Matrine Attenuates Lung Injury by Modulating Macrophage Polarization and Suppressing Apoptosis.

INTRODUCTION: Persistent lung inflammation is a characteristic of sepsis-induced lung injury. Matrine, the active ingredient from Sophora flavescens, has exhibited anti-inflammatory activities. This study investigated the effects of prophylactic administration of matrine on macrophage polarization, apoptosis, and tissue injury in a cecal ligation and puncture (CLP)-induced murine lung injury model. METHODS: Mice were randomly allocated into four groups: Sham, CLP, Sham + Matrine, and CLP + Matrine. Lung tissues were collected at 24 h post-CLP. Histopathology and immunofluorescence analysis were performed to evaluate lung injury and macrophage infiltration in the lung, respectively. Caspase-3 activities, TUNEL staining, and anti-apoptotic proteins were examined to assess apoptosis. To determine the mechanism of action of matrine, protein levels of Sirtuin 1 (SIRT1), nuclear factor κB (NF-κB), p53 and the messenger RNA levels of p53-mediated proapoptotic genes were examined to elucidate the associated signaling pathways. RESULTS: Histopathological evaluation showed that matrine prophylaxis attenuated sepsis-induced lung injury. Matrine prophylaxis attenuated sepsis-induced infiltration of the total population of macrophages in the lung. Matrine inhibited M1 macrophage infiltration, but increased M2 macrophage infiltration, thus resulting in a decrease in the proportion of M1 to M2 macrophages in septic lung. Sepsis-induced lung injury was associated with apoptotic cell death as evidenced by increases in caspase-3 activity, TUNEL-positive cells, and decreases in antiapoptotic proteins, all of which were reversed by matrine prophylaxis. Matrine restored sepsis-induced downregulation of SIRT1 and deacetylation of NF-κB p65 subunit and p53, thus inactivating NF-κB pathway and suppressing p53-induced proapoptotic pathway in septic lung. CONCLUSIONS: In summary, this study demonstrated that matrine exhibited pro-M2 macrophage polarization and antiapoptotic effects in sepsis-induced lung injury, which might be, at least partly, due to the modulation of SIRT1/NF-κB and SIRT1/p53 pathways.

Exercise training ameliorates early diabetic kidney injury by regulating the H2 S/SIRT1/p53 pathway.

Exercise training exerts protective effects against diabetic nephropathy. This study aimed to investigate whether exercise training could attenuate diabetic renal injury via regulating endogenous hydrogen sulfide (H2 S) production. First, C57BL/6 mice were allocated into the control, diabetes, exercise, and diabetes + exercise groups. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). Treadmill exercise continued for four weeks. Second, mice was allocated into the control, diabetes, H2 S and diabetes + H2 S groups. H2 S donor sodium hydrosulfide (NaHS) was intraperitoneally injected once daily for four weeks. STZ-induced diabetic mice exhibited glomerular hypertrophy, tissue fibrosis and increased urine albumin levels, urine protein- and albumin-to-creatinine ratios, which were relieved by exercise training. Diabetic renal injury was associated with apoptotic cell death, as evidenced by the enhanced caspase-3 activity, the increased TdT-mediated dUTP nick-end labeling -positive cells and the reduced expression of anti-apoptotic proteins, all of which were attenuated by exercise training. Exercise training enhanced renal sirtuin 1 (SIRT1) expression in diabetic mice, accompanied by an inhibition of the p53-#ediated pro-apoptotic pathway. Furthermore, exercise training restored the STZ-mediated downregulation of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) and the reduced renal H2 S production. NaHS treatment restored SIRT1 expression, inhibited the p53-mediated pro-apoptotic pathway and attenuated diabetes-associated apoptosis and renal injury. In high glucose-treated MPC5 podocytes, NaHS treatment inhibited the p53-mediated pro-apoptotic pathway and podocyte apoptosis in a SIRT1-dependent manner. Collectively, exercise training upregulated CBS/CSE expression and enhanced the endogenous H2 S production in renal tissues, thereby contributing to the modulation of the SIRT1/p53 apoptosis pathway and improvement of diabetic nephropathy.

[Establishment of a three-dimensional organoid culture system for mouse type 2 alveolar epithelial cells].

The purpose of this study was to establish a three-dimensional (3D) organoid culture system for type 2 alveolar epithelial (AT2) cells in mice. AT2 cells were isolated from ICR mouse lung and purified by enzymatic digestion and MicroBeads sorting. The purity of AT2 cells was determined by immunofluorescence (IF) staining using an antibody against proSPC. The AT2 differentiation was examined by IF staining with proSPC/HopX and proSPC/T1α antibodies, and proliferation of AT2 cells was assessed by EdU incorporation assays after two-dimensional (2D) culture for 8 days. In addition, AT2 cells were co-cultured with mouse lung fibroblasts (Mlg) in three-dimensional (3D) culture system. After 13 days of co-culture, the organoids were fixed in 2% paraformaldehyde for histological analysis and IF staining. The results showed that the purity of the AT2 cells was over 95%, as assessed by proSPC staining. 2D cultured AT2 cells were negative for EdU staining, which indicates that no proliferation occurs. proSPC expression was gradually disappeared, whereas T1α and HopX expression was gradually increased after 3, 5 and 8 days of culture. In 3D culture system, the alveolar organoids were formed after co-culturing AT2 cells with Mlg for 4 days. Histological analysis showed that alveolar organoids displayed a hollow morphology. proSPC was highly expressed in the peripheral cells, whereas type 1 alveolar epithelial (AT1) cells transdifferentiated from AT2 cells expressing HopX were mainly located in the interior of organoid bodies after 13 days. Some of the proSPC-positive AT2 cells located in the outer circle of alveolar organoids were stained positive for both proSPC and EdU, indicating that the AT2 cells in the alveolar organoids were proliferative. These results showed that the 3D organoid culture system of mouse AT2 cells was successfully established.

Elevated angiotensin II induces platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner during sepsis.

Thrombocytopenia is independently related with increased mortality in severe septic patients. Renin-angiotensin system (RAS) is elevated in septic subjects; accumulating studies show that angiotensin II (Ang II) stimulate the intrinsic apoptosis pathway by promoting reactive oxygen species (ROS) production. However, the mechanisms underlying the relationship of platelet apoptosis and RAS system in sepsis have not been fully elucidated. The present study aimed to elucidate whether the RAS was involved in the pathogenesis of sepsis-associated thrombocytopenia and explore the underlying mechanisms. We found that elevated plasma Ang II was associated with decreased platelet count in both patients with sepsis and experimental animals exposed to lipopolysaccharide (LPS). Besides, Ang II treatment induced platelet apoptosis in a concentration-dependent manner in primary isolated platelets, which was blocked by angiotensin II type 1 receptor (AT1R) antagonist losartan, but not by angiotensin II type 2 receptor (AT2R) antagonist PD123319. Moreover, inhibiting AT1R by losartan attenuated LPS-induced platelet apoptosis and alleviated sepsis-associated thrombocytopenia. Furthermore, Ang II treatment induced oxidative stress level in a concentration-dependent manner in primary isolated platelets, which was partially reversed by the AT1R antagonist losartan. The present study demonstrated that elevated Ang II directly stimulated platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner in sepsis-associated thrombocytopenia. The results would helpful for understanding the role of RAS system in sepsis-associated thrombocytopenia.

An Integrated Microarray Analysis Reveals Significant Diagnostic and Prognostic Biomarkers in Pancreatic Cancer.

BACKGROUND Pancreatic cancer (PAC) is a lethal cancer and it is essential to develop accurate diagnostic and prognostic biomarkers for PAC. MATERIAL AND METHODS An integrated microarray analysis of PAC was conducted to identify differentially expressed genes (DEGs) between PAC and non-tumor controls. Expression of DEGs were further confirmed by The Cancer Genome Atlas and the Genotype-Tissue Expression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein-protein integration network construction were performed to further research the biological functions of DEGs. Receiver-operating characteristic analysis and survival analysis were used to evaluate the diagnostic and prognostic value of DEGs for PAC. RESULTS Seventeen microarray datasets were downloaded from Gene Expression Omnibus to conduct the integrated microarray analysis. A total of 1136 DEGs (596 upregulated and 540 downregulated DEGs) in PAC tissues compared with non-tumor controls were identified. Pancreatic secretion (Kegg: 04972), insulin signaling pathway (Kegg: 04910), and several cancer-related pathways including pathways in cancer (Kegg: 05200), MAPK signaling pathway (Kegg: 04010), and pancreatic cancer (Kegg: 05212) were enriched for DEGs in PAC. Seven DEGs (AHNAK2, CDH3, IFI27, ITGA2, LAMB3, SLC6A14, and TMPRSS4) were found to have both great diagnostic and prognostic value for PAC. High expression of these 7 DEGs were significantly associated with poor prognosis of patients with PAC. CONCLUSIONS These 7 DEGs might be potential diagnostic and prognostic biomarkers for PAC and help uncovering the mechanism of PAC.

Downregulation of R-Spondin1 Contributes to Mechanical Stretch-Induced Lung Injury.

OBJECTIVES: The R-spondin family attenuates tissue damage via tightening endothelium and preventing vascular leakage. This study aims to investigate whether R-spondins protect against mechanical stretch-induced endothelial dysfunction and lung injury and to reveal the underlying mechanisms. DESIGN: Randomized controlled study. SETTING: University research laboratory. SUBJECTS: Patients scheduled to undergo surgery with mechanical ventilation support. Adult male Institute of Cancer Research mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Patients underwent a surgical procedure with mechanical ventilation support of 3 hours or more. Mice were subjected to mechanical ventilation (6 or 30 mL/kg) for 0.5-4 hours. Another group of mice were intraperitoneally injected with 1 mg/kg lipopolysaccharide, and 12 hours later subjected to mechanical ventilation (10 mL/kg) for 4 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 4 hours. MEASUREMENTS AND MAIN RESULTS: R-spondin1 were downregulated in both surgical patients and experimental animals exposed to mechanical ventilation. Intratracheal instillation of R-spondin1 attenuated, whereas knockdown of pulmonary R-spondin1 exacerbated ventilator-induced lung injury and mechanical stretch-induced lung vascular endothelial cell apoptosis. The antiapoptotic effect of R-spondin1 was mediated through the leucine-rich repeat containing G-protein coupled receptor 5 in cyclic stretched mouse lung vascular endothelial cells. We identified apoptosis-stimulating protein of p53 2 as the intracellular signaling protein interacted with leucine-rich repeat containing G-protein coupled receptor 5. R-spondin1 treatment decreased the interaction of apoptosis-stimulating protein of p53 2 with p53 while increased the binding of apoptosis-stimulating protein of p53 2 to leucine-rich repeat containing G-protein coupled receptor 5, therefore resulting in inactivation of p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. CONCLUSIONS: Mechanical ventilation leads to down-regulation of R-spondin1. R-spondin1 may enhance the interaction of leucine-rich repeat containing G-protein coupled receptor 5 and apoptosis-stimulating protein of p53 2, thus inactivating p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. R-spondin1 may have clinical benefit in alleviating mechanical ventilator-induced lung injury.

Probucol ameliorates EMT and lung fibrosis through restoration of SIRT3 expression.

Pulmonary fibrosis is a progressive fibrotic lung disease with a paucity of therapeutic options. Here we investigated the potential roles of probucol, a cholesterol-lowering drug with potent anti-oxidation properties, on pulmonary epithelial-mesenchymal transition (EMT) and fibrosis. We found that bleomycin-induced lung fibrosis was associated with increased transforming growth factor (TGF)-ß1, α-smooth muscle actin (α-SMA) and decreased E-cadherin expression in lung tissues, indicating EMT formation. Bleomycin treatment resulted in an induction of oxidative stress in lung tissues. Probucol treatment attenuated bleomycin-induced TGF-ß1 production, EMT and pulmonary fibrosis, meanwhile it suppressed bleomycin-induced oxidative stress. Bleomycin treatment resulted in decreases in protein expressions of Sirtuin 3 (SIRT3) in the lung, which were restored by ROS scavenger NAC and probucol treatment, suggesting that probucol might restore SIRT3 expression by suppressing bleomycin-induced oxidative stress. In the mouse alveolar type II epithelial cell line MLE-12, probucol treatment leads to an increase in SIRT3 expression in bleomycin-treated AT-II cells, which might contribute to the inhibitory effect of probucol on EMT through suppressing hypoxia inducible factor (HIF)-1α/TGF-ß1 pathway. In addition, probucol inhibited bleomycin-induced macrophage infiltration in the lung. Bleomycin decreased SIRT3 protein expression, whereas increased HIF-1α activation and TGF-ß1 release in the mouse macrophage cell line RAW264.7, which were attenuated by probucol treatment. Taken together, the present study suggests that probucol may ameliorate EMT and lung fibrosis through restoration of SIRT3 expression. The data obtained in this study provides proof for the idea that probucol may be a potential therapeutic option for the treatment of pulmonary fibrosis.

NLRP3 Inflammasome Activation Contributes to Mechanical Stretch-Induced Endothelial-Mesenchymal Transition and Pulmonary Fibrosis.

OBJECTIVES: Mechanical ventilation can induce lung fibrosis. This study aimed to investigate whether ventilator-induced lung fibrosis was associated with endothelial-mesenchymal transition and to uncover the underlying mechanisms. DESIGN: Randomized, controlled animal study and cell culture study. SETTING: University research laboratory. SUBJECTS: Adult male Institute of Cancer Research, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) knockout and wild-type mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Institute of Cancer Research, NLRP3 knockout and wild-type mice were subjected to mechanical ventilation (20 mL/kg) for 2 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 24 hours. MEASUREMENTS AND MAIN RESULTS: Mice subjected to mechanical ventilation exhibited increases in collagen deposition, hydroxyproline and type I collagen contents, and transforming growth factor-ß1 in lung tissues. Ventilation-induced lung fibrosis was associated with increased expression of mesenchymal markers (α smooth muscle actin and vimentin), as well as decreased expression of endothelial markers (vascular endothelial-cadherin and CD31). Double immunofluorescence staining showed the colocalization of CD31/α smooth muscle actin, CD31/vimentin, and CD31/fibroblast-specific protein-1 in lung tissues, indicating endothelial-mesenchymal transition formation. Mechanical ventilation also induced NLRP3 inflammasome activation in lung tissues. In vitro direct mechanical stretch of primary mouse lung vascular endothelial cells resulted in similar NLRP3 activation and endothelial-mesenchymal transition formation, which were prevented by NLRP3 knockdown. Furthermore, mechanical stretch-induced endothelial-mesenchymal transition and pulmonary fibrosis were ameliorated in NLRP3-deficient mice as compared to wild-type littermates. CONCLUSIONS: Mechanical stretch may promote endothelial-mesenchymal transition and pulmonary fibrosis through a NLRP3-dependent pathway. The inhibition of endothelial-mesenchymal transition by NLRP3 inactivation may be a viable therapeutic strategy against pulmonary fibrosis associated with mechanical ventilation.

Salidroside Attenuates Ventilation Induced Lung Injury via SIRT1-Dependent Inhibition of NLRP3 Inflammasome.

BACKGROUND: Salidroside (SDS) is the main effective ingredient of Rhodiola rosea L with a variety of pharmacologic properties. We aim to investigate the effects of SDS on ventilation induced lung injury (VILI) and explore the possible underlying molecular mechanism. METHODS: Lung injury was induced in male ICR mice via mechanical ventilation (30 ml/kg) for 4h. The mice were divided in four groups:(1) Control group; (2) Ventilation group; (3) SDS group; (4) Ventilation with SDS group. SDS (50 mg/kg) was injected intraperitoneally 1h before operation. Mouse lung vascular endothelial cells (MLVECs) were subjected to cyclic stretch for 4h. RESULTS: It was found that SDS attenuated VILI as shown in HE staining, cell count and protein content levels in BAL fluid, W/D and Evans blue dye leakage into the lung tissue. SDS treatment inhibited the activation of NLRP3 inflammasome and subsequent caspase-1 cleavage as well as interleukin (IL)-1ß secretion both in vivo and in vitro. Moreover, SDS administration up-regulated SIRT1 expression. Importantly, knockdown of SIRT1 reversed the inhibitory effect of SDS on NLRP3 inflammasome activation. CONCLUSIONS: Taken together, these findings indicate that SDS may confer protection against ventilation induced lung injury via SIRT1-de-pendent inhibition of NLRP3 inflammasome activation.

Fruit and vegetable consumption and risk of cardiovascular disease: A meta-analysis of prospective cohort studies.

A meta-analysis of prospective cohort studies was conducted to examine the relation between fruit and vegetables (FV) consumption and the risk of cardiovascular disease (CVD). We searched PubMed and EMBASE up to June 2014 for relevant studies. Pooled relative risks (RRs) were calculated and dose-response relationship was assessed. Thirty-eight studies, consisting of 47 independent cohorts, were eligible in this meta-analysis. There were 1,498,909 participants (44,013 CVD events) with a median follow-up of 10.5 years. The pooled RR (95% confidence interval) of CVD for the highest versus lowest category was 0.83 (0.79-0.86) for FV consumption, 0.84 (0.79-0.88) for fruit consumption, and 0.87 (0.83-0.91) for vegetable consumption, respectively. Dose-response analysis showed that those eating 800 g per day of FV consumption had the lowest risk of CVD. Our results indicate that increased FV intake is inversely associated with the risk of CVD. This meta-analysis provides strong support for the current recommendations to consume a high amount of FV to reduce CVD risk.

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-ß1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-ß1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

H2S Attenuates LPS-Induced Acute Lung Injury by Reducing Oxidative/Nitrative Stress and Inflammation.

BACKGROUND: Hydrogen sulfide (H2S), known as the third endogenous gaseous transmitter, has received increasing attention because of its diverse effects, including angiogenesis, vascular relaxation and myocardial protection.We aimed to investigate the role of H2S in oxidative/nitrative stress and inflammation in acute lung injury (ALI) induced by endotoxemia. METHODS: Male ICR mice were divided in six groups: (1) Control group; (2) GYY4137treatment group; (3) L-NAME treatment group; (4) lipopolysaccharide (LPS) treatment group; (5) LPS with GYY4137 treatment group; and (6) LPS with L-NAME treatment group. The lungs were analysed by histology, NO production in the mouse lungs determined by modified Griess (Sigma-Aldrich) reaction, cytokine levels utilizing commercialkits, and protein abundance by Western blotting. RESULTS: GYY4137, a slowly-releasing H2S donor, improved the histopathological changes in the lungs of endotoxemic mice. Treatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, increased anti-oxidant biomarkers such as thetotal antioxidant capacity (T-AOC) and theactivities of catalase (CAT) and superoxide dismutase (SOD) but decreased a marker of peroxynitrite (ONOO-) action and 3-nitrotyrosine (3-NT) in endotoxemic lung. L-NAME administration also suppressed inflammation in endotoxemic lung, as evidenced by the decreased pulmonary levels of interleukin (IL)-6, IL-8, and myeloperoxidase (MPO) and the increased level of anti-inflammatory cytokine IL-10. GYY4137 treatment reversed endotoxin-induced oxidative/nitrative stress, as evidenced by a decrease in malondialdehyde (MDA), hydrogenperoxide (H2O2) and 3-NT and an increase in the antioxidant biomarker ratio of reduced/oxidized glutathione(GSH/GSSG ratio) and T-AOC, CAT and SOD activity. GYY4137 also attenuated endotoxin-induced lung inflammation. Moreover, treatment with GYY4137 inhibited inducible NOS (iNOS) expression and nitric oxide (NO) production in the endotoxemia lung. CONCLUSIONS: GYY4137 conferred protection against acute endotoxemia-associated lung injury, which may have beendue to the anti-oxidant, anti-nitrative and anti-inflammatory properties of GYY4137. The present findings warrant further exploration of the clinical applicability of H2S in the prevention and treatment of ALI.

Resveratrol alleviates endotoxemia-associated adrenal insufficiency by suppressing oxidative/nitrative stress.

We have recently demonstrated that endotoxin causes oxidative stress and overproduction of nitric oxide in adrenal glands, thereby leading to adrenocortical insufficiency. The aim of this study is to investigate the effects of resveratrol, a natural plant polyphenol with anti-oxidant and anti-nitrative properties, on endotoxemia-associated adrenocortical insufficiency. Resveratrol was administered immediately before injection of lipopolysaccharide (LPS). Twenty four hours later, the adrenocorticotropic hormone (ACTH) stimulation tests was been performed to measure the plasma corticosterone level and the adrenal gland tissues were collected for histopathologic examination, and determination of malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT) activity, inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) and peroxynitrite production. Treatment with resveratrol significantly inhibited endotoxemia-induced iNOS expression, NO production, and peroxynitrite formation and also attenuated LPS-induced oxidative stress in the adrenal gland, as evidenced by the decrease of pro-oxidant biomarker (MDA), and the increases of anti-oxidant biomarkers (T-AOC, CAT and SOD activity). H&E staining demonstrated that administration of LPS resulted in increased into the adrenal gland. H&E-stained sections of adrenal glands demonstrated signs of leukocyte infiltration and hemorrhage during endotoxemia, which were significantly improved by resveratrol treatment. In addition, resveratrol reversed the LPS-induced downregulation of ACTH receptor and silent information regulator 1 (SIRT1) in adrenal gland, as well as adrenocortical hyporesponsiveness to ACTH. Resveratrol exerts protective effects against endotoxemia-associated adrenocortical insufficiency by suppressing oxidative/nitrative stress. These findings support the potential for resveratrol as a possible pharmacological agent to improve adrenocortical insufficiency resulting from oxidative/nitrative damage.

Hypoxia-induced acute lung injury is aggravated in streptozotocin diabetic mice.

PURPOSE: Hypoxia is an inevitable consequence of many respiratory diseases resulting from inadequate alveolar ventilation. As pulmonary dysfunction is recently recognized as one of the many clinical features associated with diabetes, this study aims to investigate the effect of streptozotocin (STZ)-induced diabetes on hypoxia-induced lung injury. MATERIALS AND METHODS: Mice were randomly allocated to four groups (Control, Hypoxia, Diabetes, Diabetes+Hypoxia). Control and type I diabetic (100 mg/kg STZ-treated) mice were followed for 4 weeks and finally exposed to normoxia or hypoxia (8% O2). Twelve hours later, lung tissues were collected for histopathologic examination, and determination of interleukin (IL)-1ß, IL-6, myeloperoxidase (MPO), malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, and Toll-like receptor (TLR)4 expression. RESULTS: STZ-induced diabetes aggravated histopathological changes in the lung exposed to acute hypoxia. Hypoxia increased lung MDA level but decreased T-AOC and SOD activity. STZ-induced diabetic mice presented significant increases in MDA level and SOD activity in the lung. Moreover, no difference was found in the levels of both oxidant index (MDA) and anti-oxidant indexes (T-AOC and SOD) between "Hypoxia" group and "Hypoxia plus Diabetes" group. On the other hand, STZ-induced diabetic mice presented significant increases in pulmonary neutrophil infiltration, pro-inflammatory cytokines (IL-1ß and IL-6) production, as well as TLR4 expression. Although acute hypoxia alone had no significant effect on pulmonary inflammatory markers, it profoundly increased STZ-diabetes-induced neutrophil infiltration, pro-inflammatory cytokine production, and TLR4 expression in lung tissues. CONCLUSIONS: STZ-induced diabetes may aggravate acute hypoxia-induced lung injury through enhancing pulmonary inflammatory responses.

Overweight, obesity and risk of all-cause and cardiovascular mortality in patients with type 2 diabetes mellitus: a dose-response meta-analysis of prospective cohort studies.

Overweight and obese individuals with type 2 diabetes are recommended to lose weight, but the associations between excess body weight and all-cause and cardiovascular mortality in patients with type 2 diabetes remain controversial. Therefore, we performed a dose-response meta-analysis to investigate this association. We searched PubMed and Embase through 19th October 2014 and examined the references of retrieved articles to identify relevant prospective cohort studies. A random-effect model was used to calculate the summary risk estimates. Nine studies including 13 cohorts with 161,984 participants were identified. The relative risks (RRs) of all-cause mortality in overweight and obese patients with type 2 diabetes were 0.81 (95% confidence interval (CI) 0.74-0.90) and 0.72 (95% CI 0.63-0.81) respectively, compared with the normal or non-overweight patients. Furthermore, a 5 kg/m(2) increase in body mass index was associated with a significantly reduced risk of all-cause mortality by 5% (RR 0.95, 95% CI 0.93-0.97). However, no significant association was found between obese and/or overweight and the risk of cardiovascular mortality in type 2 diabetic patients (RR 0.89; 95% CI 0.66-1.20 for overweight and RR 0.77; 95% CI 0.54-1.10 for obesity, respectively). The findings from the present meta-analysis indicate that excess body weight may be a protective factor for all-cause mortality among patients with type 2 diabetes.

Protective effect of resveratrol against endotoxemia-induced lung injury involves the reduction of oxidative/nitrative stress.

BACKGROUND: Resveratrol, a natural plant polyphenol, has received increasing attention because its varied bioactivities, including the inhibition of tumorigenesis, lipid modification and calorie-restriction. We aimed to investigate the effect of resveratrol on oxidative/nitrative stress in endotoxemia-associated acute lung injury. METHODS: Mice were injected with lipopolysaccharide (LPS, 5 mg/kg, ip). Resveratrol at a dose of 0.3 mg/kg was administered alone or immediately before injection of LPS. Twenty four hours later, lung tissues were collected for histopathologic examination, and determination of malondialdehyde (MDA), H2O2, reduced/oxidized glutathione (GSH/GSSG) ratio, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT) activity, inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) and peroxynitrite production. RESULTS: Resveratrol treatment improves histopathological changes in the lung during endotoxemia. Increased oxidative stress in endotoxemic lung was reversed by resveratrol treatment, as evidenced by the decreases of pro-oxidant biomarker (MDA and H2O2), and the increases of anti-oxidant biomarkers (GSH/GSSG ratio, T-AOC, CAT and SOD activity). Treatment with resveratrol inhibited endotoxemia-induced iNOS expression and NO production. Moreover, peroxynitrite formation in endotoxemic lung was significantly attenuated after resveratrol treatment. CONCLUSIONS: Resveratrol exerts protective effects against acute endotoxemia-associated lung injury. These beneficial effects may be due to both the anti-oxidant and anti-nitrative properties of resveratrol. These findings support the potential for resveratrol as a possible pharmacological agent to reduce acute lung injury resulting from oxidative/nitrative damage.

Hydrogen sulfide inhibits alveolar type II cell senescence and limits pulmonary fibrosis via promoting MDM2-mediated p53 degradation.

AIM: Senescence of alveolar type II (AT2) cells is an important driver of pulmonary fibrosis. This study aimed to investigate whether and how dysregulation of hydrogen sulfide (H2 S) production affected AT2 cell senescence, and then explored the effect of H2 S on the communication between AT2 and fibroblasts. METHODS: ICR mice were intratracheally administered with bleomycin (3 mg/kg). Sodium hydrosulfide (NaHS, 28 µmol/kg/d) was intraperitoneally injected for 2 weeks. The H2 S-generating enzyme cystathionine-ß-synthase (CBS) knockout heterozygous (CBS+/- ) mice were used as a low H2 S production model. RESULTS: Analysis of microarray datasets revealed downregulation of H2 S-generating enzymes in lung tissues of patients with pulmonary fibrosis. Decreased H2 S production was correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis. CBS+/- mice exhibited increased levels of p53 and p21. The numbers of AT2 cells positive for p53 and p21 were increased in CBS+/- mice as compared to control mice. H2 S donor NaHS attenuated bleomycin-induced AT2 cell senescence both in vivo and in vitro. H2 S donor suppressed bleomycin-induced senescence-associated secretory phenotype (SASP) of AT2 cells via inhibiting p53/p21 pathway, consequently suppressing proliferation and myofibroblast transdifferentiation of fibroblasts. Mechanically, H2 S suppressed p53 expression by enhancing the mouse double-minute 2 homologue (MDM2)-mediated ubiquitination and degradation of p53. CONCLUSION: H2 S inactivated p53-p21 pathway, consequently suppressing AT2 cell senescence as well as cell communication between senescent AT2 cells and fibroblasts. Aberrant H2 S synthesis may contribute to the development of pulmonary fibrosis through promoting the activation loop involving senescent AT2 cells and activated fibroblasts.

Endothelial RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of ß-catenin and ILK signaling pathways after inflammatory vascular injury.

Endothelial repair is essential for restoring tissue fluid homeostasis following lung injury. R-spondin3 (RSPO3), a secreted protein mainly produced by endothelial cells (ECs), has shown its protective effect on endothelium. However, the specific mechanisms remain unknown. To explore whether and how RSPO3 regulates endothelial regeneration after inflammatory vascular injury, the role of RSPO3 in sepsis-induced pulmonary endothelial injury was investigated in EC-specific RSPO3 knockdown, inducible EC-specific RSPO3 deletion mice, EC-specific RSPO3 overexpression mice, systemic RSPO3-administration mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in plasma from septic patients. Here we show that plasma RSPO3 levels are decreased in septic patients and correlated with endothelial injury markers and PaO2/FiO2 index. Both pulmonary EC-specific knockdown of RSPO3 and inducible EC-specific RSPO3 deletion inhibit pulmonary ECs proliferation and exacerbate ECs injury, whereas intra-pulmonary EC-specific RSPO3 overexpression promotes endothelial recovery and attenuates ECs injury during endotoxemia. We show that RSPO3 mediates pulmonary endothelial regeneration by a LGR4-dependent manner. Except for ß-catenin, integrin-linked kinase (ILK)/Akt is also identified as a novel downstream effector of RSPO3/LGR4 signaling. These results conclude that EC-derived RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of ß-catenin and ILK signaling pathways after inflammatory vascular injury.

Increased R-spondin 3 contributes to aerobic exercise-induced protection against renal vascular endothelial hyperpermeability and acute kidney injury.

AIM: Exercise training exerts protective effects against sepsis-associated multiple organ dysfunction. This study aimed to investigate whether aerobic exercise protected against sepsis-associated acute kidney injury (AKI) via modulating R-spondin 3 (RSPO3) expression. METHODS: To investigate the effects of aerobic exercise on lipopolysaccharide (LPS)-induced AKI, LPS (20 mg/kg) was intraperitoneally injected after six weeks of treadmill training. To investigate the role of RSPO3 in LPS-induced AKI, wild-type (WT) or inducible endothelial cell-specific RSPO3 knockout (RSPO3EC-/- ) mice were intraperitoneally injected with 12 mg/kg LPS. RSPO3 was intraperitoneally injected 30 min before LPS treatment. RESULTS: Aerobic exercise-trained mice were more resistant to LPS-induced body weight loss and hypothermia and had a significant higher survival rate than sedentary mice exposed to LPS. Exercise training restored the LPS-induced decreases in serum and renal RSPO3 levels. Exercise or RSPO3 attenuated, whereas inducible endothelial cell-specific RSPO3 knockout exacerbated LPS-induced renal glycocalyx loss, endothelial hyperpermeability, inflammation, and AKI. Bioinformatics analysis results revealed significant increases in the expression of matrix metalloproteinases (MMPs) in kidney tissues of mice exposed to sepsis or endotoxaemia, which was validated in renal tissue from LPS-exposed mice and LPS-treated human microvascular endothelial cells (HMVECs). Both RSPO3 and MMPs inhibitor restored LPS-induced downregulation of tight junction protein, adherens junction protein, and glycocalyx components, thus ameliorating LPS-induced endothelial leakage. Exercise or RSPO3 reversed LPS-induced upregulation of MMPs in renal tissues. CONCLUSION: Increased renal expression of RSPO3 contributes to aerobic exercise-induced protection against LPS-induced renal endothelial hyperpermeability and AKI by suppressing MMPs-mediated disruption of glycocalyx and tight and adherens junctions.

Upregulation of KLK8 contributes to CUMS-induced hippocampal neuronal apoptosis by cleaving NCAM1.

Neuronal apoptosis has been well-recognized as a critical mediator in the pathogenesis of depressive disorders. Tissue kallikrein-related peptidase 8 (KLK8), a trypsin-like serine protease, has been implicated in the pathogenesis of several psychiatric disorders. The present study aimed to explore the potential function of KLK8 in hippocampal neuronal cell apoptosis associated with depressive disorders in rodent models of chronic unpredictable mild stress (CUMS)-induced depression. It was found that depression-like behavior in CUMS-induced mice was associated with hippocampal KLK8 upregulation. Transgenic overexpression of KLK8 exacerbated, whereas KLK8 deficiency attenuated CUMS-induced depression-like behaviors and hippocampal neuronal apoptosis. In HT22 murine hippocampal neuronal cells and primary hippocampal neurons, adenovirus-mediated overexpression of KLK8 (Ad-KLK8) was sufficient to induce neuron apoptosis. Mechanistically, it was identified that the neural cell adhesion molecule 1 (NCAM1) may associate with KLK8 in hippocampal neurons as KLK8 proteolytically cleaved the NCAM1 extracellular domain. Immunofluorescent staining exhibited decreased NCAM1 in hippocampal sections obtained from mice or rats exposed to CUMS. Transgenic overexpression of KLK8 exacerbated, whereas KLK8 deficiency largely prevented CUMS-induced loss of NCAM1 in the hippocampus. Both adenovirus-mediated overexpression of NCAM1 and NCAM1 mimetic peptide rescued KLK8-overexpressed neuron cells from apoptosis. Collectively, this study identified a new pro-apoptotic mechanism in the hippocampus during the pathogenesis of CUMS-induced depression via the upregulation of KLK8, and raised the possibility of KLK8 as a potential therapeutic target for depression.