Protective Effect Of Exogenous Carbon Monoxide Releasing Molecule (CORM-2) On Pancreatic Injury Via NF-?B Signaling Pathway

Background:Sepsis refers to a systemic inflammatory response syndrome(SIRS)caused by severe infection,trauma,burn,postoperative infection and acute critical illness,accompanied by life-threatening organ dysfunction.It can further develop into severe sepsis,septic shock and multiple organ dysfunction syndrome(MODS)or even death.At present,septic shock and multiple organ dysfunction caused by sepsis have become one of the most common causes of death in intensive care unit(ICU).Because the pathogenesis of sepsis is extremely complex,even today,even with the high development of transformational medicine,clinical treatment still has little effect,and the mortality rate is still high.Therefore,it has become an important scientific problem to further explore the mechanism of sepsis and find new means of prevention and treatment.Acute pancreatitis(AP)is an inflammatory reaction that causes pancreatic enzyme to be activated in the pancreas and causes pancreatic tissue to digest,edema,bleed and even necrosis.Its clinical manifestations are diverse.The range can range from mild self-limited diseases after supportive treatment to life-threatening diseases with high complications and mortality.Sepsis is an important cause of secondary severe pancreatic injury.Acute pancreatic injury can cause a large number of inflammatory mediators to release,aggravate the inflammatory response and stress state of the body,and further aggravate the condition.Although after a large number of clinical and experimental potential therapeutic drugs or surgical treatment,about 10% of the patients died of multiple organ failure,which is partly due to the lack of pharmacological options for the treatment of AP.Studies have shown that trypsin activation,inflammation,oxidative stress and nuclear factor NF-? B are important pathophysiological components of AP.Carbon monoxide(CO)is the second important gas second messenger molecule discovered after nitric oxide.It has many physiological effects,such as relaxing vascular and bronchial smooth muscle,inhibiting platelet aggregation,inhibiting inflammatory reaction,anti-apoptosis,anti-proliferation and so on.It plays an important regulatory role in physiological processes such as nerve,respiration,circulation and pathological processes such as inhibition of acute inflammatory reaction,organ ischemia-reperfusion injury,organ transplantation rejection and so on.Carbon monoxide releasing molecule 2(CORM-2)is a newly synthesized transition metal compound which can release carbon monoxide gas.it can release carbon monoxide so it has attracted more and more attention and may be developed for medicinal purposes.Previous studies have shown that CO released by CORM-2 can significantly improve the inflammatory response of(CLP)mice with cecal ligation and perforation and LPS stimulated mice,effectively protect the important organs of mice to reduce injury,and improve the body ischemia-reperfusion.Improve the survival rate of experimental mice.However,there is no report on the protective mechanism of carbon monoxide releasing molecules in improving inflammatory response and reducing pancreatic injury,that is,cellular signaling pathway.Based on this,the purpose of this study was to analyze the protective effect of carbon monoxide on pancreatic injury induced by sepsis and severe pancreatitis in mice,and to explore the molecular mechanism of NF-? B cell pathway in this protective effect.Methods:To investigate the protective effect of CORM-2 on the pancreas of CLP mice.Clean level sixty-eight healthy male C57 BL / 6 mice(48 of which were used for survival analysis and 20 other experiments)only randomly divided into four groups: control group(Sham),CLP group,CLP + CORM-2 and CLP + iCORM-2 group.The 72 h survival rate of each group was recorded,and the blood glucose levels of mice in each group were measured at 1,2,4,6,8,10,12,16,20,24 and 36 hours.Control group started,or after the CLP respectively with 6 hours,12 hours and 24 hours of mice heart isoflurane anesthesia puncture blood,finally took pancreatic tissue cryopreserved in liquid nitrogen or with 10% formalin fixed,for follow-up study.Serum amylase and lipase levels were detected by automatic biochemical analyzer.Pancreatic tissue was stained with HE and histopathological score was performed to detect the activity of peroxidase(MPO).C57BL/6 mice were intraperitoneally injected with ranulin(50 g/kg)once every hour for 10 times,and the ANP mouse model was established.The model mice were divided into Control group,acute hemorrhagic necrotizing pancreatitis(ANP group),CORM-2 intervention group(AP+ CORM-2 group),and iCORM-2 intervention group(AP+ iCORM-2 group).After the first injection of CORM-2 or iCORM-2 for 30 min,blood and pancreatic tissue of mice in each group were collected 12 hours after the first injection.Using automatic biochemistry analyzer in the detection of serum amylase,lipase,the levels of AST,ALT,to HE staining of the pancreas,TUNEL method to detect pancreatic tissue apoptosis,weighing method to detect lung wet dry than,spectrophotometer detection in pancreatic tissue MPO activity of myeloperoxidase in the,using enzyme standard instrument in pancreatic tissue MDA(malondialdehyde)for testing.The levels of cytokines(TNF-?,IL-1,IL-6)in the plasma and pancreas of mice with severe pancreatitis were detected by ELISA,the expressions of ICAM-1 and VCAM-1 in the pancreas were detected by immunohistochemistry,and the levels of NF-B and P-I B were detected by Western blot.Results:(1)The intervention of CORM-2 could improve the survival rate of CLP model,but had no significant effect on blood glucose.Compared with Sham mice,serum amylase level began to increase at 6 h after CLP surgery,and increased significantly at 24 h after CLP surgery.At 6 h,12 h and 24 h after CLP surgery,the intervention of CORM-2 significantly reduced the serum amylase level.Serum amylase activity did not change in sepsis mice treated with iCORM-2 compared with the CLP group.Similar results were found at serum lipase levels.HE staining results of the pancreatic tissues of mice in each group showed that the pancreas of Sham group showed normal structure,while the pancreatic tissues of sepsis mice showed severe pathological damage 24 h after CLP.The pancreatic tissue of sepsis mice showed typical edema,inflammatory cell infiltration,and acinar cell necrosis.After the intervention of CORM-2,the degree of histological damage of pancreatic injury was significantly reduced,while no reduction of pancreatic injury was found in the I CORM-2 treatment group.MPO activity was significantly increased at 6 h,12 h,and 24 h after CLP compared with the sham group,and was more significant at 24 h.The treatment of CORM-2 significantly decreased MPO activity,and the treatment of CORM-2 reduced the infiltration of pancreatic neutrophils.There was no significant difference between CLP group and CLP+ iCORM-2 group.The above results suggest that the pancreatic tissue is also one of the organs vulnerable to damage in sepsis,and CORM-2 intervention has a protective effect on this damage,while the quantitative analysis of the histopathological score of the pancreas shows that CORM-2 treatment plays a protective role in clp-induced pancreatic injury compared with the CLP group.(2)A mouse model of acute haemorrhagic necrotizing pancreatitis was successfully established by continuous intraperitoneal injection of umorin.CORM-2 can significantly reduce amylase and lipase levels in serum of ANP mice induced by ranapine,reduce apoptosis of acinar cells(TUNEL),and reduce pancreatic tissue injury.CORM-2 can significantly improve the survival rate of ANP mice induced by ranapine.The above results suggest that CORM-2 can significantly protect the organ function of mice with acute hemorrhagic necrotizing pancreatitis and improve the survival rate of mice.(3)Compared with the sham group,the expression of pro-inflammatory cytokines il-6,il-1 and TNF-in the pancreatic tissue of sepsis mice was significantly increased,and the expression of il-6,il-1 and TNF-in the pancreatic tissue was decreased after CORM-2 intervention.However,iCORM-2 treatment did not affect the expression of these cytokines.Expression levels of NF-B p65 and phosphorylated-I B-in sepsis mice were significantly increased,while CORM-2 intervention inhibited expression,while iCORM-2 intervention did not.CORM-2 can obviously reduce the ANP of rain frog induced mice reduce serum levels of cytokines and pancreatic tissue(TNF alpha,beta,IL-6,IL-1)expression of CORM-2 can significantly suppress the ANP pancreatic tissue in mice induced by rain frog grain nf-kappa B p65 and phosphorylation-i kappa B-alpha predominate(p-I kappa alpha B)predominate expression level,because the nf-kappa B activation,inflammation,oxidative stress,as a result of expression products increasing.In conclusion,this study adopts the mouse model of sepsis and acute pancreatitis in mice,sepsis and pancreatitis with pancreatic inflammation has carried on the system research,and use the CORM-2 intervene,prompt the CORM-2 by inhibiting the nf-kappa B signaling pathway activation to inhibit the body's inflammatory response,thereby to sepsis and acute pancreatitis in the pancreas protection.Conclusions:It was found that ICAM-1 and VCAM-1 attached to the surface of vascular endothelial cells in pancreatic tissue of mice were significantly increased and the expression of protein NF-? B was also significantly increased in sepsis.CORM-2 intervention could significantly inhibit the above inflammatory markers.This protective effect is mediated by NF-? B signal pathway.