| ObjectiveAcute pancreatitis?AP?is a common clinical acute abdominal disease.Approximately15%to 20%of AP patients developed to severe acute pancreatitis?SAP?.SAP acute onset,rapid progress,and clinical and pathological changes are complex.The mortality of SAP is as high as 30%to 50%due to systemic inflammatory response syndrome?SIRS?during the early stages and subsequently multiple organ failure?MOF?.So it is the main cause of death in patients with clinical gastrointestinal disorders.According to 2012 revised Atlanta classification of acute pancreatitis,SAP has been redefined to be associated with persistent organ failure?lasting more than 48 h?in addition to the clinical presentation and biochemical changes of AP.Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.The pathophysiology of sepsis is divided into two stages.First,the infection and the body's immune disorders are caused by the invasion of the pathogenic bacteria.The initial symptom of body is excessive inflammatory response,which means entering cytokine storm phase.Because of sustained immune disorders,body enters the phase of immunosuppression.And the majority of patients died at this stage due to secondary infection and multiple organ dysfunction syndrome?MODS?.Recent studies showed that most patients do not die of excessive inflammation,but die of immunosuppression and multiple bacterial infections at a later stage.It has been suggested that secondary bacterial infection in SAP may be associated with its immunosuppressive state,and there is no report on whether there is a process from overinflammatory state to immunosuppressive state in the SAP animal model and the timing point of immunosuppression.It has been reported that SAP shares an indistinguishable profile of inflammation with sepsis.In our previous study,we found that the turning point of over-inflammation to immunosuppressionis was on Day 1 after surgery in CLP sepsis mouse model.Herein,whether there is a transition from hyperinflammatory response to immunosuppressive state in the SAP animal model and the timing of the immunosuppressive turning point were systematically investigated.There are several methods for the establishment of experimental animal models of AP.The bile salt-induced SAP model has been widely accepted as a representative SAP model.In this study,SAP rat model was established by a retrograde injection of sodium taurocholate?NaTc?,which could induce severe hemorrhagic necrosis of pancr eas in a short time,and reflect the pathophysiological process and mechanism of SAP.Different concentrations of sodium taurocholate can replicate SAP models with different severity.In this study,based on the establishment of SAP rat model,Pseudomonas aeruginosa?PA?was selected as the secondary against pathogen to study the immunosuppressive state of SAP rats and the transition from excessive inflammation to immunosuppression,which lay the foundation of the studying of the pathophysiological process of immunosuppressive in SAP and the treatment of different pathophysiological stages of SAP.Antimalarial artesunate?AS?is a water-soluble hemisuccinate derivative of dihydroartemisinin,which was widely used in the treatment of malaria with low toxicity and good safety.Recently,its diverse pharmacological properties such as anti-inflammatory action,enhanced antibacterial activity,and anti-tumor effects have been discovered.Our group has been committing to clinical studies of the new indications of A S.Our preliminary study has confirmed that AS has a good therapeutic effect on mouse model of LPS-tolerant and CLP immunosuppression.And AS also has a remarkable therapeutic effect on the acute pancreatitis model caused by the combination of caerulein?CR?and lipopolysaccharide?LPS?in mice.Therefore,this study performed a retrograde injection of sodium taurocholate via micro-pump through the pancreatic duct to establish the rat model of SAP.Then we did a systemic investigation on the turning point of SAP rats from over-inflammatory to immunosuppression state through the susceptibility to bacteria,cytokine levels,bacterial load and immune cell number changes.On the basis of this,we observed the therapeutic effect of artesunate on SAP immunosuppressive rats,which provide experimental evidence for further understanding of the pathophysiological process of SAP,and the treatment of SAP in different stages of pathophysiology and broaden the clinical indications of artesunate.Methods1.Establishment and evaluation of the SAP immunosuppressive rat model1.1.Establishment of SAP rat model with different mortalities:The SAP rat model was induced by retrograde injection of different doses of sodium taurocholate through the pancreatic duct.Then the 7-day mortalities were observed.The dose which can lead 40%70%rats to death was selected as the SAP model establishment method.1.2.Screening of the sublethal dose of PA:The rats were intravenously injected with different doses of PA(1.0×108,2.0×108,4.0×108,8.0×108,1.0×109,1.0×1010,and1.0×1011 CFU/kg body weight,0.2 m L/100 g body weight).The mortalitied in each group were observed for 7 days to get the sublethal dose of PA in rats.1.3.Observation of the sensitivity of SAP rats to PA challenge at different time points after model establishment:SAP rats were randomly divided into two groups:Sham group and SAP group.Two groups of rats were intravenously injected with 4×108 CFU/kg of PA?0.2 m L/100 g?on Day 1,Day 2,and Day 3 after SAP model establishment,respectively.For survival analysis,the mortality in each group was observed for 7 days.To further observe the mortality of animals at different time points within 2 days,rats were intravenously injected with 4×108 CFU/kg of PA at 0,8,12,24,36,and 48 h after SAP model establishment,respectively.For survival analysis,the mortality in each group was observed for 7 days.1.4.Confirmation of the time-point from over-inflammation to immunosuppression in SAP rat model:To confirm whether the high sensitivity of SAP rats to PA was due to excessive inflammation or immunosuppression,SAP rats were randomly divided into two groups:Sham group and SAP group.Two groups of rats were intravenously injected with 4×108 CFU/kg of PA at 0,12 and 24 h after SAP model establishment,respectively.Blood and tissues samples were taken at 2 h after PA injection for the detection of proinflammatory cytokine levels.1.5.Detection of bacterial clearance ability in SAP immunosuppressive rats:Rats were intravenously injected with 4×108 CFU/kg of PA at 24 h after SAP model establishment,and at 2 h after PA injection,the blood,lungs and spleen were collected for CFU assays.Blood or homogenates of lungs and spleen tissues were serially diluted.Subsequently,100?l of the diluents was added to MHA nutrient agar plates and cultured for 18 h.Images of the agar plates were recorded and CFU counts were obtained using a G6automated colony-counter.1.6.Detection of proinflammatory cytokines in SAP immunosuppressive rats:The levels of TNF-?and IL-1?were determined using appropriate ELISA kits.For blood samples,the serum was collected for cytokines assays.For rat tissues samples,20%tissue homogenate supernatants were collected for cytokine assays.1.7.The numbers of immune cells and the proportion of cell subtypes in SAP immunosuppressive rats:Rats were injected with 4×108 CFU/kg of PA at 24 h after SAP model establishment;and then at 2 h after the PA challenge,blood samples were collected in heparin anticoagulant tube for five classification of blood analysis using five categories of animal blood analyzer.1.8.Detection of the levels of serum amylase?AMS?and pancreatic lipase?LP?:The changes of serum amylase and pancreatic lipase were measured at different time poin ts after SAP model establishment.And the levels of serum amylase and pancreatic lipase in SAP immunosuppressive rats were also measured.2.Therapeutic effect of AS on SAP immunosuppressive rats2.1.To observe the protective effect of artesunate on SAP immunosuppressive rats with PA challenge:SAP rats were injected with 4×108 CFU/kg of PA at 24 h after SAP model establishment;and rats were treated with AS after SAP model establishment,2 times a day,with a continuous administration for 3 days.For survival analysis,the mortality in each group was observed for 7 days.2.2.To observe the effect of artesunate on the cytokines of SAP immunosuppressive rats with PA challenge:The levels of TNF-?and IL-1?were determined using appropriate ELISA kits.SAP rats were injected with 4×108 CFU/kg of PA at 24 h after SAP model establishment.And rats were treated with AS at 0,12,and 24 h after SAP model establishment,a total of 3 times.The samples were collected at 2 h after PA challenge.For blood samples,the serum was collected for cytokines assays.For rat tissues samples,20%tissue homogenate supernatants were collected for cytokine assays.2.3.To observe the effect of artesunate on blood cells of SAP immunosuppressive rats with PA challenge:SAP rats were injected with 4×108 CFU/kg of PA at 24 h after SAP model establishment.And rats were treated with AS at 0,12,and 24 h after SAP model establishment,a total of 3 times.At 2 h after PA challenge,the blood samples were collected in heparin anticoagulant tube for five classification of blood analysis using five categories of animal blood analyzer.2.4.To observe the effect of artesunate on bacterial clearance ability of SAP immunosuppressive rats with PA challenge:AS treatment was given at 0,12,and 24 h after model establishment,a total of 3 times.SAP rats were injected with 4×108 CFU/kg of PA at 24 h after SAP model establishment.And the blood,lung and spleen samples were collected at 2 h after PA challenge for CFU assays.Blood or homogenates of lungs and spleen tissues were serially diluted.Subsequently,100?l of the diluents was added to MHA nutrient agar plates and cultured for 18 h.Images of the agar plates were recorded and CFU counts were obtained using a G6 automated colony-counter.Results1.Establishment and evaluation of the SAP immunosuppressive rat model1.1.The establishment of the SAP rat model:The SAP rat model with different mortality was successfully established by different concentrations of sodium taurocholate.The SAP rat model with 48.1%mortality was treated with a retrograde injection of 5%sodium taurocholate,0.2 m L/100 g,0.2 m L/min through the pancreatic duct.1.2.Screening of the sublethal dose of PA:Different doses of PA challenge,rats had different mortalities.The dose of 4.0×108 CFU/kg was successful selected as the sublethal dose of PA,and used in subsequent experiments.1.3.The sensitivity of SAP rats to PA challenge was different at different time points after model establishment:The mortality of rats challenged with PA was different at different time points?1d,2d,3d?after SAP model establishment.SAP rats were the most susceptible to bacteria on Day 1 after the model establishment.SAP rats were given sublethal dose of PA at different time within 2 days.The result showed that the mortality of SAP+PA group was significantly higher than SAP group at 0,8,12 and 24 h after SAP model establishment?P<0.05?.But it is not clear that the high mortality of SAP+PA group was caused by whether excessive inflammation or immune suppression.1.4.Confirmation of the time-point from over-inflammation to immunosuppression in SAP rat model:The levels of TNF-?and IL-1?in SAP+PA group and Sham+PA group were much higher than those in SAP group at 0 h and 12 h after SAP model establishment,and there was no significant difference between the two groups at 0 h and 12 h?P>0.05?;indicating that SAP rats challenged with PA were died of over-inflammation at 0 h and 12 h after SAP model establishment.While the levels of TNF-?and IL-1?in SAP+PA group were slightly higher than those in Sham group but significantly lower than those in Sham+PA group?P<0.05 or 0.01?,indicating that SAP rats challenged with PA were died of immunosuppression at 24 h after model establishment.The above results indicated that 24 h after SAP model establishment was the turning point of hyperinflammatory state to immunosuppressive state.1.5.The bacterial loads of rats challenged with PA at 24 h after SAP model establishment:The results showed there was no or less bacteria in serum,lungs and spleen for NS,Sham and SAP rats without bacterial challenge.The bacte rial loads were high for Sham+PA group,however,they were much higher for SAP+PA group?P<0.01?,suggesting SAP rats had very lower clearance ability for bacteria than Sham rats at 24 h after SAP model establishment,demonstrating the time-point of 24 h after SAP model establishment was the turning point from over-inflammation to immunosuppression.1.6.Changes of proinflammatory cytokine levels in rats challenged with PA at 24 h after SAP model establishment:The levels of TNF-?and IL-1?in serum,spleen,lung and liver were significantly lower than those in Sham rats after bacterial challenge?P<0.05?,suggesting the release of proinflammatory cytokines is in a state of inhibition,again demonstrating 24 h after SAP model establishment was the turning point from over-inflammation to immunosuppression.1.7.Changes in the numbers of immune cells and the proportion of cell subtypes in rats challenged with PA at 24 h after SAP model establishment:The number of white blood cells significantly decreased in SAP immunosuppressive rats challenged with PA.And the numbers of lymphocytes and monocytes were significantly lower than those of Sham rats challenged with PA?P<0.05?,demonstrating the decreased amount of important white cells were tightly related to immunosuppression of SAP rats.1.8.Detection of the levels of serum amylase?AMS?and pancreatic lipase?LP?in SAP immunosuppressive rats and SAP rats at different time points:The activity of serum amylase in SAP rats began to rise at 2 h after the model establishment,rapidly increased to12 h peak,and then decreased rapidly,72 h down to normal levels.The activity of lipase in pancreatic tissue was similar to that of serum amylase.The activity of amylase in SAP immunosuppression rats was significantly higher than that in Sham rats?P<0.01?,and the activity of amylase in SAP+PA rats was significantly higher than that in Sham+PA rats and SAP rats?P<0.05?.The activity of lipase in pancreatic tissue of SAP immunosuppression rats was significantly higher than that of Sham rats?P<0.05?,but there was no significant difference compared with SAP+PA rats and Sham+PA rats?P>0.05?.2.Therapeutic effect of AS on SAP immunosuppressive rats2.1.Artesunate could increase the survival rate of SAP rats by 23 percentage points,and could increase the survival rate of SAP immunosuppressive rats challenged with PA by20 percentage points.2.2.Artesunate could increase the levels of TNF-?and IL-1?in SAP rats and SAP immunosuppressive rats with PA challenge.2.3.Artesunate could increase the number of white blood cells,lymphocytes and monocytes in blood of SAP rats and SAP immunosuppressive rats challenged with PA.However,the impact on the percentage of white cell fractionation is small.2.4.Artesunate could increase the bacterial loads in blood,lung and spleen of SAP immunosuppression rats challenged with PA.Conclusions1.The SAP rat model with 48.1%mortality was successfully established by retrograde injection of 5%sodium taurocholate in pancreatic duct.It has the advantages of good stability,high repeatability,simple operation and high success rate.And it is also conducive to high-volume operation.2.The turning point of over-inflammation to immunosuppression appears at 24 h after SAP model establishment.And at this turning point,SAP rats showed higher bacterial sensitivity,higher bacterial load and lower levels of proinflammatory cytokines.3.AS could significantly reduced the 7-day mortality of SAP immunosuppressive rats challenged with PA,suggesting that AS had a good therapeutic effect on SAP immunosuppressive rats.4.AS could significantly increased the level of TNF-?and IL-1?in SAP immunosuppressive rats,and significantly increased the ability of the clearance of Pseudomonas aeruginosa in SAP immunosuppressive rats. |
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