PD-1 mediates microglia polarization via the MAPK signaling pathway to protect blood-brain barrier function during cerebral ischemia/reperfusion.

BACKGROUND: Cerebral ischemia is characterized by its rapid onset and high rates of recurrence, morbidity, and mortality, with blood-brain barrier (BBB) permeability playing a vital role in brain injury. Therefore, it is important to understand the molecular mechanism which regulates the BBB during cerebral ischemia. MATERIALS AND METHODS: An in vitro model of oxygen-glucose deprivation (OGD) and an in vivo model of cerebral ischemia/reperfusion (I/R) were constructed. PD-1 overexpression vectors and vectors containing si-RNA were transfected and injected into in vitro and in vivo models. Western blotting, real-time quantitative PCR (qPCR), immunofluorescence (IF) analysis, and immunohistochemical staining were employed to evaluate the expression levels of programmed cell death-1 (PD-1), microglia M1 and M2 biomarkers, and tight junction proteins. Flow cytometry and ELISA were used to measure the levels of pro-inflammatory cytokines. The BBB permeability of brain tissues was evaluated by Evans blue dye (EBD) extravasation and transendothelial electrical resistance (TEER). Brain water content was measured to assess the extent of inflammatory exudation. The infarct volume and neurological severity score (NSS) were used to assess the severity of brain injury. Brain cell apoptosis was assessed by the TUNEL assay and hematoxylin-eosin (H&E) staining. RESULTS: PD-1 helped to convert the microglia M1 phenotype to the M2 phenotype and to reduce BBB permeability both in vitro and in vivo. Overexpression of PD-1 promoted a shift of the M1 phenotype to the M2 phenotype and reduced BBB permeability via the ERK and p38 MAPK signaling pathways. PD-1 reduced inflammatory exudation, BBB permeability, cell apoptosis, and brain injury in vivo. CONCLUSION: Our present study verified that PD-1 exerts an anti-inflammatory effect by converting the microglia M1 phenotype to the M2 phenotype, reducing BBB permeability, and thereby relieves brain injury caused by cerebral ischemia. PD-1 is potential therapeutic target for brain injury caused by cerebral ischemia.

[Predictive value of sepsis-induced coagulopathy score on 30-day mortality in septic patients].

OBJECTIVE: To evaluate the predictive effect of sepsis-induced coagulopathy (SIC) score level on the prognosis of septic patients under sepsis 3.0 criteria. METHODS: A retrospective single-center observational study was conducted on the septic patients admitted to the department of critical care medicine and the department of emergency in Guangdong Provincial People's Hospital from August 2016 to July 2021. The baseline data, laboratory indexes and SIC scores of the patients were collected on the first and fourth (4th) day after hospitalization. Whether the patients were survival within 30 days after enrollment was recorded. Univariate and multivariate Logistic regression were used to analyze the independent risk factors for 30-day mortality in septic patients. The receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of SIC score on the 30-day prognosis of septic patients. RESULTS: A total of 173 patients met the inclusion criteria including 111 (64%) survivors and 62 (36%) non-survivors. There were significant differences in lymphocyte count (LYM), sequential organ failure assessment (SOFA), oxygenation index (PaO2/FiO2) and cardiovascular SOFA score between the survival group and the non-survival group. And there were no significant differences in other indexes. On the first day of admission, there were statistically significant differences in PaO2/FiO2, cardiovascular SOFA score, LYM, SIC score between the non-survival group and the survival group. There were significant differences in international normalized ratio (INR), prothrombin activity (PTA), prothrombin time (PT), PaO2/FiO2, cardiovascular SOFA score, LYM, C-reactive protein (CRP) and procalcitonin (PCT) between the two groups on the 4th day after admission. The mortality of septic patients increased with the increase of SIC score. Binary Logistic regression analysis showed that SIC score and LYM on the 4th day after admission were independent risk factors for 30-day mortality in septic patients (both P < 0.05). The ROC curve showed that SIC score had a certain predictive value for the 30-day prognosis of septic patients [area under the ROC curve (AUC) = 0.712, 95% confidence interval (95%CI) was 0.629-0.794, P < 0.001]. The predictive value of SIC score combined with LYM was better than that of the two alone (AUC = 0.748, 95%CI was 0.688-0.828, P < 0.001). CONCLUSIONS: The SIC score has a certain predictive value for the 30-day prognosis of septic patients. The predictive value of SIC score combined with LYM is better than that of the two alone, which is expected to be a potential indicator for early assessment of the condition and prognosis of septic patients.

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.

[ß1 receptor blocker decreases the myocardial inflammation in the sepsis adult rats through inhibition of TLR4/NF-ΚB signaling pathway].

OBJECTIVE: To explore whether ß1 receptor blocker could decrease the myocardial inflammation through the Toll-like receptor 4/nuclear factor-ΚB (TLR4/NF-ΚB) signaling pathway in the sepsis adult rats. METHODS: Sixty male Wistar rats (250-300 g) aged 3 months old were allocated to four groups by random number table (n = 15): sham operation group (S group), sepsis model group (CLP group), ß1 receptor blocker esmolol intervention group (ES group), and inhibitor of the TLR4 E5564 intervention group (E5564 group). The rat sepsis model was established by cecal ligation and puncture (CLP); S group of rats underwent only an incision. Rats in S group, CLP group and E5564 group were subcutaneous injected with 0.9% sodium chloride (NaCl) 2.0 mL/kg. Besides, the rats in ES group were injected with esmolol (15 mg×kg-1×h-1) by micro pump through the caudal vein. The rats in E5564 group were injected with E5564 (0.3 mg×kg-1×h-1) by micro pump through the caudal vein 1 hour before the CLP surgery. Samples were collected 6 hours after the modelling in each group. The average arterial pressure (MAP) and cardiac output index (CI) were monitored by PU electrical conduction ECG monitor. The levels of serum cardiac troponin I (cTnI), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). The expressions of TLR4, NF-ΚB p65, IL-1ß, TNF-α in myocardial tissue was detected by Western Blot. RESULTS: There was no significant difference in MAP in each group. Compared with the S group, the CI in the CLP group was significantly decreased, the levels of serum cTnI, IL-1ß, TNF-α were significantly increased, the protein expressions of myocardial tissue TLR4, NF-ΚB p65, IL-1ß and TNF-α were significantly increased. Compared with the CLP group, the CI in the ES group and E5564 group were significantly increased (mL×s-1×m-2: 58.6±4.3, 58.9±4.4 vs. 41.2±3.9, both P < 0.01), the levels of serum cTnI, IL-1ß and TNF-α were significantly decreased [cTnI (µg/L): 1 113.81±26.64, 1 115.74±25.90 vs. 1 975.96±42.74; IL-1ß (ng/L): 39.6±4.3, 38.9±4.4 vs. 61.2±3.9; TNF-α (ng/L): 43.1±2.8, 48.7±2.6 vs. 81.3±4.4, all P < 0.01], the protein expressions of myocardial tissue NF-ΚB p65, IL-1ß, TNF-α were significantly decreased (NF-ΚB p65/ß-actin: 0.31±0.03, 0.43±0.04 vs. 0.85±0.08; IL-1ß/ß-actin: 0.28±0.05, 0.32±0.03 vs. 0.71±0.06; TNF-α/ß-actin: 0.18±0.04, 0.28±0.03 vs. 0.78±0.07, all P < 0.01), but there was no significant difference in protein expression of TLR4 (TLR4/ß-actin: 0.89±0.07, 0.87±0.09 vs. 0.95±0.09, both P > 0.05). There was no significant difference in CI, the levels of serum cTnI, IL-1ß, TNF-α, and the protein expressions of myocardial tissue TLR4, NF-ΚB p65, IL-1ß, TNF-α between ES group and E5564 group (all P > 0.05). CONCLUSIONS: ß1 receptor blocker esmolol may inhibit myocardial inflammatory response in sepsis adult rats through TLR4/NF-ΚB signaling pathway, thereby alleviating sepsis-induced myocardial injury.