Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a.

Stroke is a life-threatening disease with limited therapeutic options. Damage to the blood-brain barrier (BBB) is the key pathological feature of ischemic stroke. This study explored the role of the bradykinin (BK)/bradykinin 1 receptor (B1R) and its mechanism of action in the BBB. Human brain microvascular endothelial cells (BMECs) were used to test for cellular responses to BK by using the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining, enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, cellular permeability assays, and western blotting to evaluate cell viability, cytokine production, and reactive oxygen species (ROS) levels in vitro. A BBB induced by middle cerebral artery occlusion was used to evaluate BBB injuries, and the role played by BK/B1R in ischemic/reperfusion (I/R) was explored in a rat model. Results showed that BK reduced the viability of BMECs and increased the levels of proinflammatory cytokines (interleukin 6 [IL-6], IL-18, and monocyte chemoattractant protein-1) and ROS. Additionally, cellular permeability was increased by BK treatment, and the expression of tight junction proteins (claudin-5 and occludin) was decreased. Interestingly, Wnt3a expression was inhibited by BK and exogenous Wnt3a restored the effects of BK on BMECs. In an in vivo I/R rat model, knockdown of B1R significantly decreased infarct volume and inflammation in I/R rats. Our results suggest that BK might be a key inducer of BBB injury and B1R knockdown might provide a beneficial effect by upregulating Wnt3a.

Arterial catheterization and in-hospital mortality in sepsis: a propensity score-matched study.

BACKGROUND: Despite the extensive use of arterial catheterization (AC), clinical effectiveness of AC to alter the outcomes among patients with sepsis and septic shock has not been evaluated. The purpose of this study is to examine the association between the use of AC and in-hospital mortality in septic patients. METHODS: Adult patients with sepsis from Medical Information Mart for Intensive Care database were screened to conduct this retrospective observational study. Propensity score matching (PSM) was employed to estimate the relationship between arterial catheterization (AC) and in-hospital mortality. Multivariable logistic regression and inverse probability of treatment weighing (IPTW) were used to validate our findings. RESULTS: A total of 14,509 septic patients without shock and 4,078 septic shock patients were identified. 3,489 pairs in sepsis patients without shock and 589 pairs in septic shock patients were yielded respectively after PSM. For patients in the sepsis without shock group, AC placement was associated with increased in-hospital mortality (OR, 1.34; 95% CI, 1.17-1.54; p < 0.001). In the septic shock group, there was no significant difference in hospital mortality between AC group and non-AC group. The results of logistic regression and propensity score IPTW model support our findings. CONCLUSIONS: In hemodynamically stable septic patients, AC is independently associated with higher in-hospital mortality, while in patients with septic shock, AC was not associated with improvements in hospital mortality.

Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/TFRC and GOT1 Axis.

Sepsis can cause sepsis-associated encephalopathy (SAE), but whether SAE was induced or exacerbated by ferroptosis remains unknown. In this study, the rat sepsis model was constructed using the cecal ligation and puncture method. The blood-brain barrier (BBB) permeability was measured by Evans blue dye (EBD) in vivo. The levels of ROS, Fe ion, MDA, GSH, and GPX4 were assessed by enzyme-linked immunosorbent assay (ELISA). The exosomes isolated from serum were cultured with bEnd.3 cells for the in vitro analysis. Moreover, bEnd.3 cells cultured with 100 µM FeCl3 (iron-rich) were to simulate ferroptosis stress. The cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. A dual-luciferase reporter gene assay was performed to confirm the relationship between miR-9-5p with NEAT1, TFRC, and GOT1. In vivo, it is found that BBB permeability was damaged in model rats. Level of ROS, Fe ion, and MDA was increased, and level of GSH and GPX4 was decreased, which means ferroptosis was induced by sepsis. Exosome-packaged NEAT1 in serum was significantly upregulated in model rats. In vitro, it is found that NEAT1 functions as a ceRNA for miR-9-5p to facilitate TFRC and GOT1 expression. Overexpression of NEAT1 enhanced ferroptosis stress in bEnd.3 cells. Increased miR-9-5p alleviated sepsis-induced ferroptosis by suppressing the expression of TFRC and GOT1 both in vivo and in vitro. In conclusion, these findings suggest that sepsis induced high expression of serous exosome-derived NEAT1, and it might exacerbate SAE by promoting ferroptosis through regulating miR-9-5p/TFRC and GOT1 axis.

Optimal Targets of the First 24-h Partial Pressure of Carbon Dioxide in Patients with Cerebral Injury: Data from the MIMIC-III and IV Database.

BACKGROUND: It is generally believed that hypercapnia and hypocapnia will cause secondary injury to patients with craniocerebral diseases, but a small number of studies have shown that they may have potential benefits. We assessed the impact of partial pressure of arterial carbon dioxide (PaCO2) on in-hospital mortality of patients with craniocerebral diseases. The hypothesis of this research was that there is a nonlinear correlation between PaCO2 and in-hospital mortality in patients with craniocerebral diseases and that mortality rate is the lowest when PaCO2 is in a normal range. METHODS: We identified patients with craniocerebral diseases from Medical Information Mart for Intensive Care third and fourth edition databases. Cox regression analysis and restricted cubic splines were used to examine the association between PaCO2 and in-hospital mortality. RESULTS: Nine thousand six hundred and sixty patients were identified. A U-shaped association was found between the first 24-h PaCO2 and in-hospital mortality in all participants. The nadir for in-hospital mortality risk was estimated to be at 39.5 mm Hg (p for nonlinearity < 0.001). In the subsequent subgroup analysis, similar results were found in patients with traumatic brain injury, metabolic or toxic encephalopathy, subarachnoid hemorrhage, cerebral infarction, and other encephalopathies. Besides, the mortality risk reached a nadir at PaCO2 in the range of 35-45 mm Hg. The restricted cubic splines showed a U-shaped association between the first 24-h PaCO2 and in-hospital mortality in patients with other intracerebral hemorrhage and cerebral tumor. Nonetheless, nonlinearity tests were not statistically significant. In addition, Cox regression analysis showed that PaCO2 ranging 35-45 mm Hg had the lowest death risk in most patients. For patients with hypoxic-ischemic encephalopathy and intracranial infections, the first 24-h PaCO2 and in-hospital mortality did not seem to be correlated. CONCLUSIONS: Both hypercapnia and hypocapnia are harmful to most patients with craniocerebral diseases. Keeping the first 24-h PaCO2 in the normal range (35-45 mm Hg) is associated with lower death risk.

Comparison of clinical characteristics in patients with coronavirus disease and influenza A in Guangzhou, China.

BACKGROUND: This study aims to compare the epidemiological, clinical and laboratory characteristics between patients with coronavirus disease (COVID-19) and influenza A (H1N1), and to develop a differentiating model and a simple scoring system. METHODS: We retrospectively analyzed the data from patients with COVID-19 and H1N1. The logistic regression model based on clinical and laboratory characteristics was constructed to distinguish COVID-19 from H1N1. Scores were assigned to each of independent discrimination factors based on their odds ratios. The performance of the prediction model and scoring system was assessed. RESULTS: A total of 236 patients were recruited, including 20 COVID-19 patients and 216 H1N1 patients. Logistic regression revealed that age >34 years, temperature ≤37.5 °C, no sputum or myalgia, lymphocyte ratio ≥20% and creatine kinase-myocardial band isoenzyme (CK-MB) >9.7 U/L were independent differentiating factors for COVID-19. The area under curves (AUCs) of the prediction model and scoring system in differentiating COVID-19 from H1N1 were 0.988 and 0.962, respectively. CONCLUSIONS: There are certain differences in clinical and laboratory features between patients with COVID-19 and H1N1. The simple scoring system may be a useful tool for the early identification of COVID-19 patients from H1N1 patients.

Hypercapnia exacerbates the disruption of the blood­brain barrier by inducing interleukin­1ß overproduction in the blood of hypoxemic adult rats.

Refractory hypoxemia is the main symptom of acute respiratory distress syndrome (ARDS). Low tidal volume ventilation is routinely applied in clinical practice to correct hypoxemia, which aims to prevent ventilator­induced lung injury. However, this ventilation strategy inevitably leads to hypercapnia. Our previous study demonstrated that hypercapnia aggravated cognitive impairment in hypoxemic rats; however, the underlying mechanism remains unclear. The aim of the present study was to investigate whether hypercapnia exacerbates the blood­brain barrier (BBB) disruption through inducing interleukin (IL)­1ß overproduction in the blood of hypoxemic rats. The BBB permeability in a rat model of hypercapnia/hypoxemia was evaluated. The levels of IL­1ß in the blood of rats and human whole­blood cultures were assessed. The expression of IL­1 receptor 1 (IL­1R1), phosphorylated IL­1R1­associated kinase (p­IRAK­1) and tight junctional proteins in cerebral vascular endothelial cells was examined in vitro and in vivo. In addition, IL­1Ra, an IL­1 receptor antagonist, was used to determine whether hypercapnia affects tight junctional protein expression in hypoxic cerebral vascular endothelial cells through inducing IL­1ß overproduction. It was observed that hypercapnia alone did not disrupt the BBB, but aggravated the damage to the BBB integrity in hypoxemic rats. Hypercapnia increased IL­1ß expression in the blood of hypoxemic rats as well as in hypoxic human whole­blood cultures. IL­1R1 and p­IRAK­1 expression was increased, while that of tight junctional proteins was reduced by hypercapnia in hypoxemic cerebral vascular endothelial cells in vitro and in vivo. Additionally, the expression of tight junctional proteins was markedly increased following treatment with IL­1Ra. These results suggest that hypercapnia­induced IL­1ß overproduction in the hypoxemic blood may decrease tight junctional protein expression in cerebrovascular endothelial cells via the IL­1R1/p­IRAK­1 pathway, further disrupting BBB integrity, and eventually resulting in increased BBB permeability.

Hypertonic saline mediates the NLRP3/IL-1ß signaling axis in microglia to alleviate ischemic blood-brain barrier permeability by downregulating astrocyte-derived VEGF in rats.

INTRODUCTION: The aim of this study was to explore whether the antibrain edema of hypertonic saline (HS) is associated with alleviating ischemic blood-brain barrier (BBB) permeability by downregulating astrocyte-derived vascular endothelial growth factor (VEGF), which is mediated by microglia-derived NOD-like receptor protein 3 (NLRP3) inflammasome. METHODS: The infarct volume and BBB permeability were detected. The protein expression level of VEGF in astrocytes in a transient focal brain ischemia model of rats was evaluated after 10% HS treatment. Changes in the NLRP3 inflammasome, IL-1ß protein expression, and the interleukin-1 receptor (IL1R1)/pNF-кBp65/VEGF signaling pathway were determined in astrocytes. RESULTS: HS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulates IL-1ß expression by inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulates VEGF expression by inhibiting the phosphorylation of NF-кBp65 mediated by IL-1ß in astrocytes. CONCLUSIONS: HS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulated IL-1ß expression via inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulated VEGF expression through inhibiting the phosphorylation of NF-кBp65 mediated by IL-1ß in astrocytes.

Peri-operative risk factors for in-hospital mortality in acute type A aortic dissection.

BACKGROUND: Acute type A aortic dissection (TAAD) is cardiovascular emergency and requires surgical interventions. In-hospital mortality rate of surgical-treated TAAD patients remains high. We aim to examine the prognostic implications of peri-operative parameters to identify high-risk patient for in-hospital mortality. METHODS: A total of 264 surgically treated TAAD patients were included in this study. The association between in-hospital mortality and peri-operative parameters were examined. RESULTS: Thirty patients (11.36%) died during hospitalization. Patients with higher Apache II score had a significantly higher rate of in-hospital mortality when compared with patients scored ≤20 in unadjusted model [Score 21-25: HR =12.9 (1.7-100.8), P=0.0148; Score >25: HR =94.5 (12.6-707.6), P<0.0001]. Patients with Sbp >120 mmHg, Cr >200 mmol/L (both at admission and after surgery), BUN >8.2 mmol/L (both at admission and after surgery), AST >80 µ/L, aortic cross-clamping time >120 min and cardiopulmonary bypass time (CPBT) >230 min were also significantly related to higher rate of in-hospital mortality in univariate analysis. In multivariable analysis, APACHE II score [Score 21-25: HR =9.5 (1.2-74.4), P=0.032; Score >25: HR =51.0 (6.7-387.7), P=0.0001], AST >80 µmol/L [HR =2.3 (1.1-4.8), P=0.0251], aortic cross-clamping time >120 min (HR =2.9 (1.1-7.7), P=0.0315) remained significant in predicting TAAD in-hospital mortality. CONCLUSIONS: APACHE II score could be a useful tool to predict TAAD in-hospital mortality. AST >80 µ/L and aortic cross-clamping time >120 min were also independent predictors.

Hypertonic saline downregulates endothelial cell-derived VEGF expression and reduces blood-brain barrier permeability induced by cerebral ischaemia via the VEGFR2/eNOS pathway.

The aim of the present study was to explore the possible mechanisms by which hypertonic saline (HS) effectively ameliorates cerebral oedema via the vascular endothelial growth factor receptor 2 (VEGFR2)­mediated endothelial nitric oxide synthase (eNOS) pathway of endothelial cells in rats. A middle cerebral artery occlusion (MCAO) model in Sprague­Dawley rats and an oxygen­glucose deprivation (OGD) model in cells were used in the present study. Evans blue (EB) staining and a horseradish peroxidase flux assay were performed to evaluate the protective effect of 10% HS on the blood­brain barrier (BBB). The expression levels of vascular endothelial growth factor (VEGF), VEGFR2, zonula occludens 1 (ZO1) and occludin were quantified. The results demonstrated that 10% HS effectively reduced EB extravasation in the peri­ischaemic brain tissue. At 24 h after MCAO, the protein expression levels of VEGF and VEGFR2 in the peri­ischaemic brain tissue were downregulated following treatment with 10% HS. In vitro experiments demonstrated that the permeability of a monolayer endothelial cell barrier was decreased significantly following HS treatment. In addition, VEGF and VEGFR2 protein expression levels were increased in endothelial cells under hypoxic conditions, but that effect was suppressed by HS treatment. Furthermore, HS inhibited the downregulation of ZO1 and occludin effectively, possibly through the VEGFR2/phospholipase C γ1 (PLCγ1)/eNOS signalling pathway. In conclusion, 10% HS may alleviate cerebral oedema through reducing ischaemia­induced BBB permeability, as a consequence of inhibiting VEGFR2/PLCγ1/eNOS­mediated downregulation of ZO1 and occludin.

Presepsin level in predicting patients' in-hospital mortality from sepsis under sepsis-3 criteria.

Background: Early recognition of septic patients with poor prognosis is important for clinicians to prescribe personalized therapies which include timely fluid resuscitation therapy and appropriate antimicrobial therapy. We aimed to evaluate the effect of the presepsin level on predicting the prognosis of patients with sepsis under the sepsis-3 criteria. Methods: Patients who were diagnosed as sepsis under the sepsis-3 criteria were recruited and assigned to the survivor group and the non-survivor group according to their in-hospital mortality. The two groups' baseline characteristics were analyzed with Pearson's chi-square (χ 2) test or Kruskal-Wallis test. Binary logistic regression analysis was performed to determine the independent predictors of in-hospital mortality from sepsis. Receiver operating characteristic analysis was conducted to evaluate the efficacy of presepsin in predicting patients' in-hospital mortality from sepsis. The correlation between presepsin and the Sequential Organ Failure Assessment (SOFA) score was measured with Spearman's rank correlation coefficient. P-values of less than 0.05 were considered to indicate statistical significance. Results: Overall, 138 patients were included in this study. The presepsin level of the non-survivor group was significantly higher than that of the other group (P=0.000). Binary logistic regression showed that the presepsin level was an independent risk factor of patients' in-hospital mortality from sepsis (OR =1.221 P=0.026). The presepsin level was positively associated with the SOFA score (ρ=0.396, P=0.000). ROC curve analysis revealed the presepsin level was highly accurate in predicting patients' in-hospital mortality from sepsis (AUC =0.703, P=0.000). The AUC value of a combination of presepsin and the SOFA score was significantly larger than that of the SOFA score alone (AUC: 0.817 vs 0.793, P=0.041). Conclusions: Presepsin is a prognostic biomarker with high accuracy in predicting the prognosis of sepsis under the sepsis-3 criteria.

Long non­coding RNA DILC is involved in sepsis by modulating the signaling pathway of the interleukin­6/signal transducer and activator of transcription 3/Toll­like receptor 4 axis.

Sepsis is characterized by systemic inflammatory responses. In the present study, the role of deleted in liver cancer 1 (DILC), interleukin (IL)­6, signal transducer and activator of transcription 3 (STAT3), and Toll­like receptor 4 (TLR4) in the pathogenesis of sepsis was investigated. Reverse transcription­quantitative polymerase chain reaction analysis and western blotting were performed to evaluate the effects of lipopolysaccharide (LPS) on the expression of DILC, IL­6, STAT3, and TLR4, in addition to the effects of DILC and IL­6 on the synthesis of tumor necrosis factor (TNF­α), chemokine ligand 5 (CCL5), E­selectin and C­X­C motif chemokine receptor 1 (CXCR1). In addition, the regulatory association between DILC, IL­6, STAT3 and TLR4 was investigated. LPS reduced the expression level of DILC, and enhanced the expression of IL­6, STAT3 and TLR4. DILC directly and negatively regulated the synthesis of IL­6, as demonstrated by the markedly decreased luciferase activity in cells transfected with a wild­type DILC plasmid. On the other hand, compared with the scramble control, DILC and IL­6 small interfering (si)RNAs significantly suppressed the expression of IL­6, STAT3 and TLR4. In addition, DILC siRNA enhanced the expression of IL­6, STAT3 and TLR4, whereas the expression levels of TNF­α, CCL5, E­selectin and CXCR1 in LPS­treated THP­1 cells were downregulated following transfection with DILC and IL­6 siRNAs. In patients with sepsis, DILC expression was inhibited, although the expression levels of IL­6, STAT3 and TLR4 were upregulated. In addition, the expression levels of TNF­α, CCL5, E­selectin and CXCR1 in patients with sepsis were higher compared with normal subjects. Therefore, DILC may mediate the crosstalk between the cascades of IL­6/STAT3 and TNF­α signaling, indicating that DILC may act as a prognostic biomarker of sepsis, and may serve as a potential therapeutic target for the treatment of sepsis.

Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 in cerebral edema treatment.

BACKGROUND: Hypertonic saline (HS) has been used clinically for treatment of cerebral edema for decades. Previously we have demonstrated that HS alleviates cerebral edema via regulating water/ion channel protein and attenuating neuroinflammation. However, whether HS treatment triggers microglia polarization and its regulatory mechanism during this process is unclear. METHODS AND RESULTS: The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of neuroinflammation and microglia functions. Treatment of 10% HS not only significantly reduced infarct size and ipsilateral ischemic hemispheric brain water content (BWC) via attenuating ischemia-induction of TNF-α, IL-1ß, microglia M1 markers (iNOS, CD86) and miR-200b, but also increased neurotrophic factors such as IL-10 and IL-4, microglia M2 markers (Arg1, CD206) and Krüppel-like factor 4 (KLF4). Similar changes were confirmed in primary microglial cells subjected to hypoxia with/without HS in vitro. Importantly, overexpression of miR-200b was able to induce microglia M1 polarization via directly targeting KLF4. Restoring KLF4 expression abolished this effect. On the contrary, miR-200b inhibitor or KLF4 overexpression led to microglia M2 polarization. Mechanistically, KLF4 directly binds to promoter region of Agr1, thus inducing its transcription. Similar to treatment of HS, experimental overexpression of KLF4 in vivo exerted significant beneficial effects on ischemia-induced cerebral edema. However, knockdown of KLF4 abrogated the benefits of HS. CONCLUSIONS: Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 during its treatment of cerebral edema. This study may provide new insights of HS-related therapy for cerebral edema.

Hypercapnia induces IL-1ß overproduction via activation of NLRP3 inflammasome: implication in cognitive impairment in hypoxemic adult rats.

BACKGROUND: Cognitive impairment is one of common complications of acute respiratory distress syndrome (ARDS). Increasing evidence suggests that interleukin-1 beta (IL-1ß) plays a role in inducing neuronal apoptosis in cognitive dysfunction. The lung protective ventilatory strategies, which serve to reduce pulmonary morbidity for ARDS patients, almost always lead to hypercapnia. Some studies have reported that hypercapnia contributes to the risk of cognitive impairment and IL-1ß secretion outside the central nervous system (CNS). However, the underlying mechanism of hypercapnia aggravating cognitive impairment under hypoxia has remained uncertain. This study was aimed to explore whether hypercapnia would partake in increasing IL-1ß secretion via activating the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome in the hypoxic CNS and in aggravating cognitive impairment. METHODS: The Sprague-Dawley (SD) rats that underwent hypercapnia/hypoxemia were used for assessment of NLRP3, caspase-1, IL-1ß, Bcl-2, Bax, and caspase-3 expression by Western blotting or double immunofluorescence, and the model was also used for Morris water maze test. In addition, Z-YVAD-FMK, a caspase-1 inhibitor, was used to treat BV-2 microglia to determine whether activation of NLRP3 inflammasome was required for the enhancing effect of hypercapnia on expressing IL-1ß by Western blotting or double immunofluorescence. The interaction effects were analyzed by factorial ANOVA. Simple effects analyses were performed when an interaction was observed. RESULTS: There were interaction effects on cognitive impairment, apoptosis of hippocampal neurons, activation of NLRP3 inflammasome, and upregulation of IL-1ß between hypercapnia treatment and hypoxia treatment. Hypercapnia + hypoxia treatment caused more serious damage to the learning and memory of rats than those subjected to hypoxia treatment alone. Expression levels of Bcl-2 were reduced, while that of Bax and caspase-3 were increased by hypercapnia in hypoxic hippocampus. Hypercapnia markedly increased the expression of NLRP3, caspase-1, and IL-1ß in hypoxia-activated microglia both in vivo and in vitro. Pharmacological inhibition of NLRP3 inflammasome activation and release of IL-1ß might ameliorate apoptosis of neurons. CONCLUSIONS: The present results suggest that hypercapnia-induced IL-1ß overproduction via activating the NLRP3 inflammasome by hypoxia-activated microglia may augment neuroinflammation, increase neuronal cell death, and contribute to the pathogenesis of cognitive impairments.

Magnetic resonance spectroscopy for assessment of brain injury in the rat model of sepsis.

The diagnostic value of magnetic resonance spectroscopy (MRS), T2-weighted imaging (T2WI) and serum markers of brain injury in a rat model of sepsis were investigated. Rats were randomly divided into the control group and 6, 12 and 24 h after lipopolysaccharide-injection groups. Brain morphology and metabolism were assessed with T2WI magnetic resonance imaging (MRI) and MRS. Serum and brain tissue samples were then collected to examine the concentrations of neuron-specific enolase (NSE) and S100-ß protein. Brain T2WI showed no differences between the groups. N-acetylaspartate/choline (NAA/Cr) ratio measured by MRS showed different degrees of decrease in the sepsis groups, and serum NSE and S100-ß concentrations were increased compared with the control group. Apoptosis rates were measured in the right hippocampal area, and there were statistically significant differences between the indicated groups and the control group (p<0.05). The correlation between apoptosis rate and NAA/Cr ratio was closer than that between apoptosis rate and NSE or S100-ß (-0.925 vs. 0.434 vs. 0.517, respectively). In conclusion, MRS is a sensitive, non-invasive method to investigate complications of brain injury in septic rats, which may be utilized for the early diagnosis of brain injury caused by sepsis.

Hypertonic saline attenuates expression of Notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic BV-2 microglia.

BACKGROUND: Ischemic stroke is a major disease that threatens human health in ageing population. Increasing evidence has shown that neuroinflammatory mediators play crucial roles in the pathophysiology of cerebral ischemia injury. Notch signaling is recognized as the cell fate signaling but recent evidence indicates that it may be involved in the inflammatory response in activated microglia in cerebral ischemia. Previous report in our group demonstrated hypertonic saline (HS) could reduce the release of interleukin-1 beta and tumor necrosis factor-alpha in activated microglia, but the underlying molecular and cellular mechanisms have remained uncertain. This study was aimed to explore whether HS would partake in regulating production of proinflammatory mediators through Notch signaling. RESULTS: HS markedly attenuated the expression of Notch-1, NICD, RBP-JK and Hes-1 in activated microglia both in vivo and in vitro. Remarkably, HS also reduced the expression of iNOS in vivo, while the in vitro levels of inflammatory mediators Phos-NF-κB, iNOS and ROS were reduced by HS as well. CONCLUSION: Our results suggest that HS may suppress of inflammatory mediators following ischemia/hypoxic through the Notch signaling which operates synergistically with NF-κB pathway in activated microglia. Our study has provided the morphological and biochemical evidence that HS can attenuate inflammation reaction and can be neuroprotective in cerebral ischemia, thus supporting the use of hypertonic saline by clinicians in patients with an ischemia stroke.

Hypertonic saline protects brain endothelial cells against hypoxia correlated to the levels of epidermal growth factor receptor and interleukin-1ß.

OBJECTIVE: The aim of this study was to verify the protective effect of hypertonic saline (HS) on brain endothelial cells under hypoxic conditions and the relevant underlying mechanism. METHODS: bEnd.3 cells were treated with oxygen-glucose deprivation (OGD)-induced injury. To measure HS performance, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay, and cell apoptosis was assessed by flow cytometry and Terminal deoxynucleotidyl transferase UTP nick-end labeling staining. RNA-seq was performed to assess the expression profiles and screen the candidate genes that participated in OGD-induced injury and the HS protective effect. Quantitative real-time polymerase chain reaction (qPCR) and western blot analysis were used to confirm the expression of candidate genes, and enzyme-linked immunosorbent assay was used to measure the level of interleukin (IL)-1ß. Overexpression analyses were performed to confirm the functions of the differentially expressed genes. RESULTS: HS with a concentration of 40 mmol/L NaCl had an obvious protective effect on bEnd.3 cells after OGD-induced injury, resulting in increased cell viability and a smaller percentage of apoptotic cells. According to the RNA-seq results, epidermal growth factor receptor (EGFR) was chosen as the differentially expressed gene target in this study. The qPCR and western blot analyses further confirmed that the levels of EGFR/phosphorylated epidermal growth factor receptor and IL-1ß were enhanced after OGD-induced injury, but attenuated after treatment with 40 mmol/L of NaCl HS. Overexpressed EGFR reversed the protective effect of HS that caused low viability and high rates of apoptosis in cells. CONCLUSION: HS can protect endothelial cells against OGD-induced injury, but is affected by the expression of EGFR/p-EGFR and IL-1ß.

Hypertonic saline alleviates experimentally induced cerebral oedema through suppression of vascular endothelial growth factor and its receptor VEGFR2 expression in astrocytes.

BACKGROUND: Cerebral oedema is closely related to the permeability of blood-brain barrier, vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) all of which are important blood-brain barrier (BBB) permeability regulatory factors. Zonula occludens 1 (ZO-1) and claudin-5 are also the key components of BBB. Hypertonic saline is widely used to alleviate cerebral oedema. This study aimed to explore the possible mechanisms underlying hypertonic saline that ameliorates cerebral oedema effectively. METHODS: Middle cerebral artery occlusion (MCAO) model in Sprague-Dawley (SD) rats and of oxygen-glucose deprivation model in primary astrocytes were used in this study. The brain water content (BWC) was used to assess the effect of 10 % HS on cerebral oedema. The assessment of Evans blue (EB) extravasation was performed to evaluate the protective effect of 10 % HS on blood-brain barrier. The quantification of VEGF, VEGFR2, ZO-1 and claudin-5 was used to illustrate the mechanism of 10 % HS ameliorating cerebral oedema. RESULTS: BWC was analysed by wet-to-dry ratios in the ischemic hemisphere of SD rats; it was significantly decreased after 10 % HS treatment (P < 0.05). We also investigated the blood-brain barrier protective effect by 10 % HS which reduced EB extravasation effectively in the peri-ischemic brain tissue. In parallel to the above notably at 24 h following MCAO, mRNA and protein expression of VEGF and VEGFR2 in the peri-ischemic brain tissue was down-regulated after 10 % HS treatment (P < 0.05). Along with this, in vitro studies showed increased VEGF and VEGFR2 mRNA and protein expression in primary astrocytes under hypoxic condition (P < 0.05), but it was suppressed after HS treatment (P < 0.05). In addition, HS inhibited the down-regulation of ZO-1, claudin-5 effectively. CONCLUSIONS: The results suggest that 10 % HS could alleviate cerebral oedema possibly through reducing the ischemia induced BBB permeability as a consequence of inhibiting VEGF-VEGFR2-mediated down-regulation of ZO-1, claudin-5.

Huperzine A protects sepsis associated encephalopathy by promoting the deficient cholinergic nervous function.

Neuroinflammatory deregulation in the brain plays a crucial role in the pathogenesis of sepsis associated encephalopathy (SAE). Given the mounting evidence of anti-inflammatory and neuroprotective effects of the cholinergic nervous system, it is surprising that there is little information about its changes in the brain during sepsis. To elucidate the role of the cholinergic nervous system in SAE, hippocampal choline acetyltransferase, muscarinic acetylcholine receptor-1, acetylcholinesterase and acetylcholine were evaluated in LPS-induced sepsis rats. Expression of pro-inflammatory cytokines, neuronal apoptosis, and animal cognitive performance were also assessed. Furthermore, therapeutic effects of the acetylcholinesterase inhibitor Huperzine A (HupA) on the hippocampal cholinergic nervous function and neuroinflammation were evaluated. A deficiency of the cholinergic nervous function was revealed in SAE, accompanied with over-expressed pro-inflammatory cytokines, increase in neuronal apoptosis and brain cognitive impairment. HupA remarkably promoted the deficient cholinergic nervous function and attenuated the abnormal neuroinflammation in SAE, paralleled with the recovery of brain function. We suggest that the deficiency of the cholinergic nervous function and the abnormal neuroinflammation are synergistically implicated in the pathogenesis of SAE. Thus, HupA is a potential therapeutic candidate for SAE, as it improves the deficient cholinergic nervous function and exerts anti-inflammatory action.

[Cholinergic anti-inflammatory pathway involves in the neuroprotective effect of huperzine A on sepsis-associated encephalopathy].

Objective: To explore whether the cholinergic anti-inflammatory pathway is involved in the neuroprotective effect of acetylcholinesterase inhibitor huperzine A (HupA) on sepsis-associated encephalopathy (SAE) by observing the effect of HupA on the expressions of choline acetyltransferase (CHAT) and cholinergic muscarinic receptor M1 (CHRM1) of sepsis rats. Methods: Fifty-four male Wistar rats, 8 weeks old, were divided into three experimental groups according to random number table:control group,sepsis group, and HupA group, with 18 rats in each group. The rat model of sepsis was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS,1 mL),and the rats in control group were given the same volume of normal saline. The rats in HupA group were intraperitoneally administered with HupA 0.04 mg/kg (1 mL) at 30 minutes before model reproduction, while the rats in control group and sepsis group were treated with the same volume of saline instead. At 3,12,and 24 hours after model reproduction,6 rats in each group were sacrificed after clinical manifestation observation, and cerebral cortex tissue was collected. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1 ß) in cerebral cortex were determined with enzyme linked immunosorbent assay (ELISA),along with the measurement of neuronal apoptosis by caspase-3 and neuronal nuclear antigen (NeuN) immune double standard staining. The mRNA expressions and positive expressions of ChAT and CHRM1 were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunofluorescence methods. Results: Clinical manifestations of sepsis rats were present at 3 hours and reached a peak at 12 hours, including lethargy,vertical hair and lazy to move. Over-expression of pro-inflammatory cytokines TNF-α and IL-1ß was found in sepsis group, and apoptotic neurons marked by two fluorescences were significantly increased in sepsis rats ,in comparison with deficient ChAT and CHRM1 proteins marked by red fluorescence, and low-expressed ChAT and CHRM1 mRNA as well. The differences between sepsis group and control group were statistically significant [12-hour TNF-α (ng/L):84.97±31.84 vs.40.31 ± 10.37,12-hour IL-1 ß (ng/L):1 095.98± 127.09 vs.622.62± 117.25,12-hour ChAT mRNA (2-△△Ct):1.34 (0.67,1.86) vs.1.92 (1.12,2.87),12-hour CHRM1 mRNA (2-△△Ct):0.65±0.12 vs.1.16±0.42,all P ＜ 0.05].The septic symptoms were relieved after HupA administration,as well as the reduction of pro-inflammatory cytokines and the neuronal apoptosis, which might attribute to the increased expressions of ChAT and CHRM1.The differences between HupA group and sepsis group were statistically significant [12-hour TNF-α (ng/L):48.38 ± 12.62 vs.84.97 ± 31.84,12-hour IL-1 ß (ng/L):718.13 ± 163.33 vs.1 095.98 ± 127.09,12-hour ChAT mRNA (2-△△Ct):18.04 (17.22,19.23) vs.1.34 (0.67,1.86),12-hour CHRM1 mRNA (2-△ △Ct):1.46 ± 0.69 vs 0.65 ± 0.12,all P ＜ 0.05].The clinical manifestations and neuroinflammation mainly recovered at 24 hours. Conclusions: The cortical cholinergic neurons dysfunction and the abnormal inflammatory response are involved in the onset of SAE process. HupA plays a protective role in SAE through recovering the function of cholinergic neurons and cholinergic anti-inflammatory pathway.