Alleviation of acute pancreatitis-associated lung injury by inhibiting the p38 mitogen-activated protein kinase pathway in pulmonary microvascular endothelial cells

BACKGROUND Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis(SAP)-related acute lung injury(ALI).Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI.However,the precise mechanism associated with p38 is unclear,particularly in pulmonary microvascular endothelial cell(PMVEC)injury.AIM To determine its role in the tumor necrosis factor-alpha(TNF-α)-induced inflammation and apoptosis of PMVECs in vitro.We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI.METHODS In vitro,PMVEC were transfected with mitogen-activated protein kinase kinase 6(Glu),which constitutively activates p38,and then stimulated with TNF-α.Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels,respectively.In vivo,SAP-ALI was induced by 5%sodium taurocholate and three different doses of SB203580(2.5,5.0 or 10.0 mg/kg)were intraperitoneally injected prior to SAP induction.SAP-ALI was assessed by performing pulmonary histopathology assays,measuring myeloperoxidase activity,conducting arterial blood gas analyses and measuring TNF-α,interleukin(IL)-1βand IL-6 levels.Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration,Evans blue pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2,Bax,Bim and cle-caspase3 levels.The proteins levels of P-p38,NFκB,IκB,P-signal transducer and activator of transcription-3,nuclear factor erythroid 2-related factor 2,HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580.RESULTS In vitro,mitogen-activated protein kinase(Glu)transfection resulted in higher apoptotic rates and cytokine(IL-1βand IL-6)levels in TNF-α-treated PMVECs.In vivo,SB2035080 attenuated lung histopathological injury,decreased inflammatory activity(TNF-α,IL-1β,IL-6 and myeloperoxidase)and preserved pulmonary function.Furthermore,SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration,Evans blue accumulation,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers,apoptosis-related proteins(cle-caspase3,Bim and Bax)and endothelial microstructure.Moreover,SB203580 significantly reduced the pulmonary P-p38,NFκB,P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels.CONCLUSION p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs.

Baicalin Induces IFN-α/β and IFN-γ Expressions in Cultured Mouse Pulmonary Microvascular Endothelial Cells

We studied the effect of baicalin,an extract from Radix Scutellariae（a traditional Chinese medicine） in inducing mouse pulmonary microvascular endothelial cells（MPMVECs） to produce interferons（IFNs）.MPMVECs were cultured in vitro in the presence of different concentrations of baicalin（10,20,and 30 μg mL-1）,and the cells and the culture media were harvested at various time intervals.The proteins and mRNA levels（relative to β-actin） of IFN-α/β and IFN-γ were analyzed by RT-PCR and enzyme-linked immunosorbent assay（ELISA）.It was observed that baicalin substantially up-regulated the expression of IFN-α/β and IFN-γ.In all baicalin-treated groups,the relative levels of IFN-α/β and IFN-γ mRNAs peaked after 12 h of culturing,and IFN-α/β and IFN-γ proteins peaked after 24 h of culturing.These results suggest that baicalin can effectively induce the expression of IFNs in pulmonary microvascular endothelial cells,and thus potentially act as an antiviral compound.This study may provide background information for developing new antiviral drugs based on baicalin.

EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1,TRF2,and RAD50

Telomeres are nucleoprotein structures located at the end of each chromosome,which function in terminal protection and genomic stability.Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo.As relatively long-lived mammals based on body size,bats display unique telomeric patterns,including the upregulation of genes involved in alternative lengthening of telomeres(ALT),DNA repair,and DNA replication.At present,however,the relevant molecular mechanisms remain unclear.In this study,we performed cross-species comparison and identified EPAS1,a well-defined oxygen response gene,as a key telomeric protector in bat fibroblasts.Bat fibroblasts showed high expression of EPAS1,which enhanced the transcription of shelterin components TRF1 and TRF2,as well as DNA repair factor RAD50,conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion.Based on a human single-cell transcriptome atlas,we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation.Using in vitro-cultured human pulmonary endothelial cells,we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans.In addition,the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence.In conclusion,we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging,drawing insights from the longevity of bats.

Brain-Derived Glia Maturation Factor b Participates in Lung Injury Induced by Acute Cerebral Ischemia by Increasing ROS in Endothelial Cells

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia(ACI), we established a middle cerebral artery occlusion(MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor b(GMFB) based on quantitative analysis of the global rat serum proteome.Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was overexpressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation(OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium(CM) after OGD.We then used the CM to culture pulmonary microvascular endothelial cells(PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover,ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells.In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.

Xijiao Dihuang Decoction combined with Yinqiao Powder reverses influenza virus-induced F-actin reorganization in PMVECs by inhibiting ERM phosphorylation

Objective:It has been documented that ezrin/radixin/moesin(ERM)phosphorylation by the p38 mitogen-activated protein kinase(MAPK),Rho/ROCK,and protein kinase C(PKC)pathways leads to filamentous actin(F-actin)reorganization and microvascular endothelial cell hyperpermeability.In this study,we investigated the effects of Xijiao Dihuang Decoction combined with Yinqiao Powder(XDY)on influenza virus(IV)-induced F-actin restructuring and ERM phosphorylation regulated by the Rho/Rho kinase 1(ROCK),p38 MAPK,and PKC signaling pathways in pulmonary microvascular endothelial cells(PMVECs).Methods:Serum containing XDY(XDY-CS;13.8 g/kg)was acquired using standard protocols for serum pharmacology.Primary PMVECs were obtained from male Wistar rats and cultured.After adsorption of IV A(multiplicity of infection,0.01)for 1 h,medium with 20%XDY-CS was added to the PMVECs.The distributions of F-actin and phosphorylated ERM were determined by confocal microscopy,and F-actin expression was measured by flow cytometry.The expression levels of ROCK1,phosphorylated myosin phosphatase target-subunit(p-MYPT),phosphorylated MAPK kinase,phosphorylated p38(p-p38),phosphorylated PKC(p-PKC),and phosphorylated ERM(p-ERM)were determined by western blotting.Results:F-actin reorganization in IV-infected PMVECs was reversed by XDY-CS treatment,which was accompanied by reduced p-ERM production.The p-ERM protein accumulated at plasma membrane of PMVECs infected with IV,which was also inhibited by XDY-CS treatment.

Bench to beside:the imbalance of mutual regulation of homocysteine and hydrogen sulfide in congenital heart disease related pulmonary arterial hypertension

Background To determine the imbalance of mutual regulation of homocysteine and hydrogen sulfide(H;S)in congenital heart disease(CHD)-related pulmonary arterial hypertension(PAH)among pediatric patients,and explore possible mechanisms.Methodology and Principal Findings:In this study,we regulated homocysteine concentrations to observe the relations between homocysteine and H;S.Cell viability and activity of metabolic enzymes were determined.Cytological experiments demonstrated that exogenous or endogenous H;S both had protective effects on HPAECs and can inhibit homocysteine-induced apoptosis.The possible mechanisms were correlated with GRP78 and CHOP expressions of endoplasmic reticulum stress pathway.In addtion,we found that homocysteine and H;S were in a dynamic change,which was related to the homocysteine concentration.When the homocysteine concentrations were low(≤30μmol/L),the protective effects of H;S can resist the homocysteine-induced damage effects.However,the cytological results were different from the clinical data.Our clinical study had showed that the levels of homocysteine were higher,the levels of H;S and the OD values of cystathionine gamma-lyase(CSE)were lower in the PAH group.All the CHD-PAH patients had low homocysteine(≤30μmol/L)concentrations still lead to PAH because of decreased the protective effects of H;S due to the decreased activity of CSE.Conclusion:Homocysteine and H;S both take part in the development of CHD-PAH.Hyperhomocysteinemia may be the pathogenic factor,while H;S is the protective factor.The mutual dynamic regulations are related to the homocysteine concentration.The clinical trials and cytological experiment results have great implications for clinical practice.For patients with PAH,not only the damage of homocysteine to endothelial cells,but also we should pay attention to the decreased protection of H;S and activity of metabolic enzymes.

Low- and high-dose hydrogen peroxide regulation of transcription factor NF-E2-related factor 2

Background Reactive oxygen species （ROS） may play both physiological and pathophysiological roles. Transcription factor NF-E2-related factor 2 （Nrf2） regulates antioxidant response element （ARE）-mediated genes expression and coordinates induction of chemoprotective proteins in response to physical and chemical stresses. The exact role of Nrf2 in cellular responses to different levels of oxidative stresses remains unknown. Methods Rat pulmonary microvascular endothelial cells were cultured and treated with 0 mmol/L, 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L and 2.0 mmol/L hydrogen peroxide solution for 2 hours. Nrf2 gene expression was assayed by reverse transcription-PCR, Nrf2-ARE binding activity was assayed with electrophoretic mobility shift assay （EMSA）, and localization of Nrf2 was detected with immunohistochemistry. Results Low and moderate （0.125 mmol/L, 0.25 mmol/L and 0.5 mmol/L） doses hydrogen peroxide exposure of rat pulmonary microvascular endothelial cells led to the nuclear accumulation of Nrf2, increased activity of transcription regulation and up-regulation of ARE-medicated gene expression. In contrast, high doses of hydrogen peroxide （1 mmol/L 2 mmol/L） exposure of the cells led to the nuclear exclusion of Nrf2, decreased activity transcription regulation and down-regulation of ARE-mediated gene expression. Conclusion Low and moderate doses of hydrogen peroxide play protective roles by increasing transcription activity of Nrf2, whereas high- dose hydrogen peroxide plays a deleterious role by decreasing transcription activity of Nrf2.

The Notch pathway attenuates burn-induced acute lung injury in rats by repressing reactive oxygen species

Background:Acute lung injury(ALI)is a common complication following severe burns.The underlying mechanisms of ALI are incompletely understood;thus,available treatments are not sufficient to repair the lung tissue after ALI.Methods:To investigate the relationship between the Notch pathway and burn-induced lung injury,we established a rat burn injury model by scalding and verified lung injury via lung injury evaluations,including hematoxylin and eosin(H&E)staining,lung injury scoring,bronchoalveolar lavage fluid and wet/dry ratio analyses,myeloperoxidase immunohistochemical staining and reac-tive oxygen species(ROS)accumulation analysis.To explore whether burn injury affects Notch1 expression,we detected the expression of Notch1 and Hes1 after burn injury.Then,we extracted pulmonary microvascular endothelial cells(PMVECs)and conducted Notch pathway inhibition and activation experiments,via aγ-secretase inhibitor(GSI)and OP9-DLL1 coculture,respectively,to verify the regulatory effect of the Notch pathway on ROS accumulation and apoptosis in burn-serum-stimulated PMVECs.To investigate the regulatory effect of the Notch pathway on ROS accumulation,we detected the expression of oxidative-stress-related molecules such as superoxide dismutase,nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(NOX)2,NOX4 and cleaved caspase-3.NOX4-specific small interfering RNA(siRNA)and the inhibitor GKT137831 were used to verify the regulatory effect of the Notch pathway on ROS via NOX4.Results:We successfully established a burn model and revealed that lung injury,excessive ROS accumulation and an inflammatory response occurred.Notch1 detection showed that the expression of Notch1 was significantly increased after burn injury.In PMVECs challenged with burn serum,ROS and cell death were elevated.Moreover,when the Notch pathway was suppressed by GSI,ROS and cell apoptosis levels were significantly increased.Conversely,these parameters were reduced when the Notch pathway was activated by OP9-DLL1.Mechanistically,the inhibition of NOX4 by siRNA and GKT137831 showed that the Notch pathway reduced ROS production and cell apoptosis by downregulating the expression of NOX4 in PMVECs.Conclusions:The Notch pathway reduced ROS production and apoptosis by downregulating the expression of NOX4 in burn-stimulated PMVECs.The Notch-NOX4 pathway may be a novel therapeutic target to treat burn-induced ALI.