF of patients with SAH. CCR2 levels were associated with patient outcomes at the 6-month follow-up. CONCLUSION: CCR2 expression was upregulated in both in vitro and in vivo SAH models. Additionally, inhibition of CCR2, at least partly through the PI3K/AKT pathway, alleviated neuroinflammation and neuronal apoptosis in vivo and in vitro. CCR2 levels in the CSF have a moderate diagnostic value for 6-month outcome prediction in patients with SAH.

Low shear stress induces macrophage infiltration and aggravates aneurysm wall inflammation via CCL7/CCR1/TAK1/ NF-κB axis.

BACKGROUND: This study aimed to elucidate the mechanism by which wall shear stress (WSS) influences vascular walls, accounting for the susceptibility of intracranial aneurysms (IAs) to rupture. METHOD: We collected blood samples from the sacs of 24 ruptured and 28 unruptured IAs and analyzed the expression of chemokine CCL7 using enzyme-linked immunosorbent assay (ELISA). Univariate and multivariate logistic regression analyses were employed to assess clinical data, aneurysm morphology, and hemodynamics in both groups. Pearson correlation analysis investigated the relationship between CCL7 expression in aneurysm sac blood and WSS. Additionally, we established a bionic cell parallel plate co-culture shear stress model and a mouse low shear stress (LSS) model. The model was modulated using CCL7 recombinant protein, CCR1 inhibitor, and TAK1 inhibitor. We further evaluated CCL7 expression in endothelial cells and the levels of TAK1, NF-κB, IL-1ß, and TNF-α in macrophages. Subsequently, the intergroup differences in expression were calculated. RESULTS: CCL7 expression was significantly higher in the ruptured group compared to the unruptured group. Hemodynamic analysis indicated that WSS was an independent predictor of the risk of aneurysm rupture. A negative linear correlation was observed between CCL7 expression and WSS. Upon addition of CCL7 recombinant protein, upregulation of CCR1 expression and increased levels of p-TAK1 and p-p65 were observed. Treatment with CCR1 and TAK1 inhibitors reduced inflammatory cytokine expression in macrophages under LSS conditions. Overexpression of TAK1 significantly alleviated the inhibitory effects of CCR1 inhibitors on p-p65 and inflammatory cytokines. CONCLUSION: LSS prompts endothelial cells to secrete CCL7, which, upon binding to the macrophage surface receptor CCR1, stimulates the release of macrophage inflammatory factors via the TAK1/NF-κB signaling pathway. This process exacerbates aneurysm wall inflammation and increases the risk of aneurysm rupture.

Blocking P2RX7 Attenuates Ferroptosis in Endothelium and Reduces HG-induced Hemorrhagic Transformation After MCAO by Inhibiting ERK1/2 and P53 Signaling Pathways.

Hyperglycemia is a risk factor for poor prognosis after acute ischemic stroke and promote the occurrence of hemorrhagic transformation (HT). The activation of P2RX7 play an important role in endotheliocyte damage and BBB disruption. Ferroptosis is a novel pattern of programmed cell death caused by the accumulation of intracellular iron and lipid peroxidation, resulting in ROS production and cell death. This study is to explore the mechanism of P2RX7 in reducing HT pathogenesis after acute ischemic stroke through regulating endotheliocyte ferroptosis. Male SD rats were performed to establish middle cerebral artery occlusion (MCAO) model injected with 50% high glucose (HG) and HUVECs were subjected to OGD/R treated with high glucose (30 mM) for establishing HT model in vivo and in vitro. P2RX7 inhibitor (BBG), and P2RX7 small interfering RNAs (siRNA) were used to investigate the role of P2RX7 in BBB after MCAO in vivo and OGD/R in vitro, respectively. The neurological deficits, infarct volume, degree of intracranial hemorrhage, integrity of the BBB, immunoblotting, and immunofluorescence were evaluated at 24 h after MCAO. Our study found that the level of P2RX7 was gradually increased after MCAO and/or treated with HG. Our results showed that treatment with HG after MCAO can aggravate neurological deficits, infarct volume, oxidative stress, iron accumulation, and BBB injury in HT model, and HG-induced HUVECs damage. The inhibition of P2RX7 reversed the damage effect of HG, significantly downregulated the expression level of P53, HO-1, and p-ERK1/2 and upregulated the level of SLC7A11 and GPX4, which implicated that P2RX7 inhibition could attenuate oxidative stress and ferroptosis of endothelium in vivo and in vitro. Our data provided evidence that the P2RX7 play an important role in HG-associated oxidative stress, endothelial damage, and BBB disruption, which regulates HG-induced HT by ERK1/2 and P53 signaling pathways after MCAO.

Genetic modification of miR-34a enhances efficacy of transplanted human dental pulp stem cells after ischemic stroke.

Human dental pulp stem cells (hDPSCs) promote recovery after ischemic stroke; however, the therapeutic efficacy is limited by the poor survival of transplanted cells. For in vitro experiments in the present study, we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion. We found that miRNA-34a-5p (miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs. Inhibition of miR-34a facilitated the proliferation and antioxidant capacity and reduced the apoptosis of hDPSCs. Moreover, dual-luciferase reporter gene assay showed WNT1 and SIRT1 as the targets of miR-34a. In miR-34a knockdown cell lines, WNT1 suppression reduced cell proliferation, and SIRT1 suppression decreased the antioxidant capacity. Together, these results indicated that miR-34a regulates cell proliferation and antioxidant stress via targeting WNT1 and SIRT1, respectively. For in vivo experiments, we injected genetically modified hDPSCs (anti34a-hDPSCs) into the brains of mice. We found that anti34a-hDPSCs significantly inhibited apoptosis, reduced cerebral edema and cerebral infarct volume, and improved motor function in mice. This study provides new insights into the molecular mechanism of the cell proliferation and antioxidant capacity of hDPSCs, and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

Diabetes or calcium channel blocker contribute to cerebral hemodynamics after bypass surgery in adult patients with moyamoya disease.

Background: Moyamoya disease (MMD) is a teratogenic and lethal disease. However, existing studies do not sufficiently indicate the impact factors. Therefore, we investigated the different impact factors on cerebral hemodynamics after revascularization in patients with MMD. Methods: We retrospectively collected the clinical data of 233 adult patients with MMD who underwent revascularization surgery in the Department of Neurosurgery, Renmin Hospital of Wuhan University, from January 2015 to June 2021 for this retrospective cohort study. We analyzed the effects on hemodynamic improvement of age, sex, stroke type, early symptoms, Suzuki stage, history of hypertension, history of diabetes, and history of hyperlipidemia in patients with MMD. We also evaluated the efficacy of different revascularization strategies and we verified the effect of computed tomography perfusion (CTP) in evaluating cerebral hemodynamics. Results: The CTP values demonstrated that δ cerebral blood volume (CBV) values were significantly higher in the combined group [1.01 (0.87-1.75)] relative to those in the indirect group [1.34 (1.01-1.63); P=0.027]. There was no statistical significance in the improvement of clinical symptoms and clinical prognosis between the indirect and combined groups. Patients with MMD with diabetes [δ mean transit time (MTT), 0.49 (0.35-0.70) vs. 0.72 (0.52-0.87); P<0.001] or calcium channel blocker (CCB) [δCBV, 1.46 (1.10-1.83) vs. 1.12 (0.93-1.54); P=0.001] had better cerebral hemodynamics than patients in non-diabetic group or non-CCB group after revascularization. Conclusions: We didn't find differences in clinical outcome between indirect and combined revascularization in patients with MMD. we demonstrated that CTP values can be used as a way to detect postoperative cerebral hemodynamic changes in MMD patients. Interestingly, we found that MMD patients with diabetes or CCB showed better cerebral perfusion after revascularization.

CXCR4-BTK axis mediate pyroptosis and lipid peroxidation in early brain injury after subarachnoid hemorrhage via NLRP3 inflammasome and NF-κB pathway.

C-X-C chemokine receptor type 4 (CXCR4) is critical for homeostasis of the adaptive and innate immune system in some CNS diseases. Bruton's tyrosine kinase (BTK) is an essential kinase that regulates inflammation in immune cells through multiple signaling pathways. This study aims to explore the effect of CXCR4 and BTK on neuroinflammation in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Our results showed that the expression of CXCR4 and p-BTK increased significantly at 24 h after SAH in vivo and in vitro. Ibrutinib improved neurological impairment, BBB disruption, cerebral edema, lipid peroxidation, neuroinflammation and neuronal death at 24 h after SAH. Inhibition of BTK phosphorylation promoted the in vitro transition of hemin-treated proinflammatory microglia to the anti-inflammatory state, inhibited the p-P65 expression and microglial pyroptosis. NLRP3 deficiency can significantly reduce pyroptosis in SAH mice. Moreover, CXCR4 inhibition can suppress NLRP3-mediated pyroptosis, NF-κB activation and NOX2 expression in vitro, and ibrutinib can abolish CXCR4-aggravated BBB damage and pyroptosis in EBI after SAH. The levels of CXCR4 in CSF of SAH patients is significantly increased, and it is positively correlated with GSDMD and IL-1ß levels, and have a moderate diagnostic value for outcome at 6-month follow-up. Our findings revealed the effect of CXCR4 and P-BTK on NLRP3-mediated pyroptosis and lipid peroxidation after SAH in vivo and in vitro, and the potential diagnostic role of CXCR4 in CSF of SAH patients. Inhibition of CXCR4-BTK axis can significantly attenuate NLRP3-mediated pyroptosis and lipid peroxidation by regulating NF-κB activation in EBI after SAH.

Taurine attenuates neuronal ferroptosis by regulating GABAB/AKT/GSK3ß/ß-catenin pathway after subarachnoid hemorrhage.

Ferroptosis, characterized by lipid peroxidation and intracellular iron accumulation, has been reported to be involving in the pathophysiological of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Although taurine reportedly yields neuroprotective effects in multiple central neurological diseases and can attenuated neuron damage after stroke, its role in EBI after SAH remains unclear. The present study indicated that taurine levels in cerebrospinal fluid were significantly reduced in SAH patients, which suggested that taurine treatment after SAH could improve neurological impairment, oxidative stress, iron accumulation, BBB integrity and neuronal ferroptosis in the SAH model in vivo. Taurine could attenuate MDA levels and ROS accumulation and regulate the expression of SLC7A11 and GPX4 and the AKT/GSK3ß pathway in vitro. GABAB receptor inhibition and Ly294002 could reverse the therapeutic effects of taurine and significantly downregulate the levels of p-AKT, p-GSK3ß, ß-catenin, SLC7A11 and GPX4. The protective effects of taurine on SLC7A11 and GPX4 expression were reversed by ICG001 treatment in vitro. Taken together, our findings revealed that taurine could improve neurological function and alleviate cerebral edema, oxidative stress and BBB disruption after SAH, which reduced neuronal ferroptosis by regulating the GABAB/AKT/GSK3ß/ß-catenin signaling pathway.

Dental pulp stem cell transplantation facilitates neuronal neuroprotection following cerebral ischemic stroke.

OBJECTIVES: This study aimed to identify and evaluate the intracranial transplantation of dental pulp stem cells (DPSCs) as a possible ischemic stroke therapy that mitigates neuronal death/apoptosis. MATERIALS AND METHODS: DPSCs were isolated from the impacted third molars of healthy volunteers and then intracranially injected at 24 h post-ischemic stroke to Sprague Dawley rats that had been subjected to 2 h of middle cerebral artery occlusion. Neurological functional deficits were assessed using the modified neurological severity score (mNSS), and cerebral edema was quantified using brain water content. Neuronal death/apoptosis was indicated by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, NeuN immunofluorescence and immunohistochemistry, and Western blot analysis of the protein expression of anti-apoptotic indicator of Bcl-2 and apoptotic indicators of Bax and caspase 3. RESULTS: DPSC transplantation could ameliorate neurological dysfunction and brain edema, reduce infarct volume, decrease the percentage of TUNEL-positive nuclei, increase the number and percentage of NeuN-positive cells in ischemic penumbra, increase the ratio of Bcl-2 and Bax and down-regulate the production of caspase 3 in the cortical infarct zone. CONCLUSIONS: DPSC therapy via intracranial injection exerted remarkably neuroprotection mainly by inhibiting neuronal death/apoptosis.

A web-based dynamic nomogram for rupture risk of posterior communicating artery aneurysms utilizing clinical, morphological, and hemodynamic characteristics.

Background: Predicting rupture risk is important for aneurysm management. This research aimed to develop and validate a nomogram model to forecast the rupture risk of posterior communicating artery (PcomA) aneurysms. Methods: Clinical, morphological, and hemodynamic parameters of 107 unruptured PcomA aneurysms and 225 ruptured PcomA aneurysms were retrospectively analyzed. The least absolute shrinkage and selection operator (LASSO) analysis was applied to identify the optimal rupture risk factors, and a web-based dynamic nomogram was developed accordingly. The nomogram model was internally validated and externally validated independently. The receiver operating characteristic (ROC) curve was used to assess the discrimination of nomogram, and simultaneously the Hosmer-Lemeshow test and calibration plots were used to assess the calibration. Decision curve analysis (DCA) and clinical impact curve (CIC) were used to evaluate the clinical utility of nomogram additionally. Results: Four optimal rupture predictors of PcomA aneurysms were selected by LASSO and identified by multivariate logistic analysis, including hypertension, aspect ratio (AR), oscillatory shear index (OSI), and wall shear stress (WSS). A web-based dynamic nomogram was then developed. The area under the curve (AUC) in the training and external validation cohorts was 0.872 and 0.867, respectively. The Hosmer-Lemeshow p > 0.05 and calibration curves showed an appropriate fit. The results of DCA and CIC indicated that the net benefit rate of the nomogram model is higher than other models. Conclusion: Hypertension, high AR, high OSI, and low WSS were the most relevant risk factors for rupture of PcomA aneurysms. A web-based dynamic nomogram thus established demonstrated adequate discrimination and calibration after internal and external validation. We hope that this tool will provide guidance for the management of PcomA aneurysms.

The eIF4A Inhibitor Silvestrol Blocks the Growth of Human Glioblastoma Cells by Inhibiting AKT/mTOR and ERK1/2 Signaling Pathway.

The most frequently identified central nervous system tumor in adults is glioblastoma multiforme (GBM). GBM prognosis remains poor despite multimodal treatment, i.e., surgery and radiation therapy with concurrent temozolomide-based chemotherapy. Silvestrol, an eIF4A inhibitor, has been demonstrated to be able to kill tumor cells in previous studies. In this study, it was found that silvestrol considerably attenuated the proliferative potential of U251 and U87 glioma cells and reduced expression of cyclin D1. In addition, silvestrol reduced the level of ERK1/2 and decreased the levels of AKT phosphorylation. Unfortunately, the effect of silvestrol in inhibiting GBM cells was greatly reduced with hypoxia, and the downregulation in AKT/mTOR and ERK1/2 were also rescued with an upregulation of HIF1α, which warranted further research. Taken together, silvestrol exerted antitumor effects in GBM cells by inhibiting the AKT/mTOR and ERK1/2 signaling cascades.

Different Hemodynamic Characteristics and Resulting in Different Risks of Rupture Between Wide-Neck and Narrow-Neck Aneurysms.

Objective: This study aimed to determine the ruptured rate and hemodynamic difference between wide-neck aneurysms (WNAs) and narrow-neck aneurysms (NNAs), as well as the hemodynamic parameters of risk factors for aneurysm rupture. Methods: A total of 121 cases of intracranial aneurysms (IAs) were studied retrospectively between January 2019 and April 2021 at Renmin Hospital of Wuhan University. Intracranial aneurysms were classified into four types: ruptured wide-neck aneurysms (RWNAs), unruptured wide-neck aneurysms (UWNAs), ruptured narrow-neck aneurysms (RNNAs), and unruptured narrow-neck aneurysms (UNNAs). The Chi-square test was used to compare differences in rupture ratios. The clinical characteristics and hemodynamics were analyzed statistically to reveal the rupture risk factors. Moreover, significant parameters were subjected to binary logistic regression analysis to identify the independent predictive factors. The receiver operating characteristic (ROC) curve was performed to obtain cutoff values. Results: WNAs ruptured more frequently than NNAs (P = 0.033). Ruptured intracranial aneurysms (RIAs) were characterized by significantly higher intra-aneurysmal pressure (IAP), wall shear stress (WSS), wall shear stress gradient (WSSG), and lower normalized wall shear stress (NWSS) than unruptured intracranial aneurysms (UIAs). RWNAs had higher IAP, WSS, and lower NWSS than UWNAs (P < 0.05). RNNAs had higher IAP, Streamwise WSSG and lower NWSS compared to UNNAs (P < 0.05). Binary logistic regression revealed that IAP and WSS were independent predictive risk factors for WNAs rupture, with cut-off values of 405.5 and 6.66 Pa, respectively. Also, IAP was an independent predictive risk factor for NNA rupture, with a cut-off value of 255.3 Pa. Conclusions: Wide-neck aneurysms and narrow-neck aneurysms have diverse hemodynamics, which prompts a higher rupture ratio for WNAs. IAP could characterize the rupture risk in both WNAs and NNAs independently, but WSS could only predict the rupture risk in WNAs. This research might assist neurosurgeons with fostering a more sensible strategy for the treatment of IAs.

Andrographolide Attenuates Blood-Brain Barrier Disruption, Neuronal Apoptosis, and Oxidative Stress Through Activation of Nrf2/HO-1 Signaling Pathway in Subarachnoid Hemorrhage.

Andrographolide (Andro), a diterpene of the labdane family extracted from the Asian plant Andrographis paniculata, is neuroprotective against stroke and Alzheimer's disease. However, whether Andro protected the brain against subarachnoid hemorrhage (SAH) was still unknown. Thus, we explored whether Andro attenuated blood-brain barrier (BBB) disruption and neuronal apoptosis and inhibited oxidative stress to protect the brain against SAH both in vitro and in vivo and detected underlying mechanisms of Andro's neuroprotective effects in the present study. Oxyhemoglobin (OxyHb)-treated neuronal PC12 cells were used as an in vitro model. An in vivo model was established using Sprague-Dawley rats. Moreover, we used an inhibitor of heme oxygenase-1 (HO-1) (ZnPPIX) in vitro and in vivo experiments to evaluate whether the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade acted as one protective molecular mechanism of Andro against SAH. Our results revealed that, in vitro, Andro increased cell viability, inhibited apoptosis, and activated Nrf2/HO-1 cascade of neuronal PC12 cells treated with OxyHb. In vivo, Andro attenuated the neurological dysfunction, neuronal apoptosis, BBB disruption, brain edema, and oxidative stress and activated the Nrf2/HO-1 pathway. ZnPPIX reversed the effects of Andro in vitro and in vivo. Our research suggested that Andro alleviated BBB disruption, neuronal apoptosis, and oxidative stress in SAH, possibly via the Nrf2/HO-1 signaling pathway.