Phosphoinositide 3 - Kinase Signaling Pathways Involved In Lung Injury Repair And Mechanism Is Discussed In This Paper

Part1Preventive Effects of Phosphatidylinositol3-kinases Inhibitors on Lipopolysaccharide induced Acute Lung Injury in MiceObjective:To investigated the preventive effects of Phosphatidylinositol3-kinases (PI3K) inhibitors and compared the efficacy of intratracheal and intragastrical deliveries on lung edema and gas volume, leukocyte recruitment, and chemokine production at4and24h after LPS induced ALI in mice. In vitro studies were further conducted to investigate whether the PI3K inhibitor has direct effects on epithelial cells and neutrophils.Methods:Male CD-1mice aged6-8weeks were selected and administered3different PI3K inhibitors (LY294002, GDC0941and SHBM1009) either intranasally or intragastrically once a day for3days before intratracheal instillation of lipopolysaccharide at4h and24h. Animal bodyweight (BW) was measured both before the experiment and at termination. After termination, the lung weight/body weight (LW/BW) ratio, gas volume, and total number of leukocytes and levels of KC in Brochoalveolar lavage fluid (BALF) were measured. Excised lung gas volume (ELGV) was measured based on the Archimedes principle. Effects of SHBM1009on lipopolysaccharide (LPS)-induced capillary permeability were evaluated by Evans blue dye, leukocyte distribution and activation were measured. The number of neutrophils and macrophages in the lung tissue was measured as the average cell number per cross-section of lungs based on evaluating10lung areas per specimen. For the in vitro study, the CD-1mouse airway epithelial cells and circulating neutrophils were isolated, purified, and cultured cultured in the presence or absence of LPS at1mg/mL and SHBM1009at1or10mg/mL. The production of KC and LTB4was evaluated using ELISA kit at3,6,12, and24h after LPS challenge. Neutrophil adhesion was measured. The direct inhibitory effects of LY294002and SHBM1009at different concentrations on the superoxide generation from mouse neutrophils stimulated by LPS at1mg/mL for24h were measured as described previously.Results:LW/BW ratio in animals pretreated with vehicle and challenged with LPS were significantly higher than those challenged with PBS at4and24h (P<0.01), which was prevented by intranasal or intragastrical pretreatment with LY294002, intragastrically with GDC-0941, and intranasally with SHBM1009(P<0.05and P<0.01, respectively). Levels in animals with LY294002(intranasally at24h) and GDC-0941(intranasally at4h and intragastrically at4and24h) were still significantly higher than those without LPS (P<0.05for both). Intranasal pretreatment with either LY294002or SHBM1009significantly prevented LPS-induced increase of ELGV at4or24h after LPS challenge (P<0.05). Intranasal pretreatment with all PI3K inhibitors significantly prevented LPS-induced leukocyte influx in BALF at4h (P<0.05and P<0.01, respectively). Intranasal pretreatment with LY294002or SHBM1009at4or24h showed partially preventive effects on LPS-induced KC production (P<0.05). LPS significantly increased Evans blue dye leakage in the airway and lung (P<0.01vs control subjects), which was prevented by intranasal delivery of SHBM1009(P<0.05vs animals with vehicle and LPS). Pretreatment with SHBM1009prevented LPS-increased number of neutrophils (P<0.01) and macrophages (P<0.05). Levels of KC in the mouse epithelial cell supernatant were significantly higher from3h after LPS challenge compared with the control or those treated with SHBM1009at1or10mg/mL (P<0.05and P<0.01, respectively). The adhesion rate of mouse neutrophils stimulated by KC, LTB4, or LPS was inhibited by SHBM1009at different concentrations. The inhibitory potency of SHBM1009depended on stimuli. LY294002or SHBM1009significantly inhibited superoxide production from LPS-stimulated mouse neutrophils, and the50%inhibitory dose of SHBM1009was much lower than that of LY294002.Conclusion:Local delivery of PI3K inhibitors played more effective roles in the prevention of endotoxin-induced lung injury than the systemic delivery. The preventive effects of PI3K inhibitors varied most likely because of chemical properties, targeting sites, and pharmacokinetics. The protective effects of PI3K inhibitors on ALI may through direct inhibition of airway epithelial cells, neutrophils, and macrophages. PI3K may be a therapeutic target for acute lung injury, and local delivery of PI3K inhibitors may be one of the optimal approaches for the therapy. Part2Roles and mechanism of phosphoinositide3-kinase in acute and chronic lung inflammation, tissue injury, remodeling and emphysema in ratsObjective:To evaluate the therapeutic effects of SHBM1009, a new PI3K/mTOR inhibitor, on chronic lung inflammation, tissue injury and remodeling7and28days after the intratracheal instillation of pancreatic elastase (PE), and potential mechanisms by which PI3K may be involved in the inflammation or biological functions of airway epithelial cells or pulmonary myofibroblasts.Methods:Adult male Wister rats, weighing180-200g, were intratracheally instilled with PE (0.2mL,100IU) or PBS, followed by7or28days of continuously intranasal instillation of SHBM1009or budesonide (the reference drug). The animals were divided randomly into six experimental groups:1) animals were challenged with vehicle and treated with vehicle at0.2ml;2) animals were challenged with PE and treated with vehicle;3) animals were challenged with vehicle and treated with SHBM1009at10mg-kg-1,4and5) animals were challenged with PE and treated with SHBM1009at1and10mg-kg-1or6) animals were challenged with PE and treated with budesonide at1mg-kg-1, respectively. There were12animals per group and6in each time point. Lung weight, body weight and lung density were measured for each rat. Excised lung gas volume (ELGV) was measured by Archimedes’ principle. The bronchoalveolar lavage fluid was harvested for evaluating cell count, protein concentration, levels of IL-1β and TGF-β1. HE staining, scanning electron microscopy and Masson staining were used to evaluate lung morphometry. Besides, cellular biological functions were monitored and determined with Cell-IQ, an integrated, fully automated system which can monitor and record the cell number, proliferation, differentiation, apoptosis or movement without use of labels or markers. The cells were plated onto24-well plates with an appropriate density and challenged with PE at concentrations of0.03,0.30,1.00, or2.00U/ml, respectively, to evaluate damaging effects of PE per se. In order to determine protective and dose-associated effects of PI3K inhibitors, cells were co-cultured with BEZ235at concentrations of0.01,0.10,1.00or10μM, or SHBM1009at concentrations of0.01,0.10,1.00, or10μM, respectively, under PE at1.00U/ml. The effects of BEZ235or SHBM1009at concentrations of0.01,0.10, or1.00μM were furthermore evaluated under PE challenge at0.3U/ml.Results:Intratracheal instillation of SHBM1009at the dose of lmg/kg and budesonide can prevent PE-increased lung weights on7and28days, respectively. Values of lung densities in animals with PE and vehicle7days after the challenge were significantly higher than those without PE, which were significantly attenuated by the treatment with SHBM1009or budesonide (p<0.01). PE instillation increased the lung density at7days while decreased it at28days, while the treatment with SHBM100910mg/kg and budesonide can significantly attenuate such reduction (p<0.01). Levels of total protein in BALF significantly increased7days after the challenge with PE (p<0.01), which was prevented by the treatment with SHBM1009at1or10mg/kg and budesonide (p<0.01or0.05, respectively). Treatment with SHBM1009or budesonide significantly inhibited PE-increased inflammatory cells in BALF on both7and28days (p<0.05or0.01, respectively). Levels of IL-1in BALF or serum harvested from animals with PE challenge and vehicle treatment were significantly higher than from those without PE on7and28days (p<0.01, Fig2C), and were significantly attenuated by treatment with SHBM1009and budesonide (p<0.05or0.01, respectively). For the morphologic changes, alveolar walls and airspace were compromised and enlarged, the thickness of remained alveolar walls increased, and the number of monocytes with the wall elevated in PE-challenged animals treated with vehicle. PE-induced lung injury was protected by the treatment with SHBM1009or Budesonide, while therapeutic effects of SHBM1009at10mg/kg were better than at1mg/kg. Intratracheal instillation of PE induced a significant increase in the area of collagen-positive staining in the lung, while the treatment with SHBM1009or budesonide significantly inhibited the deposition of collagen. Total number of lung epithelial cells significantly decreased when the co-culture with PE in a dose-dependent pattern. PE (1U/ml)-induced reductions of total cell number were partially prevented by the addition of BEZ at0.1-1.0uM or SHBM1009at0.01-10uM, while SHBM1009at0.01-1.0uM or BEZ at0.01and0.1uM fully prevented from decreased total cell number by PE at0.3U/ml. Altered number of stable, differentiated and death cells co-cultured with PE alone or PE at1.0or0.3U/ml plus BEZ or SHBM1009at the different concentrations was similar to the pattern of total cell number.Conclusion:PI3K plays a critical role in the development of both acute and chronic lung injury and the progress of tissue remodeling and emphysema. The mechanisms include promoting proliferation and differentiation of epithelial cells, inhibiting the proliferation and differential of fibroblasts and collagen deposition. PI3K inhibitors may be considered as one of new therapeutic alternatives for chronic lung diseases e.g. COPD or emphysema, or at least as a candidate of therapeutic combination. Part3Protective effects of keratinocyte growth factor-2on warm ischemia reperfusion induced lung injury in ratsObjectives:The purpose of this study was to investigate whether KGF-2had a beneficial effect on IR-induced ALI. In addition, the effect of KGF-2on endothelial cells was also investigated to provide insight into the possible molecular mechanisms involved in the action of KGF-2.Methods:SD rats were randomly divided into seven experimental groups (n=15):1) Sham group:animals undergoing a sham operation and pre-treated with vehicle;2) IR group:animals with I/R-ALI and pre-treated with vehicle;3),4) and5) KGF-2low, middle, and high treatment group:animals were intratracheally instilled with2.5mg/kg,5mg/kg and10mg/kg KGF-272hours prior to IR surgery;7) dexamethasone (DXM) group:DXM at a dose of5mg/kg was administered introperitoneally2hours prior to IR. Keratinocyte growth factor-2(2.5,5, or10mg/kg) was administered intratracheally to rats3days before surgery. Then the left lung in rats was subjected to ischemia for60minutes and reperfusion for as long as180minutes. Lung morphology, blood gas analysis, total cell number and protein concentration in the bronchoalveolar lavage fluid were measured to evaluate the lung injury. The protective effects of keratinocyte growth factor-2on human pulmonary microvascular endothelial cells and related mechanisms were evaluated in vitro. Cell-IQ was applied to monitoring biological behavior; Brdu proliferation assay was used to assess cell proliferation; measurement of the permeability of FITC-labeled albumin and transendothelial electrical resistence to assesse barrier function of the endothelial.Results:Pre-treatment with keratinocyte growth factor-2at the doses of2.5mg and-5mg/kg effectively inhibited lung edema, inflammatory cell infiltration, protein exudation and the release of inflammatory cytokines. In vitro study demonstrated that keratinocyte growth factor-2could inhibit endothelial cell apoptosis, enhancing migration, and maintaining the integrity of the blood-gas barrier. Phosphoinositide3-kinase inhibitors attenuated the protective effect of Keratinocyte growth factor-2in endothelial cells.Conclusion:Keratinocyte growth factor-2pre-treatment reduced ischemia reperfusion-induced acute lung injury. The protective effects of keratinocyte growth factor-2may contribute to its protective effects on both epithelial and endothelial cells.