The Role And Mechanism Of EUK-134 In The Treatment Of Acute Pancreatitis

Acute Pancreatitis(AP)is a clinically common acute abdominal disorder of the digestive system with consistently high clinical morbidity.In recent years,the treatment of AP has been developing in a multidisciplinary direction.However,there is still a deficiency of specific drugs for early treatment except supportive treatment,fluid therapy,analgesia and treatment for etiology.The pathogenesis of acute pancreatitis has not been fully elucidated,but the destruction of acinar cell homeostasis plays an important role.Oxidative stress(OS)is an abnormal pathological state of the body,where the oxidative and antioxidant system of the tissues and cells are unbalanced,and a large number of highly active and toxic products are released,resulting in tissue damage and cell death.Reactive oxygen species(ROS),the main product of oxidative stress reaction,will react with unsaturated fatty acids in the cell membrane and organelle membrane,leading to organelle damage and cell death.Mitochondria are important places for energy metabolism of cells,and the destruction of mitochondrial homeostasis will directly affect cell metabolism and inflammatory process.EUK-134 is a highly effective synthetic antioxidant with similar effects to superoxide dismutase and catalase.At present,the antioxidant effect of EUK-134 has played an important role in alleviating tissue and organ damage in mice with shock and alleviating acute lung injury.In this study,the role of EUK-134 in the treatment of acute pancreatitis and its related mechanism were investigated through in vitro and in vivo experiments.Part 1.EUK-134 alleviates acute pancreatitis in mice induced by caeruleinObjective: Oxidative stress plays an important role in the course and progression of AP.This study was conducted to investigate the therapeutic effect of the antioxidant EUK-134 on acute pancreatitis induced by caerulein in mice.Methods: The C57BL/6 mice were selected for modeling,and 25 7-week-old male mice were randomly divided into blank control group,caerulein modeling group,low-dose treatment group,medium-dose treatment group and high-dose treatment group.Mice in the caerulein modeling group and in the treatment group were induced by intraperitoneal injection of caerulein(50 μg/kg)and mice in the blank control group were injected with the same amount of normal saline.Mice in the treatment group were intraperitoneally injected with low(1 mg/kg),medium(5 mg/kg)and high(10 mg/kg)doses of EUK-134 before the first injection and after the fifth injection.The mice were sacrificed 12 hours after the first injection of caerulein,and blood samples were collected to detect the levels of the propionate aminotransferase(ALT),the aspartate aminotransferase(AST),the total cholesterol(TC)and the triglyceride(TG).Pancreas,liver,kidney and lung tissues were taken for H&E staining,and pathological experts were invited to evaluate the degree of pancreatic tissue injury according to the pathological scale of AP.TUNEL was used to detect apoptotic signals in pancreatic tissues of each group.The expressions of IL-1β,IL-6,TNF-a in pancreatic tissue were detected by q RT-PCR.The severity of inflammation and the safety of EUK-134 treatment in each group were comprehensively evaluated.Results: Compared with the control group,the pancreatic tissue of mice in the caerulein group and the treatment group showed different degrees of pancreatic edema,inflammatory cell infiltration and acicular cell necrosis,indicating that the caerulein group induced acute pancreatitis mouse model was successfully established.H&E staining of pancreatic tissue of mice in the treatment group showed less pancreatitis,which was manifested as decreased acinus cell necrosis,decreased inflammatory cell infiltration,and decreased edema.The pancreatic tissue damage in the high-dose and medium-dose groups was more mild than that in the low-dose group,but there was no difference between the medium-dose and the high-dose groups,and there was obvious extrapancreatic tissue damage in the high-dose group.TUNEL staining showed decreased apoptosis in the treatment group.The results of q RT-PCR showed that the expression of inflammatory cytokines was significantly decreased in the treatment group.In terms of safety,compared with the control group,medium dose EUK-134 did not cause structural changes or inflammatory reactions in liver,kidney and lung tissues,and serum ALT,AST,TC and TG were not elevated abnormally.Conclusions: Medium dose EUK-134 can significantly reduce the pancreatic injury and the expression of inflammatory cytokines in mice with acute pancreatitis induced by caerulein,with no toxic or side effects.Part 2.In vivo study of EUK-134 alleviating acute pancreatitis in mice by regulating NF-κB signaling pathway and inhibiting oxidative stress in pancreatic tissueObjective: To determine the changes of oxidative stress level and NF-κB pathway in pancreatic tissue of mice with acute pancreatitis after EUK-134 treatment.Methods: Pancreatic tissues were obtained from the mouse model of acute pancreatitis induced by caerulein and the experimental mouse model treated with EUK-134.The Antioxidant enzyme,such as superoxide dismutase(SOD)and catalase(CAT),and the products,such as malondialdehyde(MDA)were detected to comprehensively evaluate the oxidative stress level of pancreatic tissues.Mitochondrial ultrastructure was observed under electron microscope and NF-κB pathway was detected by western blot.Results: After treatment,compared with the caerulein group,the activities of SOD and CAT were increased,and the product MDA was decreased,which showed that the pancreatic oxidative stress level of mice in the treatment group was decreased,and the activities of SOD2 in mitochondrial in the pancreatic tissue was increased in the treatment group.The mitochondria ultrastructure in pancreatic tissue was observed by electron microscopy,which showed that the mitochondrial damage was serious in the CAE group,but not in the EUK-134 group.Western blot showed that phosphorylation of NF-κB pathway protein decreased in the treatment group.Conclusions: When acute pancreatitis occurs,the level of oxidative stress in pancreatic tissue of mouse increases,activating the NF-κB pathway and aggravating inflammation.EUK-134 can reduce the level of oxidative stress and inhibit the activation of NF-κB pathway in acute pancreatitis,and reduce the level of tissue inflammation.Part 3.In vitro study of EUK-134 alleviating inflammation in pancreatic acinar cells by maintaining mitochondrial homeostasisObjective: To determinate the mechanism of EUK-134 alleviating inflammation in acinar cells was investigated in vitro.Methods: AR42 J cells(pancreatic acinus cell line)were stimulated by CCK to detect oxidative stress signal,mitochondrial ultrastructure and mitochondrial permeability transition pore under,and to detect NF-κB pathway.The appropriate concentration of EUK-134 was obtained by CCK-8 assay,and the changes of oxidative stress level in acinar cells,NF-κB pathway signal and mitochondrial homeostasis were detected after treatment with EUK-134.AR42 J cells were transfected with the specific knockdown expression plasmid of SOD2,a key oxidative stress enzyme in mitochondria.The transfected cells were stimulated with CCK and EUK-134.The changes of oxidative stress level and NF-κB pathway signal were compared when SOD2 expression was reduced and after EUK-134 treatment.Results: The oxidative stress level of pancreatic acinar cells was significantly increased after CCK induction,and the mitochondria was damaged seriously.After EUK-134 treatment,the oxidative stress level of pancreatic acinar Cells was significantly decreased compared with that of CCK group.The mitochondrial ridge and mitochondrial membrane structure were only slightly changed,the mitochondrial channel was not abnormally open,and the activation of NF-κB pathway protein was significantly decreased.The expression of SOD2 in transfected cells was decreased,and the inflammation was significantly increased after CCK stimulation,and the activation of NF-κB pathway was enhanced.However,the inflammation was significantly relieved after EUK-134 treatment,and the activation of NF-κB pathway was weakened.Conclusions: EUK-134 can reduce the level of oxidative stress and the expression of inflammation mediator in acinar cells after stimulation,maintain mitochondrial homeostasis,and thus reduce the level of inflammation in acinar cells.SOD2 is an important oxidative defense barrier of pancreatic acinar cells.Summing up the above research,we can come to the conclusions as follows:The disruption of dynamic balance of pancreatic acinus cells plays an important role in the occurrence and development of acute pancreatitis.During inflammation,when the oxidation and antioxidant effects are unbalanced in tissues and cells,damaged pancreatic acinus cells and activated immune cells will release a large number of oxidative intermediates,and a lots of reactive oxygen species will cause disruption of mitochondrial homeostasis and activate NF-κB signaling pathway,which would amplify the inflammatory cascade.The antioxidant EUK-134 alleviates acute pancreatitis by inhibiting oxidative stress in pancreatic acinar cells,maintaining mitochondrial homeostasis,and inhibiting the activation of NF-κB pathway.This study further clarified the important role of oxidative stress in the occurrence and development of acute pancreatitis,and discussed the efficacy and mechanism of antioxidant EUK-134 in the treatment of acute pancreatitis,providing theoretical basis for potential clinical application.