| Research background and purposeAcute pancreatitis(AP)is a local inflammatory reaction caused by the abnormal activation of pancreatic enzymes in pancreatic acinar cells and the subsequent self-digestion of pancreatic parenchyma,about 20% of the mild acute pancreatitis(MAP)can progress to severe acute pancreatitis(SAP).Different from self-limited MAP,the widely inflammation and necrosis of the pancreas are the main characteristics of SAP with a tendency to complicate and severe,it can be made of local inflammation rapid progress to systemic inflammatory response syndrome(SIRS)in a short period of time,and often accompanied by heart,lung,kidney and intestinal injury causing multiple organ failure(MOF),the case fatality rate is as high as 15-30%.Among them,SAP-associated cardiac injury(SACI),also known as "pancreatic heart syndrome",is one of the most serious complications of SAP intensification.It can manifest as abnormal cardiac function,toxic myocarditis and even heart failure.At present,there is no effective method of prevention and treatment on SACI,thus it has become an urgent clinical problem to actively explore its mechanism and find reliable prevention and treatment measures.It has been confirmed that in the course of SAP,oxidative stress caused by the systemic inflammatory response is closely related to the occurrence and development of SACI.At the early stage of SAP,pancreatic acinar cells and activated inflammatory cells release a large number of myocardial inhibitors(a class of cytokines and inflammatory mediators with toxic effects on heart).These cardiac inhibitory factors will cause cardiomyocytes to release a large number of oxygen free radicals.When the scavenging ability of the body to free radicals is exceeded,the biological macromolecules in the cell(such as lipids,proteins,DNA,etc.)will be rapidly oxidized,causing d amage to the structure and function of cardiomyocytes,leading to ventricular remodeling and abnormal cardiac function.Some scholars have found that membrane permeable oxygen free radical scavenger(tempol)can effectively reduce myocardial injury in AP rats,further confirming the relationship between SACI and oxidative stress.In summary,if the systemic inflammatory response of SAP and its accompanying oxidative stress can be alleviated,it is expected to be an effective treatment for SACI.In the early stage of SAP,the systemic inflammatory response may lead to the accumulation of pancreatitis associated ascitic fluid(PAAF)in the abdominal cavity with varying degrees.Aiming at PAAF,our center has done a lot of clinical and basic research,which proves that timely drainage of PAAF to the outside of the body through abdominal paracentesis drainage(APD)is an important part in the treatment of SAP.Timely removal of PAAF rich in various inflammatory mediators and toxic substances through APD can effectively reduce the degree of inflammation and oxidative stress in the body,delay or avoid the occurrence of multi-organ failure,and exert the protective effect to important organs.In addition,animal studies have demonstrated that APD can alleviate SAP-related lung and intestine injury.Based on this,we speculate that early implementation of APD in SAP is expected to have a significant therapeutic effect on SACI.Studies have shown that oxidative stress is closely related to the abnormal function of oxidative kinase.It has been reported that the overactivation of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase is not only an important mechanism for the increase of oxidative stress level in the body,but also a major source of reactive oxide species(ROS)production in the cardiovascular system under pathological conditions,which is closely related to the occurrence and development of cardiovascular diseases.Given the close relationship between SACI and oxidative stress and the activatio n of various inflammatory mediators on NADPH oxidase,the activation of NADPH oxidase may also be involved in myocardial oxidative damage caused by SAP.Therefore,we hypothesized that APD may play a protective role on cardiac tissue by reducing the activation of NADPH oxidase and the generation of oxygen free radicals.If this hypothesis is true,how does APD mitigate oxidative damage to the myocardium caused by NADPH oxidase activation? Clarification of these issues will provide a new theoretical basis fo r the mechanism of APD in treating SAP.High mobility group box-1 protein(HMGB1)is a highly conserved nuclear protein,which is mainly bound to intruclear chromosomes in physiological state.It was found that HMGB1 can be passively released from the necrotic pancreatic tissue and actively secreted by infiltrated mononuclear/macrophagic cells to extracellular in AP,so PAAF contains high concentration of HMGB1.Extracellular HMGB1 act as an important damage-related model molecule directly participate in the inflammatory cascade amplification and intensification of pancreatitis,which plays an important role in the injury of pancreas and distant organs.It has been proved that HMGB1 can activate NADPH oxidase by acting on damage related molecular pattern receptors,causing an increase in intracellular ROS content.While the high concentration of HMGB1 in PAAF will cause the secondary increase of HMGB1 concentration in circulation after being reabsorbed by peritoneum,and further activate NADPH oxidase in cardiac tissue.Therefore,whether early implementation of APD drainage PAAF can reduce the circulating concentration of HMGB1 by reducing its reabsorption,and reduce the activation of NADPH oxidase in the cardiac tissue mediated by HMGB1,so as to play a protective role in the heart? Further research is needed.In view of the above problems,we first used 5% sodium taurocholate retrograde injection to prepare the myocardial injury model of SAP rats,and carried out APD on SAP rats,aiming at the following research: 1.whether APD has a protective effect on SACI;2.the role of NADPH oxidase activation in SACI and its related molecular mechanisms;3.whether APD can regulate the expression of NADPH oxidase in cardiac tissue and reduce oxidative stress damage.In addition,on the basis of successfully establishing the rat model of mild pancreatitis,the PAAF of SAP rats and the PAAF with neutralizing HMGB1 were injected into the abdominal cavity of mild pancreatitis rats respectively,to further verify the role of HMGB1 in PAAF in the course of SAP and the relevant mechanism of APD in the treatment of SACI.Objective,methods and resultsPart ?: the effect of APD on myocardial injury in SAP ratsObjective: observe the therapeutic effect of APD on SAP rats and its protective effect on myocardial injuryMethods1.Effect of APD on the mortality of SAP ratsTo study the effect of APD on the mortality of SAP rats,a total of 75 SD male rats were randomly divided into Sham group,SAP group,and SAP+APD group,with 15 rats in each group.SAP rat model was prepared by fine needle puncture of biliopancreatic duct and retrograde injection of 5% sodium taurocholate.In SAP+APD group,after successful SAP modeling,the rubber drainage tube with external negative pressure drainage ball was placed in the right lower abdomen with 0.5 cm indwelled in the abdominal cavity and fixed in the abdominal wall,the abdomen was closed after conventional disinfection.In sham group,the pancreas was turned over several times after laparotomy,and a 0.5 cm long rubber drainage tube was placed in the abdominal cavity and then closed the abdomen.The mortality of each group was recorded and ascites were measured 24 hours after the operation.2.The protective effect of APD on SACIA total of 45 SD male rats were randomly divided into Sham group,SAP group,and SAP+APD group,with 15 rats in each group,and the molding method is the same as before.Electrocardiogram(ECG)was detected 8 hours after modeling,cardiac ultrasound and arterial blood pressure were detected 24 hours after modeling,and then rats were sacrificed to collect ascites,serum and heart tissue for detection: HE staining was used to observe the pathological changes of myocardial tissue and to calculate the pathological score,the dry-wet weight ratio of myocardial tissue was calculated,the concentrations of serum myocardial enzyme spectrum,TNF-? and IL-1? were detected by Elisa,Tunel method was used to detect myocardial cell apoptosis,and western blot was used to determine the expression of Bax,Bcl-2 and caspase-3.Results1.At 24 hours after modeling,the mortality rate of the rats in the SAP group was 40.0%,and the average ascites volume was 9.5±1.7ml.The mortality rate of the rats in the SAP+APD group was 16.0%,and the average ascites volume was 6.5±1.4ml,which was significantly lower than those in the SAP group.2.The ECG of SAP rats presented obvious abnormalities,while the abnormal rate of ECG in SAP+APD group was significantly reduced,and ST-segment elevation was observed occasionally.24 hours after modeling,compared with Sham group,the cardiac function of SAP group was significantly impaired,mainly manifested as decreased myocardial systolic function,decreased ejection fraction and significantly decreased arterial blood pressure.The impaired cardiac function in the SAP+APD group were significantly improved compared with the SAP group.3.Compared with Sham group,the serum concentrations of myocardial enzymes,amylase activity,TNF-? and IL-1? in SAP group were significantly increased.The above indexes in the SAP+APD group were significantly lower than those in the SAP group.Histopathological observation of myocardial tissue: compared with Sham group,rats in SAP group were observed to have myocardial cell turbidity,accompanied by partial dissolution degeneration,and mononuclear cell infiltration in local areas,consistent with early myocarditis,and the pathological score and wet/dry weight ratio were significantly increased.Compared with the SAP group,the pathological changes of myocardial tissue in SAP+APD rats were significantly reduced,and the pathological score and wet/dry weight ratio were significantly declined.4.In the SAP group,myocardial cell apoptosis was observed,the protein expression of Bax and Cleaved caspase-3 were significantly increased,and Bcl-2 expression was significantly decreased.The myocardial cell apoptosis in the SAP+APD group was significantly declined,and the imbalance between pro-apoptotic proteins and anti-apoptotic proteins was significantly improved.ConclusionsThe method of retrograde injection of sodium taurocholate into the cholangiopancreatic duct can be used to prepare the myocardial injury model of SAP rats.Secondly,APD can significantly reduce the death rate of SAP rats,reduce the pathological damage of cardiac tissue,and improve the abnormal cardiac function.Part two: The role of NADPH oxidase in the effects of APD on SACIObjective: to clarify the role of NADPH oxidase in SACI,and to confirm that APD alleviates myocardial oxidative injury by regulating NADPH oxidase expression.1.The role of NADPH oxidase in SACIMethods(1)In vivo experiment,a total of 60 SD male rats were randomly divided into Sham group,SAP group,SAP plus apocynin group(SAP-APO group)and apocynin control group(APO-CON group),with 15 in each group.The injection of 10% dimethyl sulfoxide(DMSO)dissolved apocynin(20 mg/kg)through the rat caudal vein in SAP-APO group and APO-CON group rats was given 30 min before the model was established,and the Sham group and SAP group rats injected the same volume of DMSO at the same time.Cardiac ultrasound was detected 24 hours after modeling,and then rats were sacrificed to collect serum,pancreas and heart tissue for detection: immunohistochemistry and western blot to detect the protein expression of myocardial NADPH oxidase,photometric method to detect the NADPH oxidase activity and colorimetric method to detect oxidative stress related indicators,DHE staining to detect myocardial tissue ROS levels,HE staining to observe the myocardial tissue and pancreatic tissue pathological changes and pathological grading,Elisa method to detect the serum myocardial enzyme spectrum,serum inflammatory factors and pancreatic enzyme activity,Tunel method to detect myocardial cell apoptosis,western blot to determine the expression of Bax,Bcl-2 and caspase-3,and the protein expression and phosphorylation levels of the MAPK signaling pathway(ERK1/2,JNK,p38).(2)In vitro simulation experiments,H9C2 cardiomyocytes were first cultured in medium containing 5% and 10% normal rat serum or SAP rat serum.Cell viability was detected by cck-8 method at 3,6,12,and 24 hours after the experiment to determine the optimal serum intervention concentration and intervention time.Then H9C2 cardiomyocytes were divided into normal rat serum groups(NS group),SAP rats serum group(SS group),SAP rats serum plus apocynin group(SA group)rats and normal rats serum plus apocynin group(NA group).Detection: the ROS content of cardiomyocytes was detected by DHE staining,the cardiomyocytes apoptosis was detected by flow cytometry,and the protein expression and phosphorylation levels of ERK1/2,JNK and p38 were detected by western blot.2.Effect of APD on cardiac NADPH oxidase expression in SAP ratMethodsA total of 30 SD male rats were randomly divided into Sham group,SAP group,and SAP+APD group,with 10 rats in each group.The modeling methods were the same as before.Cardiac tissue was collected 24 hours after modeling for detection: protein expression of NADPH oxidase was detected by western blot,activity of NADPH oxidase was detected by spectrophotometry,ROS content in cardiac tissue was detected by DHE staining,and indicators related to oxidative stress were detected by colorimetry.Results1.After 24 hours of modeling,the expression and activity of NADPH oxidase protein in cardiac tissue of SAP rats were significantly increased compared with Sham group rats.Compared with the SAP group,the cardiac dysfunction in the SAP-APO group was significantly reduced,the serum concentration of myocardial enzyme spectrum,myocardial histopathological score,ROS content and levels of oxidative stress-related indicators were significantly declined,and the myocardial cell apoptosis was also significantly reduced.At the same time,compared with the SAP group,the expression of pro-apoptotic proteins in myocardial tissue was significantly reduced,the expression of anti-apoptotic proteins was significantly increased,and the protein expression and phosphorylation levels of the MAPK signaling pathways ERK1/2,JNK and p38 were also significantly reduced.While there was no significant difference between SAP group and SAP group in the pathological score of pancreatic tissue,but the level of serum inflammatory factors decreased to a certain extent.In addition,there was no statistical difference between the indicators of Sham group and APO-CON group.2.In the in vitro simulation experiment,it was determined that the optimal intervention condition was 12 h stimulation with medium containing 10%SAP rat serum.Compared with NS group,the ROS content of cardiomyocytes in SS group was significantly increased,the number of apoptosis was significantly increased,and the protein expression and phosphorylation levels of ERK1/2,JNK and p38 were significantly increased.Compared with the SS group,the SA group showed a different degree of reduction in the above indicators.There was no significant difference between SS group and NA group.3.After 24 hours of modeling,the protein expression and activity of NADPH oxidase in cardiac tissue of rats in SAP+APD group were significantly lower than those in SAP group,ROS content was significantly reduced,and the levels of oxidative stress-related indicators were also significantly declined.ConclusionsThe above experimental results indicated that the over-activation of NADPH oxidase plays an important role in SACI,and early application of APD can inhibit the abnormal activation of NADPH oxidase in SAP rat myocardial tissue and reduce the oxidative damage in myocardial tissue.Part three: The role of HMGB1 in the effects of APD on SACIObjective: to confirm that APD decreased the expression of NADPH oxidase in myocardial tissue by regulating HMGB1Methods1.A total of 30 SD male rats were randomly divided into Sham group,SAP group and SAP+APD group,with 10 rats in each group.The rats were sacrificed 24 hours after modeling,and serum,ascites and pancreatic tissues were collected for detection: Elisa method was used to detect the activity of serum trypsin and the concentration of HMGB1 in serum and ascites.The pathological changes of pancreatic tissues were observed by HE staining and pathological scores were made.2.A total of 36 SD male rats were used to establish a model of mild pancreatitis by intraperitoneal continuous injection of cerulein,and then rats were randomly divided into 6 groups(6 in each group):(1)MAP control group(CN group);(2)PAAF injection group(PI group)(3)PAAF plus anti-HMGB1 neutralizing antibody 50?g injection group(PIH 50 group);(4)PIH 100 group;(5)PIH 200 group;(6)PAAF plus control lgY injection group(PIC group).Serum and heart tissue were collected 8 hours after injection for detection: serum HMGB1 content was detected by Elisa,m RNA levels of NADPH oxidase in myocardial tissue were detected by RT-PCR,and protein expression was detected by western blot.Results1.Compared with the SAP group,APD can significantly alleviate pancreatic tissue damage,reduce inflammatory cell infiltration,and decline serum trypsin activity and HMGB1 concentration.In addition,high concentration of HMGB1 was found in the PAAF of rats in the SAP group.2.After the MAP rats were injected with PAAF intraperitoneally,it was found that the serum HMGB1 level in PI group was significantly higher than that in CN group,with increased mRNA and protein expression of Nox-2 and m RNA expression of Nox-4.With the gradual increase of the anti-HMGB1 neutralizing antibody dose,the serum HMGB1 concentration of rats decreased significantly when the neutralizing antibody dose reached 200 ug,and the mRNA and protein expressions of Nox-2 and Nox-4 m RNA expressions also decreased significantly.In addition,there was no significant difference between the indicators of PIC group and PI group.ConclusionsEarly application of APD can significantly improve the pathological damage of the pancreas in SAP rats,effectively reduce the release and reabsorption of HMGB1 into the blood circulation,and thus inhibit the expression of NADPH oxidase in cardiac tissue mediated by HMGB1.Summary1.In the course of SAP,early implementation of APD has a significant protective effect on SACI.Specifically,it can reduce the serum concentration of myocardial enzyme spectrum,alleviate the degree of myocardial tissue damage,decline the occurrence of myocardial cell apoptosis,and improve the abnormality of cardiac function.2.The excessive production of ROS in the cardiac tissue caused by the overactivation of NAPDH oxidase is the initiator of SACI.The production and accumulation of ROS can cause myocardial cell apoptosis and activation of MAPK signaling pathway,resulting in impairment of cardiac function,while inhibition of NADPH oxidase activity can significantly alleviate the degree of myocardial injury caused by SAP.3.PAAF produced during SAP contains high concentration of HMGB1,which can enter the blood circulation of the body through peritoneal reabsorption and other ways,and up-regulate the expression of NADPH oxidase in cardiac tissue.In the early stage of SAP,implementation of APD can effectively inhibit the activation of HMGB1 mediated NADPH oxidase signaling pathway by reducing the release and reabsorption of HMGB1,so as to reduce the oxidative damage of myocardium and play a protective role on heart.(Fig 1)... |
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