| Acute pancreatitis is caused by a variety of etiologys,resulting in trypsin activation,local inflammation of the pancreas as the main feature,with or without other organs dysfunction.Severe acute pancreatitis should be accompanied with persistent organ failure,according to the Atlanta classification standard,about 10% patients are classified as severe acute pancreatitis,with high mortality,acute lung injury is a severe complication of acute panreatitis,in severe acute pancreatitis,acute lung injury and acute respiratoy distress syndrome mortality are 30%~40%,for acute severe pancreatitis associated lung injury,there is no specific treatment at present.NF-?B is a key of transcription factor consist of P50,P52,REL,P65 and REL-B,which control a variety of inflammatory genes expression,such as TNF-?,IL-6,and IL-8,induced nitric oxide synthase.Water channel protein is a kind of small protein,promoting water transit quickly,AQP5 can react water transfer of lung tissue and the severity of lung edema,Curcumin is a plant polyphenols extracted from the zingiberaceae plants yellow spice,regulating transcription factors such as the NF-?B,adhesion molecules,enzymes,etc.It plays an important role in inflammation and cancer.Diphenyl difluoroketone EF24 is a kind of curcumin synthetic analogues,It was shown that curcumin analogues EF24 inhibit the NF-?B activity,reduce proinflammatory cytokines TNF-?,IL-6 mRNA expression and secretion,play a role in inflammatory response.The study have shown that giving EF24 treatment in hemorrhagic shock rats models can inhibit IL-1 receptor pathway,reducing the concentration of serum TNF-? and IL-6,reduce hemorrhagic shock cause lung inflammatory.The purpose of this study is to explore the effect of diphenyl difluoroketone EF24 on the lung injury in severe pancreatitis.Objective: Experiment aimed to explore the effect and mechanism of biphenyl fluorineketone EF24 on pancreatitis lung injury,histopathology changes of SAP pancreas and lung tissues were observed by Hematoxylin and Eosin(HE)staining,lung wet-to-dry weight ratios were tested,centrifugal cells in bronchoalveolar lavage fluid were observed by Wright staining,the expression of lung tissue AQP5 mRNA,p65 mRNA were detected by Real Time PCR,the expression of AQP5 protein and p65 protein were detected by western-blot,concentration of serum IL-6,TNF-? and concentration of bronchoalveolar lavage fluid TNF-? were tested by ELISA,which can provide new ideas for treatment of lung injury in severe pancreatitis.Methods:1 All the 60 Sprague-Dawley(SD)rats were divided into control group(CON),severe acute pancreatitis group(SAP)and EF24 group(EF24)randomly.Then each group was further divided into two subgroups(6h group and 12 h group)according to time points,10 rats in each group.2 SAP group rats and EF24 group rats were respectively injected intraperitoneally the same dose of 20% L-arginine(3.0 g/kg)2 times,each time interval for 1 hour.The CON group rats were injected with L-argnine an equal dose of 0.9% NaCl 2 times intraperitoneally,each time interval for 1 hour,1 hour after the accomplishment of modeling,EF24 group rats were injected intraperitoneally with biphenyl florineketone EF24(0.4 mg/kg).CON group and SAP group rats were respectively injected EF24 an equal dose of polyethylene glycol(PEG)400(PEG:0.9%=1:1)intraperitoneally.3 The pancreas,lung tissues and blood were taken at 6 or 12 hours after modeling.The histopathology changes of pancreatic tissues and lung tissues were observed by Hematoxylin and Eosin(HE)staining.Lung wet-to-dry weight ratios were tested,centrifugal cells in bronchoalveolar lavage fluid were observed by Wright staining.The levels of serum IL-6 and TNF-? and the levels of TNF-? of bronchoalveolar lavage fluid were evaluated by ELISA.The expression of lung tissue AQP5 mRNA,p65 mRNA and protein were detected respectively by Real-Time PCR and Western-blot.4 Analyzed by a commercially available statistical software package(SPSS 21.0),ALL date were presented as mean±SD(standard deviation),P<0.05 with statistical difference.Results:1 HE staining: SAP group,EF24 group compared with CON group respectively,the severity of pancreas increased.With the time developed,the pancreas and lung damage increased gradually.On the basis of Mayer,Osman scoring criteria,SAP group and EF24 group lung tissues damage aggravated.Compared with the SAP group,the degree of lung damage in EF24 group reduced;2 Lung wet dry weight ratio: compared with CON group respectively,SAP group,EF24 group lung wet dry weight ratio at each point increased significantly,with statistical difference(all P<0.01).EF24 group compared with SAP group,EF24 group lung wet dry weight ratio significantly reduced at each point,with statistical difference(P<0.01);Compared between the two subgroups,no significant difference was found between the subgroups in CON group(P>0.05),in SAP group and EF24 group,lung wet dry weight ratio of 12 h group were higher than 6h group significantly,with statistical difference(respectively P<0.01,P<0.05);3 Alveolar lavage fluid cell count: SAP group,EF24 group for each time point respectively compared with CON group,SAP group,EF24 group alveolar lavage fluid total cell count increased obiviously,with statistical difference(all P<0.01);EF24 group compared with SAP group,the alveolar lavage fluid total cell count of EF24 group decreased at each point,with statistical difference(P<0.01);no significant difference was found between the subgroups in CON group(P>0.05);In the SAP group and EF24 group,alveolar lavage fluid total cell count of 12 h group increased significantly compared with 6h group,with statistical difference(all P<0.01);SAP group,EF24 group respectively compared with CON group,the macrophage count and lymphocyte count were higher than CON group significantly at each point,with statistical difference(all P<0.01);EF24 group compared with SAP group,EF24 group macrophage count and lymphocyte count were lower significantly,with statistical difference(P<0.01);4 The levels of serum IL-6: compared with CON group respectively,the levels of serum IL-6 in SAP group and EF24 group increased significantly at each point,with statistical difference(all P<0.01);EF24 group compared with SAP group,the levels of serum IL-6 in EF24 group significantly reduced at each point,with statistical difference(P<0.01);Compared beteen the subgroups,no significant differences were found in CON group and EF24 group(all P>0.05).In SAP group,the levels of serum IL-6 in 12 h group significantly were higher than 6h group,with statistical difference(P<0.01);5 The levels of serum TNF-?: the SAP group,the EF24 group respectively compared with CON group,the levels of serum TNF-? increased significantly at each point,with statistical difference(all P<0.01);EF24 group compared with SAP group,the levels of serum TNF-? in EF24 group significantly reduced at each point,with statistical difference(P<0.01);Compared between the two subgroups,no significant difference was found between the subgroups in CON group and EF24 group(all P>0.05),In SAP group,the levels of serum TNF-? of 12 h group was higher than 6h group significantly,with statistical difference(P<0.01);6 The levels of Alveolar lavage fluid TNF-?: SAP group,EF24 group compared with CON group respectively,the levels of Alveolar lavage fluid TNF-? increased significantly at each point,with statistical difference(all P<0.01);EF24 group compared with SAP group,the levels of Alveolar lavage fluid TNF-? in EF24 group significantly reduced at each point,with statistical difference(P<0.01);Compared between the two subgroups,no significant difference were found between the subgroups in CON group and EF24 group(all P>0.05),In SAP group,the cotent of Alveolar lavage fluid TNF-? of 12 h group more than 6h group significantly,with statistical difference(P<0.01);7 The expression of lung AQP5 protein: SAP group,EF24 group compared with CON group respectively,the expression of AQP5 protein decreased significantly in each subgroup,with statistical difference(all P<0.01);EF24 group compared with SAP group,the expression of lung AQP5 protein in EF24 group was higher than SAP group significantly,with statistical difference(P<0.01);Comparison beteen the subgroups,no significant difference were found between the subgroups in CON group,EF24 group(all P>0.05),the 12 h group was lower than 6h group in SAP group significantly,with statistical difference(P<0.05);8 The expression of lung p65 protein: compared with CON group respectively,the expression of lung p65 protein in SAP group and EF24 group increased significantly in each subgroup,with statistical difference(all P<0.01);EF24 group decreased significantly compared with the SAP group in each subgroup,with statistical difference(P<0.01);Comparison beteen the subgroups,no significant difference were found between the subgroups in CON group and EF24 group(all P>0.05),the expression of lung p65 protein at 12 h was higher than 6h in SAP group significantly,with statistical difference(P<0.01);9 The expression of lung AQP5 mRNA: compared with CON group respectively,the expression of lung AQP5 mRNA in SAP group and EF24 group decreased significantly in each subgroup,with statistical difference(all P<0.01);EF24 group increased significantly comapared with SAP group in each subgroup,with statistical difference(P<0.01);Comparison between the subgroups,no significantly difference were found between the subgroups in CON group,EF24 group(all P>0.05),the 12 h group was lower than 6h group in SAP group significantly,with statistical difference(P<0.05);10 The expression of lung p65 mRNA: compared with CON group respectively,the expression of lung p65 mRNA in SAP group and EF24 group increased significantly in each subgroup,with statistical difference(all P<0.01);the expression of lung p65 mRNA in EF24 group was lower than SAP group,with statistical difference(P<0.01);Comparison between the subgroup,no significantly difference were found between the subgroups in CON group,EF24 group(all P>0.05),in SAP group at 12 h was higher than at 6h significantly,with statistical difference(P<0.05).Conclusion:1 EF24 could up-regulate the expression of AQP5,down-regulate the expression of p65,reduce SAP associated with lung inflammation,had a protective effect for SAP associated with lung injury.2 EF24 reduced the expression of the promote inflammation factor TNF-?,IL-6,alleviated SAP associated with lung inflammation reaction.3 The mechanism of EF24 involving in reducing SAP associated with lung injury might be inhibiting NF-?B activation and down-regulating the expression of proinflammatory factor TNF-?,IL-6,up-regulating the expression of AQP5. |
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