| Acute lung injury and acute respiratory distress syndrome are major causes of acute respiratory failure in critically ill patients, responsible for more than 40% mortality, even though there are significant improvements in supportive care. Previous studies demonstrated that bone marrow-derived mesenchymal stem cells (BMSCs) could attenuate experimental acute lung injury induced by lipopolysaccharide (LPS), bleomycin, ventilation or ischemia-reperfusion. BMSCs could be engrafted as type I and Ⅱ epithelial cells or endothelial cells in the injured lung for the repair. BMSCs were also found to modulate the inflammation and reduce lung injury through the release of paracrine factors. BMSCs could attenuate sepsis by releasing prostaglandin E2, which reprogrammed macrophages to increase interleukin-10 production. IL-1 receptor antagonist (IL-1ra) could also mediate the anti-inflammatory and anti-fibrotic effect of BMSCs during bleomycin-induced lung injury.Adrenergic receptor agonists, especially β-adrenergic agonists, have the potential role in the treatment of acute lung injury by accelerating alveolar fluid re-absorption and suppressing inflammation. Those agonists could increase DNA synthesis of embryonic stem cells and BMSCs, and were involved in the protective role against oxidative stresses in mesenchymal stem cells.The present study investigated the effects and the possible mechanism of BMSCs transplantation in a rat model of aspiration induced lung injury. Then we studied the effects of adrenergic receptor agonists and antagonists in biological behaviors of BMSCs, including a and β adrenergic receptor agonist adrenaline, α-agonist norepinephrine, a-antagonist phentolamine, P-agonist isoproterenol, or β-antagonist propranolol. We also studied the role of adrenaline in migration of BMSCs in ex-vivo models of LPS-induced lung injury, and explored whether adrenaline could help BMSCs modulate inflammation through release of paracrine cytokines. Preventive roles of adrenaline-stimulated BMSCs in acute lung injury were furthermore investigated in an experimental model of acute lung injury induced by the intratracheal instillation of LPS. Part 1The study on the role and mechanism of bone marrow-derived mesenchymal stem cells alleviating lung injury in a rat model of acid aspirationThe objective of this study was to investigate the effects and the possible mechanism of bone marrow derived mesenchymal stem cells (BMSCs) transplantation in a rat model of aspiration induced lung injury.The experiment was carried out in Bio medical Research Center of Zhongshan Hospital BMSCs cultures were obtained from bone marrow of Sprague-Dawley (SD) rats.24 SD rats were randomly divided into 3 groups:Control, Injury and Injury+ BMSCs groups, in which hydrochloric acid (HC1) (1.2 ml/kg, pH=1.5) or the same volume of phosphate buffered saline (PBS) were instilled into trachea. Then,5x106 BMSCs or 0.5 ml PBS were injected into jugular vein of rats. Rats were exsanguinated 6 h after injury. Arterial blood gas, wet/dry ratio and histological changes of lung tissue were determined. Bronchoalveolar lavage fluid (BALF) and serum were collected for the measurement of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6) and Interleukin-10 (IL-10) by Enzyme-linked immuno sorbent assay (ELISA). In vitro, lung cells from normal rats and from HC1 injury rats were co-cultured with BMSCs in a Transwell system (8 um pore size).36 h later, the numbers of migrated BMSCs were counted. In addition, lung cells from HC1 injured rats were co-cultured with BMSCs in either a standard single well or in a Transwell (0.4 um pore size). Control wells were prepared with only lung cells from normal rats or HC1 injured rats. The cell culture supernatants were then collected after the 6 h incubation to assay for the levels of TNF-a, IL-6 and IL-10 by ELISA. Comparisons among multiple groups were performed using 1-way analysis of variance (ANOVA). Comparisons between groups were performed using independent-sample/test. Our results found that BMSCs transplantation attenuated histological lung injury and hypoxia caused by HC1 instillation. Injury+BMSCs group had decreased wet/dry ratio compared with Injury group. BMSCs administration mediated a down-regulation of inflammation by deceasing TNF-a concentration while increasing IL-10 in BALF and serum. In vitro coculture studies of BMSCs with lung cells provided evidence that lung cells stimulated BMSCs migration towards injured lung. Besides, the anti-inflammatory effect of BMSCs was not cell contact dependent.In conclusion, BMSCs transplantation prevented acid aspiration induced lung injury caused by HC1 instillation, in which the effects were partly driven by the paracrine effects and anti-inflammatory role of BMSCs.Part 2The study on the participation of adrenergic receptors in the proliferation of bone marrow-derived mesenchymal stem cellsThe objective of this study was to evaluate the effects of adrenergic receptor agonists or antagonists on biological behaviors of BMSCs.BMSCs were stimulated with PBS or adrenaline at concentrations of 0-100 μ.M. BMSCs were also stimulated with a-adrenergic receptor agonist (norepinephrine), P-adrenergic receptor agonist (isoproterenol), or in combination of adrenaline with phentolamine or propranolol, respectively, to compare with cells stimulated with vehicle or adrenaline alone. The cell biological behaviors including the cell proliferation, death and movement were measured by the real-time cell monitoring system, using a Cell-IQ cell culturing platform. Images were captured at 5 min intervals for 72 h. Increased percentages of numbers of total cells, number of divided cells, number of dead cells and cell movement length were calculated.Total number of BMSCs or divided cells significantly increased from 6 h and reached the peak at 18-24 h or at 36 h, respectively, after the stimulation of adrenaline at 10 uM, as compared to control (P< 0.05). Adrenaline at 1 μM did not affect proliferation, while adrenaline at 100 μM decreased it. Adrenaline at different concentrations did not influence dead cell number and cell movement. As a result,10 μ,M was chosen as the concentration used in latter studies. Norepinephrine and isoproterenol had similar stimulatory effects on proliferation of BMSCs, but effects were relatively weaker than adrenaline. The two adrenergic agonists-norepinephrine and isoproterenol did not affect cell death and movement. Phentolamine and propranolol had similar inhibitory effects on proliferation of BMSCs, when used in combination with adrenaline. The two adrenergic antagonists did not affect dead cell number and cell movement.We concluded that adrenaline at 10 μM enhanced proliferation of BMSCs through both a and β adrenergic receptors.Part 3The study on the role and mechanism of adrenaline-stimulated bone marrow-derived mesenchymal stem cells preventing lung injury in LPS-induced lung injuryWe conducted this study to investigate the preventive role of adrenaline-stimulated bone marrow-derived mesenchymal stem cells (BMSCs) on lipopolysaccharide (LPS)-induced lung injury.Suspensions of lung cells from animals with or without LPS-induced injury were co-cultured with BMSCs or adrenaline stimulated BMSCs in a Transwell system (8 um pore size). The numbers of migrated BMSCs were counted 36 h later. Sliced lung tissue from animals with or without LPS-induced injury was co-cultured with BMSCs or adrenaline stimulated BMSCs in a 6-well plate. The numbers of BMSCs’ migration towards and adhesion to lung tissue were counted after co-culture for 24 h. LPS-stimulated alveolar macrophages were co-cultured with BMSCs (with adrenaline stimulation or not) in Transwell (0.4 um pore size).for 6 h. The cell culture supernatants were then collected to measure the levels of TNF-a, IL-1β, IL-6, IL-10, IL-13, angioietin-1, keratocyte growth factor (KGF) and IL-1 receptor antagonist (IL-1ra) by ELISA. Real-time quantitative PCR analysis was conducted to measure mRNA expression levels of angiopoietin-1, KGF, IL-lra, IL-10 and IL-13 from BMSCs. Western blot was used to measure intracellular protein expression levels of IL-10, IL-13, KGF and IL-1ra. A preliminary animal experiment was conducted to validate the findings in ex-vivo study. Animals were pretreated intravenously with BMSCs, adrenaline-stimulated BMSCs, conditioned medium of BMSCs, adrenaline or vehicle 24 h before the intra-tracheal instillation of LPS or the same volume of PBS. Histological changes were determined 24 h after LPS or vehicle instillation. We found that adrenaline at 10μM promoted the migration of BMSCs towards LPS-injured lung cells. Besides, it also promoted BMSCs’ migration towards and adhesion to injured lung tissue. Adrenaline-stimulated BMSCs down-regulated the inflammation of LPS stimulated macrophages by decreasing TNF-a and IL-1 β or increasing IL-10, probably through the expression and secretion of several paracrine factors including angiopoietin-1, KGF, IL-1ra, IL-10 and IL-13. Adrenaline reduced the extent of injury in LPS injured rats.In conclusion, our data indicated that adrenaline stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation.Conclusions1. BMSCs transplantation prevented acid aspiration induced lung injury caused by HC1 instillation, in which the effects were partly driven by the paracrine effects and anti-inflammatory role of BMSCs.2. Adrenaline at 10 μM enhanced proliferation of BMSCs through both a and β adrenergic receptors.3. Adrenaline stimulated BMSCs might contribute to the prevention from acute lung injury through the promotion of proliferation and migration towards injured lung, and modulation of inflammation.Novelty of this study1. Our results demonstrated for the first time that injured lung tissue stimulated BMSCs’ migration and adhesion, which we thought resulted from humoral factors produced by injured lung.2. Our findings were the first evidence that adrenaline could promote the proliferation and migration of BMSCs, and help modulate inflammation in LPS injured rat lung.The potential application of this studyAdrenergic receptor agonists might be used to stimulate BMSCs or other types of MSCs for transplantation in patients with lung injury, with a great possibility of increasing engraftment rate and improving injury repair. |
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