| Background and objectives: Severe acute pancreatitis (SAP) is a commonclinical disease with acute onset, rapid progress, multiple complications, and highfatality rate. Systemic inflammatory response syndrome (SIRS) is an earlymanifestation when SAP grows into multiple organ dysfunction syndrome (MODS), andSIRS is a major reason to the high fatality rate of SAP. Inflammatory overreaction willresult in immune function decline, organic infection, impaired functions, and lead tohigher incidence of complications[1,2], which will aggravate SAP. Therefore, the key tolower the fatality rate of SAP is to block the progress from SIRS to MODS.Polymorphonuclear neutrophils (PMN) are important effector cells of inflammatoryreaction, and play a key role in the occurrence, development and turnover ofinflammation[4]. The abnormity or delayed apoptosis of PMN is a mainpathophysiological course of SIRS. Calcium ions as the most common signaltransduction factor in human body, play an important role in cell division, growth anddeath, and regulate the physiological activities of various cells and tissues, so thedisequilibrium of calcium homeostasis is a key step in cell apoptosis.The objectives of this study are to build SAP-affected SIRS rat models, to studythe calcium ion concentration variation inside PMN cells and mitochondria, toinvestigate the effects of Emodin and Dexamethasone on calcium ion concentrationvariation and the mechanisms, to determine whether calcium homeostasis is thetherapeutic target of Emodin and Dexamethasone, and to provide theoretical basis forclinical treatment of SAP.Methods: SIRS rat models of severe acute pancreatitis were built via retrogradeinjection of1.5%sodium deoxycholate solution through biliopancreatic duct.Stage1, grouping and modeling: Healthy SD rats were randomly divided intoGroup A (sham operation), Group B (SAP SIRS model control), Group C (Emodinintervention); Groups C1-C3(final Emodin concentrations1,5and10ug/ml), with10 rats in each group. In-vitro intervention:24h after modeling, peripheral blood wascollected to separate PMN, and then all groups were intervened: Group A (normalsaline), Group B (normal saline), Group C1(1ug/ml Emodin), Group C2(5ug/mlEmodin), and Group C3(10ug/ml Emodin). Then24h after intervention, PMN werecollected and PMN apoptosis rate was detected for each group.Stage2: grouping and modeling: Healthy SD rats were randomly divided into5groups: Sham-operated group (Group SO), SAP SIRS model control group (GroupMCO), Dexamethasone intervened group (Group DEX), Emodin intervened group(Group EMD, final concentration5ug/ml, determined from the results in stage1), andEmodin+Dexamethasone intervened group (Group EAD), with20rats in each group(the latter4groups together were called Group SAP.SIRS). In-vitro intervention:24hafter modeling, the PMN separated by peripheral blood in the SO group were dividedinto2halves, viz. sham-operated control group (Group SOC) and sham-operatedcalcium antagonist group (Group SOCA). All groups were intervened: Group SOC(normal saline), Group SOCA (final Lacidipine concentration1μg/L, final ethanolconcentration <0.2%), Group MCO (normal saline), Group DEX (final concentration0.1umol/ml), Group EMD (final concentration5μg/L, final ethanol concentration<0.1%), and Group EAD. Then24h after intervention, PMN were collected, andapoptosis rate and intracellular and intramitochondrial calcium ion concentrationchanges were detected for each group.ResultsStage1: Test PMN apoptosis percentage in each group was detected by using flowcytometry: the percentage of PMN apoptosis of Group A was significantly higher thanGroup B (P<0.01); Group C1and Group C2were both higher than Group B (P<0.01),further more, Group C2was apparently higher than Group C1(P<0.05); PMN inGroup C3were basically in the advanced stage of apoptosis or the stage of secondarynecrosis.Stage2:1.Test PMN apoptosis percentage in each group was detected by usingflow cytometry(1) Group SO: The percentage of PMN apoptosis of Group SOC was significantlyhigher than Group SOCA(P<0.01).(2) Group SAP.SIRS: Compared with Group MCO, the percentage of PMNapoptosis of Group DEX, Group EMD, and Group EAD was significantly higher (P<0.05), and the increasing degree was the lowest in Group DEX. The percentage ofPMN apoptosis of Group EMD and Group EAD were both higher than Group DEX(P<0.05).There was no difference between Group EMD and Group EAD (p>0.05).(3) Group SO and Group SAP.SIRS: The percentage of PMN apoptosis of GroupMCO was significantly lower than Group SOC(P<0.01). Both of Group EMD andGroup EAD were higher than Group SOC (P<0.05). Group DEX was lower than GroupSOC (P<0.05).2.Test intracellular calcium ion concentration variations detected by using laserscanning and confocal microscope.(1) Group SO: The intracellular calcium concentration of Group SOCA wassignificantly lower than Group SOC (P<0.01).(2) Group SAP.SIRS: Compared with Group MCO, the intracellular calciumconcentration of Group DEX, Group EMD, and Group EAD was significantly higher(P<0.05), and the increasing degree was the lowest in Group DEX. Both of Group EMDand Group EAD were higher than Group SOC (P<0.05).There was no differencebetween Group EMD and Group EAD (p>0.05).(3) Group SO and Group SAP.SIRS: The intracellular calcium concentration ofGroup MCO was significantly lower than Group SOC(P<0.01). Both of Group EMDand Group EAD were higher than Group SOC (P<0.05). Group DEX was lower thanGroup SOC (P<0.05).3.Test intramitochondrial calcium ion concentration variations detected by usinglaser scanning and confocal microscope(1) Group SO: The intramitochondrial calcium concentration of Group SOCA wassignificantly lower than Group SOC (P<0.01).(2) Group SAP.SIRS: Compared with Group MCO, the intramitochondrial calciumconcentration of Group DEX, Group EMD, and Group EAD was significantly higher(P<0.05), and the increasing degree was the lowest in Group DEX. Both of Group EMDand Group EAD were higher than Group SOC (P<0.05).There was no differencebetween Group EMD and Group EAD (p>0.05).(3) Group SO and Group SAP.SIRS: The intramitochondrial calcium concentrationof Group MCO was significantly lower than Group SOC(P<0.01). Both of Group EMDand Group EAD were higher than Group SOC (P<0.05). Group DEX was lower thanGroup SOC (P<0.05). Conclusions: Neutrophils apoptosis rates in SAP SIRS rat models weresignificantly lower than those of normal rats, probably due to the calcium homeostasisin neutrophils. Emodin could promote the apoptosis of PMN in SIRS rat of severe acutepancreatitis and inhibit the delayed apoptosis by increasing intracellular andintramitochondrial calcium ion concentrations. Though Dexamethasone could increaseintracellular and intramitochondrial calcium ion concentrations and promote theapoptosis of PMN, the effects were lower than Emodin. The combination ofDexamethasone and Emodin did not show any synergetic effect. |
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