[Effects of Notch signaling pathway and vascular endothelial growth factor gene on the functions of endothelial cells derived from rat bone marrow mesenchymal stem cells].

OBJECTIVE: To explore the effects of Notch signaling pathway and the vascular endothelial growth factor [VEGF(165)] gene on the functions of endothelial cells derived from rat bone marrow mesenchymal stem cells (MSCs). METHODS: Isolated and cultivated rat bone marrow MSCs in vitro, then the cells were treated by VEGF165 and basic fibroblast growth factor (bFGF) for 2 weeks to induce them to differentiate into endothelial cells. The gene of VEGF165 was transfected into differentiated endothelial cells to promote the functions of the cells. The receptor Notch1 and the ligand Jagged1 of the Notch signaling were detected by reverse transcription-polymerase chain reaction (RT-PCR) before and after the transfection. γ-secretase inhibitor L-685458 was used to block Notch pathway. Migration ability of cells was detected by scarification test. Cells were inoculated on semisolid gel to study their ability of forming capillary-like structure. RESULTS: After transfection, VEGF165 mRNA could be detected on the differentiated endothelial cells. The expression of Jagged1 mRNA was up regulated(1.08 ± 0.01 vs. 1.01 ± 0.02,P < 0.01) and there was no change in Notch1 mRNA(0.60 ± 0.02 vs. 0.59 ± 0.01,P > 0.05). The ability of migration was enhanced (number of cells on the scratched area:46.45 ± 4.46 vs. 41.61 ± 1.42,P < 0.05), and the ability of forming capillary-like structure on semisolid gel showed no change (cells classification: 3.00 ± 0.89 vs. 2.00 ± 0.89,P > 0.05). After the transfection, using the γ-secretase inhibitor L-685458 to block the Notch signaling transduction, the ability of migration of the differentiated endothelial cells (number of cells on the scratched area: 51.72 ± 3.47 vs. 46.45 ± 4.46,P < 0.05), and that of forming capillary--like structure (cells classification: 4.17 ± 0.75 vs. 3.00 ± 0.89, P < 0.05), was also further enhanced. CONCLUSION: Transfection of the gene of VEGF165 into the differentiated endothelial cells can reinforce the function of these cells, and when Notch signaling was blocked, this effect can be further amplified.

[Effect of salvianolic acid B on TGF-beta1-induced human embryonic lung fibroblast's biological behavior].

OBJECTIVE: To investigate the effect of salvianolic acid B (SAB) on the proliferation of human embryonic lung fibroblast MRC-5, and the secretion of procollagen I and endogenous transforming growth factor-beta1, (TGF-beta1). METHODS: The MRC-5 cells were randomly divided into four groups as follows: the control group: cells cultured with DMEM but with no TGF-beta1, or SAB; the TGF-beta1, group: cell cultured with 10 ng/mL TGF-beta1; the SAB1 group: cell cultured with medium with 10 ng/mL TGF-beta1 and 1 pmol/L SAB; the SAB2 group: cell cultured with medium with 10 ng/mL TGF-beta1, and 10 pmol/L SAB. The proliferation of cells was assayed by MTT incorporation. The concentration of amino-terminal propeptide of type I procollagen (PINP), a marker of collagen synthesis, was measured by radioimmunoassay. The endogenous TGF-beta1, levels were measured using ELISA. RESULTS: The optical density, procollagen I contents, and endogenous TGF-beta1, levels significantly increased when compared with those of the control group (P<0.05). Compared with the TGF-beta1, group, the optical density was obviously lowered, the procollagen I contents and endogenous TGF-beta1, levels significantly decreased in the SAB1 group and the SAB2 group, and better in the SAB2 group, showing statistical difference (P<0.05). CONCLUSIONS: SAB could inhibit the proliferation of MRC-5 cells induced by TGF-beta1 and attenuate the roles of secreting collagen and endogenous TGF-beta1. It had the potential of postponing or delaying the progressive developing of pulmonary fibrosis.

[The role of Notch signaling during the differentiation of rat bone marrow mesenchymal stem cells into endothelial cells].

OBJECTIVE: To research the role of Notch signaling during the differentiation of bone marrow mesenchymal stem cells (MSCs) into endothelial cells and its effect on the functions of the differentiated cells. METHODS: Rat bone marrow MSCs were isolated and cultured in vitro, then the cells were treated with vascular endothelial growth factor (VEGF165) and basic fibroblast growth factor (bFGF) for 2 weeks to induce it to differentiate into endothelial cells. The differentiated cells were identified by fluorescence immunoassay. The receptors and ligands of the Notch signaling were detected by reverse transcription-polymerase chain reaction (RT-PCR) before and after the differentiation. γ-secretase inhibitor was used to block Notch pathway. Migration ability of cells were assessed by scarification test. Cells were inoculated on semisolid gel to study their ability of forming the capillary-like structure. RESULTS: After inducing MSCs to differentiate into endothelial cells by VEGF165 and bFGF, MSCs gained the characteristics of the endothelial cells with expression of CD31 and Flk1. There were Notch1 mRNA and Jagged1 mRNA expressions in rat bone marrow MSCs. The expression changes in the receptor Notch1 were not statistically significant on the differentiated cells (0.59±0.01 vs. 0.59±0.01, P>0.05), but there was a trend towards an increase of Jagged1 mRNA (1.01±0.02 vs. 0.99±0.03, P>0.05). When Notch pathway was blocked, the differentiated cells' migration ability was increased (number of cells on the scratched area: 44.61±4.34 vs. 40.06±2.43, P<0.05), and the ability of forming capillary-like structure was also increased (cells classification: 3.67±0.82 vs. 2.00±0.89, P<0.01). CONCLUSION: Notch signaling may have an important role during the differentiation of MSCs into endothelial cells. The function of differentiated cells were strengthened when Notch pathway was blocked.

The timing of continuous renal replacement therapy initiation in sepsis-associated acute kidney injury in the intensive care unit: the CRTSAKI Study (Continuous RRT Timing in Sepsis-associated AKI in ICU): study protocol for a multicentre, randomised controlled trial.

INTRODUCTION: Acute kidney injury (AKI) is one of the most common organ dysfunction in sepsis, and increases the risk of unfavourable outcomes. Renal replacement therapy (RRT) is the predominant treatment for sepsis-associated AKI (SAKI). However, to date, no prospective randomised study has adequately addressed whether initiating RRT earlier will attenuate renal injury and improve the outcome of sepsis. The objective of the trial is to compare the early strategy with delayed strategy on the outcomes in patients with SAKI in the intensive care unit (ICU). METHODS AND ANALYSIS: This is a large-scale, multicentre, randomised controlled trial about SAKI. In total, 460 patients with sepsis and evidence of AKI stage 2 of Kidney Disease Improving Global Outcomes (KDIGO) will be recruited and equally randomised into the early group and the delay group in a ratio of 1:1. In the early group, continuous RRT (CRRT) will be started immediately after randomisation. In the delay group, CRRT will initiated if at least one of the following criteria was met: stage 3 of KDIGO, severe hyperkalaemia, pulmonary oedema, blood urea nitrogen level higher than 112 mg/dL after randomisation. The primary outcome is overall survival in a 90-day follow-up period (90-day all-cause mortality). Other end points include 28-day, 60-day and 1-year mortality, recovery rate of renal function by day 28 and day 90, ICU and hospital length of stay, the numbers of CRRT-free days, mechanical ventilation-free days and vasopressor-free days, the rate of complications potentially related to CRRT, CRRT-related cost, and concentrations of inflammatory mediators in serum. ETHICS AND DISSEMINATION: The trial has been approved by the Clinical Research and Application Institutional Review Board of the Second Affiliated Hospital of Guangzhou Medical University (2017-31-ks-01). Participants will be screened and enrolled from patients in the ICU with SAKI by clinicians, with no public advertisement for recruitment. Results will be disseminated in research journals and through conference presentations. TRIAL REGISTRATION: NCT03175328.

[The effects of the plasma levels of serotonin and neuropeptide on ventilated patients with Yitongshu acupuncture points injection].

OBJECTIVE: To evaluate the change in the plasma levels of serotonin (5-hydroxytryptamine) and neuropeptide (beta-endorphin, beta-EP) after injection of Yitongshu into Zusanli points in patients under mechanical ventilation. METHODS: Twenty-eight patients undergoing mechanical ventilation were randomly divided into two groups: midazolam combined with fentanyl group (control group, n=14) and midazolam combined with fentanyl and Yitongshu group (Yitongshu group, n=14). The drugs were given to the patients continuously intravenously with an injection pump in an even rate, with the dosage adjusted to reach the sedative target of visual analog score (VAS)< or =3-4 and Ramsay 2-4. Yitongshu injection (4 ml) was injected into the Zusanli point on both sides 11 hours or 23 hours after administration of the durgs in Yitongshu group. The hemodynamic and respiratory parameters, including mean arterial pressure (MAP), heart rate (HR), respiratory rate (RR), pulse oxygen saturation (SpO(2)), oxygenation index (PaO(2)/FiO(2)) and pressure airway (Paw), were recorded, and the sedation levels (VAS and Ramsay) were evaluated before sedation and 1, 12, 24 hours after sedation in these patients. The plasma levels of 5-hydroxytryptamine and beta-EP were examined before sedation, 12 hours and 24 hours after sedation. RESULTS: Compared with that before sedation, HR and VAS score were significantly lower, and Ramsay score was significantly higher in both groups. MAP was significantly lower at 1 hour, and RR at 12 hours and 24 hours , as well as the Paw at 24 hours, and the PaO(2)/FiO(2) was significantly higher at 24 hours. The level of 5-hydroxytryptamine at 12 hours and 24 hours in Yitongshu group [(101.45+/-14.67) ng/L, (104.86+/-11.74) ng/L] was significantly higher than that in control group [(61.57+/-10.62) ng/L, (59.86+/-8.64) ng/L, both P<0.05]. But the level of beta-EP showed no difference between two groups [control group: (162.72+/-38.44) ng/L at 12 hours, (151.83+/-24.54) ng/L at 24 hours; Yitongshu group: (169.35+/-28.10) ng/L at 12 hours, (159.41+/-15.89) ng/L at 24 hours, both P>0.05]. CONCLUSION: Yitongshu injection can reduce the plasma level of 5-hydroxytryptamine in ventilated patients, but with no effect on beta-EP.

TGF-beta1 induces human bronchial epithelial cell-to-mesenchymal transition in vitro.

The subepithelial fibrosis component of airway remodeling in asthma is mediated through induction of transforming growth factor-beta1 (TGF-beta1) expression with consequent activation of myofibroblasts to produce extracellular matrix proteins. The number of myofibroblasts is increased in the asthmatic airway and is significantly correlated with the thickness of lamina reticularis. However, much is still unknown regarding the origin of bronchial myofibroblasts. Emerging evidence suggests that myofibroblasts can derive from epithelial cells by an epithelial-to-mesenchymal transition (EMT). In this study we investigated whether TGF-beta1 could induce bronchial epithelial EMT in the human bronchial epithelial cell. Cultured human bronchial epithelial cells, 16HBE-14o, were stimulated with 10 ng/ml TGF-beta1. Morphologic changes were observed and stress fiber by actin reorganization was detected by indirect immunostaining. The expression of alpha-SMA (alpha-smooth muscle actin) and the epithelial cell marker E-cadherin were detected in those 16HBE-14o cells after TGF-beta1 stimulation for 72 h, using immunostaining and RT-PCR. The contents of collagen I were determined by radioimmunoassay, and the levels of endogenous TGF-beta1 were measured with ELISA. Human bronchial epithelial cells stimulated with TGF-beta1 were converted from a "cobblestone" epithelial structure into an elongated fibroblast-like shape. Incubation of human bronchial epithelial cells with TGF-beta1 induced de novo expression of alpha-SMA, increased formation of stress fiber by F-actin reorganization, and loss of epithelial marker E-cadherin. Moreover, a significant increase in the levels of collagen I and endogenous TGF-beta1 released from bronchial epithelial cells stimulated with TGF-beta1 were observed. These results suggested that human bronchial epithelial cells, under stimulation of TGF-beta1, underwent transdifferentiation into myofibroblasts.

[Clinical analysis of 190 cases of outbreak with atypical pneumonia in Guangzhou in spring, 2003].

OBJECTIVE: To study methods of diagnosis and treatment for atypical pneumonia (Severe Acute Respiratory Syndrome), outbreak of the illness in Guangzhou during Jan. - Mar., 2003. METHODS: 190 cases with atypical pneumonia were analyzed, and the cases were admitted in Guangzhou municipal First Hospital and Guangzhou municipal Eighth Hospital. RESULTS: Patients were infected by close quarters contacting each other. All patients manifest high fever, and accompany by dyspnea, cough, palpitate, weakness, headache and swirl. Other 46 cases were accompanied by diarrhea. Most of patients, manifestation of lungs was negative. Chest X-ray, shadow of lungs were light in beginning, and change to severity slowly or suddenly during 5 - 10 days. Of these cases, 36 cases develop to ARDS and 11 cases died with severity ARDS. Using general antibiotic was of no effect for the illness. Continual positive airway pressure (CPAP) and glucocorticoid was required that can control deprivation of the disease when toxicosis symptom of patients was severity and shadow of lungs diffuse more and more. CONCLUSION: Infectivity of the illness is evidence and spread by airway. Using general antibiotic was of no effect for the illness. Continual positive airway pressure (CPAP) and glucocorticoid are effective for control of the disease.