Trimethylamine N-oxide predicts stroke severity in diabetic patients with acute ischaemic stroke and is related to glycemic variability.

BACKGROUND AND PURPOSE: The present study analyzed the relationship between circulating trimethylamine N-oxide (TMAO) levels and stroke severity in diabetic patients with acute ischaemic stroke. A further aim was to investigate whether higher TMAO levels were associated with platelet aggregation and glycemic variability. METHODS: This was a cross-sectional analysis of 108 patients with type 2 diabetes mellitus (DM) undergoing acute ischaemic stroke and 60 healthy controls. Fasting plasma TMAO was measured using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry. RESULTS: Plasma TMAO levels of patients with acute ischaemic stroke were significantly higher than those of healthy controls. Amongst stroke patients, 50 were defined as undergoing mild stroke, and their plasma TMAO levels were lower compared to those with moderate to severe stroke. Platelet aggregation and mean amplitude of glycemic excursions were both correlated with plasma TMAO levels and these relationships remained significant in multiple linear regression analyses. Moreover, in streptozotocin-induced diabetic rats fed a diet enriched with choline to increase TMAO synthesis, platelet aggregation was significantly increased in the DM + choline and fluctuating DM (FDM) + choline groups compared to the control group. This increase was abolished in rats receiving oral antibiotics, which markedly reduced plasma TMAO levels. Importantly, compared with the DM + choline group, the FDM + choline group displayed significantly elevated TMAO levels and higher platelet aggregation. CONCLUSIONS: Our results demonstrated that higher plasma TMAO levels were associated with stroke severity and suggested a novel link between plasma TMAO levels and glycemic variability in diabetic patients with acute ischaemic stroke.

Is compromised intestinal barrier integrity responsible for the poor prognosis in critically ill patients with pre-existing hyperglycemia?

BACKGROUND: Compromised intestinal barrier integrity can be independently driven by hyperglycemia, and both hyperglycemia and intestinal barrier injury are associated with poor prognosis in critical illness. This study investigated the intestinal barrier biomarkers in critically ill patients, to explore the role of compromised intestinal barrier integrity on the prognosis of critically ill patients with pre-existing hyperglycemia. METHODS: This was a retrospective observational study. The relationships between intestinal barrier biomarkers and glycated hemoglobin A1c (HbA1c), fasting blood glucose (FBG), indicators of clinical characteristics, disease severity, and prognosis in critically ill patients were investigated. Then the metrics mentioned above were compared between survivors and non-survivors, the risk factors of 90-day mortality were investigated by logistic regression analysis. Further, patients were divided into HbA1c < 6.5% Group and HbA1c ≥ 6.5% Group, metrics mentioned above were compared between these two groups. RESULTS: A total of 109 patients with critical illness were included in the study. D-lactate and lipopolysaccharide (LPS) were associated with sequential organ failure assessment (SOFA) score and 90-day mortality. LPS was an independent risk factor of 90-day mortality. DAO, NEU (neutrophil) proportion, temperature, lactate were lower in HbA1c ≥ 6.5% Group while D-lactate, LPS, indicators of disease severity and prognosis showed no statistical difference between HbA1c < 6.5% Group and HbA1c ≥ 6.5% Group. CONCLUSIONS: Intestinal barrier integrity is associated with the disease severity and prognosis in critical illness. Compromised intestinal barrier integrity might be responsible for the poor prognosis in critically ill patients with pre-existing hyperglycemia.

Anti-Hyperglycemic Agents in the Adjuvant Treatment of Sepsis: Improving Intestinal Barrier Function.

Intestinal barrier injury and hyperglycemia are common in patients with sepsis. Bacteria translocation and systemic inflammatory response caused by intestinal barrier injury play a significant role in sepsis occurrence and deterioration, while hyperglycemia is linked to adverse outcomes in sepsis. Previous studies have shown that hyperglycemia is an independent risk factor for intestinal barrier injury. Concurrently, increasing evidence has indicated that some anti-hyperglycemic agents not only improve intestinal barrier function but are also beneficial in managing sepsis-induced organ dysfunction. Therefore, we assume that these agents can block or reduce the severity of sepsis by improving intestinal barrier function. Accordingly, we explicated the connection between sepsis, intestinal barrier, and hyperglycemia, overviewed the evidence on improving intestinal barrier function and alleviating sepsis-induced organ dysfunction by anti-hyperglycemic agents (eg, metformin, peroxisome proliferators activated receptor-γ agonists, berberine, and curcumin), and summarized some common characteristics of these agents to provide a new perspective in the adjuvant treatment of sepsis.

[Successful treatment of acute right heart failure under the guidance of point-of-care ultrasound: a case report].

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.

[Evaluation of extravascular lung water in patients with acute respiratory distress syndrome by syndecan-1 combined with lung ultrasonography].

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.

[Effects of Hippo pathway on differentiation, proliferation, migration of bone marrow mesenchymal stem cells in vitro].

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 type II alveolar epithelial cells (AEC II) 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 AEC II 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 AEC II 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/ß-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/ß-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]. CONCLUSIONS: Activation of Hippo pathway could enhance differentiation of BMSCs to AEC II, promote proliferation and ability of horizontal migration and homing BMSCs to conditioned medium of ARDS lung tissue in vitro.

[Effects of Hippo signaling pathway on lung injury repair by mesenchymal stem cells in acute respiratory distress syndrome].

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-1ß, 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/ß-actin: 0.51±0.12, 0.68±0.10 vs. 0.27±0.08, Occludin/ß-actin: 0.49±0.19, 0.79±0.11 vs. 0.25±0.08, all P < 0.05), TP, ALB, IL-1ß, 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-1ß (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.

Mesenchymal stem cells with downregulated Hippo signaling attenuate lung injury in mice with lipopolysaccharide­induced acute respiratory distress syndrome.

Mesenchymal stem cell (MSC)­mediated repair of injured alveolar epithelial cells is a promising potential cure for acute respiratory distress syndrome (ARDS); however, the repairing effect of MSCs is limited by poor homing and differentiation. Our previous study revealed that the inhibition of the Hippo signaling pathway promotes the proliferation, migration and differentiation of MSCs in vitro, leading to the hypothesis that MSCs with downregulated Hippo signaling could further ameliorate lipopolysaccharide (LPS)­induced ARDS in vivo. In the current study, mouse bone marrow­derived MSCs (mMSCs) with downregulated Hippo signaling were constructed by shRNA­mediated knockdown of large tumor suppressor kinase 1 (Lats1) and were intratracheally administered to LPS­induced mouse models of ARDS. The inhibition of Hippo signaling increased the retention of mMSC in ARDS lung tissue and their differentiation toward alveolar type II epithelial cells. Furthermore, mMSCs with downregulated Hippo signaling led to a decreased lung wet weight/body weight ratio, decreased total protein and albumin concentrations in bronchoalveolar lavage fluid, decreased levels of proinflammatory factors and increased levels of anti­inflammatory factors. Finally, mMSCs with downregulated Hippo signaling improved pathological changes and decreased pulmonary fibrosis in lungs of mice with ARDS. These results suggest that the inhibition of the Hippo signaling pathway in mouse mMSCs by knockdown of Lats1 could further improve the protective effects of mMSCs against epithelial damage and the therapeutic potential of mMSCs on mice with ARDS.

[Changes in neutrophil function in septic liver injury and its effect on prognosis: a prospective observational study].

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.

The initial glycemic variability is associated with early neurological deterioration in diabetic patients with acute ischemic stroke.

The association between glycemic variability and early neurological deterioration (END) in acute ischemic stroke remains unclear. This study attempted to explore whether initial glycemic variability increases END in diabetic patients with acute ischemic stroke. We enrolled type 2 diabetic patients undergoing acute ischemic stroke from November 2015 to November 2016. A total of 336 patients within 72 h from stroke onset were included. The serum glucose levels were checked four times per day during the initial 3 hospital days. The standard deviation of blood glucose (SDBG) values and the mean amplitude of glycemic excursions (MAGE) were calculated for glycemic variability. END was defined as an increase in the National Institutes of Health Stroke Scale (NIHSS) ≥ 2 points between hospital days 0 and 5. The frequencies of END and HbA1c were significantly different in subjects grouped according to tertiles of MAGE (9.09, 12.07 and 50.00%, p < 0.001 for END; 7.36 ± 1.91, 7.83 ± 1.93 and 8.56 ± 1.79, p < 0.001 for HbA1c). Compared to patients without END, patients with END had significantly higher HbA1c levels (8.30 ± 1.92 vs 7.80 ± 1.93, p = 0.043), increased SDBG (3.42 ± 1.14 vs 2.60 ± 0.96, p < 0.001), and increased MAGE (6.46 ± 2.09 vs 4.59 ± 1.91, p < 0.001). In a multivariable logistic regression, stroke etiology (OR 0.675; 95% CI 0.485-0.940, p = 0.020), baseline NIHSS (OR 1.086; 95% CI 1.004-1.175, p = 0.040), and MAGE (OR 1.479; 95% CI 1.162-1.882, p = 0.001) were significantly associated with END. Initial glycemic variability is associated with END in diabetic patients with acute ischemic stroke.

[Effects of Hippo signaling on anti-oxidative stress of mesenchymal stem cells in vitro].

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 H2O2 treatment and the effect of oxidative stress induced by H2O2 on survival of mMSCs was evaluated using methyl thiazolyl tetrazolium (MTT) assay. The effect of oxidative stress induced by H2O2 on 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 H2O2 in 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 H2O2 in 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]. CONCLUSIONS: Hippo signaling could affect survival and capability of anti-oxidative stress of mMSCs via modulation of Bcl-2/Bax ratio in vitro.

Modulation of GSK-3ß/ß-Catenin Signaling Contributes to Learning and Memory Impairment in a Rat Model of Depression.

Background: It is widely accepted that cognitive processes, such as learning and memory, are affected in depression, but the molecular mechanisms underlying the interactions of these 2 disorders are not clearly understood. Recently, glycogen synthase kinase-3 beta (GSK-3ß)/ß-catenin signaling was shown to play an important role in the regulation of learning and memory. Methods: The present study used a rat model of depression, chronic unpredictable stress, to determine whether hippocampal GSK-3ß/ß-catenin signaling was involved in learning and memory alterations. Results: Our results demonstrated that chronic unpredictable stress had a dramatic influence on spatial cognitive performance in the Morris water maze task and reduced the phosphorylation of Ser9 of GSK-3ß as well as the total and nuclear levels of ß-catenin in the hippocampus. Inhibition of GSK3ß by SB216763 significantly ameliorated the cognitive deficits induced by chronic unpredictable stress, while overexpression of GSK3ß by AAV-mediated gene transfer significantly decreased cognitive performance in adult rats. In addition, chronic unpredictable stress exposure increased the expression of the canonical Wnt antagonist Dkk-1. Furthermore, chronic administration of corticosterone significantly increased Dkk-1 expression, decreased the phosphorylation of Ser9 of GSK-3ß, and resulted in the impairment of hippocampal learning and memory. Conclusions: Our results indicate that impairment of learning and memory in response to chronic unpredictable stress may be attributed to the dysfunction of GSK-3ß/ß-catenin signaling mediated by increased glucocorticoid signaling via Dkk-1.

[Under-expression of LATS1 promotes the differentiation, proliferation and migration of mesenchymal stem cells by inhibition the Hippo signaling pathway in vitro].

OBJECTIVE: To explore the effects of under-expression of large tumor suppressor 1 (LATS1) on activation of Hippo signaling pathway and differentiation, proliferation, migration of bone marrow mesenchymal stem cells (mMSCs) of mice in vitro. METHODS: mMSCs of C57BL/6 mice were divided into normal control (MSC) group, empty vector control (MSC-GFP) group, LATS1-over-expressing (MSC-LATS1) group, empty vector without LATS1 shRNA control (MSC-shControl) group and LATS1-under-expressing (MSC-shLATS1) group. Lentiviral vectors with activated, inactivated LATS1 (the key molecule of Hippo signaling pathway) modifications and empty vectors were constructed and were used to infect mMSCs in vitro. The transduction efficiencies mediated by the lentiviral vectors were evaluated by fluorescence microscopy and flow cytometry. The mRNA expression of LATS1 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expressions of LATS1, YAP (p-YAP), 14-3-3 were quantified by Western Blot to evaluate the activation of Hippo signaling pathway. Osteogenic and adipogenic differentiation of mMSCs were evaluated through measurement of Runx2, OSX and C/EBPα, PPAR-γ mRNA by qRT-PCR, as well as Alizarin Red S and Oil red O staining. Proliferation of mMSCs was evaluated using methy thiazdyl tetrazolium (MTT) assay. The scratch test and Transwell chamber test were used to analyze the horizontal and vertical migration ability of mMSCs. RESULTS: The transduction efficiencies mediated by the lentiviral vectors were 94.74%-96.10%. Compared with MSC-GFP group, the activation of Hippo signaling pathway was promoted in MSC-LATS1 group [LATS1 mRNA (2-Δ ΔCT): 4.37±0.21 vs. 1.20±0.04, LATS1 protein (gray value): 2.21±0.06 vs. 1.09±0.10, p-YAP/YAP protein (gray value): 1.51±0.13 vs. 0.98±0.05, 14-3-3 protein (gray value): 1.92±0.18 vs. 1.10±0.09, all P < 0.05], osteogenic and adipogenic differentiation of mMSCs were decreased in MSC-LATS1 group [mineralization (A value): 0.13±0.02 vs. 0.40±0.03, Runx2 mRNA (2-Δ ΔCT): 0.51±0.02 vs. 0.98±0.09, OSX mRNA (2-Δ ΔCT): 0.41±0.04 vs. 1.04±0.09, lipid accumulation (A value): 0.10±0.02 vs. 0.25±0.03, C/EBPα mRNA (2-Δ ΔCT): 0.33±0.03 vs. 1.11±0.09, PPAR-γ mRNA (2-Δ ΔCT): 0.29±0.02 vs. 1.04±0.10, all P < 0.05], the proliferation rate of mMSCs at 4-7 days was decreased in MSC-LATS1 group and so were the horizontal and vertical migration of mMSCs [wound healing rate: (18.65±3.53)% vs. (40.29±1.87)%, migrated cells (cells/MP): 35.99±6.18 vs. 103.67±17.77, both P < 0.05]. Compared with MSC-shControl group, the activation of Hippo signaling pathway was inhibited in MSC-shLATS1 group [LATS1 mRNA (2-Δ ΔCT): 0.16±0.01 vs. 0.98±0.03, LATS1 protein (gray value): 0.38±0.03 vs. 1.04±0.07, p-YAP/YAP protein (gray value): 0.58±0.04 vs. 1.05±0.06, 14-3-3 protein (gray value): 0.14±0.02 vs. 1.02±0.09, all P < 0.05], osteogenic and adipogenic differentiation of mMSCs were increased in MSC-shLATS1 group [mineralization (A value): 0.93±0.13 vs. 0.44±0.05, Runx2 mRNA (2-Δ ΔCT): 1.44±0.12 vs. 0.95±0.04, OSX mRNA (2-Δ ΔCT): 1.67±0.06 vs. 1.10±0.11, lipid accumulation (A value): 0.47±0.06 vs. 0.28±0.04, C/EBPα mRNA (2-Δ ΔCT): 3.98±0.61 vs. 0.99±0.10, PPAR-γ mRNA (2-Δ ΔCT): 3.05±0.36 vs. 0.98±0.14, all P < 0.05], the proliferation rate of mMSCs at 3-7 days was increased in MSC-shLATS1 group and so were the horizontal and vertical migration of mMSCs [wound healing rate: (80.18±6.98)% vs. (46.18±1.01)%, migrated cells (cells/MP): 212.69±41.21 vs. 115.87±35.15, both P < 0.05]. CONCLUSIONS: Under-expression of LATS1 promotes the differentiation, proliferation, migration of mMSCs by inhibition of Hippo signaling pathway in vitro.

Blood oxygen level-dependent signals via fMRI in the mood-regulating circuit using two animal models of depression are reversed by chronic escitalopram treatment.

BACKGROUND: People exposed to stressful experience are at increased risk of the development of depression. A number of functional imaging studies have found disturbances in the mood-regulating circuit of the stress-exposed depressed patients, although few animal imaging studies have been undertaken addressing the brain functional changes of depression. METHODS: Two rat models of depression: maternal separation (MS) and chronic unpredictable mild stress (CUMS), imitating early life stress and adult stress respectively, were administered with escitalopram. The differences in functional brain changes were determined by blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI). RESULTS: Increased BOLD activation was observed in some brain regions of MS and CUMS animals, such as the bilateral hypothalamus, limbic system, hippocampus and frontal lobe, which were parts of mood-regulating circuit. Furthermore, the MS- and CUMS-induced increases in BOLD activation were partially attenuated by chronic escitalopram treatment. CONCLUSIONS: These results suggested hyperactivation of mood-regulating circuit at baseline in the depressed animals exposed to stressful experience, and escitalopram can at least partially reverse these effects.

The Role of the Two-Pore Domain Potassium Channel TREK-1 in the Therapeutic Effects of Escitalopram in a Rat Model of Poststroke Depression.

AIM: Poststroke depression (PSD) is one of the most common neuropsychiatric complications after stroke. TREK-1, a two-pore-domain potassium channel, has been implicated in the pathogenesis of stroke and depression. The aim of this study was to investigate whether TREK-1 plays a role in the therapeutic effects of the selective serotonin reuptake inhibitor (SSRI) escitalopram in a rat PSD model. METHODS: The whole-cell patch-clamp technique was performed to assess the effect of escitalopram on recombinant TREK-1 currents in HEK293 cells. The expression of TREK-1 mRNA and protein was measured in the hippocampus and prefrontal cortex (PFC), and neural stem cell (NSC) proliferation was detected in the hippocampal dentate gyrus (DG) in PSD rats after 3 weeks of escitalopram administration. RESULTS: Escitalopram reversibly inhibited TREK-1 currents in a concentration-dependent manner. Chronic treatment with escitalopram significantly reversed the reductions in weight gain, locomotor activity, and sucrose preference in PSD rats. The expressions of TREK-1 mRNA and protein were significantly increased in hippocampal CA1, CA3, DG, and PFC in PSD rats, with the exception of TREK-1 mRNA in hippocampal CA1. NSC proliferation was significantly decreased in hippocampal DG of PSD rats. Escitalopram significantly reversed the regional increases of TREK-1 expression and the reduction of hippocampal NSC proliferation in PSD rats. CONCLUSION: TREK-1 plays an important role in the therapeutic effects of the SSRI escitalopram in PSD model, making TREK-1 an attractive candidate molecule for further understanding the pathophysiology and treatment of PSD.

Fluoxetine regulates neurogenesis in vitro through modulation of GSK-3ß/ß-catenin signaling.

BACKGROUND: It is generally accepted that chronic treatment with antidepressants increases hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. Recently, glycogen synthase kinase-3 beta (GSK-3ß)/ß-catenin signaling was shown to be involved in the mechanism of how antidepressants might influence hippocampal neurogenesis. METHODS: The aim of this study was to determine whether GSK-3ß/ß-catenin signaling is involved in the alteration of neurogenesis as a result of treatment with fluoxetine, a selective serotonin reuptake inhibitor. The mechanisms involved in fluoxetine's regulation of GSK-3ß/ß-catenin signaling pathway were also examined. RESULTS: Our results demonstrated that fluoxetine increased the proliferation of embryonic neural precursor cells (NPCs) by up-regulating the phosphorylation of Ser9 on GSK-3ß and increasing the level of nuclear ß-catenin. The overexpression of a stabilized ß-catenin protein (ΔN89 ß-catenin) significantly increased NPC proliferation, while inhibition of ß-catenin expression in NPCs led to a significant decrease in the proliferation and reduced the proliferative effects induced by fluoxetine. The effects of fluoxetine-induced up-regulation of both phosphorylation of Ser9 on GSK-3ß and nuclear ß-catenin were significantly prevented by the 5-hydroxytryptamine-1A (5-HT1A) receptor antagonist WAY-100635. CONCLUSIONS: The results demonstrate that fluoxetine may increase neurogenesis via the GSK-3ß/ß-catenin signaling pathway that links postsynaptic 5-HT1A receptor activation.

A rat brain MRI template with digital stereotaxic atlas of fine anatomical delineations in paxinos space and its automated application in voxel-wise analysis.

This study constructs a rat brain T2 -weighted magnetic resonance imaging template including olfactory bulb and a compatible digital atlas. The atlas contains 624 carefully delineated brain structures based on the newest (2005) edition of rat brain atlas by Paxinos and Watson. An automated procedure, as an SPM toolbox, was introduced for spatially normalizing individual rat brains, conducting statistical analysis and visually localizing the results in the Atlas coordinate space. The brain template/atlas and the procedure were evaluated using functional images between rats with the right side middle cerebral artery occlusion (MCAO) and normal controls. The result shows that the brain region with significant signal decline in the MCAO rats was consistent with the occlusion position.

Learning and memory alterations are associated with hippocampal N-acetylaspartate in a rat model of depression as measured by 1H-MRS.

It is generally accepted that cognitive processes, such as learning and memory, are affected in depression. The present study used a rat model of depression, chronic unpredictable mild stress (CUMS), to determine whether hippocampal volume and neurochemical changes were involved in learning and memory alterations. A further aim was to determine whether these effects could be ameliorated by escitalopram treatment, as assessed with the non-invasive techniques of structural magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Our results demonstrated that CUMS had a dramatic influence on spatial cognitive performance in the Morris water maze task, and CUMS reduced the concentration of neuronal marker N-acetylaspartate (NAA) in the hippocampus. These effects could be significantly reversed by repeated administration of escitalopram. However, neither chronic stress nor escitalopram treatment influenced hippocampal volume. Of note, the learning and memory alterations of the rats were associated with right hippocampal NAA concentration. Our results indicate that in depression, NAA may be a more sensitive measure of cognitive function than hippocampal volume.

Hippocampal neurochemistry is involved in the behavioural effects of neonatal maternal separation and their reversal by post-weaning environmental enrichment: a magnetic resonance study.

Exposure to early life stress results in behavioural changes, and these dysfunctions may persist throughout adulthood. In this study, we investigated whether hippocampus volume and neurochemical changes were involved in the appearance of these effects in the maternal separation (MS) animal model using the noninvasive techniques of structural magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Sprague-Dawley rats exposed to MS for 180 min from postnatal days (PND) 2-14 demonstrated decreased sucrose preference, increased immobility in the forced swimming test (FST), and impaired memory in the Morris water maze in adulthood. Environmental enrichment (EE) (PND 21-60) could ameliorate the effects of MS on sucrose preference and learning and memory but not on immobility in the FST. In addition, EE significantly increased N-acetylaspartate (NAA) of MS animals. However, we did not find an effect of MS or EE on hippocampal volume. These results indicate the involvement of hippocampal neurochemistry in the behavioural changes that result from early stressful life events and their modification by post-weaning EE. Thus changes in NAA, as a measure of neuronal integrity, appear to be a sensitive correlate of these behavioural effects.

Fluoxetine attenuates the inhibitory effect of glucocorticoid hormones on neurogenesis in vitro via a two-pore domain potassium channel, TREK-1.

RATIONALE: Sustained stress and elevated glucocorticoid reduces neurogenesis, whereas chronic treatment with antidepressants increases neurogenesis and blocks the effects of stress. Recently, TREK-1, a two-pore domain (K(2)p) potassium channel, has been shown to be involved in the mechanisms of major depression. OBJECTIVES: This study aimed to investigate whether TREK-1 is involved in the alteration of neurogenesis according to glucocorticoids and antidepressants. RESULTS: The present study addressed the expression of TREK-1 in neural stem cells (NSCs) and found TREK-1 was only associated with NSC proliferation. Bupivacaine and curcumin, two strong TREK-1 channel inhibitors, significantly increased embryonic NSC viability and proliferation while transfection of hTREK-1 decreased cell proliferation in embryonic NSCs. Dexamethasone, a glucocorticoid hormone receptor agonist, upregulated both protein and mRNA levels of TREK-1 leading to decreased NSC proliferation which could be reversed by bupivacaine. Fluoxetine, a serotonin reuptake inhibitor antidepressant that has been previously found to inhibit TREK-1 channels, robustly, could attenuate the upregulation of TREK-1 expression and the inhibition of NSC proliferation induced by dexamethasone. CONCLUSIONS: Taken together, these data suggest that TREK-1 is associated with NSC proliferation and probably is a modulator of the effect that fluoxetine attenuates the inhibitory neurogenesis induced by glucocorticoid hormones.