Intrahippocampal injection of IL-1ß upregulates Siah1-mediated degradation of synaptophysin by activation of the ERK signaling in male rat.

Interleukin-1ß (IL-1ß) has been described to exert important effect on synapses in the brain. Here, we explored if the synapses in the hippocampus would be adversely affected following intracerebral IL-1ß injection and, if so, to clarify the underlying molecular mechanisms. Adult male Sprague-Dawley rats were divided into control, IL-1ß, IL-1ß + PD98059, and IL-1ß + MG132 groups and then sacrificed for detection of synaptophysin (syn) protein level, synaptosome glutamate release, and synapse ultrastructure by western blotting, glutamate kit and electron microscopy, respectively. These rats were tested by Morris water maze for learning and memory ability. It was determined by western blotting whether IL-1ß exerted the effect of on syn and siah1 expression in primary neurons via extracellular regulated protein kinases (ERK) signaling pathway. Intrahippocampal injection of IL-1ß in male rats and sacrificed at 8d resulted in a significant decrease in syn protein, damage of synapse structure, and abnormal release of neurotransmitters glutamate. ERK inhibitor and proteosome inhibitor treatment reversed the above changes induced by IL-1ß both in vivo and in vitro. In primary cultured neurons incubated with IL-1ß, the expression level of synaptophysin was significantly downregulated coupled with abnormal glutamate release. Furthermore, use of PD98059 had confirmed that ERK signaling pathway was implicated in synaptic disorders caused by IL-1ß treatment. The present results suggest that exogenous IL-1ß can suppress syn protein level and glutamate release. A possible mechanism for this is that IL-1ß induces syn degradation that is regulated by the E3 ligase siah1 via the ERK signaling pathway.

MiRNA-338-3p Inhibits Neuroinflammation in the Corpus Callosum of LCV-LPS Rats Via STAT1 Signal Pathway.

Neuroinflammation is a common characteristic of intracranial infection (ICI), which is associated with the activation of astrocytes and microglia. MiRNAs are involved in the process of neuroinflammation. This study aimed to investigate the potential mechanism by which miR-338-3p negatively modulate the occurrence of neuroinflammation. We here reported that the decreased levels of miR-338-3p were detected using qRT-PCR and the upregulated expression of TNF-α and IL-1ß was measured by ELISA in the cerebrospinal fluid (CSF) in patients with ICI. A negative association between miR-338-3p and TNF-α or IL-1ß was revealed by Pearson correlation analysis. Sprague-Dawley (SD) rats were injected with LPS (50 µg) into left cerebral ventricule (LCV), following which the increased expression of TNF-α and IL-1ß and the reduction of miR-338-3p expression were observed in the corpus callosum (CC). Moreover, the expression of TNF-α and IL-1ß in the astrocytes and microglia in the CC of LCV-LPS rats were saliently inhibited by the overexpression of miR-338-3p. In vitro, cultured astrocytes and BV2 cells transfected with mimic-miR-338-3p produced less TNF-α and IL-1ß after LPS administration. Direct interaction between miR-338-3p and STAT1 mRNA was validated by biological information analysis and dual luciferase assay. Furthermore, STAT1 pathway was found to be implicated in inhibition of neuroinflammation induced by mimic miR-338-3p in the astrocytes and BV2 cells. Taken together, our results suggest that miR-338-3p suppress the generation of proinflammatory mediators in astrocyte and BV2 cells induced by LPS exposure through the STAT1 signal pathway. MiR-338-3p could act as a potential therapeutic strategy to reduce the neuroinflammatory response. Diagram describing the cellular and molecular mechanisms associated with LPS-induced neuroinflammation via the miR-338-3p/STAT1 pathway. LPS binds to TLRs on astrocytes or microglia to activate the STAT1 pathway and upregulate the production of pro-inflammatory cytokines. However, miR-338-3p inhibits the expression of STAT1 and reduces the production of inflammatory mediators.

Extracellular vesicles derived from lung cancer cells exposed to intermittent hypoxia upregulate programmed death ligand 1 expression in macrophages.

PURPOSE: Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), compromises immune surveillance through the upregulation of programmed cell death-1 ligand (PD-L1). Tumor-released extracellular vesicles (EVs) have been reported to modulate immunosuppressive activities. We investigated whether or not EVs derived from intermittent hypoxic lung cancer cells can alter the expression of PD-L1 in macrophages. METHODS: The expression of PD-L1+monocytes from 40 patients with newly diagnosed non-small-cell lung cancer (NSCLC) and with (n=21) or without (n=19) OSA were detected. Plasma EVs isolated from NSCLC patients with moderate-severe OSA (n=4) and without OSA (n=4) were co-cultured with macrophages. A549 cells were exposed to normoxia or IH (48 cycles of 5 min of 1% O2 hypoxia, followed by 5 min of normoxia). EVs were isolated from cell supernatant and were co-cultured with macrophages differentiated from THP-1. PD-L1 and hypoxia-inducible factor-1 α (HIF-1α) expressions were measured by flow cytometry, immunofluorescence, and Western blot analysis. RESULTS: PD-L1+monocytes were elevated in NSCLC patients with OSA and increased with the severity of OSA and nocturnal desaturation. PD-L1+ macrophages were induced by EVs from NSCLC patients with OSA and positively correlated with HIF-1α expressions. EVs from IH-treated A549 can promote PD-L1 and HIF-1α expression in macrophages and the upregulation of PD-L1 expression was reversed by specific HIF-1α inhibitor. CONCLUSION: IH can enhance the function of EVs derived from lung cancer cells to aggravate immunosuppressive status in macrophages. HIF-1α may play an important role in this process.

A nomogram incorporating functional and tubular damage biomarkers to predict the risk of acute kidney injury for septic patients.

BACKGROUND: Combining tubular damage and functional biomarkers may improve prediction precision of acute kidney injury (AKI). Serum cystatin C (sCysC) represents functional damage of kidney, while urinary N-acetyl-ß-D-glucosaminidase (uNAG) is considered as a tubular damage biomarker. So far, there is no nomogram containing this combination to predict AKI in septic cohort. We aimed to compare the performance of AKI prediction models with or without incorporating these two biomarkers and develop an effective nomogram for septic patients in intensive care unit (ICU). METHODS: This was a prospective study conducted in the mixed medical-surgical ICU of a tertiary care hospital. Adults with sepsis were enrolled. The patients were divided into development and validation cohorts in chronological order of ICU admission. A logistic regression model for AKI prediction was first constructed in the development cohort. The contribution of the biomarkers (sCysC, uNAG) to this model for AKI prediction was assessed with the area under the receiver operator characteristic curve (AUC), continuous net reclassification index (cNRI), and incremental discrimination improvement (IDI). Then nomogram was established based on the model with the best performance. This nomogram was validated in the validation cohort in terms of discrimination and calibration. The decision curve analysis (DCA) was performed to evaluate the nomogram's clinical utility. RESULTS: Of 358 enrolled patients, 232 were in the development cohort (69 AKI), while 126 in the validation cohort (52 AKI). The first clinical model included the APACHE II score, serum creatinine, and vasopressor used at ICU admission. Adding sCysC and uNAG to this model improved the AUC to 0.831. Furthermore, incorporating them significantly improved risk reclassification over the predictive model alone, with cNRI (0.575) and IDI (0.085). A nomogram was then established based on the new model including sCysC and uNAG. Application of this nomogram in the validation cohort yielded fair discrimination with an AUC of 0.784 and good calibration. The DCA revealed good clinical utility of this nomogram. CONCLUSIONS: A nomogram that incorporates functional marker (sCysC) and tubular damage marker (uNAG), together with routine clinical factors may be a useful prognostic tool for individualized prediction of AKI in septic patients.

Hypercapnia Exacerbates the Blood-Brain Barrier Disruption Via Promoting HIF-1a Nuclear Translocation in the Astrocytes of the Hippocampus: Implication in Further Cognitive Impairment in Hypoxemic Adult Rats.

Hypercapnia in combination with hypoxemia is usually present in severe respiratory disease in the intensive care unit (ICU) and can lead to more severe cognitive dysfunction. Increasing evidence has indicated that the compromised blood-brain barrier (BBB) in the hippocampus in hypoxemia conditions can result in cognitive dysfunction. However, the role and underlying mechanism of hypercapnia in the BBB disruption remains poorly known. A rat model of hypercapnia was first established in this study by intubation and mechanical ventilation with a small-animal ventilator. After this, the cognitive function of the experimental rats was assessed by the Morris water maze test. The BBB permeability was evaluated by the Evans Blue (EB) test and brain water content (BWC). Western blot analysis was carried out to detect the protein expressions of total and nuclear hypoxia-inducible factor-1α (HIF-1α), matrixmetalloproteinase-9 (MMP-9) and Aquaporins-4 (AQP-4) in the hippocampus tissue. Double immunofluorescence further verified the protein expression of different biomarkers was localized in the astrocytes of the hippocampus. Hypercapnia alone did not disrupt the BBB, but it could further enhance the BBB permeability in hypoxemia. Concomitantly, up-regulation of nuclear HIF-1α, AQP-4, MMP-9 protein expression along with increased degradation of the occludin and claudin-5 proteins was found in the hypercapnia rat model, while the total HIF-1α remained unchanged. Interestingly, these changes were independent of the acidosis induced by hypercapnia. Of note, after premedication of 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α nuclear translocation), the disrupted BBB could be restored resulting in improvement of the cognitive impairment. Meanwhile, accumulation of nuclear HIF-1α, protein expression of AQP-4 and MMP-9 and protein degradation of the occludin and claudin-5 were decreased. Thus, our study demonstrated that hypercapnia can further disrupt the BBB through promoting HIF-1α nuclear translocation and up-regulation of AQP-4 and MMP-9 in hypoxemia. It is therefore suggested that the cascade of hypercapnia-induced nuclear HIF-1α protein translocation in hypoxia-activated astrocytes may be a potential target for ameliorating cognitive impairment.

A nomogramic model based on clinical and laboratory parameters at admission for predicting the survival of COVID-19 patients.

BACKGROUND: COVID-19 has become a major global threat. The present study aimed to develop a nomogram model to predict the survival of COVID-19 patients based on their clinical and laboratory data at admission. METHODS: COVID-19 patients who were admitted at Hankou Hospital and Huoshenshan Hospital in Wuhan, China from January 12, 2020 to March 20, 2020, whose outcome during the hospitalization was known, were retrospectively reviewed. The categorical variables were compared using Pearson's χ2-test or Fisher's exact test, and continuous variables were analyzed using Student's t-test or Mann Whitney U-test, as appropriate. Then, variables with a P-value of ≤0.1 were included in the log-binomial model, and merely these independent risk factors were used to establish the nomogram model. The discrimination of the nomogram was evaluated using the area under the receiver operating characteristic curve (AUC), and internally verified using the Bootstrap method. RESULTS: A total of 262 patients (134 surviving and 128 non-surviving patients) were included in the analysis. Seven variables, which included age (relative risk [RR]: 0.905, 95% confidence interval [CI]: 0.868-0.944; P < 0.001), chronic heart disease (CHD, RR: 0.045, 95% CI: 0.0097-0.205; P < 0.001, the percentage of lymphocytes (Lym%, RR: 1.125, 95% CI: 1.041-1.216; P = 0.0029), platelets (RR: 1.008, 95% CI: 1.003-1.012; P = 0.001), C-reaction protein (RR: 0.982, 95% CI: 0.973-0.991; P < 0.001), lactate dehydrogenase (LDH, RR: 0.993, 95% CI: 0.990-0.997; P < 0.001) and D-dimer (RR: 0.734, 95% CI: 0.617-0.879; P < 0.001), were identified as the independent risk factors. The nomogram model based on these factors exhibited a good discrimination, with an AUC of 0.948 (95% CI: 0.923-0.973). CONCLUSIONS: A nomogram based on age, CHD, Lym%, platelets, C-reaction protein, LDH and D-dimer was established to accurately predict the prognosis of COVID-19 patients. This can be used as an alerting tool for clinicians to take early intervention measures, when necessary.

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.

Evaluation of a combination "lymphocyte apoptosis model" to predict survival of sepsis patients in an intensive care unit.

BACKGROUND: A major challenge in sepsis intervention is unclear risk stratification. We postulated that a panel of biomarkers of lymphocyte apoptosis and immune function, termed the "lymphocyte apoptosis model," would be an effective tool for predicting 28-day survival for sepsis patients. METHODS: A total of 52 consecutive sepsis patients were enrolled. Peripheral blood samples were collected on day 1 of admission for quantification of biomarkers of lymphocyte apoptosis and immune function, including lymphocyte count, lymphocyte apoptotic percentage, expression on monocyte HLA-DR, CD4+/CD8+ T cell ratio, T helper type 1 to type 2 ratio (Th1/Th2), cytochrome c levels, and various proinflammatory cytokine levels. Sepsis severity was classified using Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores. Survival was assessed at 28 days. RESULTS: Compared with survivors, non-survivors had significantly higher lymphocyte apoptotic percentages and plasma cytochrome c levels and significantly lower lymphocyte counts, Th1/Th2 ratios, and HLA-DR expression on day 1 of admission. Multivariate analysis identified cytochrome c levels (odds ratio [OR]1.829, p = 0.025), lymphocyte apoptotic percentage (OR 1.103, p = 0.028), lymphocyte count (OR 0.150, p = 0.047), and HLA-DR expression (OR 0.923, p = 0.021) as independent predictors of 28-day mortality. A logistic regression equation incorporating the independent risk factors predicted 28-day mortality with greater accuracy than did the APACHE II score or single components biomarkers. CONCLUSIONS: The "lymphocyte apoptosis model" may be useful for risk stratification and predicting prognosis of sepsis patients.

Microglia-derived IL-1ß contributes to axon development disorders and synaptic deficit through p38-MAPK signal pathway in septic neonatal rats.

BACKGROUND: Axon development plays a pivotal role in the formation of synapse, nodes of Ranvier, and myelin sheath. Interleukin-1ß (IL-1ß) produced by microglia may cause myelination disturbances through suppression of oligodendrocyte progenitor cell maturation in the septic neonatal rats. Here, we explored if a microglia-derived IL-1ß would disturb axon development in the corpus callosum (CC) following lipopolysaccharide (LPS) administration, and if so, whether it is associated with disorder of synapse formation in the cerebral cortex and node of Ranvier. METHODS: Sprague-Dawley rats (1-day old) in the septic model group were intraperitoneally administrated with lipopolysaccharide (1 mg/kg) and then sacrificed for detection of IL-1ß, interleukin-1 receptor (IL-1R1), neurofilament-68, neurofilament-160, and neurofilament-200, proteolipid, synaptophysin, and postsynaptic density 95 (PSD95) expression by western blotting and immunofluorescence. Electron microscopy was conducted to observe alterations of axonal myelin sheath and synapses in the cortex, and proteolipid expression was assessed using in situ hybridization. The effect of IL-1ß on neurofilament and synaptophysin expression in primary neuron cultures was determined by western blotting and immunofluorescence. P38-MAPK signaling pathway was investigated to determine whether it was involved in the inhibition of IL-1ß on neurofilament and synaptophysin expression. RESULTS: In 1-day old septic rats, IL-1ß expression was increased in microglia coupled with upregulated expression of IL-1R1 on the axons. The expression of neurofilament-68, neurofilament-160, and neurofilament-200 (NFL, NFM, NFH) and proteolipid (PLP) was markedly reduced in the CC at 7, 14, and 28 days after LPS administration. Simultaneously, cortical synapses and mature oligodendrocytes were significantly reduced. By electron microscopy, some axons showed smaller diameter and thinner myelin sheath with damaged ultrastructure of node of Ranvier compared with the control rats. In the cerebral cortex of LPS-injected rats, some axo-dendritic synapses appeared abnormal looking as manifested by the presence of swollen and clumping of synaptic vesicles near the presynaptic membrane. In primary cultured neurons incubated with IL-1ß, expression of NFL, NFM, and synaptophysin was significantly downregulated. Furthermore, p38-MAPK signaling pathway was implicated in disorder of axon development and synaptic deficit caused by IL-1ß treatment. CONCLUSIONS: The present results suggest that microglia-derived IL-1ß might suppress axon development through activation of p38-MAPK signaling pathway that would contribute to formation disorder of cortical synapses and node of Ranvier following LPS exposure.

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.

IL-1ß induces hypomyelination in the periventricular white matter through inhibition of oligodendrocyte progenitor cell maturation via FYN/MEK/ERK signaling pathway in septic neonatal rats.

Neuroinflammation elicited by microglia plays a key role in periventricular white matter (PWM) damage (PWMD) induced by infectious exposure. This study aimed to determine if microglia-derived interleukin-1ß (IL-1ß) would induce hypomyelination through suppression of maturation of oligodendrocyte progenitor cells (OPCs) in the developing PWM. Sprague-Dawley rats (1-day old) were injected with lipopolysaccharide (LPS) (1 mg/kg) intraperitoneally, following which upregulated expression of IL-1ß and IL-1 receptor 1 (IL-1R1 ) was observed. This was coupled with enhanced apoptosis and suppressed proliferation of OPCs in the PWM. The number of PDGFR-α and NG2-positive OPCs was significantly decreased in the PWM at 24 h and 3 days after injection of LPS, whereas it was increased at 14 days and 28 days. The protein expression of Olig1, Olig2, and Nkx2.2 was significantly reduced, and mRNA expression of Tcf4 and Axin2 was upregulated in the developing PWM after LPS injection. The expression of myelin basic protein (MBP) and 2',3'-cyclic-nucleotide 3"-phosphodiesterase (CNPase) was downregulated in the PWM at 14 days and 28 days after LPS injection; this was linked to reduction of the proportion of myelinated axons and thinner myelin sheath as revealed by electron microscopy. Primary cultured OPCs treated with IL-1ß showed the failure of maturation and proliferation. Furthermore, FYN/MEK/ERK signaling pathway was involved in suppression of maturation of primary OPCs induced by IL-1ß administration. Our results suggest that following LPS injection, microglia are activated and produce IL-1ß in the PWM in the neonatal rats. Excess IL-1ß inhibits the maturation of OPCs via suppression of FYN/MEK/ERK phosphorylation thereby leading to axonal hypomyelination.

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.

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.

LncRNA xist regulates sepsis associated neuroinflammation in the periventricular white matter of CLP rats by miR-122-5p/PKCη Axis.

Background: Neuroinflammation is a common feature of many neurological diseases, and remains crucial for disease progression and prognosis. Activation of microglia and astrocytes can lead to neuroinflammation. However, little is known about the role of lncRNA xist and miR-122-5p in the pathogenesis of sepsis-associated neuroinflammation (SAN). This study aims to investigate the role of lncRNA xist and miR-122-5p in the pathogenesis of SAN. Methods: Levels of miR-122-5p and proinflammatory mediators were detected in the cerebrospinal fluid (CSF) of patients with intracranial infection (ICI) by ELISA and qRT-PCR. miRNA expression in the periventricular white matter (PWM) in rats was analyzed by high-throughput sequencing. Levels of lncRNA xist, miR-122-5p and proinflammatory mediators in the PWM were measured using qRT-PCR and western blot. Bioinformatics analysis was used to predict the upstream and downstream of miR-122-5p. The interaction between miR-122-5p and its target protein was validated using luciferase reporter assay. BV2 and astrocytes were used to detect the expression of lncRNA xist, miR-122-5p. Results: The level of miR-122-5p was significantly decreased in the CSF of ICI patients, while the expression of IL-1ß and TNF-α were significantly upregulated. Furthermore, it was found that the expression of IL-1ß and TNF-α were negatively correlated with the level of miR-122-5p. A high-throughput sequencing analysis showed that miR-122-5p expression was downregulated with 1.5-fold changes in the PWM of CLP rats compared with sham group. Bioinformatics analysis found that lncRNA xist and PKCη were the upstream and downstream target genes of miR-122-5p, respectively. The identified lncRNA xist and PKCη were significantly increased in the PWM of CLP rats. Overexpression of miR-122-5p or knockdown of lncRNA xist could significantly downregulate the level of PKCη and proinflammatory mediators from activated microglia and astrocytes. Meanwhile, in vitro investigation showed that silencing lncRNA xist or PKCη or enhancing the expression of miR-122-5p could obviously inhibit the release of proinflammatory mediators in activated BV2 cells and astrocytes. Conclusion: LncRNA xist could regulate microglia and astrocytes activation in the PWM of CLP rats via miR-122-5p/PKCη axis, further mediating sepsis associated neuroinflammation.

Is metoclopramide beneficial for the postpyloric placement of nasoenteric tubes? A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Metoclopramide is frequently prescribed as an adjuvant for the postpyloric placement of nasoenteric tubes (NETs). However, a recent meta-analysis showed that metoclopramide was not beneficial in adults. Thus, this study aimed to reevaluate the effect of metoclopramide on the postpyloric placement of NETs. METHODS: A systematic search of PubMed, Embase, the Cochrane Library, Web of Science, Chinese National Knowledge Infrastructure (CNKI), and Wanfang data was conducted up to August 2020 for randomized controlled trials (RCTs) comparing metoclopramide with placebo or no intervention. Trial sequential analysis (TSA) was used for the primary outcomes (the success rate of the postpyloric placement of NETs). RESULTS: Seven eligible RCTs that included 520 participants were identified. The results of the pooled effect sizes showed that metoclopramide significantly facilitated the postpyloric placement of NETs (relative risk [RR], 1.48; 95% CI, 1.11-1.97; P = .007; I2 = 37%). However, the risk-of-bias assessment and the TSA results indicated that the qualities of the RCTs and the sample sizes were insufficient to confirm the efficacy of metoclopramide. Further subgroup analysis revealed that successful postpyloric placement was more pronounced in studies in which spiral NETs were employed (RR, 1.85; 95% CI, 1.41-2.43; P < .001; I2 = 0%). Additionally, overall adverse events were minimal. CONCLUSIONS: The evidence accumulated so far was not strong enough to demonstrate metoclopramide's beneficial effects on the postpyloric placement of NETs. Further high-quality, large-sample RCTs are required to elucidate the effects of metoclopramide.

Gut Microbiota Mediates the Susceptibility of Mice to Sepsis-Associated Encephalopathy by Butyric Acid.

Purpose: Neuroinflammation plays an important part in the pathophysiology of sepsis-associated encephalopathy (SAE). Gut microbiota and gut brain axis are considered as important mediators in the development of neurological diseases. The aim of this study was to investigate the role of intestinal microbiota in sepsis-related brain injury and to explore the underlying mechanisms. Methods: Mouse model of SAE was established using cecal ligation and puncture (CLP). Based on the mouse mortality and the associated time of death, light SAE (LSAE) and severe SAE (SSAE) were classified. Fecal microbiota transplantation (FMT) was performed to verify the role of intestinal microbiota. Feces of mice in the two groups which collected before operation were sequenced for 16S and targeted short chain fatty acids. Results: Intestinal microbiota from SSAE and LSAE mice displayed diverse functions. Interestingly, LSAE mice produced more butyric acid compared with SSAE mice. In the in vivo experiments, sodium butyrate (NaB) reduced the high oxidative stress levels in mice hippocampus and conferred a marked survival superiority to sepsis mice. In addition, NaB prevented the increase in intracellular reactive oxygen species (ROS) generation and inducible nitric-oxide synthase expression in LPS-stimulated primary microglia. The GPR109A/Nrf2/HO-1 signaling pathway was found to be involved in the activation of antioxidant response of primary microglia induced by sodium butyrate. Conclusion: Our findings indicate a crucial role of gut microbiota in the susceptibility to SAE. Butyrate, a metabolite of intestinal microbiota, may have a neuroprotective effect in the process of sepsis by GPR109A/Nrf2/HO-1 pathway.

Nomogram to predict survival outcome of patients with veno-arterial extracorporeal membrane oxygenation after refractory cardiogenic shock.

OBJECTIVE: : This study aims to develop a nomogram model to predict the survival of refractory cardiogenic shock (RCS) patients that received veno-arterial extracorporeal membrane oxygenation (VA-ECMO). METHODS: A total of 235 and 209 RCS patients were supported with VA-ECMO from January 2018 to December 2019 in Guangdong Provincial People's Hospital, and from January 2020 to December 2020 in four third-grade and class-A hospitals were a development cohort (DC) and validation cohort (VC), respectively. Finally, 137 and 98 patients were included in the DC and VC. Multivariate logistic regression analysis was used to identify variables, and only these independent risk factors were used to establish the nomogram model. The receiver operating characteristic curve (ROC), calibration plot, decision curve, and clinical impact curves were used to evaluate the nomogram's discriminative ability, predictive accuracy, and clinical application value. RESULTS: Pre-ECMO cardiogenic arrest (pre-ECA), lactate (Lac), inotropic score (IS), and modified nutrition risk in the critically ill score (mNUTRIC score) were incorporated into the nomogram. This showed good discrimination in the DC, with an area under ROC (AUROC) and a 95% confidence interval (CI) of 0.959 (0.911-0.986). The AUROC (95% CI) of the VC was 0.928 (0.858-0.971). The calibration plots of the DC and VC presented good calibration results. The decision curve and clinical impact curve of the nomogram provided improved benefits for RCS patients. CONCLUSIONS: This study established a prediction nomogram composed of pre-ECA, Lac, IS, and mNUTRIC scores that could help clinicians to predict the survival probability at hospital discharge precisely and rapidly for RCS patients that received VA-ECMO.

Influence of Melatonin on Behavioral and Neurological Function of Rats with Focal Cerebral Ischemia-Reperfusion Injury via the JNK/FoxO3a/Bim Pathway.

OBJECTIVE: To investigate the influence of melatonin on behavioral and neurological function of rats with focal cerebral ischemia-reperfusion injury via the JNK/FoxO3a/Bim pathway. METHODS: One hundred and twenty healthy male SD rats were randomized into the model group (Model: the middle cerebral artery occlusion (MCAO) model was constructed and received an equal volume of normal saline containing 5% DMSO), sham operation group (Sham: received no treatment except normal feeding), and low, medium, and high dose of melatonin group (L-MT, M-MT, and H-MT intraperitoneally injected 10, 20, and 40 mg/kg melatonin 30 min after IR, respectively), with 24 rats in each group. Following 24 h of reperfusion, the rats in each of the above groups were tested for neurological deficit symptoms and behavioral changes to screen the rats included in the study. HE and TUNEL stainings were performed to observe pathological changes. Levels of oxidative stress-related indexes, inflammatory factor-related indexes, nuclear factor-κB p65 (NF-κB p65), and interferon-γ (IFN-γ) in the rat brain were measured by ELISA. The JNK/FoxO3a/Bim pathway-related proteins as well as Bcl-2, Caspase-3, and Bax were examined using Western blot. RESULTS: Detection of behavioral indicators showed that the MACO model was successfully constructed in rats. L-MT, M-MT, and L-MT groups presented reduced malondialdehyde (MDA), reactive oxygen species (ROS), tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, IL-1ß, IFN-γ, NF-κB p65, and apoptosis compared with the Model group (P < 0.05), and the improvement degree was better in the M-MT group versus the L-HT group. Bcl-2 protein expression in the brain tissue of L-MT, M-MT, and H-MT groups increased significantly, while Bax, Caspase-3, p-JNK, p-FoxO3a, and Bim protein expression declined markedly, versus the Model group (P < 0.05). The changes of indexes were greater in the M-MT group compared with that in the L-MT group. No significant difference was observed in all the above indexes between the M-MT group and the H-MT group (P > 0.05). CONCLUSIONS: In the MACO rat model, melatonin can effectively reduce Bax and Caspase-3 levels by modulating the JNK/FoxO3a/Bim pathway, inhibit neuronal apoptosis, and alleviate neurological deficits by reducing the release of proinflammatory mediators, with anti-inflammatory and antioxidant effects. In addition, 20 mg/kg is the optimal melatonin concentration.

Sepsis-Induced Gut Dysbiosis Mediates the Susceptibility to Sepsis-Associated Encephalopathy in Mice.

Sepsis-associated encephalopathy (SAE) is common in septic patients and is associated with adverse outcomes. The gut microbiota has been recognized as a key mediator of neurological disease development. However, the exact role of the gut microbiota in regulating SAE remains elusive. Here, we investigated the role of the gut microbiota in SAE and its underlying mechanisms. Cecal ligation and puncture (CLP) was conducted to induce sepsis in mice. Neurological scores were recorded to distinguish SAE-resistant (SER) (score of >6 at 36 h postoperatively) from SAE-susceptible (SES) (score of ≤6 at 36 h postoperatively) mice. 16S rRNA gene sequencing and metabolomics analyses were used to characterize the gut microbiota in the two groups. Fecal microbiota transplantation was performed to validate the role of the gut microbiota in SAE progression. The gut microbiota was more severely disrupted in SES mice than in SER mice after sepsis modeling. Interestingly, mice receiving postoperative feces from SES mice exhibited more severe cortical inflammation than mice receiving feces from SER mice. Indole-3-propionic acid (IPA), a neuroprotective molecule, was more enriched in feces from SER mice than in feces from SES mice. IPA alleviated CLP-induced anxiety and spatial memory impairment in septic mice. Moreover, IPA markedly inhibited NLRP3 inflammasome activation and interleukin-1ß (IL-1ß) secretion in lipopolysaccharide-stimulated microglia. These responses were attenuated after antagonizing the aryl hydrocarbon receptor. Our study indicates that the variability in sepsis-induced gut dysbiosis mediates the differential susceptibility to SAE in CLP-induced experimental sepsis mice, and microbially derived IPA is possibly involved in SAE development as a neuroprotective compound. IMPORTANCE The bidirectional interactions between the gut microbiota and sepsis-associated encephalopathy (SAE) are not well characterized. We found that the gut microbiota was more severely disturbed in SAE-susceptible (SES) mice than in SAE-resistant (SER) mice after sepsis modeling. Mice gavaged with postoperative feces from SES mice exhibited more severe neuroinflammation than mice gavaged with feces from SER mice. The gut microbiota from SER mice enriched a neuroprotective metabolite, IPA, which appeared to protect mice from SAE. The potential underlying mechanism of the protective effect of IPA may be mediated via the inhibition of NLRP3 inflammasome activation and IL-1ß secretion in microglia. These anti-inflammatory effects of IPA may be regulated by aryl hydrocarbon receptors. These results enhance our understanding of the role of the intestinal microbiota in sepsis. In particular, gut microbiota-derived IPA may serve as a potential therapeutic agent to prevent neuroinflammation in SAE.

Indole-3-Propionic Acid as a Potential Therapeutic Agent for Sepsis-Induced Gut Microbiota Disturbance.

The effects of using gut microbiota metabolites instead of live microorganisms to modulate sepsis-induced gut dysbiosis remain largely unknown. We assessed the effects of microbiota metabolite indole-3-propionic acid (IPA) on gut microbiota in mice during sepsis. Sepsis models were constructed by cecal ligation and puncture (CLP) methods. Fecal microbiota composition analysis was performed to characterize the gut microbiota composition. Fecal microbiota transplantation was performed to validate the roles of gut microbiota on sepsis progression. IPA-treated mice exhibited lower serum inflammatory mediator levels and a higher survival rate than those of saline-treated mice after modeling of sepsis, which were negated in the presence of antibiotics. Compared with saline-treated mice after modeling, IPA-treated mice showed a markedly different intestinal microbiota composition, with an enrichment of Bifidobacteriaceae family and a depletion of Enterobacteriaceae family. Mice gavaged with postoperative feces from IPA-treated animals displayed better survival than mice gavaged with feces from saline-treated animals. Overall, these data suggest that IPA offers a microbe-modulated survival advantage in septic mice, indicating that some microbiota metabolites could replace live microorganisms as potential options for regulation of sepsis-induced gut dysbiosis. IMPORTANCE The role of gut microbiota in the pathophysiology of sepsis is gaining increasing attention and developing effective and safe sepsis therapies targeting intestinal microorganisms is promising. Given the safety of probiotic supplementation or fecal microbiota transplantation in critically ill patients, identifying an abiotic agent to regulate the intestinal microbiota of septic patients is of clinical significance. This study revealed that IPA, a microbiota-generated tryptophan metabolite, ameliorated sepsis-induced mortality and decreased the serum levels of proinflammatory cytokines by modulating intestinal microbiota. Although IPA did not increase the abundance and diversity of the microbiota of septic mice, it significantly decreased the number of Enterobacteriaceae family. These findings indicate that a specific microbiota metabolite (e.g., IPA) can mediate the intestinal microbiota apart from FMT or probiotics.