Objective:To investigate the influence of trimethylamine N-oxide(TMAO)on the development of early neurological deterioration(END)in diabetic patients with acute ischemic stroke.Methods:In this cross-sectional study, 108 type 2 diabetes patients with acute ischemic stroke treated at the Department of Neurology in the Affiliated Wuxi People’s Hospital of Nanjing Medical University between October 2019 and November 2020 were consecutively recruited.END was defined as an increase in the National Institutes of Health Stroke Scale(NIHSS)≥ 2 points and exclusion of intracranial hemorrhage or bleeding transformation in cranial imaging evaluation within 5 days of initial deterioration of neurological dysfunction.The patients were divided into 2 groups, an END(n=36)group and a non-END group(n=72). Fasting plasma TMAO was measured using isotope dilution liquid chromatography coupled to tandem mass spectrometry.Results:Of the 108 patients, 36(33.3%)were diagnosed with END, and their plasma TMAO levels were significantly higher compared with patients without END( Z=-3.500, P<0.001). For prediction of END, the area under the ROC curve for plasma TMAO levels was 0.707(95% CI: 0.603-0.811, P<0.001). The frequencies of END in subjects grouped via tertiles of TMAO were 22.2%, 19.4% and 58.3%, respectively, with significant differences between the 3 groups( χ2=14.979, P=0.001). Univariate analysis showed that elevated TMAO( OR=1.160, 95% CI: 1.050-1.282, P=0.004)was associated with END.A multivariate logistic regression model further confirmed the association between TMAO and END( OR=1.145, 95% CI: 1.033-1.269, P=0.010). Conclusions:Increased plasma TMAO levels are associated with END in diabetic patients with acute ischemic stroke.

Objective:To explore the clinical value of peripheral blood neutrophil-to-lymphocyte ratio (NLR) combined with neutrophil extracellular traps (NETs) in predicting liver injury in patients with sepsis.Methods:A prospective observational study was conducted. The patients who met the diagnostic criteria for sepsis 3.0 admitted to the Department of Intensive Care Unit in Wuxi People’s Hospital Affiliated to Nanjing Medical University from March 2019 to June 2020 were selected as the research objects. The basic informations of the patients were recorded. NLR based on blood routine at admission was calculated. PicoGreen fluorescence quantitative detection kit was used to detect the quantitative level of free DNA (cf-DNA/NETs) in the peripheral plasma of patients at admission. According to whether liver injury occurred, the patients were divided into the sepsis without liver injury group and sepsis with liver injury group. Binary Logistics regression analysis was used to predict the risk factors of sepsis with liver injury. The receiver operating characteristic (ROC) curve was drawn. The value of NLR, NETs, NLR combined with NETs in predicting liver injury in patients with sepsis was analyzed.Results:A total of 122 patients with sepsis were enrolled, of which 45 patients suffered from septic liver injury, with an incidence rate of 36.89%. The NLR of the sepsis wth liver injury group was (21.63 ± 4.71), the NLR of the sepsis without injury injury group was (15.03 ± 4.71), and the NETs level of the sepsis with liver injury group was (505.86 ± 250.05) μg/L, the level of NETs in the sepsis without liver injury group was (179.27 ± 67.20) μg/L, and the differences between the two groups were statistically significant (all P<0.05). Binary logistic regression analysis found that NLR ( OR=1.470, 95% CI: 1.121-1.926, P<0.05) and NETs ( OR=1.018, 95% CI: 1.005-1.030, P<0.05) at admission were the independent risk factors for liver injury in patients with sepsis. The best cut-off value of NLR was 16.68, and the best cut-off value of NETs was 317 μg/L. The sensitivity of combined application of NLR and NETs to predict liver injury in patients with sepsis was 77.78%, specificity was 98.70%, the area under the curve was 0.930, and the Youden Index was 0.765. Conclusions:The peripheral blood NLR and NETs levels are independent risk factors for liver injury in patients with sepsis. The combined application of NLR and NETs has a certain predictive value for liver injury in patients with sepsis. Taking NLR of 16.68 and NETs of 317 μg/L as the cut-off values, they can be used as early warning indicators to predict liver injury in patients with sepsis.

In recent years, more and more attention has been paid to the evaluation and management of right heart function, for which point-of-care unltrasound provides more opportunities. A patient with acute right heart failure after tricuspid valve replacement was successfully treated in department of critical care medicine of Wuxi People's Hospital Affiliated to Nanjing Medical University. This patient showed typical manifestations of acute right heart failure by point-of-care ultrasound. The overall right ventricular systolic function was weakened, and the right ventricle was enlarged. Ratio of the diameter for right ventricle to left ventricle was greater than 1. In the parasternal short-axis view, the right ventricle was oval, and ventricular septum was convex to the left ventricle. The preload of left ventricular was low and the left ventricular diastolic function was limited. Under the guidance of point-of-care ultrasound, the patient's condition tended to improve after treatments such as strengthening the heart, adjusting the preload and afterload of the left and right ventricles, improving renal blood perfusion, and respiratory support. The right ventricle was smaller than before, the systolic function of right ventricle and diastolic function of left ventricle were improved. The successful treatment experience of this case is summarized for reference.

Objective:To investigate the value of plasma syndecan-1 (SDC-1) combined with lung ultrasonography in evaluating the degree of extravascular lung water in patients with acute respiratory distress syndrome (ARDS).Methods:From July 2018 to July 2019, 50 patients with ARDS admitted to the department of intensive care unit of Wuxi People's Hospital Affiliated to Nanjing Medical University were enrolled. After admission, pulse indicator continuous cardiac output (PiCCO) catheter was established for all patients. PiCCO indexes, including extravascular lung water index (EVLWI) and pulmonary vascular permeability index (PVPI) were monitored by one doctor. Another doctor performed lung ultrasound examination, and calculated the sum of the number of B-lines under 10 ultrasound sections of upper blue point, lower blue point, diaphragm point, Plaps point and rear blue point of both lungs. Then the level of plasma SDC-1 was detected by enzyme linked immunosorbent assay (ELISA). Pearson correlation method was used to analyze the correlation between the number of ultrasonic B-lines, plasma SDC-1 level and EVLWI and PVPI. Taking 10 mL/kg EVLWI as the boundary value, the degree of pulmonary edema in patients with ARDS was divided into mild pulmonary edema and severe pulmonary edema. The receiver operator characteristic curve (ROC curve) was drawn, and the number of B-lines, SDC-1 and the predictive value of the combination of the above two indicators on the severity of pulmonary edema in patients with ARDS were analyzed.Results:The cardiac index (CI) and central venous pressure (CVP) of 50 patients with ARDS were (46.84±6.00) mL·s -1·m -2 and (8.12±1.80) mmHg (1 mmHg = 0.133 kPa), cardiogenic pulmonary edema was excluded. In 50 patients with ARDS, EVLWI was (10.82±2.92) mL/kg, PVPI was 3.02±0.69, the number of ultrasound B-lines was 40.90±13.05, and plasma SDC-1 was (568.25±118.14) μg/L. Pearson correlation analysis showed that the number of ultrasound B-lines in patients with ARDS was significantly positively correlated with EVLWI and PVPI ( r1 = 0.802, r2 = 0.799, both P < 0.01). Plasma SDC-1 was also positively correlated with EVLWI and PVPI ( r1 = 0.732, r2 = 0.576, both P < 0.01). ROC curve analysis showed that the number of B-lines and SDC-1 had good predictive value for the severity of pulmonary edema in patients with ARDS. The area under ROC curve (AUC) and 95% confidence interval (95% CI) were 0.891 (0.803-0.979) and 0.875 (0.772-0.978), respectively. When the cut-off of B-lines was 40.50, the sensitivity and specificity were 82.1% and 86.4%, respectively. When the cut-off of SDC-1 was 559.37 μg/L, the sensitivity and specificity were 85.7% and 81.8%, respectively. Combining the number of B-lines with SDC-1 could further improve the predictive value of pulmonary water in patients with ARDS. The AUC (95% CI) was 0.958 (0.890-1.000), and the sensitivity and specificity were 92.9% and 91.8%, respectively. Conclusions:The level of plasma SDC-1 and the number of pulmonary ultrasonic B-lines have a good correlation with the degree of extravascular lung water in patients with ARDS. The combined application of the two noninvasive indexes can be used to evaluate the degree of extravascular lung water in patients with ARDS.

Objective: To explore the changes in polymorphonuclear neutrophils (PMN) function in peripheral blood of patients with sepsis and liver injury and its prognostic value. Methods: A prospective observational study was conducted. The patients who met the criteria of Sepsis-3 admitted to intensive care unit (ICU) of Wuxi People's Hospital Affiliated to Nanjing Medical University from March to August in 2019 were enrolled as the research objects, and the patients were divided into sepsis without liver injury group and sepsis with liver injury group; non-sepsis patients who were hospitalized at the same time were enrolled as non-sepsis group; and the healthy people in the physical examination center were enrolled as healthy control group. The gender, age, white blood cell (WBC), PMN and procalcitonin (PCT) were recorded when the patients were admitted to ICU as well as the people on the day of physical examination. The acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) and sequential organ failure assessment (SOFA) scores were calculated. The 28-day mortality was recorded. The quantitative level of neutrophil extracellular traps (NETs) which reflected by circulating free DNA (cf-DNA/NETs) in peripheral plasma was determined by PicoGreen fluorescence quantitative detection; the qualitative level of NETs was detected by immunofluorescence staining. PMN was extracted from the healthy control group, sepsis without liver injury group and sepsis with liver injury group and cultured in vitro, the quantitative level of cf-DNA/NETs in cell supernatant was determined by PicoGreen fluorescence quantitative detection. The patients were divided into two groups according to 28-day outcome of sepsis patients with liver injury. Receiver operating characteristic (ROC) curve was plotted, and the area under ROC curve (AUC) was calculated to analyze the prognostic value of NETs in sepsis patients with liver injury. Results: Finally, 21 sepsis patients without liver injury, 15 sepsis patients with liver injury, 20 with non-sepsis and 20 with healthy examination were enrolled. There was no significant difference in gender or age among the four groups, indicating that the patients in each group were comparable. The levels of cf-DNA/NETs in peripheral blood, WBC and PMN of the sepsis with and without liver injury groups were significantly higher than those of the healthy control group and non-sepsis group, PCT, APACHE Ⅱ score, SOFA score and 28-day mortality were significantly higher than those of the non-sepsis group, and the levels of cf-DNA/NETs in peripheral blood, PCT and 28-day mortality of the sepsis with liver injury group were significantly higher than those of the sepsis without liver injury group [cf-DNA/NETs (μg/L): 481.60±275.86 vs. 169.76±57.05, PCT (μg/L): 11.29 (1.79, 67.10) vs. 1.11 (0.19, 4.09), 28-day mortality: 73.3% (11/15) vs. 38.1% (8/21), all P < 0.05]. The results of PMN in vitro showed that there was no NETs in normal culture of healthy control group, and a small amount of NETs was detected in sepsis with and without liver injury groups. After stimulation of PMN stimulator phorbol-12-myristate-13-acetic acid (PMA), more NETs were produced in neutrophils of three groups compared with normal culture. Quantitative analysis showed that the level of cf-DNA/NETs in cell supernatant of the sepsis with liver injury group was significantly higher than that of the sepsis without liver injury group (μg/L: 1 872.29±258.44 vs. 1 313.55±147.45, P < 0.01). In 15 sepsis patients with liver injury, 4 patients survived for 28 days (26.7%) and 11 died (73.3%). The cf-DNA/NETs level of the dead group on the day of admission was significantly higher than that of the survival group (μg/L: 582.36±160.05 vs. 241.17±96.14, P < 0.05). ROC curve analysis showed that the AUC of NETs level in peripheral blood for predicting 28-day death of sepsis patients with liver injury was 0.932 [95% confidence interval (95%CI) was 0.787-1.000]; when the best cut-off value was 266.81 μg/L, the sensitivity was 90.9%, the specificity was 75.0%, and the approximate index was 0.659. Conclusions The function of NETs in sepsis patients with liver injury has been further changed. The level of peripheral blood NETs has a certain guiding value for the prognosis of sepsis patients with liver injury.

Objectives To determine the effect of Hippo signaling pathway on lung injury repair of bone-marrow derived mesenchymal stem cells (mMSCs) in murine lipopolysaccharide (LPS) induced acute respiratory distress syndrome (ARDS).Methods C57BL/6 mouse bone marrow-derived MSC (mMSCs) cell lines with low expression of large tumor suppressor 1 (LATS 1) were constructed by lentiviral vector transfection.ARDS was modeled by intratracheally injection of 2 mg/mL lipopolysaccharide (LPS)50 μL.C57BL/6 mice were randomly(random number) divided into four groups (n=36):normal control group,ARDS group,ARDS mice transplanted with mMSCs transfected with blank lentivirus vector (MSC-shcontrol group) or sh-LATS 1 lentivirus vector (MSC-shLATS 1 group).Mice were sacrificed at 3,7,and 14 d after modeling,and lung tissue and bronchoalveolar lavage fluid (BALF) were collected.Near-infrared fluorescence imaging,immunofluorescence staining and Western blot were used to evaluate retention and differentiation of mMSCs in lung tissue.Lung tissue wet weight/body weight ratio (LWW/BW) and total protein (TP) and albumin (ALB) in BALF were determined to reflect pulmonary edema.The expression of Occludin protein in lung epithelium was tested by Western blot.The levels of interleukins (IL-1β,IL-6,and IL-10) in BALF were assessed by enzyme-linked immunosorbent assay (ELISA).Lung injury score and pulmonary fibrosis score in lung tissue were assessed.Results The retention ofmMSCs at 3,7 and 14 d in the MSC-shLATS1 group were significantly higher than those in the MSC-shcontrol group [(26.25±4.58) vs (12.13±3.75) cells/HP,(20.49±3.86) vs (9.97±2.76) cells/HP,and (13.77±3.55) vs (6.89±2.10) cells/HP.all P＜0.05],so was the differentiation of mMSCs into type Ⅱ alveolar epithelial cells at 14 d [(64.12±15.29)％ vs (19.64±3.71)％,P＜0.05].LWW/BW and TP and ALB in BALF at 3 and 14 d in the MSC-shLATS1 group [(9.85±1.51),(6.11±0.83) (mg/g) and (5.12±0.87),(3.05±0.87) (mg/mL)and (0.44±0.18),(0.33±0.04) (mg/mL)] were higher than those in the MSC-shcontrol group [(14.88± 1.74),(8.04±l.70)(mg/g) and (8.08±1.72),(5.94±1.20) (mg/mL) and (0.71±0.21),(1.07±0.29) (mg/mL)] (all P＜0.05),so was the relative expression of Occludin protein[(0.79±0.11) vs (0.49±0).19),(P＜0.05)].The levels of IL-1β and IL-6(pg/mL) in BALF in the MSC-shLATS1 group [(60.11±8.58),(101.74±11.55)] was lower than those in the MSC-shcontrol group [(99.26±14.32),(145.54±13.29)] (all P＜0.05),but the levels of IL-10 in BALF in the MSC-shLATS 1 group (316.65±37.88)pg/mL was higher than those in the MSC-shcontrol group (190.83±22.61)pg/mL (P＜0.05).Lung injury scores at 3 and 14 d in the MSC-shLATS1 group [(7.18±1.12),(3.33±0.49)] was lower than those in the MSC-shcontrol group [(9.72±1.45),(5.11±0.86)] (all P＜0.05),so was pulmonary fibrosis score at 14 d [(0.68±0.12) vs (1.47±0.18),P＜0.05].Conclusion Inhibition of Hippo signaling pathway through underexpression of LATS1 could improve the therapeutic effects of mMSCs in murine LPS-induced ARDS.

Objective To explore the effects of Hippo pathway on differentiation, proliferation, and migration of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods BMSCs of C57BL/6 mice were identified using fluorescence-activated cellsorting analysis and the capabilities of osteogenic, chondrogenic and adipogenic differentiation were evaluated. The differentiation of BMSCs to typeⅡalveolar epithelial cells (AECⅡ) was induced by indirect co-culture with mouse lung epithelial cells (MLE-12) and small airway epithelial cell growth medium (SAGM). The Hippo pathway was regulated by 2-deoxy-D-glucose (2-DG) and 9E1, the effects of 2-DG and 9E1 on the expression of BMSCs surface proteins (SPB, SPC and SPD) mRNA and pro-SPC protein were detected by real time quantitative polymerase chain reaction (qRT-PCR) and Western Blot. The effect of Hippo pathway on differentiation of BMSCs to AECⅡ cells was evaluated. The effect of Hippo pathway on the proliferation of BMSCs was evaluated by methyl thiazolyl tetrazolium (MTT) assay (intervention of 0.1, 0.5, 1.0, 5.0 mmol/L 2-DG). The scratch test and Transwell chamber test were used to analyze the effect of Hippo pathway on migration ability of BMSCs to conditioned medium of acute respiratory distress syndrome (ARDS) lung tissue. Results 2-DG could activate Hippo pathway in a dose-dependent manner and promote the differentiation to AECⅡ and proliferation of BMSCs, the maximum effects were observed after 5 mmol/L of 2-DG treatment [SPB mRNA (2-ΔΔCT): 2.42±0.28 vs. 1.89±0.11, SPC mRNA (2-ΔΔCT): 8.06±0.68 vs. 6.59±0.79, SPD mRNA (2-ΔΔCT): 6.45±0.37 vs. 5.27±0.28, pro-SPC/β-actin: 5.80±1.86 vs. 4.93±1.18, proliferation rate:(145.46±18.18)% vs. (98.91±4.36)%, all P < 0.05], but 9E1 could reverse those effects through inhibition of Hippo pathway [SPB mRNA (2-ΔΔCT): 1.32±0.17 vs. 1.89±0.11, SPC mRNA (2-ΔΔCT): 3.91±0.34 vs. 6.59±0.79, SPD mRNA (2-ΔΔCT): 3.38±0.25 vs. 5.27±0.28, pro-SPC/β-actin: 2.48±0.17 vs. 4.93±1.18, proliferation rate: (80.00±7.27)% vs. (98.91±4.36)%, all P < 0.05]. The ability of horizontal migration [wound healing: (27.17±3.53)% vs. (52.45±6.52)%, P < 0.05] and homing BMSCs to conditioned medium of ARDS lung tissue [cell count (fold, relative to control): 2.77±0.21 vs. 1.90±0.19, P < 0.05] were increased after activation of Hippo pathway by 2-DG treatment, but those effects were reversed after inhibition of Hippo pathway by 9E1 treatment [wound healing: (79.89±8.42)% vs. (52.45±6.52)%, cell count (fold, relative to control): 1.69±0.13 vs. 1.90±0.19, both P < 0.05]. Conclusion Activation of Hippo pathway could enhance differentiation of BMSCs to AECⅡ, promote proliferation and ability of horizontal migration and homing BMSCs to conditioned medium of ARDS lung tissue in vitro.

OBJECTIVE: To investigate the effects of Hippo signaling pathway on lung injury repair of mesenchymal stem cells (MSC) in acute respiratory distress syndrome (ARDS) and its mechanism. METHODS: Mouse bone marrow-derived MSC (mMSCs) cell lines with low expression of large tumor suppressor 2 (LATS2) were constructed by lentiviral vector transfection. Male C57BL/6 mice aging 6-8 weeks old were divided into four groups according to random number table (n = 36). The ARDS animal model (ARDS group) was reproduced by intratracheally injection of 2 g/L lipopolysaccharide (LPS) 50 μL, the normal saline (NS) control group was injected with an equal volume of NS. After 4 hours of model reproduction, 5×10⁴ mMSCs transfected with blank lentivirus vector (MSC-shcontrol group) or shLATS2 lentivirus vector (MSC-shLATS2 group) were transplanted intratracheally, while NS control group and ARDS group were injected with equal volume of phosphate buffered saline (PBS). Mice were sacrificed at 3, 7, and 14 days after modeling, and lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Near-infrared fluorescence imaging, immunofluorescence staining and Western Blot were used to track mMSCs in lung tissue. Retension and proportion of mMSC differentiation into type II alveolar epithelial cells (AEC II) were evaluated. Lung tissue wet weight/body weight ratio (LWW/BW) and total protein (TP) and albumin (ALB) in BALF were determined to reflect pulmonary edema. The expression of Occludin protein in lung epithelium was tested by Western Blot to reflect permeability of epithelium. The levels of interleukins (IL-1β, IL-6, IL-10) in BALF were assessed by enzyme-linked immunosorbent assay (ELISA) to reflect lung inflammation. Hematoxylin-eosin (HE) staining and modified Masson staining were carried out, and the scores were assessed to reflect lung injury and evaluate pulmonary fibrosis. RESULTS: The signal intensity of isolated lung fluorescence images at 3 days in the MSC-shLATS2 group was significantly higher than that in the MSC-shcontrol group (fluorescence intensity: 0.039±0.005 vs. 0.017±0.002, P < 0.05), the number of green fluorescent protein (GFP)-positive cells in lung tissue was also significantly higher than that in the MSC-shcontrol group (cells/HP: 29.65±6.98 vs. 17.50±4.58, P < 0.05), but they all decreased with time; and the proportion of mMSCs differentiated into AEC II was significantly increased [(64.12±15.29)% vs. (19.64±3.71)%, P < 0.05]. Compared with the NS control group, the levels of surface active protein C (SPC) and Occludin protein in the ARDS group were significantly decreased, LWW/BW ratio and TP, ALB and inflammatory factors levels in BALF were significantly increased; extensive alveolar and interstitial edema, hemorrhage and diffuse inflammatory cell infiltration were found in lung tissue, and the lung injury score was significantly increased; collagen fibers were deposited in alveolar septum and alveolar cavity, and pulmonary fibrosis score was also increased significantly. Compared with the ARDS group, the expression levels of SPC and Occludin at 14 days in the MSC-shcontrol group and the MSC-shLATS2 group were significantly increased (SPC/β-actin: 0.51±0.12, 0.68±0.10 vs. 0.27±0.08, Occludin/β-actin: 0.49±0.19, 0.79±0.11 vs. 0.25±0.08, all P < 0.05), TP, ALB, IL-1β, IL-6 levels in BALF at 3 days were significantly decreased [TP (g/L): 8.08±1.72, 5.12±0.87 vs. 12.55±2.09; ALB (g/L): 0.71±0.21, 0.44±0.18 vs. 1.18±0.29, IL-1β (ng/L): 99.26±14.32, 60.11±8.58 vs. 161.86±25.17, IL-6 (ng/L): 145.54±13.29, 101.74±11.55 vs. 258.79±27.88, all P < 0.05], and IL-10 was significantly increased (ng/L: 190.83±22.61, 316.65±37.88, both P < 0.05). Furthermore, all the above parameters in the MSC-shLATS2 group were significantly improved as compared with those in the MSC-shcontrol group (all P < 0.05). LWW/BW ratio in the MSC-shLATS2 group was significantly lower than that in the ARDS group and the MSC-shcontrol group (mg/g: 9.85±1.51 vs. 16.78±1.92, 14.88±1.74, both P < 0.05). CONCLUSIONS Inhibiting Hippo signaling pathway by low expression of LATS2 could promote the retention of mMSCs in lung tissue and differentiation into AEC II cells of ARDS mice, improve pulmonary edema and alveolar epithelial permeability, regulate pulmonary inflammatory response, and alleviate pathological damage and fibrosis of lung tissue.
Animals ; Hippo Signaling Pathway ; Lung Injury/prevention & control* ; Male ; Mesenchymal Stem Cells/metabolism* ; Mice ; Mice, Inbred C57BL ; Protein Serine-Threonine Kinases/metabolism* ; Respiratory Distress Syndrome/metabolism* ; Signal Transduction

OBJECTIVE: To explore the changes in polymorphonuclear neutrophils (PMN) function in peripheral blood of patients with sepsis and liver injury and its prognostic value. METHODS: A prospective observational study was conducted. The patients who met the criteria of Sepsis-3 admitted to intensive care unit (ICU) of Wuxi People's Hospital Affiliated to Nanjing Medical University from March to August in 2019 were enrolled as the research objects, and the patients were divided into sepsis without liver injury group and sepsis with liver injury group; non-sepsis patients who were hospitalized at the same time were enrolled as non-sepsis group; and the healthy people in the physical examination center were enrolled as healthy control group. The gender, age, white blood cell (WBC), PMN and procalcitonin (PCT) were recorded when the patients were admitted to ICU as well as the people on the day of physical examination. The acute physiology and chronic health evaluation II (APACHE II) and sequential organ failure assessment (SOFA) scores were calculated. The 28-day mortality was recorded. The quantitative level of neutrophil extracellular traps (NETs) which reflected by circulating free DNA (cf-DNA/NETs) in peripheral plasma was determined by PicoGreen fluorescence quantitative detection; the qualitative level of NETs was detected by immunofluorescence staining. PMN was extracted from the healthy control group, sepsis without liver injury group and sepsis with liver injury group and cultured in vitro, the quantitative level of cf-DNA/NETs in cell supernatant was determined by PicoGreen fluorescence quantitative detection. The patients were divided into two groups according to 28-day outcome of sepsis patients with liver injury. Receiver operating characteristic (ROC) curve was plotted, and the area under ROC curve (AUC) was calculated to analyze the prognostic value of NETs in sepsis patients with liver injury. RESULTS: Finally, 21 sepsis patients without liver injury, 15 sepsis patients with liver injury, 20 with non-sepsis and 20 with healthy examination were enrolled. There was no significant difference in gender or age among the four groups, indicating that the patients in each group were comparable. The levels of cf-DNA/NETs in peripheral blood, WBC and PMN of the sepsis with and without liver injury groups were significantly higher than those of the healthy control group and non-sepsis group, PCT, APACHE II score, SOFA score and 28-day mortality were significantly higher than those of the non-sepsis group, and the levels of cf-DNA/NETs in peripheral blood, PCT and 28-day mortality of the sepsis with liver injury group were significantly higher than those of the sepsis without liver injury group [cf-DNA/NETs (μg/L): 481.60±275.86 vs. 169.76±57.05, PCT (μg/L): 11.29 (1.79, 67.10) vs. 1.11 (0.19, 4.09), 28-day mortality: 73.3% (11/15) vs. 38.1% (8/21), all P < 0.05]. The results of PMN in vitro showed that there was no NETs in normal culture of healthy control group, and a small amount of NETs was detected in sepsis with and without liver injury groups. After stimulation of PMN stimulator phorbol-12-myristate-13-acetic acid (PMA), more NETs were produced in neutrophils of three groups compared with normal culture. Quantitative analysis showed that the level of cf-DNA/NETs in cell supernatant of the sepsis with liver injury group was significantly higher than that of the sepsis without liver injury group (μg/L: 1 872.29±258.44 vs. 1 313.55±147.45, P < 0.01). In 15 sepsis patients with liver injury, 4 patients survived for 28 days (26.7%) and 11 died (73.3%). The cf-DNA/NETs level of the dead group on the day of admission was significantly higher than that of the survival group (μg/L: 582.36±160.05 vs. 241.17±96.14, P < 0.05). ROC curve analysis showed that the AUC of NETs level in peripheral blood for predicting 28-day death of sepsis patients with liver injury was 0.932 [95% confidence interval (95%CI) was 0.787-1.000]; when the best cut-off value was 266.81 μg/L, the sensitivity was 90.9%, the specificity was 75.0%, and the approximate index was 0.659. CONCLUSIONS The function of NETs in sepsis patients with liver injury has been further changed. The level of peripheral blood NETs has a certain guiding value for the prognosis of sepsis patients with liver injury.
APACHE ; Hepatic Insufficiency/diagnosis* ; Humans ; Liver ; Neutrophils ; Organ Dysfunction Scores ; Prognosis ; Prospective Studies ; ROC Curve ; Retrospective Studies ; Sepsis/diagnosis*

Objective To explore the effects of Hippo signaling on anti-oxidative stress of mouse marrow mesenchymal stem cells (mMSCs) in vitro. Methods mMSCs derived from C57BL/6 mice were identified using fluorescence-activated cell sorting analysis and the capabilities of osteogenic, chondrogenic and adipogenic differentiation were evaluated. 2-deoxy-D-glucose (2-DG) or XMU-MP-1 was used to modulate Hippo signaling. Oxidative stress was induced by H2O2treatment and the effect of oxidative stress induced by H2O2on survival of mMSCs was evaluated using methyl thiazolyl tetrazolium (MTT) assay. The effect of oxidative stress induced by H2O2on Hippo signaling and the effect of Hippo signaling on capability of anti-oxidative stress of mMSCs were analyzed through apoptosis-regulated proteins (Bcl-2 and Bax) using Western Blot. Results Hippo signaling was activated by 2-DG in a concentration-dependent manner and the effect was most prominent by 5 mmol/L of 2-DG [compared with the blank control group, large tumor suppressor 1 (LATS1) protein (grey value): 2.33±0.25 vs. 0.98±0.03, phosphorylated Yes-associated protein (p-YAP)/YAP protein ratio (grey value): 2.30±0.35 vs. 1.01±0.05, 14-3-3 protein (grey value):2.19±0.40 vs. 0.99±0.04, all P < 0.05]; Hippo signaling was inhibited by 100 nmol/L of XMU-MP-1 [compared with the blank control group, LATS1 protein (grey value): 0.69±0.10 vs. 0.98±0.03, p-YAP/YAP protein ratio (grey value):0.65±0.06 vs. 1.01±0.05, 14-3-3 protein (grey value): 0.75±0.11 vs. 0.99±0.04, all P < 0.05]. Death of mMSCs was induced by H2O2in a concentration-dependent manner and the minimal effective concentration was 0.1 mmol/L [compared with the blank control group, survival rate of mMSCs: (81.25±11.85)% vs. (100.44±12.39)%, P < 0.05]. Inhibition of Hippo signaling was induced by H2O2in a concentration-dependent manner and the minimal effective concentration was also 0.1 mmol/L [compared with the blank control group, LATS1 protein (grey value): 0.75±0.06 vs. 1.01±0.09, p-YAP/YAP protein ratio (grey value): 0.69±0.05 vs. 0.98±0.05, both P < 0.05], those effects might associate with reduction of Bcl-2/Bax ratio (grey value: 0.48±0.18 vs. 1.06±0.09, P < 0.05). Compared with the treatment of 0.1 mmol/L of H2O2, activation of Hippo signaling by 5 mmol/L of 2-DG [ LATS1 protein (grey value):0.95±0.05 vs. 0.64±0.06, p-YAP/YAP protein ratio (grey value): 0.87±0.03 vs. 0.45±0.16, both P < 0.05] improved survival of mMSCs [(92.80±9.43)% vs. (75.47±9.43)%, P < 0.05] through an increase of Bcl-2/Bax ratio (grey value:1.14±0.16 vs. 0.77±0.12, P < 0.05); however, inhibition of Hippo signaling by 100 nmol/L of XMU-MP-1 [ LATS1 protein (grey value): 0.39±0.03 vs. 0.64±0.06, p-YAP/YAP protein ratio (grey value): 0.28±0.04 vs. 0.45±0.16, both P < 0.05] decreased survival of mMSCs [(57.54±4.59)% vs. (75.47±9.43)%, P < 0.05] through an decrease of Bcl-2/Bax ratio (grey value: 0.63±0.20 vs. 0.77±0.12, P < 0.05). Compared with normal lung tissue, acute respiratory distress syndrome (ARDS) lung tissue markedly activate Hippo signaling in mMSCs [LATS1 protein (grey value): 1.71± 0.08 vs. 1.00±0.10, p-YAP/YAP protein ratio (grey value): 2.46±0.39 vs. 1.01±0.04, 14-3-3 protein (grey value):2.27±0.52 vs. 1.01±0.08, all P < 0.05]. Conclusion Hippo signaling could affect survival and capability of anti-oxidative stress of mMSCs via modulation of Bcl-2/Bax ratio in vitro.