| Severe acute pancreatitis(SAP)is the most severe type of acute pancreatitis with organ failure lasting more than 48 hours.SAP often develops into severe acute pancreatitis associated acute lung injury(SAP-ALI)and acute respiratory distress syndrome(ARDS)in acute pancreatitis,with a mortality rate of 30%-40%.Symptomatic and supportive therapy such as fluid replacement and anti-infection is still the main treatment method at present.The lack of specific treatment is the main reason for the high mortality of SAP-ALI patients.Therefore,it is necessary to further study the mechanism in the occurrence and development of SAP-ALI.Colchicine(COL)is an immunomodulatory drug that acts by inhibiting microtubule formation and tubulin polymerization,and is widely used in various rheumatic diseases(such as gout and Beh?et’s disease).In recent years,studies have found that colchicine can inhibit the expression of selectin,leukocyte migration,exocytosis and phagocytosis.In addition,colchicine reduces interleukin release to exert anti-inflammatory effects.Since the specific pathogenesis and disease progression of SAP-ALI are still unclear,basic research is urgently needed to provide a theoretical basis for the treatment of SAPALI.We tried to build a SAP-ALI rat model to explore the effect of colchicine preintervention on SAP-ALI rats.Part Ⅰ: Effects of colchicine on the severity of lung injury in rats with severe acute pancreatitis Objective: To observe the effect of colchicine on the severity of lung injury in rats with severe acute pancreatitis.Methods: To observe the effect of colchicine on SAP-ALI rats,45 SD male rats were selected and randomly divided into 3 groups(15 rats in each group),respectively:(1)Sham group;(2)Severe acute pancreatitis associated lung injury group(SAP-ALI group);(3)Colchicine group(Colchicine group).Rats in the colchicine medication group were given colchicine working solution(0.5 mg/kg/d)by gavage,while the sham operation group and the severe acute pancreatitis associated lung injury group were given the same volume of normal saline by gavage in the colchicine intervention group.The SAP-ALI rat model was induced by retrograde biliopancreatic duct injection of 5% sodium taurocholate.SD rats were sacrificed 24 hours after surgery.The condition of the rats was observed and recorded 24 hours after operation.The H&E staining of pancreas and lung tissues,the determination of plasma amylase,lipase,and lactate dehydrogenase content,and the pathological score of pancreas and lung tissues were used to determine whether the model was successful.To explore the intervention effect of colchicine on rat SAPALI model.Results: 24 hours after the operation,the rats in the SAP-ALI group showed lethargy,decreased muscle tone,deep breathing and shortness of breath compared with the rats in the colchicine administration group.Compared with the rats in the colchicine administration group,the rats in the SAP-ALI group had more severe necrosis and edema of the pancreatic tissue,and hemorrhage in the lung tissue was more obvious.The results of H&E staining showed that the cell necrosis,inflammatory cell infiltration and hemorrhage in the pancreas and lung tissues of the rats in the SAP-ALI group were significantly higher than those in the colchicine treatment group,and the pathological scores were higher than those in the colchicine intervention group.The results of statistical analysis showed that the plasma amylase,lipase and lactate dehydrogenase contents of the rats in the SAP-ALI group were significantly higher than those in the colchicine intervention group.Conclusion: The above results suggest that colchicine can reduce the severity of SAPALI in rats,the degree of edema and hemorrhage of pancreas and lung tissue,the scope of necrosis of pancreatic tissue,and the content of amylase,lipase and lactate dehydrogenase in circulation.Part Ⅱ: The role of inflammation in the protection of severe acute pancreatitis associated lung injury in rats by colchicine Objective: To observe the effect of colchicine on inflammation in the pancreas and lung tissues of SAP-ALI rats.Methods: Colchicine administration and modeling methods were the same as the first part.SD rats were sacrificed 24 hours after surgery.The expression of inflammatory cells markers in pancreas and lung tissues were detected by immunohistofluorescence.Immunohistochemistry detects activation of inflammatory pathways in pancreatic and lung tissues.Western Blot detection of iNOS,MPO,pNF-κB p65,NF-κB,p-STAT3,STAT3,p-AKT and AKT protein expression in pancreatic tissues.Plasma levels of proinflammatory cytokines were detected by ELISA.Results: Compared with the Sham group,the expression levels of MPO,CD68 and iNOS in the pancreas and lung tissues of the SAP-ALI group were significantly increased.The activities of MPO,CD68 and iNOS in the pancreas and lung tissues of rats in the colchicine intervention group were significantly lower than those in the untreated SAPALI group.The activation of inflammatory pathways NF-κB p65,AKT and STAT3 in the colchicine intervention group was significantly lower than that in the SAP-ALI group.Compared with the SAP-ALI group,the plasma levels of inflammatory cytokines TNF-α,IL-6 and IL-1β were significantly decreased in the colchicine intervention group.Conclusion: Colchicine can reduce the inflammatory response in the SAP-ALI rat model,and reduce the severity of SAP-ALI by reducing the infiltration of inflammatory cells,inhibiting the activation of inflammatory pathways,and reducing the production of proinflammatory cytokines in plasma.Part Ⅲ: The role of apoptosis in the protection of severe acute pancreatitis associated lung injury by colchicine in rats Objective: To observe the effect of colchicine on apoptosis of pancreas and lung tissues in SAP-ALI rats.Methods: Colchicine administration and modeling methods were the same as the first part.SD rats were sacrificed 24 hours after surgery.The expression of cleaved caspase 3in pancreas and lung tissues was detected by immunohistochemistry.TUNEL detected the number of apoptotic cells in pancreas and lung tissues.Western Blot detected the expression of apoptosis-related proteins Bcl2,Bax and Cleaved caspase 3 in pancreatic tissues.Results: Compared with the Sham group,the expression levels of pro-apoptosis-related proteins Bax and Cleaved caspase 3 in the pancreas and lung tissues of the SAP-ALI group were significantly increased,and TUNEL showed that the number of apoptosis cells in the pancreas and lung tissues increased.Compared with the SAP-ALI model group,the expression of the anti-apoptotic protein Bcl2 in the pancreas and lung tissues of the colchicine intervention group increased,the expression of the pro-apoptotic proteins Bax and Cleaved caspase 3 decreased,and the number of apoptotic cells decreased.Conclusion: Colchicine can reduce the number of apoptotic cells and the expression of pro-apoptosis-related proteins in the pancreas and lung tissues,thereby reducing the damage of the pancreas and lung tissues in the SAP-ALI model.Part Ⅳ: The role of oxidative stress in the protection of severe acute pancreatitis associated lung injury by colchicine in rats Objective: To investigate colchicine attenuating the severity of SAP-ALI rat model by inhibiting oxidative stress.Methods: Colchicine administration and modeling methods were the same as the first part.SD rats were sacrificed 24 hours after surgery.The expressions of 4-HNE,Nrf2 and HO-1 in pancreas and lung tissues were detected by immunohistochemistry.ROS staining was used to detect reactive oxygen species in pancreas and lung tissues.Western Blot detection of 4-HNE,Nrf2 and HO-1 protein expression in pancreatic tissues.Biochemical detection of SOD,MDA and GSH content in plasma.Results: Compared with the Sham group,the content of 4-HNE in the pancreas and lung tissues of the rats in the SAP-ALI group increased,the protein expression levels of Nrf2 and HO-1 were significantly decreased,and the detection of ROS showed that the reactive oxygen species in the pancreas and lung tissues increased.Compared with the SAP-ALI model group,the Nrf2/HO-1 protein expression in the pancreas and lung tissues of the colchicine intervention group was increased,the 4-HNE content was decreased,and the production of reactive oxygen species was also significantly decreased.After colchicine intervention,the contents of SOD and GSH in the plasma of rats were increased,and the contents of MDA were decreased.Conclusion: Colchicine can reduce the levels of oxidative stress products in the pancreas and lung tissues of SAP-ALI rats,reduce the production of reactive oxygen species in the pancreas and lung tissues,and promote the expression of Nrf2/HO-1 pathway.Figure [16] Table [2] Reference [56]... |
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