Abnormal Mechanisms Of Neutrophil Apoptosis Mediated By Endoplasmic Reticulum Signal Pathway In Severe Acute Pancreatitis And Protective Effect Of Emodin

Background:Acute pancreatitis(AP)is a common acute abdominal disease.Clinically,AP can be classified as mild acute pancreatitis(MAP),moderately severe acute pancreatitis(MSAP)or severe acute pancreatitis(SAP).SAP is extremely dangerous.The reason that SAP is difficult to treat is that it has two peak time points at which the risk of mortality is high,the first one occurs owing to early multiple organ dysfunction syndromes(MODS)that are usually caused by the systemic inflammatory response syndrome(SIRS),and the second one occurs owing to late MODS,which are usually caused by necrosis in the pancreas and sepsis.Therefore,the prevention of SIRS is undoubtedly important in the early treatment of SAP.However,the exact mechanism of SIRS is not yet clear.There is still no effective treatment for SIRS.Emodin(1,3,8-trihydroxy-6-methyl-anthraquinone)is the main active component of rhubarb.Many findings support the idea that emodin has benefits as a pharmaceutical treatment for inflammation and reduce the release of inflammatory mediators,and has therapeutic effect on SIRS.However,the exact mechanisms of emodin's therapeutic effect on SIRS are poorly understood.This study aims to investigate mechanisms of SAP inducing SIRS and mechanisms of emodin's therapeutic effect on SIRS induced by SAP.Objective:1.To explore the abnormal apoptosis of neutrophils and its probable mechanism in SAP with SIRS by clinical experiment.2.To further clarify the molecular mechanism of abnormal neutrophil apoptosis in SAP with SIRS and corresponding protective effect and mechanism of emodin by cellular level experiment.3.To verify the protective effect and mechanisms of emodin on SAP with SIRS by animal level experiment,and to provide theoretical basis for the application of emodin in treatment of SAP with SIRS.Methods:1.Part one.The healthy volunteers were assigned to a CON group,the patients with SIRS caused by SAP were assigned to an SAP group.Blood samples were taken,and the level of TNF-?,IL-6 and CRP were tested.Neutrophils were isolated.Both neutrophil apoptotic rates and the level of caspases 4,3 were detected.2.Part two.To clarify the neutrophil apoptotic changes in SAP rats and to explore the pro-apoptotic effect and mechanisms of emodin,rats were randomly divided into 3groups,one group received sham-operation,other two groups received model-operation.Neutrophils from rats that received sham-operation were assigned to an SO group,and neutrophils from rats that received model-operation were assigned to an SAP group and an EM group respectively.After treatment,neutrophil apoptotic rates were detected,protein expression level of Bi P and calpain 1 were examined,endoplasmic reticulum(ER)Ca²⁺and cytosolic Ca²⁺were determined,the protein activity of calpain1 was measured.To clarify the regulatory mechanism of emodin on Ca²⁺homeostasis,rats were randomly divided into 3 groups,each group received model-operation,neutrophils from rats of the 3 groups were assigned to an SAP group,an EM group and an EH group respectively.After treatment,ER Ca²⁺and cytosolic Ca²⁺in neutrophils were determined.To confirm that emodin activates calpain 1 via elevating cytosolic Ca²⁺,rats were randomly divided into 3 groups,each group received model-operation,neutrophils from rats of the 3 groups were assigned to an SAP group,an EM group and an EB group respectively.After treatment,protein activity of calpain 1 in neutrophils was determined.To confirm that emodin induce neutrophil apoptosis via calpain 1-caspase12-caspase 3 pathway,rats were randomly divided into 5 groups,one group received sham-operation,other four groups received model-operation.Neutrophils from rats that received sham-operation were assigned to an SO group,and neutrophils from rats that received model-operation were assigned to an SAP group,a PD group,an EM group and a PE group respectively.After treatment,calpain1 activity,caspases 12,3 levels in neutrophils and neutrophil apoptotic rates in each group were examined.3.Part three.To explore the effect of emodin on ER Ca²⁺and cytosolic Ca²⁺in neutrophils in vivo studies,rats were randomly divided into an SO group,an SAP group and an EM group.After treatment,blood samples were taken,and neutrophils were isolated so that ER Ca²⁺and cytosolic Ca²⁺distributions could be examined in various group.To confirm that emodin induces neutrophil apoptosis via Ca²⁺-calpain 1-caspase12-caspase 3 pathway,by which alleviating SIRS and reducing mortality,rats were randomly divided into an SO group,an SAP group,a PD group,an EM group and a PE group.After treatment,blood samples were taken,and neutrophils were isolated.Protein expression level and protein activity of calpain 1 were examined,caspases 12,3 levels were measured,neutrophil apoptotic rates were detected.Histological socores of pancrease,serum level of TNF-??IL-6,SIRS scores,survival rate were also examined.Result:1.Part one.The serum level of TNF-??IL-6?CRP is obviously higher in group SAP than those in group CON.The neutrophil apoptotic rates,protein expression level of cleaved-caspases 4,3 is obviously lower in group SAP than those in group CON.2.Part twoNeutrophil apoptotic rates in group SO,SAP,EM:Compared with group SO,neutrophil apoptotic rates decreased in group SAP.Compared with group SAP,neutrophil apoptotic rates increased in group EM.Bi P protein expression in group SO,SAP,EM:The expression levels of Bi P were comparable between group SO and group SAP.Compared with group SO and group SAP,the expression levels of Bi P increased significantly in group EM.ER Ca²⁺and cytosolic Ca²⁺levels in group SO,SAP,EM,EH:ER Ca²⁺levels were obviously elevated and cytosolic Ca²⁺levels were obviously reduced in group SAP relative to the levels in group SO;ER Ca²⁺levels were obviously reduced and cytosolic Ca²⁺levels were obviously elevated in group EM relative to levels in group SAP;ER Ca²⁺levels were obviously elevated and cytosolic Ca²⁺were obviously reduced in group EH relative to levels in group EM.The changes of protein expression level of calpain1 in group SO,SAP,EM and the changes of protein activity level of calpain1 in group SO,SAP,EM,EB:Protein expression levels of calpain1 did not show significant changes among any of the groups.However,calpain1 activity levels was suppressed in group SAP relative to levels in group SO,and enhanced in group EM relative to levels in group SAP,and suppressed in group EB relative to levels in group EM.Protein activity level of calpain1 in group SO,SAP,PD,EM,PE:Compared with group SO,calpain1 activity decreased in group SAP and group PD,there is no difference in calpain1 activity between group SAP and group PD.Compared with group SAP,calpain1 activity increased significantly in group EM.Compared with group EM,calpain1 activity decreased significantly in group PE.Cleaved-caspases 12,3 levels in group SO,SAP,PD,EM,PE:Compared with group SO,cleaved-caspases 12,3 levels decreased in group SAP and group PD,there is no difference in cleaved-caspases 12,3 levels between group SAP and group PD.Compared with group SAP,cleaved-caspases 12,3 levels increased significantly in group EM.Compared with group EM,cleaved-caspases 12,3 levels decreased significantly in group PE.Neutrophil apoptotic rates in group SO,SAP,PD,EM,PE:Compared with group SO,neutrophil apoptotic rates decreased in group SAP and group PD,there is no difference in neutrophil apoptotic rates between group SAP and group PD.Compared with group SAP,neutrophil apoptotic rates increased significantly in group EM.Compared with group EM,neutrophil apoptotic rates decreased significantly in group PE.3.Part three.ER Ca²⁺and cytosolic Ca²⁺levels in group SO,SAP,EM:Compared with group SO,ER Ca²⁺levels increased in group SAP.Compared with group SAP,ER Ca²⁺levels decreased significantly in group EM.Compared with group SO,cytosolic Ca²⁺levels decreased in group SAP.Compared with group SAP,cytosolic Ca²⁺levels increased in group EM.Protein activity levels of calpain1 in group SO,SAP,PD,EM,PE:Compared with group SO,calpain1 activity levels decreased in group SAP and group PD,there is no difference in calpain1 activity levels between group SAP and group PD.Compared with group SAP,calpain1 activity levels increased significantly in group EM.Compared with group EM,calpain1 activity levels decreased significantly in group PE.Cleaved-caspases 12,3 levels in group SO,SAP,PD,EM,PE:Compared with group SO,cleaved-caspases 12,3 levels decreased in group SAP and group PD,there is no difference in cleaved-caspases 12,3 levels between group SAP and group PD.Compared with group SAP,cleaved-caspases 12,3 levels increased significantly in group EM.Compared with group EM,cleaved-caspases 12,3 levels decreased significantly in group PE.Neutrophil apoptotic rates in group SO,SAP,PD,EM,PE:Compared with group SO,neutrophil apoptotic rates decreased in group SAP and group PD,there is no difference in neutrophil apoptotic rates between group SAP and group PD.Compared with group SAP,neutrophil apoptotic rates increased significantly in group EM.Compared with group EM,neutrophil apoptotic rates decreased significantly in group PE.Pancreatic pathologic presentation in group SO,SAP,PD,EM,PE:Compared with group SO,pancrease from the rats in group SAP and group PD presented with severe degree of damaged lobules,hemorrhaging,necrosis and infiltration by neutrophils and monocytes.There is no difference in pancreatic pathologic scores between group SAP and group PD.Compared with group SAP,pancrease from the rats in group EM and group PE presented with mild degree of damaged lobules,hemorrhaging,necrosis and infiltration of neutrophils and monocytes.There is no significant difference in pancreatic pathologic scores between group EM and group PE.Serum levels of TNF-??IL-6 in group SO,SAP,PD,EM,PE:Compared with group SO,serum levels of TNF-??IL-6 increased in group SAP and group PD,there is no difference in serum levels of TNF-??IL-6 between group SAP and group PD.Compared with group SAP,serum levels of TNF-??IL-6 decreased significantly in group EM.Compared with group EM,serum levels of TNF-??IL-6 increased significantly in group PE.SIRS scores in group SO,SAP,PD,EM,PE:Compared with group SO,SIRS scores increased in group SAP and group PD,there is no difference in SIRS scores between group SAP and group PD.Compared with group SAP,SIRS scores decreased significantly in group EM.Compared with group EM,SIRS scores increased significantly in group PE.The survival rate difference among group SO,SAP,PD,EM,PE:Compared with group SO,the survival rate decreased in group SAP and group PD.Compared with group SAP,the survival rate increased significantly in group EM.Compared with group EM,the survival rate decreased significantly in group PE.Conclusions:1.Delayed neutrophil apoptosis is an important factor that leads to SIRS in patients with SAP.Delayed neutrophil apoptosis occurred via downregulation of cleaved caspase3,rather than downregulation of transcription and translation of caspase 3.The signaling pathway underlying delayed neutrophil apoptosis is mediated by caspase 4.2.Neutrophil apoptosis is delayed in SIRS in rats with SAP,and emodin is able to induce neutrophil apoptosis in vitro studies.The mechanism underlying this effect is relying on Ca²⁺-calpain 1-caspase 12-caspase 3 pathway.3.Delayed neutrophil apoptosis leads to SIRS and increases motality in rats with SAP.Emodin has protective effect against SIRS and decreases motality in rats with SAP.The mechanism underlying this effect includes the following steps:emodin increases cytosolic Ca²⁺levels via depletion of ER Ca²⁺levels.Calpain1 is activated by increased cytosolic Ca²⁺levels.Activated calpain1 cleaves caspase 12,and the activation of ER-resident caspase 12 causes the activation of cytoplasmic caspase3.Activated caspase 3 induce the neutrophil apoptosis.Increased neutrophil apoptosis reduce the release of inflammatory mediators,such as TNF-?,IL-6,by which SIRS is suppressed and modality is reduced.