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.

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.

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.

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.

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.

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.

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.

Hypertonic saline alleviates cerebral edema by inhibiting microglia-derived TNF-α and IL-1ß-induced Na-K-Cl Cotransporter up-regulation.

BACKGROUND: Hypertonic saline (HS) has been successfully used clinically for treatment of various forms of cerebral edema. Up-regulated expression of Na-K-Cl Cotransporter 1 (NKCC1) and inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) has been demonstrated to be closely associated with the pathogenesis of cerebral edema resulting from a variety of brain injuries. This study aimed to explore if alleviation of cerebral edema by 10% HS might be effected through down-regulation of inflammatory mediator expression in the microglia, and thus result in decreased NKCC1 expression in astrocytes in the cerebral cortex bordering the ischemic core. METHODS: The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of NKCC1, TNF-α and IL-1ß expression using Western blotting, double immunofluorescence and real time RT-PCR, and the model also was used for evaluation of brain water content (BWC) and infarct size. SB203580 and SP600125, specific inhibitors of the p38 and JNK signaling pathways, were used to treat primary microglia cultures to determine whether the two signaling pathways were required for the inhibition of HS on microglia expressing and secreting TNF-α and IL-1ß using Western blotting, double immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The effect of TNF-α and IL-1ß on NKCC1 expression in primary astrocyte cultures was determined. In addition, the direct inhibitory effect of HS on NKCC1 expression in primary astrocytes was also investigated by Western blotting, double immunofluorescence and real time RT-PCR. RESULTS: BWC and infarct size decreased significantly after 10% HS treatment. TNF-α and IL-1ß immunoexpression in microglia was noticeably decreased. Concomitantly, NKCC1 expression in astrocytes was down-regulated. TNF-α and IL-1ß released from the primary microglia subjected to hypoxic exposure and treatment with 100 mM HS were decreased. NKCC1 expression in primary astrocytes was concurrently and progressively down-regulated with decreasing concentration of exogenous TNF-α and IL-1ß. Additionally, 100 mM HS directly inhibited NKCC1 up-regulation in astrocytes under hypoxic condition. CONCLUSIONS: The results suggest that 10% HS alleviates cerebral edema through inhibition of the NKCC1 Cotransporter, which is mediated by attenuation of TNF-α and IL-1ß stimulation on NKCC1.