Homeobox B4 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on endotoxin-associated acute lung injury in rats.

BACKGROUND: Alveolar capillary endothelial cell (EC) injury has a pivotal role in driving acute respiratory distress syndrome (ARDS) progression and maintaining endothelial homeostasis. A previous ex vivo study revealed that overexpression of homeobox B4 (HOXB4) in bone marrow mesenchymal stem cells (BMSCs) enhanced protection against lipopolysaccharide (LPS)-induced EC injury by activating the Wnt/ß-catenin pathway. This in vivo study was performed to verify whether BMSCs overexpressing HOXB4 exert similar protective effects on LPS-induced acute lung injury (ALI) in an animal model. METHODS: The ALI rat model was established by intraperitoneal injection of LPS. Wildtype BMSCs or BMSCs overexpressing HOXB4 were then injected via the tail vein. The lung characteristics of rats were visualized by computed tomography. Lung histopathological characteristics and collagen deposition were assessed by hematoxylin-eosin and Masson's staining, respectively, which were combined with the lung wet/dry ratio and proinflammatory factor levels in bronchoalveolar lavage fluid to further evaluate therapeutic effects. Expression of ß-catenin and VE-cadherin was assessed by western blotting and immunofluorescence. RESULTS: Compared with wildtype BMSCs, overexpression of HOXB4 optimized the therapeutic effects of BMSCs, which manifested as improvements in lung exudation and histopathological features, reduced lung collagen deposition, amelioration of lung permeability, attenuation of lung inflammation, and enhanced expression of ß-catenin and VE-cadherin proteins. CONCLUSIONS: HOXB4-overexpressing BMSCs optimized the protective effect against LPS-induced ALI by partially activating Wnt/ß-catenin signaling.

Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced endothelial cell injury via activating PI3K/AKT signaling pathway.

BACKGROUND: Lung endothelial barrier injury plays an important role in the pathophysiology of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) therapy has shown promise in ARDS treatment and restoration of the impaired barrier function. It has been reported that Wnt5a shows protective effects on endothelial cells. Therefore, the study aimed to investigate whether overexpression of Wnt5a could promote the protective effects of MSCs on Lipopolysaccharide (LPS)-induced endothelial cell injury. METHODS: To evaluate the protective effects of MSCs overexpressing Wnt5a, we assessed the migration, proliferation, apoptosis, and angiogenic ability of endothelial cells. We assessed the transcription of protective cellular factors using qPCR and determined the molecular mechanism using Western blot analysis. RESULTS: Overexpression of Wnt5a upregulated the transcription of protective cellular factors in MSCs. Co-culture of MSCWnt5a promoted endothelial migration, proliferation and angiogenesis, and inhibited endothelial cell apoptosis through the PI3K/AKT pathway. CONCLUSIONS: Overexpression of Wnt5a promoted the therapeutic effect of MSCs on endothelial cell injury through the PI3K/AKT signaling. Our study provides a novel approach for utilizing genetically modified MSCs in the transplantation therapy for ARDS.

Overexpression of FoxM1 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on acute respiratory distress syndrome.

BACKGROUND: Injury of alveolar epithelial cells and capillary endothelial cells is crucial in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) are a promising cell source for ALI/ARDS treatment. Overexpression of Fork head box protein M1 (FoxM1) facilitates MSC differentiation into alveolar type II (AT II) cells in vitro. Moreover, FoxM1 has been shown to repair the endothelial barrier. Therefore, this study explored whether overexpression of FoxM1 promotes the therapeutic effect of bone marrow-derived MSCs (BMSCs) on ARDS by differentiation of BMSCs into AT II cells or a paracrine mechanism. METHODS: A septic ALI model was established in mice by intraperitoneal administration of lipopolysaccharide. The protective effect of BMSCs-FoxM1 on ALI was explored by detecting pathological variations in the lung, total protein concentration in bronchoalveolar lavage fluid (BALF), wet/dry (W/D) lung weight ratio, oxidative stress levels, cytokine levels, and retention of BMSCs in the lung. In addition, we assessed whether FoxM1 overexpression promoted the therapeutic effect of BMSCs on ALI/ARDS by differentiating into AT II cells using SPC-/- mice. Furthermore, the protective effect of BMSCs-FoxM1 on lipopolysaccharide-induced endothelial cell (EC) injury was explored by detecting EC proliferation, apoptosis, scratch wounds, tube formation, permeability, and oxidative stress, and analyzing whether the Wnt/ß-catenin pathway contributes to the regulatory mechanism in vitro using a pathway inhibitor. RESULTS: Compared with BMSCs-Vector, treatment with BMSCs-FoxM1 significantly decreased the W/D lung weight ratio, total BALF protein level, lung injury score, oxidative stress, and cytokine levels. With the detected track of BMSCs-FoxM1, we observed a low residency rate and short duration of residency in the lung. Notably, SPC was not expressed in SPC-/- mice injected with BMSCs-FoxM1. Furthermore, BMSCs-FoxM1 enhanced EC proliferation, migration, and tube formation; inhibited EC apoptosis and inflammation; and maintained vascular integrity through activation of the Wnt/ß-catenin pathway, which was partially reversed by XAV-939. CONCLUSION: Overexpression of FoxM1 enhanced the therapeutic effect of BMSCs on ARDS, possibly through a paracrine mechanism rather than by promoting BMSC differentiation into AT II cells in vivo, and prevented LPS-induced EC barrier disruption partially through activating the Wnt/ß-catenin signaling pathway in vitro.

Overexpression of FoxM1 Enhanced the Protective Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Lipopolysaccharide-Induced Acute Lung Injury through the Activation of Wnt/ß-Catenin Signaling.

Background: Mesenchymal stem cell- (MSC-) based cell and gene therapies have made remarkable progress in alleviating acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the benefits of Forkhead box protein M1 (FoxM1) gene-modified MSCs in the treatment of ALI have not been studied. Methods: We evaluated the therapeutic effects of FoxM1-modified MSCs in ALI mice induced by lipopolysaccharide (LPS) by quantifying the survival rate, lung weight ratio (wet/dry), and contents of bronchoalveolar lavage fluid. In addition, microcomputed tomography, histopathology, Evans Blue assay, and quantification of apoptosis were performed. We also explored the underlying mechanism by assessing Wnt/ß-catenin signaling following the treatment of mice with FoxM1-modified MSCs utilizing the Wnt/ß-catenin inhibitor XAV-939. Results: Compared with unmodified MSCs, transplantation of FoxM1-modified MSCs improved survival and vascular permeability; reduced total cell counts, leukocyte counts, total protein concentrations, and inflammatory cytokines in BALF; attenuated lung pathological impairments and fibrosis; and inhibited apoptosis in LPS-induced ALI/ARDS mice. Furthermore, FoxM1-modified MSCs maintained vascular integrity during ALI/ARDS by upregulating Wnt/ß-catenin signaling, which was partly reversed via a pathway inhibitor. Conclusion: Overexpression of FoxM1 optimizes the treatment action of MSCs on ALI/ARDS by inhibiting inflammation and apoptosis and restoring vascular integrity partially through Wnt/ß-catenin signaling pathway stimulation.

Ghrelin pretreatment enhanced the protective effect of bone marrow-derived mesenchymal stem cell-conditioned medium on lipopolysaccharide-induced endothelial cell injury.

BACKGROUND: Lung endothelial barrier injury plays a crucial role in the pathophysiology of acute respiratory distress syndrome. It has been demonstrated that bone marrow-derived mesenchymal stem cells-conditioned medium (BMSCs-CM) and ghrelin have a protective effect. This study investigated if ghrelin pretreatment enhanced the protective effect of BMSCs-CM on lipopolysaccharide (LPS)-induced endothelial cell injury. METHODS: BMSCs were isolated from rat bone marrow, expanded, then phenotypically tested for mesenchymal stem cell-identifying criteria by flow cytometry. The effects of the conditioned medium derived from ghrelin-pretreated BMSCs (BMSCs-ghrelin-pretreated-CM) on LPS-injured endothelial cells were evaluated by migration, apoptosis, permeability, and pro-inflammatory factor (e.g., tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6) assays in endothelial cells. Further, AKT/GSK3ß pathway activation in endothelial cells was examined by Western blot, and the gene expression profiles of ghrelin-pretreated BMSCs were examined by RNA sequencing. RESULTS: BMSCs-ghrelin-pretreated-CM had a greater protective effect on LPS-induced endothelial cell injury than BMSCs-CM by improving cell migration, alleviating apoptosis, and reducing endothelial permeability and the release of pro-inflammatory factors in endothelial cells. The mechanism is partly related to AKT/GSK3ß pathway activation after BMSCs-ghrelin-pretreated-CM treatment. There were five upregulated candidate genes (Wnt5a [i.e., Wnt Family Member 5A], S100b [i.e., S100 Calcium-Binding Protein B], Bmp2 [i.e., Bone Morphogenetic Protein 2], Id4 [i.e., Inhibitor Of DNA Binding 4], and PTHLH [i.e., Parathyroid Hormone Like Hormone]) in BMSCs after ghrelin treatment, and all were associated with AKT pathway activation and endothelial function. CONCLUSIONS: Ghrelin pretreatment enhanced the protective effect of BMSCs-CM on LPS-induced endothelial cell injury, partly by activating the AKT/GSK3ß pathway.

Overexpression of HOXB4 Promotes Protection of Bone Marrow Mesenchymal Stem Cells Against Lipopolysaccharide-Induced Acute Lung Injury Partially Through the Activation of Wnt/ß-Catenin Signaling.

PURPOSE: Pulmonary vascular endothelial cell (EC) injury is recognized as one of the pathological factors of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Bone marrow mesenchymal stem cell (BMSC)-based cytotherapy has attracted substantial attention over recent years as a promising therapeutic approach for ALI/ARDS; however, its use remains limited due to inconsistent efficacy. Currently, gene modification techniques are widely applied to MSCs. In the present study, we aimed to investigate the effect of BMSCs overexpressing Homeobox B4 (HOXB4) on lipopolysaccharide (LPS)-induced EC injury. METHODS: We used LPS to induce EC injury and established EC-BMSC coculture system using transwell chambers. The effect of BMSCs on ECs was explored by detecting EC proliferation, apoptosis, migration, tube formation, and permeability, and determining whether the Wnt/ß-catenin pathway is involved in the regulatory mechanism using XAV-939, inhibitor of Wnt/ ß-catenin. RESULTS: As compared to BMSCWT, BMSCHOXB4 coculture promoted EC proliferation, migration, and tube formation after LPS stimulation and attenuated LPS-induced EC apoptosis and vascular permeability. Mechanistically, BMSCHOXB4 coculture prevented LPS-induced EC injury by activating the Wnt/ß-catenin pathway, which is partially reversible by XAV-939. When cocultured with BMSCHOXB4, pro-inflammatory factors were dramatically decreased and anti-inflammatory factors were greatly increased in the EC medium compared to those in the LPS group (P<0.05). Additionally, when compared to BMSCWT coculture, the BMSCHOXB4 coculture showed an enhanced modulation of IL-6, TNF-α, and IL-10, but there was no statistically significant effect on IL-1ß and IL-4. CONCLUSION: Coculturing of BMSCHOXB4 prevented LPS-induced EC injury by reversing the inactivation of the Wnt/ß-catenin signaling pathway. An in vivo study remains warranted to ascertain whether engraftment of BMSCHOXB4 can be an attractive strategy for the treatment of ALI/ARDS.

Ghrelin protects against lipopolysaccharide-induced acute respiratory distress syndrome through the PI3K/AKT pathway.

Pulmonary endothelial barrier dysfunction is a major pathophysiology observed in acute respiratory distress syndrome (ARDS). Ghrelin, a key regulator of metabolism, has been shown to play protective roles in the respiratory system. However, its effects on lipopolysaccharide (LPS)-induced pulmonary endothelial barrier injury are unknown. In this study, the effects of ghrelin on LPS-induced ARDS and endothelial cell injury were evaluated in vivo and in vitro. In vivo, mice treated with LPS (3 mg/kg intranasal application) were used to establish the ARDS model. Annexin V/propidium iodide apoptosis assay, scratch-wound assay, tube formation assay, transwell permeability assay, and Western blotting experiment were performed to reveal in vitro effects and underlying mechanisms of ghrelin on endothelial barrier function. Our results showed that ghrelin had protective effects on LPS-induced ARDS and endothelial barrier disruption by inhibiting apoptosis, promoting cell migration and tube formation, and activating the PI3K/AKT signaling pathway. Furthermore, ghrelin stabilized LPS-induced endothelial barrier function by decreasing endothelial permeability and increasing the expression of the intercellular junction protein vascular endothelial cadherin. LY294002, a specific inhibitor of the PI3K pathway, reversed the protective effects of ghrelin on the endothelial cell barrier. In conclusion, our findings indicated that ghrelin protected against LPS-induced ARDS by impairing the pulmonary endothelial barrier partly through activating the PI3K/AKT pathway. Thus, ghrelin may be a valuable therapeutic strategy for the prevention or treatment of ARDS.

Association of Diabetes and Admission Blood Glucose Levels with Short-Term Outcomes in Patients with Critical Illnesses.

BACKGROUND: Association of diabetes and admission glucose on the short-term prognosis in patients with critical illnesses are currently ambiguous. We aimed to determine whether diabetes and admission glucose affects short-term prognosis of critically ill patients. METHODS: We performed a retrospective analysis of data on 46,476 critically ill patients from the critical care database. Association of diabetes with 28-day mortality was assessed by inverse probability weighting based on the propensity score. Smoothing splines and threshold effect analysis were applied to explore the relationship between admission glucose and clinical outcomes. RESULTS: Of the 33,680 patients enrolled in the study, 8,701 (25.83%) had diabetes. In the main analysis, the 28-day mortality was reduced by 29% (hazard ratio (HR)=0.71, 95% confidence interval (CI) 0.67-0.76) in patients with diabetes compared to those without diabetes. The E-value of 2.17 indicated robustness to unmeasured confounders. Significant interactions were observed for glucose at ICU admission, admission type, and insulin use (Interaction P <0.05). A V-shaped relationship was observed between admission glucose and 28-day mortality in non-diabetic patients, with the lowest 28-day mortality corresponding to a glucose level of 101.75 mg/dl (95% CI 94.64-105.80 mg/dl), and admission hypoglycemia or hyperglycemia should be avoided, especially in patients admitted to the surgical intensive care unit (SICU), cardiac surgery recovery unit (CSRU), and coronary care unit (CCU); for diabetic patients, elevated admission glucose does not appear to be associated with a poor prognosis and perhaps may be beneficial except for CCU and CSRU. CONCLUSION: The non-detrimental effect of diabetes on the short-term prognosis of critically ill patients was further confirmed, which would reduce 28-day mortality by approximately 29%. For non-diabetic patients, the admission glucose level corresponding to the lowest 28-day mortality was 101.75 mg/dl (95% CI 94.64-105.80 mg/dl); however, for diabetics, the appropriate admission glucose threshold remains unresolved.

Overexpression of FoxM1 promotes differentiation of bone marrow mesenchymal stem cells into alveolar type II cells through activating Wnt/ß-catenin signalling.

BACKGROUND: Acute respiratory distress syndrome (ARDS) becomes a serious challenge in critical care medicine due to the lack of effective therapy. As the damage of alveolar epithelium is a characteristic feature of ARDS, inducing mesenchymal stem cells (MSCs) to differentiate into alveolar epithelial cells turns out to be a promising therapy for ARDS, but the differentiation efficiency is yet to be improved. The study aimed to investigate the effect of overexpressing FoxM1 on MSCs' differentiation into alveolar epithelial cells. METHODS: MSCs were isolated from mouse bone marrow, followed by transfected with lentivirus carrying the FoxM1 plasmid. Small airway epithelial cell growth medium was used as a culture system for inducing MSCs' differentiation into alveolar epithelial cells. Differentiation efficiency was assessed by detecting the expression levels of specific markers of alveolar epithelial cells mainly using quantitative reverse-transcription polymerase chain reaction and Western blot. To examine whether Wnt/ß-catenin signalling was involved in the regulation mechanism, a specific inhibitor of the pathway XAV-939 was used and nuclear and cytoplasmic proteins were also analysed respectively. Co-immunoprecipitation was performed to examine the potential interaction between FoxM1 and ß-catenin. RESULTS: Overexpressing FoxM1 statistically significantly increased the expression levels of specific markers of type II alveolar epithelial cells prosurfactant protein C and surfactant protein B, which was partially reversed by XAV-939 treatment, while the expression levels of specific marker of type I alveolar epithelial cells aquaporin 5 did not change significantly. Overexpressing FoxM1 also increased the nuclear translocation of ß-catenin and its transcriptional activity. A direct interaction between FoxM1 and ß-catenin was found in co-immunoprecipitation assay. CONCLUSION: Overexpression of FoxM1 could improve the efficiency of MSCs' differentiation into type II alveolar epithelial cells partly by activating Wnt/ß-catenin signalling.

Ghrelin attenuates sepsis-induced acute lung injury by inhibiting the NF-κB, iNOS, and Akt signaling in alveolar macrophages.

Ghrelin has proven to be protective against sepsis-induced acute lung injury (ALI) via anti-inflammatory effects. However, its mechanisms remain poorly understood. Alveolar macrophages (AMs) play a key role in mediating inflammatory responses during sepsis-induced ALI by secretion of cytokines and chemokines. This study was undertaken to investigate whether ghrelin suppresses inflammatory effects of AMs and therefore may help to attenuate sepsis-induced ALI. A sepsis model in rats was achieved using cecal ligation and puncture. Ghrelin treatment markedly improved histopathological changes in the lungs and reduced pulmonary inflammation in septic rats. NF-κB translocation and p-Akt and inducible nitric oxide synthase (iNOS) activities in AMs from septic rats were suppressed by ghrelin. In vitro data indicated that ghrelin decreased the levels of LPS-induced IL-1ß, TNF-α, and IL-6, NF-κB translocation, and iNOS and Akt activities of AMs. Furthermore, the NF-κB/iNOS pathway or Akt signaling was positively correlated with LPS-induced inflammatory production of AMs in vitro. In conclusion, ghrelin exerts a protective role against sepsis-induced ALI probably by reducing the production of inflammatory cytokines from AMs via inhibition of the NF-κB/iNOS pathway or Akt signaling.

Effects of Ghrelin on iNOS-Derived NO Promoted LPS-Induced Pulmonary Alveolar Epithelial A549 Cells Apoptosis.

BACKGROUND/AIMS: In the process of abnormal apoptosis of pulmonary alveolar type II epithelial A549 cells in acute respiratory distress syndrome (ARDS), inducible nitric oxide synthase (iNOS) activity in the lung, nitric oxide (NO) production, and the level of protein S-nitrosylation were increased. However, the role of excessive NO production in sepsis-induced ARDS is controversial. Additionally, ghrelin is a growth hormone that exerts an inhibitory role in cell apoptosis. We examined the effect of NO and S-nitrosylation on apoptosis of A549 cells induced by Lipopolysaccharide (LPS) and molecular mechanism underlying the anti-apoptotic effect of ghrelin in this process. METHODS: Flow cytometry and qPCR were used to detect lentiviral infection efficiency and iNOS gene level, respectively. Extracellular and intracellular NO levels were observed by Griess assay kit and DAF-FM DA. Mitochondrial transmembrane potential, apoptosis rate and SNO levels were determined by flow cytometry, Biotin-Switch method and immunofluoresence staining. The expression of iNOS, apoptotic proteins and JNK were assessed by immunoblot analysis. RESULTS: The results showed about two times increase in iNOS expression and intracellular NO levels response to LPS exposure at 24 hours (P< 0.05), while not in extracellular NO levels. NO donors, S-nitroso-N-acetylpenicillamine (SNAP) significantly raised (36.7%, P< 0.05; 38.4%, P< 0.05; 41.8%, P< 0.05) extracellular NO levels without influencing the intracellular NO levels. LPS increased the apoptosis rate (42.4%±2.6% vs 2.8%±1%, P< 0.05) of A549 accompanied by increased Bax levels and decreased Bcl-2 levels through activating JNK signaling, which was reversed when we diminished the iNOS expression in A549 cells using lentiviral vectors encoding iNOS shRNA in the presence of LPS (24.8%±3.8% vs 42.4%±2.6%, P< 0.05). However, the apoptosis rate was increased when SNAP was added (38.8%±1.3% vs 24.8%±3.8%, P< 0.05). Furthermore, we investigated whether ghrelin exert a protective role against LPS-induced apoptosis and the potential mechanism involved in. Ghrelin alone appeared to decrease iNOS expression (32.3%, P< 0.05; 42.3%, P< 0.05), which showed no signifiant difference between LPS+ghrelin group and LPS group. However, this study showed that ghrelin decreased the intracellular NO production (38.9%, P< 0.05), protein S-nitrosylation levels (33.5%, P< 0.05), Bax protein expression (70.2%, P< 0.05), whereas increasing Bcl-2 protein expression (14.1%, P< 0.05) and mitochondrial transmembrane potential (∆ΨM) (20.7%, P< 0.05) in the presence of LPS. CONCLUSION: The data suggested that NO derived from iNOS induced by LPS stimulation exerts an important role in promoting apoptosis of A549 cells, and ghrelin abolished intracellular NO production and protein S-nitrosylation levels, abrogating the apoptosis of A549 cells partly through inhibiting mitochondrial-dependent pathways.

Effects of ghrelin on the apoptosis of human neutrophils in vitro.

Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation and the diffuse infiltration of neutrophils into the alveolar space. Neutrophils are abundant, short-lived leukocytes that play a key role in immune defense against microbial infections. These cells die via apoptosis following the activation and uptake of microbes, and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter pathogens. Apoptosis is essential for the removal of neutrophils from inflamed tissues and for the timely resolution of neutrophilic inflammation. Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue receptor, produced and secreted mainly from the stomach. Previous studies have reported that ghrelin exerts anti-inflammatory effects in lung injury through the regulation of the apoptosis of different cell types; however, the ability of ghrelin to regulate alveolar neutrophil apoptosis remains largely undefined. We hypothesized that ghrelin may have the ability to modulate neutrophil apoptosis. In this study, to examine this hypothesis, we investigated the effects of ghrelin on freshly isolated neutrophils in vitro. Our findings demonstrated a decrease in the apoptotic ratio (as shown by flow cytometry), as well as in the percentage of cells with decreased mitochondrial membrane potential (ΔΨm) and in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick­end labeling-positive rate, accompanied by an increased B-cell lymphoma 2/Bax ratio and the downregulation of cleaved caspase-3 in neutrophils following exposure to lipopolysaccharide (100 ng/ml). However, pre-treatment with ghrelin at a physiological level (100 nM) did not have a notable influence on the neutrophils in all the aforementioned tests. Our findings suggest that ghrelin may not possess the ability to modulate the neutrophil lifespan in vitro.

Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/ß-catenin signaling and suppresses lung inflammation.

Alveolar macrophages (AMs) undergo increased apoptosis during sepsis-induced acute respiratory distress syndrome (ARDS). Ghrelin exhibits an antiapoptotic effect in several cell types and protects against sepsis-induced ARDS in rats; however, the molecular mechanisms underlying this antiapoptotic effect remain poorly understood. In this study, we first examined the antiapoptotic effect of ghrelin on lipopolysaccharide (LPS)-stimulated AMs in vitro. In AMs, GH secretagogue receptor-1a (GHSR-1a), the ghrelin receptor, was expressed, and treatment of AMs with ghrelin markedly reduced LPS-induced apoptosis, mitochondrial transmembrane potential decrease, and cytochrome c release. These effects of ghrelin were mediated by GHSR-1a because a GHSR-1a-targeting small interfering RNA abolished the antiapoptotic action of ghrelin. LPS treatment activated the c-Jun N-terminal kinase (JNK) signaling pathway but inhibited the Wnt/ß-catenin pathway. Interestingly, combined LPS-ghrelin treatment reduced JNK activation and increased Wnt/ß-catenin activation. Furthermore, like ghrelin treatment, the addition of the JNK inhibitor SP600125 or the glycogen synthase kinase-3ß inhibitor SB216763 rescued AMs from apoptosis. We also demonstrated that ghrelin altered the balance of Bcl-2-family proteins and inhibited caspase-3 activity. Next, we investigated whether ghrelin protected against septic ARDS in vivo. Sepsis was induced in male rats by performing cecal ligation and puncture; administration of ghrelin reduced sepsis-induced AMs apoptosis, pulmonary injury, protein concentrations in the bronchoalveolar lavage fluid, the lung neutrophil infiltration, and wet to dry weight ratio. However, administration of a specific ghrelin-receptor antagonist, [D-Lys-3]-GH-releasing peptide-6, abolished the beneficial effects of ghrelin. Collectively our results suggest that ghrelin exerts an antiapoptotic effect on AMs at least partly by inhibiting JNK and activating the Wnt/ß-catenin pathway and thereby helps alleviate septic ARDS in rats.

Bone marrow mesenchymal stem cells protect alveolar macrophages from lipopolysaccharide-induced apoptosis partially by inhibiting the Wnt/ß-catenin pathway.

Apoptosis of alveolar macrophages (AMs) plays a pathogenic role in acute lung injury (ALI) and its severe type, acute respiratory distress syndrome (ARDS). Mesenchymal stem cells (MSCs) are promising therapeutic cells for preventing apoptosis and eliminating cellular injury. We investigated the effects of rat bone marrow mesenchymal stem cells (BMSCs) on lipopolysaccharide (LPS)-induced apoptosis in AMs using transwell experiments, and examined the underlying mechanisms LPS induced AMs apoptosis in a dose- and time-dependent fashion, whereas BMSCs reduced AMs apoptosis when co-cultured at appropriate ratios. BMSCs decreased expression of cleaved caspase-3 and the pro-apoptotic protein, Bax, whilst increased levels of the anti-apoptotic protein, Bcl-2, prolonging the lifespan of AMs in vitro. Promotion of AMs survival by BMSCs required down-regulation of p-GSK-3ß and ß-catenin in AMs. The anti-apoptosis action of BMSCs was reversed by SB216763, a specific inhibitor of GSK-3ß that also activates Wnt/ß-catenin signaling. In conclusion, BMSCs can attenuate AM apoptosis partially by suppressing the Wnt/ß-catenin pathway.

Ghrelin attenuates sepsis-associated acute lung injury oxidative stress in rats.

This study investigated the effect of ghrelin on oxidative stress in septic rat lung tissue. Male Sprague-Dawley rats were divided into sham-operation, sepsis, and ghrelin groups. Sepsis was induced by cecal ligation and puncture. Ghrelin was administered intraperitoneally at 3 and 15 h post-operation. Bronchoalveolar lavage was performed to collect alveolar macrophages (AMs). Inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in alveolar macrophages and iNOS protein levels were measured by reverse transcription PCR (RT-PCR) and Western blot. Pulmonary pathology was analyzed and nitrotyrosine expression was examined by immunohistochemistry. Plasma superoxide dismutase (SOD) and lung wet/dry weight were measured. In the sepsis group, iNOS mRNA expression in AMs was 1.33 ± 0.05, 1.44 ± 0.08, and 1.57 ± 0.11 at 6, 12, and 20 h post-surgery, respectively, and were higher compared with the sham-operation group (p<0.05). No increase was observed at longer time points. iNOS mRNA expression in the sepsis group was lower compared with the ghrelin group (2.27 ± 0.37) (p<0.05) at 20 h post-surgery. iNOS protein levels in the ghrelin group (0.87 ± 0.03, p<0.05) were lower than in the sepsis group at 20 h. Ghrelin group pathological scores were lower than in the sepsis group (p<0.05). Plasma SOD was slightly non-significantly decreased in the ghrelin group. No difference was observed in lung wet/dry weight ratios between sepsis and ghrelin groups. iNOS mRNA expression in AMs was elevated between 6 and 20 h after cecal ligation and puncture (CLP), but did not progress. Ghrelin attenuated pulmonary iNOS protein expression and tended to increase plasma SOD activity. Ghrelin suppressed pulmonary nitrosative stress in septic rats, but did not improve lung wet/dry weight ratios.

[The effects of rosiglitazone combined ceftazidime on peroxisome proliferator activated receptor γ activity and interleukin in septic rats].

OBJECTIVE: To observe the effects of rosiglitazone (RSG) and ceftazidime (CAZ) on peroxisome proliferator activated receptor γ (PPARγ) activity in nucleated cells and interleukin (IL-4, IL-6) levels in plasma in septic rats. METHODS: According to randomized digital table, 180 male Sprague-Dawley (SD) rats were assigned to control group, sham operation group, sepsis group, CAZ group, RSG group and combined CAZ and RSG group. Sepsis model was established by cecal ligation and puncture (CLP). Drugs were administered by intraperitoneal injection at 3-hour post-operation, once every 12-hour. The PPARγ activity in nucleated cells and IL-4, IL-6 levels in plasma were detected by enzyme linked immunosorbent assay (ELISA) at 12, 24 and 48 hours post-operation. RESULTS: There was no difference in PPARγ activity and levels of IL-4 and IL-6 at each time point post-operation between control group and sham operation group. Compared with control group and sham operation group, PPARγ activity [absorbance (A) value] in nucleated cells in sepsis group, where downward trend was seen as time went on, significantly reduced (0.263±0.017 vs. 0.292±0.005, 0.294±0.007, both P<0.05). PPARγ activity was significantly higher in CAZ group, RSG group and CAZ + RSG group than in sepsis group (0.282±0.008, 0.336±0.020, 0.347±0.007 vs. 0.263±0.017, all P<0.05), CAZ + RSG group>RSG group >CAZ group (both P<0.05). Plasma IL-6 and IL-4 levels were higher in sepsis group than in control group and sham operation group (IL-6: 436.77±62.28 ng/L vs. 45.11±10.42 ng/L, 42.28±7.54 ng/L; IL-4: 89.24±25.06 ng/L vs. 41.34±7.08 ng/L, 41.49±7.27 ng/L, all P<0.05) and reached peak at 24 hours and 48 hours post-operation, respectively. Compared with sepsis group, IL-6 and IL-4 levels in CAZ group, RSG group and CAZ + RSG group were significantly decreased (IL-6: 273.48±12.13 ng/L, 317.64±14.10 ng/L, 253.94±13.57 ng/L vs. 436.77±62.28 ng/L; IL-4: 59.12±7.03 ng/L, 68.37±8.28 ng/L, 53.81±8.34 ng/L vs. 89.24±25.06 ng/L, all P<0.05), CAZ + RSG group < CAZ group < RSG group (all P<0.05). CONCLUSION: In septic rats, PPARγ activity in nucleated cells was decreased. On the basis of effective antibiotic treatment, RSG might play a role in improving PPARγ activity in nucleated cells and reducing the levels of inflammation mediators and anti-inflammatory in plasma.

[The relationship of peroxisome proliferator activated receptor γ ligands activity in nucleated cell and interleukin-6 level in septic rats].

OBJECTIVE: To observe the relationship between activity of peroxisome proliferator activated receptor γ (PPARγ) in nucleated cell and level of pro-inflammatory mediator interleukin-6 (IL-6) in plasma of rats with sepsis. METHODS: According to the random number table, 90 male Sprague-Dawley (SD) rats were randomly divided into three groups, namely control group, sham operation group and sepsis group. Each group was further divided into three subgroups according to postoperative time points, i.e. 12, 24 and 48-hour subgroups. Each subgroup consisted of 10 rats. Sepsis was reproduced by cecal ligation and puncture (CLP). The PPARγ activity in nucleated cells and IL-6 level in plasma were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The PPARγ activity in nucleated cells was significantly decreased at 12, 24 and 48 hours in sepsis group (A value: 0.279±0.004, 0.264±0.009, 0.245±0.012) compared with control group (0.292±0.007, 0.293±0.004, 0.293±0.005) and sham operation group (0.295±0.008, 0.295±0.006, 0.294±0.007), while the IL-6 level was significantly increased in sepsis group (ng/L: 365.25±15.53, 507.16±20.86, 437.89±25.09) compared with control group (43.54±11.10, 48.82±10.62, 42.96±9.52) and sham operation group (42.43±6.77, 40.32±6.48, 44.10±9.36, all P<0.05). When septic condition became worse, the PPARγ activity in nucleated cells of sepsis group lowered, and IL-6 level was gradually elevated after operation, reaching the peak at 24 hours, and then gradually lowered, and the difference of the value between any two time points was all statistically significant (all P<0.05). There was a negative correlation between the PPARγ activity in nucleated cells and IL-6 level in 12-hour subgroup of sepsis group (r=-0.703, P=0.023). CONCLUSION: In septic rats, the PPARγ activity in nucleated cells was lowered while the pro-inflammatory mediator IL-6 level in plasma elevated, and there was a negative correlation between PPARγ activity and IL-6 level.

Role of TNF-α, sTNF-R55 and sTNF-R75 in inflammation of acute exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Although some recent studies have demonstrated the important role of tumor necrosis factor-α (TNF-α) and soluble TNF receptors (sTNF-R) in inflammation of chronic obstructive pulmonary disease, the exact roles of TNF-α and sTNF-R as well as their interaction remained unclear. OBJECTIVES: To study changes in levels of systemic and airway local TNF-α and sTNF-R (sTNF-R55, sTNF-R75) in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), and clarify the relationship between these mediators and airflow limitation in AECOPD patients. METHODS: TNF-α, sTNF-R55 and sTNF-R75 levels in induced sputum and plasma as well as lung functions were examined in 48 AECOPD patients before and after treatment. Samples from 28 healthy volunteers served as controls. RESULTS: Compared to healthycontrols, both pre- and posttreatment levels of TNF-α, sTNF-R55 and sTNF-R75 in induced sputum and plasma of COPD patients were higher. In patients with AECOPD, posttreatment TNF-α levels significantly decreased compared to pretreatment levels (0.6 ± 0.46 vs. 0.82 ± 0.35 µg/l in plasma, p < 0.01; 0.48 ± 0.27 vs. 0.82 ± 0.34 µg/l in sputum, p < 0.001). While posttreatment sTNF-R55 and sTNF-R75 levels increased in both kinds of samples, mediator levels in plasma and lung functions were unrelated (p > 0.05). sTNF-R55 and sTNF-R75 levels in induced sputum were positively correlated with lung functions (p < 0.05), while TNF-α levels were negatively correlated with forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to FEV1 predicted value. CONCLUSIONS: Inflammatory and anti-inflammatory mediators were imbalanced in the airways of AECOPD patients. It was local inflammation but not systemic inflammation that was closely related to airflow limitation.

[Oxidative stress status in patients with chronic obstructive pulmonary disease and its relation to glucocorticoid receptor levels].

OBJECTIVE: To study changes in the levels of systematic and airway local oxidative stress in patients in different stages of chronic obstructive pulmonary diseases (COPD), and explore the association between oxidative stress and glucocorticoid receptor (GR) level in the peripheral blood leukocytes. METHODS: The levels of malonaldehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in induced sputum and plasma, as well as GR levels in peripheral blood leukocytes and plasma levels of cortisol and adrenocorticotrophic hormone (ACTH), were examined in 33 patients with acute exacerbations of COPD (AECOPD, group A), 27 with stable COPD (group B), and 28 healthy volunteers (including 15 smokers as group C, and 15 nonsmokers as group D). RESULTS: MDA level in induced sputum and plasma decreased, whereas the levels of GSH, SOD and GSH-PX increased significantly in the order of groups A, B, C, and D (P<0.05). The activity of SOD in induced sputum and plasma were significantly lower in group C than in group D. No significant difference was noted in the other oxidative stress indices between groups C and D (P>0.05). The plasma levels of cortisol and ACTH showed no significant difference between the 4 groups, while the GR level in peripheral blood leukocytes increased significantly in the order of groups A, B, C and D (1565-/+719, 2069-/+488, 2739-/+926, and 4793 -/+1415 U, respectively, P<0.05). After controlling for the factor of smoking status, the plasma and sputum SOD activity were both positively correlated to GR, with the partial correlation coefficient of 0.512 and 0.564, respectively (P<0.001). CONCLUSION: Patients in different stages of COPD, especially those with AECOPD, may sustain systematic and local oxidation and anti-oxidation imbalance. Decreased SOD activity may contribute to GR level decrement in peripheral blood leukocytes in these patients.

[A case control study on the impact of CYP450 MSPI and GST-M1 polymorphisms on the risk of lung cancer].

OBJECTIVE: To study the correlation of polymorphisms of CYP1A1 MSPI and glutathiones S-transferase (GST-M1) independently and in combination with the risk of lung cancer. METHODS: A case control study which included 91 cases of lung cancer and 138 controls collected from the First Affiliated Hospital of Sun Yat-sen University of Medical Sciences, Guangzhou Tumor Hospital and The Red Cross Hospital of Guangzhou or conmunity area. All subjects were investigated with a uniform questionnaire. Blood samples were collected from all cases and controls for detecting CYP1A1 MSPI and GST-M1 polymorphisms which were analyzed by PCR and RFLP. RESULTS: It showed that there was no significant difference in frequencies of this genotypes of CYP1A1 MSPI between the two groups. The frequency of GST-M1 null (0/0) genotype was higher in the case group than in the control group, with an OR of 1.38 (95% CI 0.81 - 2.38), but there was no statistical significance. However, combination of several genotypes was strongly associated with lung cancer. There was a synergistic interaction between the m2m2 genotype of CYP1A1 MSPI and GST-M1 (0/0) genotype, with an OR of 2.47 (95% CI 1.03 - 5.90). CONCLUSION: The combination of two genetic polymorphisms significantly increases the risk of lung cancer.