[Effect of proprotein convertase subtilisin/kexin type 9 on platelet activation associated with sepsis].

OBJECTIVE: To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis. METHODS: (1) Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. (2) Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting. RESULTS: (1) Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (µg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×109/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients (n = 20) was significantly higher than that in the non-thrombocytopenia patients (n = 37; µg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients (r = -0.340, P = 0.010). (2) Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×109/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/ß-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. CONCLUSIONS: PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.

[Effect of continuous blood purification on immunity and endothelial cell function in patients with sepsis].

OBJECTIVE: To explore the effect of continuous blood purification (CBP) on the immunity and endothelial cell function of patients with sepsis. METHODS: A prospective study was conducted. The patients aged ≥ 18 years old and meeting the diagnostic criteria of sepsis admitted to the department of critical care medicine of Binzhou Medical University Hospital from March 2019 to October 2020 were selected as the research subjects, and the patients were divided into standard treatment group and CBP treatment group according to random number table method. Both groups were given standard treatment including initial fluid resuscitation, infection source control and antibiotics according to the 2016 international guidelines for the management of sepsis and septic shock. CBP treatment group was additionally given continuous veno-venous hemofiltration (CVVH) at a dose of 25-30 mL×kg-1×h-1 and blood flow rate of 150-200 mL/min for more than 20 hours a day for 3 days. The clinical data of patients including blood lactic acid (Lac), procalcitonin (PCT), lymphocyte count (LYM), acute physiology and chronic health evaluation II (APACHE II) score, sequential organ failure assessment (SOFA) score were recorded before treatment and 1 day and 3 days after treatment. At the same time, the venous blood was collected, and the immune function related indexes [interleukins (IL-4, IL-7), programmed death receptor-1 (PD-1), programmed death ligand-1 (PD-L1), interferon-γ (IFN-γ)] and endothelial cell injury related markers [soluble thrombomodulin (sTM), angiopoietin-2 (Ang-2), von Willebrand factor (vWF), heparan sulfate (HS), syndecan-1 (SDC-1)] levels in serum were determined by enzyme-linked immunosorbent assay (ELISA). The length of intensive care unit (ICU) stay of patients in the two groups was recorded, and the outcomes of patients in the two groups were followed up for 28 days. RESULTS: Finally, 20 patients were enrolled in the standard treatment group, and 19 patients were enrolled in the CBP treatment group. There were no significant differences in gender, age and infection site between the two groups. The length of ICU stay in the standard treatment group was (10±5) days, and 5 patients died and 15 patients survived after 28 days. The length of ICU stay in the CBP treatment group was (9±4) days, and 8 patients died and 11 patients survived after 28 days. There were no significant differences in the length of ICU stay and number of patients who died within 28 days between the two groups (both P > 0.05). There were no significant differences in the Lac, PCT, LYM, APACHE II score, SOFA score and immune function and endothelial cell injury related indexes before treatment and 1 day after treatment between the two groups. After 3 days of treatment, the Lac, PCT, APACHE II score and SOFA score of the CBP treatment group were significantly lower than those before treatment, and pro-inflammatory and anti-inflammatory cytokines such as IFN-γ and IL-4, apoptosis-related indicators such as PD-1 and IL-7, and endothelial injury related factors such as sTM, SDC-1 and HS were significantly improved compared with the pre-treatment, the improvement degree of the above indicators was more obvious than that of the standard treatment group, and LYM was significantly higher than that of the standard treatment group (×109/L: 1.3±0.3 vs. 0.9±0.4, P < 0.05), IL-4, IFN-γ, IFN-γ/IL-4 ratio, IL-7, PD-1, sTM, SDC-1, HS, and Ang-2 were significantly lower than those of the standard treatment group [IL-4 (ng/L): 2.8 (1.5, 3.2) vs. 3.3 (2.7, 5.2), IFN-γ (ng/L): 6.3 (5.4, 106.5) vs. 217.9 (71.4, 517.1), IFN-γ/IL-4 ratio: 3.7 (1.8, 70.3) vs. 59.1 (18.3, 124.9), IL-7 (ng/L): 4.6 (3.2, 5.1) vs. 6.3 (5.2, 8.0), PD-1 (µg/L): 0.04 (0.03, 0.06) vs. 0.08 (0.05, 0.12), sTM (µg/L): 4.9 (4.3, 7.4) vs. 8.7 (6.0, 10.8), SDC-1 (µg/L): 0.6 (0.3, 1.1) vs. 0.9 (0.8, 2.5), HS (ng/L): 434.8 (256.2, 805.0) vs. 887.9 (620.1, 957.3), Ang-2 (ng/L): 934.0 (673.3, 1 502.1) vs. 2 233.9 (1 472.5, 3 808.4)], the differences were statistically significant (all P < 0.05). CONCLUSIONS: CBP treatment can eliminate the patient's immunosuppressive state, reduce a variety of endothelial injury markers and the degradation of glycocalyx, but cannot decrease the 28-day death risk or shorten the length of ICU stay.

Association between plasma glycocalyx component levels and poor prognosis in severe influenza type A (H1N1).

Influenza A virus infection causes a series of diseases, but the factors associated with disease severity are not fully understood. Disruption of the endothelial glycocalyx contributes to acute lung injury in sepsis, but has not been well studied in H1N1 influenza. We aim to determine whether the plasma glycocalyx components levels are predictive of disease severity in H1N1 influenza. This prospective observational study included 53 patients with influenza A (H1N1) during the influenza season, and 30 healthy controls in our hospital. Patients were grouped by severity and survival. We collected clinical data and blood samples at admission. Inflammatory factors (tumor necrosis factor-α, interleukin-6, interleukin-10) and endothelial glycocalyx components (syndecan-1, hyaluronan, heparan sulfate) were measured. The plasma levels of syndecan-1, hyaluronan, and heparan sulfate were significantly higher in patients with severe influenza A (H1N1) than in mild cases. Syndecan-1 and hyaluronan were positively correlated with disease severity, which was indicated by the APACHE II and SOFA scores and lactate levels, and negatively correlated with albumin levels. At a cutoff point ≥ 173.9 ng/mL, syndecan-1 had a 81.3% sensitivity and 70.3% specificity for predicting of 28-day mortality. Kaplan-Meier analysis demonstrated a strong association between syndecan-1 levels and 28-day mortality (log-rank 11.04, P = 0.001). Elevated plasma levels of syndecan-1 has a potential role in systemic organ dysfunction and may be indicative of disease severity in patients with influenza A (H1N1).

[Significance of high mobility group box 1, von Willebrand factor and other cytokines in the evaluation of severity and prognosis of sepsis patients].

OBJECTIVE: To explore the value of high mobility group box 1 (HMGB1), von Willebrand factor (vWF) and other cytokines in predicting the severity and prognosis of sepsis patients. METHODS: Patients with sepsis and septic shock who ≥ 18 years old and met the Sepsis-3 diagnostic criteria admitted to the department of critical care medicine of Binzhou Medical University Hospital from January to June 2019 were taken as the research objects. The healthy individuals for regular health examination in the same period were taken as the control. The basic information, acute physiology and chronic health evaluation II (APACHE II) and sequential organ failure assessment (SOFA) scores were recorded. The venous blood was taken within 24 hours after the patients were diagnosed. The levels of HMGB1, vWF, tumour necrosis factor-α (TNF-α), interleukin-10 (IL-10), soluble thrombomodulin (sTM), vascular endothelial growth factor receptor 2 (VEGFR-2), angiopoetin-2 (Ang-2) and other cytokines in serum were determined by enzyme linked immunosorbent assay (ELISA). Differences among patients with sepsis, septic shock, healthy physical examinees, and patients who died in 28-day and those who survived, were compared. Spearman rank correlation method was used to analyze the correlation among each cytokine and APACHE II, SOFA scores. The receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of cytokines on the prognosis of patients with sepsis/septic shock. Logistic regression was used to analyze the risk factors of 28-day death. RESULTS: Eleven patients with sepsis, 25 patients with septic shock and 30 healthy individuals were enrolled. Among the patients with sepsis/septic shock, 15 died in 28-day and 21 survived. The serum levels of TNF-α, IL-10, HMGB1, vWF, sTM and VEGFR-2 in patients with sepsis were significantly higher than those in the healthy control group. The levels of TNF-α, IL-10, HMGB1, vWF, sTM in septic shock group were higher than those in the sepsis group, while the Ang-2 level decreased significantly. The serum levels of TNF-α, IL-10, HMGB1, vWF and sTM in the death group were higher than those in the survival group, while Ang-2 was lower than the survival group. Spearman correlation analysis showed that HMGB1, TNF-α, sTM, IL-10, vWF were positively correlated with APACHE II score when patients with sepsis/septic shock were enrolled (r values were 0.652, 0.666, 0.445, 0.430 and 0.355, respectively, all P < 0.05), and HMGB1, TNF-α also positively correlated with SOFA score (r values were 0.433, 0.479, both P < 0.05). Ang-2 was negatively correlated with APACHE II and SOFA scores (r values were -0.519, -0.440, both P < 0.05). ROC curve analysis showed that the predictive value of HMGB1, vWF, IL-10, sTM for 28-day death in patients with sepsis/septic shock were higher than the APACHE II score [the area under ROC curve (AUC) and 95% confidence interval (95%CI): 0.946 (0.870-1.000), 0.902 (0.790-1.000), 0.877 (0.745-1.000), 0.868 (0.734-1.000) vs. 0.846 (0.700-0.991)]. Logistic regression analysis showed that APACHE II score, vWF, sTM, and IL-10 were independent risk factors for 28-day death in patients with sepsis/septic shock (ß values were 4.731, 0.407, -7.058, -0.887, all P < 0.05). CONCLUSIONS: HMGB1, vWF, IL-10, sTM and other cytokines all can be used to evaluate the severity and prognosis of sepsis patients.

Low molecular weight heparin may prevent acute lung injury induced by sepsis in rats.

The purpose of this study was to assess the protective effect of low molecular weight heparin (LMWH) on acute lung injury (ALI) in rats induced by sepsis. Rat ALI model was reproduced by cecal ligation and puncture (CLP). All rats were randomly divided into three groups (n=50): control group (A), ALI group (B), and LMWH-treated group (C). Group A received a sham operation and the other groups underwent CLP operation. Groups A and B accepted intraperitoneal injection (i.p.) of normal saline (NS) at a dose of 2.0 ml kg(-1) and ceftriaxone (30 mg kg(-1)), group C was intraperitoneally injected with additional LMWH (150 U kg(-1)) except saline and ceftriaxone. Blood was collected and lung tissue was harvested at the designated time points for analysis. The lung specimens were harvested for morphological studies, immunohistochemistry examination. Lung tissue edema was evaluated by tissue water content. The levels of lung tissue myeloperoxidase (MPO) were determined. Meanwhile, the nuclear factor-kappa B (NF-κB) activation, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) levels, high mobility group box 1 (HMGB1) and intercellular adhesion molecule-1 (ICAM-1) protein levels in the lung were studied. There was a significant difference in each index between groups A and B (P<0.05). Treatment with LMWH significantly decreased the expression of TNF-α, IL-1ß, HMGB1 and ICAM-1 in the lungs of ALI rats. Similarly, treatment with LMWH dramatically diminished sepsis-induced neutrophil sequestration and markedly reduced the enhanced lung permeability. In the present study, LMWH administration inhibited the nuclear translocation of NF-κB in the lungs. These data suggest that LMWH attenuates inflammation and ameliorates lung pathology in CLP-induced sepsis in a rat model.