The Mechanism Of Severe Hypertriglyceridemia Aggravates Acute Lung Injury In Acute Pancreatitis

Acute pancreatitis(AP)is one of the most common acute abdominal diseases in internal medicine,and it manifests as a sudden inflammatory disease involving pancreatic exocrine functions.The main cause of AP includes gallstone,alcohol,hypertriglyceridemia and so on.The deterioration of AP could induce systemic inflammatory response and cause distant organ failures.It’s reported that the annual incidence of AP is 40-70/100,000 people and most of the patients are presented with acute interstitial edema pancreatitis,which has a mild disease course.Apart from the mild acute pancreatitis(MAP),there is still 10-20%patients suffering from uncontrollable systemic inflammatory response syndrome(SIRS)or infected pancreatic necrosis(IPN)and developing into severe acute pancreatitis(SAP).The common complication of SAP in clinic is multiple organ dysfunction syndrome(MODS),which mainly showing as acute lung injury(ALI),acute kidney injury(AKI)and acute hepatic failure(AHF)and the mortality rate could reach up to 10-40%.SAP patients usually experience two death peaks,and the former one is predominantly caused by SIRS and the following MODS,while the later one commonly comes from IPN,sepsis and the combined MODS.At present,the incidence of hypertriglyceridemic acute pancreatitis(HTG-AP)is accelerating year by year in China,and hypertriglyceridemia(HTG)has been the second leading cause of AP.According to previous clinical researches,the severity of AP is positively correlated with the serum triglyceride(TG)levels.What’s more,the incidence of MODS in AP patients is significantly higher than that in biliary pancreatitis patients,and HTG is an independent risk factor for ALI and AKI.Lung is the most easily affected distant organ in AP and 40-70%of SAP patients are complicated with ALI,the mortality rate of which could be as high as 60%.Hence,there is an urgent need to explore the underlying mechanism of HTG on the exacerbation of ALI in AP patients and it can provide basis theory for important strategy of clinical prevention and treatment of SAP.The mechanism of HTG aggravating AP related lung injury remains unclear.The latest research manifested that the NLRP3 inflammasome pathway plays important role in pathogenesis of ALI and free fatty acid(FFA)can activate the NLRP3 inflammasome pathways.Therefore,we put forward such a hypothesis in our study:FFA created by TG lipolysis could aggravate HTG-AP related lung injury by activating NLRP3 inflammasome pathway.There has reported a variety of HTG-AP animal models at present,including lipoprotein lipase(LPL)knockout mice,Apolipoprotein-C3(Apo-C3)transgenic mice and high-fat diet feeding hamster,while all of them have different degrees of defects and own limits in their application.Hence,in order to further investigate the mechanism of HTG-AP,there is an urgent need to establish a novel HTG-AP animal model.In this study,we establish a novel HTG-AP mice model by using poloxamer 407(P-407)combined with Caerulein(Cae).Through the use of two severe hypertriglyceridemia(SHTG)mice model(P-407 induced and GPIHBP1 knockout(GPIHBP1-/-)mice)separately,we aim to observe dynamically the effect of HTG on AP related ALI or lipopolysaccharide(LPS)induced ALI,at the same time,we try to explain the possible mechanism from the view of NLRP3 inflammasome pathway.Our study could be divided into two parts:Part one:Development of a novel model of hypertriglyceridemic acute pancreatitis in miceObjective:the morbidity and mortality rates of HTG-AP increased rapidly over the last decade.However,an appropriate animal model was lacking to recapitulate this complicated human disease.This study aimed to establish a novel mice HTG-AP model.Methods:6 to 8 weeks C57BL/6 mice were selected as experimental animals,they were divided into four groups randomly:PBS group,P-407 group,PBS+Cae group and P-407+Cae group.SHTG model was induced by intraperitoneal injection of P-407 every other day,and then inducing AP by Cae intraperitoneal injection.After 12 hours,mice were executed in anesthetic condition,then blood and pancreatic tissue samples were collected.We aimed to observe the effect on AP in different durations and extents of HTG.What’s more,a very low dose Cae(5μg/kg)was used to stimulate P-407 induced SHTG mice and their control mice,through which we could explore the influence of SHTG to susceptibility of AP.Finally,we detect the distribution of immune cells in pancreas tissue of SHTG mice by flow cytometry.Results:In our study,serum TG levels of P-407 induced mice were elevated in a dose-dependent manner,and high dose P-407(0.5g/kg)could induce serum TG concentration at a level higher than 5000mg/dl steadily.The pancreatic and pulmonary injuries were much severer in HTG mice than the control mice when injected with conventional dose Cae(50 μg/kg).What’s more,the severity of AP was positively correlative with the duration and extent of HTG.In addition,we found that a very low dose Cae(5 μg/kg)could induce pancreatic injury in SHTG mice,mainly manifesting as pancreatic edema and inflammatory cells infiltration,while there was no obvious pathological injury in normal mice.Finally,we observed that HTG leaded to the increased infiltrations of CD45+F4/80+cells and CD45+Gr-1+cells in mice pancreatic tissues by using flow cytometry.Conclusion:we have developed a novel mice HTG-AP model,which was steady and controllable and could mimic the physiological,histological and clinical features of human HTG-AP.Part two:The mechanism of severe hypertriglyceridemia aggravates acute lung injury in acute pancreatitisObjective:On the basis of our novel HTG-AP mice model,we aim to observe the effect of SHTG on AP related ALI and explore the possible underlying mechanism.Methods:P-407 induced SHTG mice,GPIHBP1 knoclout(GPIHBP1-/-)mice and their control mice were used.Intraperitoneal inject Cae(50μg/kg)to build AP related ALI model or intraperitoneal inject LPS(5mg/kg)to build ALI model.Serum,bronchoalveolar lavage fluid(BALF),pancreatic and pulmonary tissues samples were collected.In addition,human line A549 was applied in vitro studies.LPS(20μg/ml)was used to build model of lung epithelial cell damage and oleic acid(OA)were administration to observe effect of FFA in cell damage.What’s more,MCC950,which is specific inhibitors of NLRP3 inflammasome pathway,is applied to explore mechanism.Results:1.Either in Cae induced AP related ALI model or LPS induced ALI model,SHTG both exacerbated the severity of ALI in mice,and the levels of PALF inflammatory cytokines in SHTG mice,such as TNF-α、MCP-1、IL-1β、IL-6 elevated to varying degrees in comparison with the control mice.Immunohistochemical showed expressions of MPO and CD68 in pulmonary tissues of SHTG mice are remarkable higher than the control mice.Protein and mRNA levels of NLRP3、AS C、Caspase-1 and IL-1β in mice pulmonary tissues elevated obviously after ALI were induced,especially in SHTG mice.2.In vitro experiment,we found that incube A549 cells with OA simply cannot cause cell damage,but OA can aggravate cell damage induced by LPS and lead to protein and mRNA levels of NLRP3、ASC、Caspase-1 and IL-1β of A549 cell increased,this effect could be counteracted by MCC950.Conclusion:SHTG could exacerbate AP related ALI and LPS induced ALI by activating NLRP3 inflammasome pathway of lung epithelial cell.