| Background Severe acute pancreatitis (SAP) continues to be a clinical challenge. Current estimates rank SAP as one of the most common gastrointestinal cause of death, with an high mortality rate in those with multi-organ failure (MOF). Although persistent organ failure, presence of local complications, and/or death define SAP, death from AP occurs predominantly in patients with persistent organ failure such as respiratory failure. Acute lung injury (ALI) caused by SAP may result in pulmonary edema and pleural effusion, which is lethal. Until now, there has been a lack of understanding of the underlying mechanisms for SAP associated acute lung injury, pulmonary capillary leakage and pulmonary edema leakage channels. However, there has also been a lack of effective treatment. Extensive literatures have been recently reported that bone marrow mesenchymal stem cells (BMSCs) can promote the repair of lung with ALI, and improve lung function. This study aimed to elucidate these underlying mechanisms of pulmonary edema and pulmonary capillary leakage in an acute lung injury rat model caused by severe acute pancreatitis, and evaluate the effect of treatment with allogeneic BMSCs.1. Establishment of a severe acute pancreatitis model in ratsObjective We aimed to establish a stable and reliable SAP model with symptoms of lung injury, which was conveniently used to study ALI and ARDS caused by SAP.Methods Sprague-Dawley rats were randomized into the experimental group, the control group and the sham operation group. SAP model was prepared through retrograde injection of 5% taurocholic acid. After injection, the pancreatic duct was ligated in the experimental group, but not in the control group. We examined SAP rats for any pancrease tissue damage at 3,6,12 and 24hours.Results A SAP model with symptoms of acute lung injury in rats was established successfully.48-hour survival rate of animals in the experimental group was 10%, in the control group was 40% and 100% of the sham group. Through the serum amylase and histological observation, pancrease tissue damage of the experimental group were significantly higher than of the control group and sham group.Conclusion Injury process and pathogenesis of SAP with symptoms of lung injury in clinical could be reproduced in this animal model. A good experimental animal model was provided for the studies of SAP lung injury.2. The characteristics of pulmonary edema and pulmonary leak in rat model of severe acute pancreatitisObjective We aimed to evaluate the severity of lung injury and the the characteristics of pulmonary edema caused by lung leakage, and to evaluate the experimental animal models, which was conveniently used to study ALI caused by SAP.Methods Sprague-Dawley rats were randomized into the experimental group and the sham operation group. SAP model was prepared through retrograde injection of 5% taurocholic acid. After injection, the pancreatic duct was ligated in the experimental group, but not injection in the sham operation group. We examined SAP rats for any early lung tissue damage at 3,6,12 and 24hours.Results A typical clinical manifestation with shortness of breath and frothy sputum so on was shown in SAP model in rats. Through the lung W/D, histological and electron microscopy observation, pulmonary edema and lung tissue damage of the experimental group were significantly higher than of the sham group.Conclusion Injury process and pathogenesis of ALI or ARDS caused by SAP in clinical could be reproduced in this animal model. A good experimental animal model was provided for the pulmonary capillary leakage studies of SAP lung injury.3. Mechanisms of pulmonary capillary leakage in an acute lung injury rat model of severe acute pancreatitisObjective We aimed to examine leakage pathways and aquaporin 1 and 5 expression in lung tissues of rats with early acute lung injury caused by SAP and to analyse mechanisms of pulmonary capillary leakage in pulmonary edema development.Methods Sprague-Dawley rats were randomized into the SAP group and the sham operation group. SAP model was prepared through retrograde injection of 5% taurocholic acid. We examined SAP rats for any early lung tissue injury, lung visceral pleura, lung ultrastructure damage with lanthanum nitrate tracer and MMP9, AQP1 and 5 expression in lung tissues at different time points (3,6,12, and 24hours) during disease development.Results Electron microscope found that serious destructions were observed in lung capillary endothelia, basal membranes, alveolar epithelia and lung pleura in SAP animals. Immunofluorescence staining revealed that AQP1 and 5 expression in the SAP lung tissues gradually decreased from 6 hours compared with controls. but MMP9 expression gradually increased compared with controls. Real-time RT-PCR analysis showed that AQP1 and 5 mRNA levels of the SAP rats significantly decreased 6hours post-experiment (P<0.001). MMP9 mRNA levels obviously increased and reached at the peak was at 12 hours (p<0.05). Western blotting analysis showed that AQP1 and 5 expression significantly decreased from 6hours (p<0.001). MMP9 protein expression significantly increased from 6 hours, and the peak of protein expression was at 24 hours(p<0.05).Conclusions Severe pulmonary edema and pleural effusion with ALI caused by SAP was determined by mechanisms of pulmonary capillary leakage, which were widen gap for damaged tight junction between endothelia or epithelia, channells in endothelia for cracks across the membrane, basement membrane dissolution and even fracture of lung pleura fibrous with sift-shaped holes. Decreased lung AQP1 and 5 expression may be related to pulmonary edema development and increased severity of lung injury and pulmonary edema, which provides an additional mechanism for pancreatitis-associated lung injury.4. Culture and purification of BMSCs and identification of BMSCsObjective We aimed to primarily culture, to purify BMSCs, and to identify BMSCs according to surface markers for BMSCs transplantation.Methods Bone marrow samples were collected from healthy young male Sprague-Dawley rats in sterility condition. Both lower limbs of the rats were removed and the medullary cavities were fushed with MSC medium, which consisting of Dulbecco's modified Eagle's medium (DMEM), low glucose supplemented with 10% fetal bovine serum(FBS), 100U/ml penicillin, 100mg/ml streptomycin and 2mmol/L L-glutamine. After resuspended, the cells were plated at a density of 5x10' cells/mL. Cultures were maintained in a humidified atmosphere with 5%CO2 at 37℃Adherence of BMSCs was observed. After passage 3, surface markers(CD29+, CD90+ CD34-, CD45-) were detected with flowcytometry. Surface markers(CD44+ CD29+) were detected with immunocytochemistry.Results The fifth passage BMSCs in vitro maintained a strong and stable proliferation and cytological characteristics. Identification with flowcytometry showed that the third passage BMSCs with immunophenotype CD29+ CD90+ CD34-CD45- were up to 98%. Immunocytochemical staining showed that P3 BMSCs with CD44+ CD29+ accounted for 98% or more. It was found that the P3 BMSCs was pure enough.Conclusion Affer serial passages, BMSCs are purified. Purity of P3 BMSCs is over 98%. BMSCs maintained a strong and stable proliferation and cytological characteristics during culture.5. Therapeutic benefits by allogeneic BMSCs on pulmonary capillary leakage in an acute lung injury rat model of severe acute pancreatitisObjective We aimed to observe the therapeutic benefits by allogeneic BMSCs on pulmonary capillary leakage in an acute lung injury rat model of severe acute pancreatitis and to analyze the possible mechanismsMethods Sprague-Dawley rats were randomized into the Control-SAP group, BMSCs-SAP group and MP-SAP group. SAP model was prepared through retrograde injection of 5% taurocholic acid. After SAP were induced successfully, BMSCs-SAP rats were therapied with BMSCs. MP-SAP rats were dealed with methylprednisolone sodium succinate(MP). Control-SAP animals were without treatment but only injected with L-DMEM. We examined SAP rats for any lung tissue injury, lung visceral pleura, lung ultrastructure damage with lanthanum nitrate tracer and MMP9, AQP1 and 5 expression in lung tissues at different time points (6 and 24hours) during disease development.Results In BMSCs-SAP group, electron microscope found that minor destructions were observed in lung capillary endothelia, basal membranes, alveolar epithelia and lung pleura than in Control-SAP animals. Real-time RT-PCR analysis and Western blotting analysis showed that AQP1 and 5 expression of the BMSCs-SAP group rats significantly increased from 12hours compared with Control-SAP group, but decreased compared with MP-SAP group (p<0.05). However, MMP9 expression of the BMSCs-SAP group rats significantly decreased from 12hours compared with Control-SAP group, and increased compared with MP-SAP group (p<0.05)Conclusions Transplanted allogeneic BMSCs inhibited inflammatory mediators (IL-1βand TNF-α) to block the inflammation pathway, to repress the expression of MMP9, and to reduce inflammatory injury in pulmonary capillary endothelia, basement membrane and pulmonary epithelia, for protecting the integrity of pulmonary capillary endothelial barrier and alveolar epithelial barrier. At the same time, BMSCs inhibited inflammatory mediators to maintain the normal expression of AQP1 and AQP5, which improved the transfer efficiency of pulmonary interstitial and alveolar edema fluid. Our results showed that the therapeutic effects of allogeneic BMSCs transplantation on pulmonary capillary leakage in a rat model of SAP was significantly positive. The short-term therapeutic effects of methylprednisolone sodium succinate was more obvious than the effects of BMSCs in SAP acute phase, but BMSCs transplantation treatment might have more advantages for replacement of damaged cells and tissue repair.SummaryIn conclusion, severe pulmonary edema and pleural effusion with ALI was determined by the ruinous damages in capillary endothelial barrier, alveolar epithelial barrier and lung pleura. Then ruptured capillary endothelia, incomplete capillary basal membranes, separated conjunction of pulmonary epithelia or pore-like structure in lung pleura played critical roles in the quickly, persistent and irreversible leaking progression of ALI and the development of its associated pulmonary complications in a bile induced model of SAP in rats. Otherwise, decreased lung AQP1 and 5 transferring efficiency due to down-regulated expression of these two proteins may be related to pulmonary edema development and increase severity of lung injury and pulmonary edema, which provides an additional mechanism for pancreatitis-associated lung injury.Our study showed that the therapeutic effects of allogeneic BMSCs transplantation on pulmonary capillary leakage in a rat model of SAP was significantly positive. The possible mechanisms were involved as the followings. BMSCs inhibited inflammatory mediators (IL-1βand TNF-α) to block the inflammation pathway, to repress the expression of MMP9, and to reduce inflammatory injury in pulmonary capillary endothelia, basement membrane and pulmonary epithelia, for protecting the integrity of pulmonary capillary endothelial barrier and alveolar epithelial barrier. At the same time, BMSCs inhibited inflammatory mediators to maintain the normal expression of AQP1 and AQP5, which improved the transfer efficiency of pulmonary interstitial and alveolar edema fluid. |
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