| ALI is one common complication caused by various reasons such as:trauma, infection, sepsis, extracorporeal circulation and extensive burns. ALI and ARDS are considered two continuous stages according to pathological process. ARDS is characterized by severe hypoxaemia and progressive dyspnoea. However, hypoxaemia is difficult to be reversed. The patients with ALI always evolve into ARDS or even more severe condition, if not treated timely.Alveolar-capillary membrane is always damaged by various inflammatory factors including interleukin-1 β (IL-1β), tumor necrosis factor-a (TNF-a) and interferon-y (IFN-y). On account of inflammatory insult, pulmonary edema appears and diffusion dysfunction of carbondioxide and oxygen will ensue as the promoted permeability of alveolar-capillary membrane. Pulmonary compliance will be lowered owing to the reduction of alveolar surfactant secreted by type Ⅱ alveolar epithelial cell (AEC-Ⅱ); therefore, pulmonary atelectasis and respiratory failure readily occur.So far, the effective therapeutic approaches for ALI and ARDS include respiratory support, conservative fluid therapy and infection prevention. In addition, pharmacological therapies including inhaled surfactant, nitric oxide, glucocorticoids and antioxidants pentoxifylline has been evaluated effectively more or less in reducing mortality of ALI. And more, Prognosis varies depending upon numerous causes involved. If the primary inflammation is not controlled in time, prognosis of ALI and ARDS is generally poor. If bone marrow transplantation was complicated by ARDS, mortality is always 100%. At this situation, innovative therapies are therefore needed. To date, cell-based therapy and gene therapy are considered as attractive approaches and MSC-based therapy is considered as a more promising method.Mesenchymal stem cells (MSCs) were found originally in bone marrow. Currently, in addition to bone marrow, MSCs could also be isolated from placenta, umbilical cord, amniotic fluid, and so on. MSCs are fibroblast-like morphology and generally evaluated by three major biological criteria:self-duplication, immunoregulation and differentiation into different cells, such asosteoblasts, adipocytes, cartilage cells, nerve cells and myocardial cells. M SCs positively express cell surface markers including CD29, CD90 and CD 105 and negatively express CD34, CD45 surface molecules. MSCs was reported to possess more multifunctions such as homing to inflammatory sites following injury, differentiating into various cell types in conditioned setting, secreting multiple soluble factors that are capable of repairing injured cells and attenuating injury and performing immunoregulation. To date, there have been more than 475 clinical trials of mesenchymal stem cell registered in Clinical Trials.gov.Although MSCs have been evidenced to play an important role via various functions in treating ALI, the specific mechanisms remain unclear. Currently, MSC engraftment and differentiation, immunoregulation, restoring epithelial protein permeability and improving alveolar fluid clearance (AFC) were considered the main mechanisms. Nevertheless, the rate of MSC differentiation into pulmonary epithelium proved lower. And more, it was reported that MSCs exerted an important role more or less in regulating inflammatory response.AFC is the resolution to clear pulmonary edema by alveolar epitheliums.AFC is always damaged in inflammatory situation. The level of AFC impairment has significant prognostic value in determining morbidity and mortality. Both alveolar epithelial cell type Ⅰ (AT-Ⅰ) and AT-Ⅱ participate in the process of AFC and especially AT-Ⅱ play an important role in mediating AFC. Apical sodium channels and aquaporin of AT-Ⅱ as well as the basolateral sodium-potassium adenosine triphosphatase (Na-K-ATPase) were involved in transporting sodium and pulmonary residual fluid from alveoli to pulmonary interstitium. So, it is important to make clear the specific mechanism by which MSCs promote impaired AFC in ALI via paracrine function.Methods1. MSC culture and identification.Sprague Dawley (SD) rat were sacrificed under anesthesia. MSCs were isolated from the BM of the tibiae and femurs of 8-week old male rat. As cells reached 80-90% confluence, MSCs were passaged every 3-4 days by trypsinization and used for experiments between 3-6 passage. MSCs were cultured with Dulbecco’s Modified Eagle medium (DMEM)containing 1% glutamine, 100u/ml penicillin, streptomycin and 2% Fetal Bovine Serum in a humidified incubator (5% CO2 and 37 ℃) under sterile conditions. MSCs were identified with phenotypic markers by flow cytometry. MSCs were cultured with adipogenic or osteogenic induction medium (Cyagen Biosciences, Guangzhou, China) until they reached 90% confluent. MSCs were stained with oil red 0 or alizarin red in culture plate.2. Primary culture of AT-Ⅱ cells.AT-II cells were isolated form male SD rat as previously researches 13, which were cultured with DMEM/F12 containing 2% FBS, 100U/ml penicillin and 100U/ml streptomycin. Cells were identified by surfactant protein A (SP-A) and goat anti-rabbit secondary antibody by immunofluorescence technique. Images were taken by confocal microscopy system.3. Building co-culture system and proliferation assay.AT-Ⅱ cells were seeded into each upper compartment of transwell while MSCs were plated in bottom compartment with no direct cell contact. To injury the cells, AT-Ⅱ cells were exposed to the three major proinflammatory cytokines(Cytomix) containing TNF-α, IL6 and IL1-β (1.7ng/ml,87.6ng/ml and 4.4ng/ml, respectively) which were determined according to the previous study.4. Cell proliferation assay.Cell proliferation assay was carried out according to the protocol using a CCK8 kit under different conditions:1) AT-Ⅱ cells alone,2) Cytomix-exposed AT-Ⅱ cells,3) Cytomix-exposed AT-Ⅱ cells+ MSCs. The optical density (OD) was then measured with amicroplate reader at 450nm.5. KGF concentration detected with ELISA.Concentrations of KGF secreted by MSCs were measured in the supernatant of culture medium with rat KGF specific ELISA kit as MSCs were exposed to cytomix in comparison to KGF concentration in normal medium at 1/2h,6h,24h and 72h (n=3 per group). MSCs were starved using serum-free medium for 24h before cytomixexposure. Based on lipofectamine 2000, MSCs were transfected with SiRNA to knock down KGF expression according to the protocol. Additionally, mock transfection was performed meanwhile. MSCs transfected with cy5-labeled negative SiRNA was imaged by confocal microscopy. The knocking down efficiency was evaluated by KGF secretion in supernatant of MSCs, MSCs transfected with KGF SiRNA (MSCs-SiRNA) and MSCs transfected with only lipofectamine (MSCs-lipof) were measured at 48h after transfection.6. MSC tracing study.ALI rat model were built by intravenous administration of lOmg/kg LPS (lipopolysaccharide) dissolved in normal saline. MSCs were digested into single cells with 0.25% trypsase and stained with cell membrane dye Dil. Dil-stained MSCs (5×105 cells) were injected through rat tail vein 4h later following LPS administration. Rats were sacrificed to determine the quantity of cells left in lung after MSC injection at different time points with small animal imaging technology at 15min,2h,24h and 72h after MSC administration. Next, lung frozen sections (6μm in thickness) were made at 15min,24h and 72h to determine the locations where MSCs distributed. Meanwhile, cell nuclei were stained with Dapi.7. Histopathology and wet/dry analysis.All 7-8 week old male rats were randomly assigned to different groups. Rats without LPS administration were injected with Phosphate Buffered Saline (PBS) or MSCs for possible inflammatory reaction caused by PBS or MSCs. Rats intravenously injected with 10mg/kg LPS were followed by PBS, MSCs, MSCs-SiRNA or MSCs-lipof (5×105 cells) respectively injected 4 hour later. Rats were sacrificed at 72h after LPS injection. Meanwhile left lung lobes were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm thick sections which were stained with hematoxylin and eosin (HE) and images were taken with LEICACTR4000 microscope. Concurrently, right rat lung lobes were excised and the wet weight was recorded. After right lung lobes were placed in an incubator at 65℃ for 3 days, ratio of wet/dry was determined finally.8. Detection of SP-A, al and β1 subunits at protein and gene level.AT-Ⅱ cells were seeded on each upper compartment in 6-well transwell, while MSCs were plated in each bottom compartment. Therapeutic effects were evaluated at the following experimental situations:1) AT-Ⅱ cells with PBS (control group), 2)cytomix-exposed AT-Ⅱ cells with PBS,3) AT-Ⅱ cells with MSCs,4) cytomix-exposed AT-Ⅱ cells with MSCs,5) cytomix-exposed AT-Ⅱ cells with MSCs-siRNA,6) cytomix-exposed AT-Ⅱ cells with MSCs-Lipof. After co-culture for 72h, total protein was extracted from AT-Ⅱ cells and western blot analysis was carried out according to the protocol. Proteins of al and β1 subunits were evaluated by western blot. Meanwhile protein expressions of p-AKT、AKT、p-mTOR and mTOR in PI3K pathway were evaluated as well. Expressions of α1 and β1 subunits at gene level were analyzed by two step quantitative real time PCR (qPCR). Total RNA was isolated from AT-Ⅱ cells after co-culture for 48h using Trizol reagent according to manufacturer’s instructions.9. Statistical analysisAll experimental groups were carried out at least three times for each group. Results are expressed as the mean ± SD. Comparison was made by, one sample t test, One-Way ANOVA or ANOVA of repeated measuring method for multiple-comparison test with spss13.0. (P<0.05 was considered statistically significant).Results1. Rat MSCs are adherent and spindle-like cells, which differentiated into the predominant mesenchymal lineages:adipocytes and osteocytes. MSCs started from a single cell to a colony in a culture plate. Rat MSCs positively expressed the cell surface markers CD29、CD44、CD90 and negatively express CD34 and CD45 by flow cytometry. Primary culture of rat AT-ⅡCells are adherent cell like round stone in culture plate, which were identified with SP-A binded by FITC-labeled secondary antibody. Green fluorescence from AT-Ⅱ Cells was detected in cell membrane by confocal fluorescence microscope.2. CCK8 assay demonstrated that exposure to cytomix resulted in down-regulated proliferation of AT-Ⅱ cells compared with AT-Ⅱ cells without inflammatory exposure at 1,2 and 3 day. However, proliferation of injured AT-Ⅱ cells was ameliorated by MSCs.3. Concentration of KGF was detected higher as MSCs were stimulated by cytomix versus at normal situation, suggesting MSCs produced more KGF in response to cytomix(TNF-α、IL6 and IL1-β). However, KGF concentration was detected lower as cytomix-exposed AT-II cells were treated with MSCs in comparison to that of cytomix-exposed MSCs.4. KGF concentration was detected higher as MSCs were cultured under inflammatory situation versus in normal medium at 1/2h,6h and 24h. KGF secretion approached the highest level at 6h. But we found that MSCs produced less KGF at 72h under inflammatory situation versus in normal medium. Cy5-labeled negative SiRNA in cytoplasm surrounded Dapi-stained nuclei in MSCs, which showed that majority of MSCs were transfected with SiRNA. Additionally, data revealed that the secreted KGF was significantly down regulated after transfection with KGF SiRNA. KGF secretion by MSCs-lipof made no difference with the section by MSCs.5. MSC tracing in lung after intravenous injection was determined by small animal imaging technology. Signal intensity of injected MSCs reached the highest level at 15min. Majority of injected MSCs was lost at 72h versus 2h and 24h. Meanwhile, distribution of injected MSCs according to lung frozen sections at 15min, 24h and 72h showed that Dapi-labelednuclei were surrounded by DiI-labeled cytomembrane and MSCs got clustered at 15min in lung tissue which scattered at 24h and 72h.6. LPS injection to rats resulted in marked inflammatory injury according to HE-stained lung sections. MSC administration reduced the lung injury, which was attenuated by MSCs-SiRNA. MSCs-lipof improved the inflammatory reaction also, which suggested that mock tranfection had no obvious side effect on MSCs. Meanwhile, we did not found the inflammatory reaction caused by PBS or MSCs. LPS administration to rat resulted in increased ratio of Wet/dry versus rats injected with PBS. Wet/dry decreased lower as LPS-exposed rats were treated by MSCs, which returned as LPS-exposed rats were treated with MSCs-SiRNA. Meanwhile, MSC or PBS injection did not lead to increased ratio.7. Western blot assay was performed at 48h after co-culture. As shown, expressions of protein aland (β1 were decreased lower after inflammatory situation. Co-culture with MSCs increased the expression of al and β1 subunit in cytomix-exposed AT-Ⅱ cells. Expression of al subunit protein was decreased as cytomix-exposed AT-Ⅱ cells were cultured with MSCs-SiRNA but we did not found the decreased expression of β1 subunit. Simultaneously, expressions of al and β1 subunit at gene level were assessed at 48h under different conditions. Exposure to cytomix leaded to the decreased mRNA level of al and β1 subunit, which were increased as injuried MSCs were treated with MSCs. Meanwhile, the increased expression of al subunit mRNA was down regulated by MSCs-SiRNA. But we did not found decreased expression of β1 subunit by MSCs-SiRNA. Expression of al or β1 at mRNA or protein level made no difference as cytomix-exposed AT-II cells were treated with MSCs-lipof in comparison to MSCs.Conclusion1. MSCs produced more KGF under inflammatory condition versus under normal condition. Dil-labeled MSCs reached the highest level at 15min after injection. Majority of injected MSCs were lost significantly at 72h.2. LPS administration to rats resulted in the marked inflammatory reaction and increased ratio of wet/dry. Treatment with MSCs following LPS reduced inflammatoryinfiltrates and septal thickening. MSCs might reduce the injury via enhancing AFC.3. Therapeutic benefits were provided by MSCs-secreted KGF and mainly mediated by up-regulated al but not β1 subunit of Na+/K+-ATPase in AT-Ⅱ cells.4. MSCs might increase AT-Ⅱ cell proliferation and expression of al subunit via a KGF-dependent PI3K/Akt/mTOR pathway. |
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