| Background:Severe acute pancreatitis(SAP)is a sudden inflammatory disease of the pancreas with a variable clinical course,and acute lung injury(ALI)is the most common complication of distant organs in the early stage of SAP.The leading cause of death in SAP is ALI and its severe form,acute respiratory distress syndrome(ARDS).When SAP occurs,local damage to the pancreas and impaired microcirculation leads to ischemia and hypoxia of the intestinal mucosa,resulting in damage to the function of the intestinal barrier and the entry of bacteria,endotoxins and associated virulence factors into the lung via lymphatic channels and the body circulation,triggering a cytokine storm that activates multiple intracellular inflammation-related signaling pathways and releases large amounts of inflammatory factors,resulting in diffuse interstitial lung oedema and alveolar edema and pulmonary respiratory dysfunction,ultimately leading to ALI.Emodin(EMO),the main active ingredient of Chinese medicinal rhubarb,has a wide range of pharmacological effects,including anti-inflammatory,anti-cancer,anti-viral,anti-bacterial,anti-allergic,anti-osteoporosis,diuretic,immunosuppressive and neuroprotective.Currently,there are numerous experimental investigations on the use of EMO to treat ALI.However,the pharmacological mechanism of the protective effect of EMO in the pathological process of severe acute pancreatitis-associated acute lung injury(SAP-ALI)is not fully understood and needs to be further explored.Objective:1.To investigate the role of the signal transducer and activator of transcription 3(STAT3)/CCAAT enhancer binding protein β(C/EBPβ)/cyclooxygenase-2(COX-2)signaling pathway in the pathogenesis of SAP-ALI.2.To investigate whether EMO exerts a therapeutic effect by modulating the STAT3/C/EBPβ/COX-2 signaling pathway.Methods:Male SD(Sprague-Dawley)rats were randomly divided into the sham operation group(SO),severe acute pancreatitis group(SAP),adeno-associated virus negative control group(SAP+AAV-shNC,sh-NC),adeno-associated virus silent STAT3 group(SAP+AAV-shSTAT3,sh-STAT3),adeno-associated virus silent C/EBPβ group(SAP+AAV-shC/EBPβ,sh-C/EBPβ)and emodin group(EMO)(n=10 rats per group).SAP models were established by retrograde injection of 5%sodium taurocholate(STC)into the biliopancreatic duct.The peri-pancreatic tissues of rats in the SO group were loosened after laparotomy.After induction of SAP,emodin was intragastrically administered to the EMO group.Four weeks before modeling,rats in the sh-NC group,sh-STAT3 group,and sh-C/EBPβ group were treated with adeno-associated viruses(AAV)AAV-shNC,AAV-shSTAT3,and AAV-shC/EBPβ intratracheal drips,respectively.The severity of model injury was assessed by blood gas analysis,pancreatic and lung histopathological scores,wet/dry(W/D)ratio of lung tissue and by measuring serum amylase activity,inflammatory factor(IL-β,TNF-α,IL-6)levels using ELISA.Levels of oxidative stress indicators such as malondialdehyde(MDA),glutathione(GSH),and 4-hydroxynonenal(4-HNE)were tested according to the kit instructions.The gene and protein expression levels of STAT3,C/EBPβ,and COX-2 were detected at the transcriptional and translational levels using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and western blot(WB)analysis,respectively.The BCA protein assay was used to analyse the total protein content of bronchoalveolar lavage fluid(BALF).Meanwhile,apoptosis was evaluated by TUNEL staining.In addition,immunofluorescence staining was used to further analyse the expression of target proteins on the signaling pathway.Results:1.Compared with the SO group,rats in the SAP group showed a more than 3-fold increase in serum amylase activity,significantly higher levels of serum inflammatory factors IL-6,IL-1β,and TNF-α(p<0.001),severe damage to pancreatic and lung tissues(p<0.001)and higher W/D ratio in lung tissues(p<0.001),indicating the success of this experimental model.Adeno-associated viral silencing of STAT3 and C/EBPβsignificantly reduced edema and inflammatory infiltration in rat lung tissues(p<0.001),decreased lung W/D ratio(p<0.05,p<0.01,respectively),decreased serum amylase activity(p<0.001)and serum inflammatory factor IL-1β,IL-6,and TNF-α levels.The mRNA expression levels of STAT3,C/EBPβ,and COX-2 in the lung tissue of rats in the SAP group and sh-NC group were elevated,and these three genes were significantly down-regulated in the sh-STAT3 group and sh-C/EBPβ group.WB results showed the same trend,suggesting that knocking down STAT3 or C/EBPβ can effectively reduce the severity of SAP-ALI.Compared with the SO group,the levels of oxidative stress indicators 4-HNE and MDA in lung tissue showed different degrees of elevation in the SAP and sh-NC groups(p<0.001),accompanied by depletion of GSH(p<0.01).In contrast,the levels of 4-HNE and MDA decreased in the sh-STAT3 and sh-C/EBPp groups(p<0.001),as well as the levels of GSH increased(p<0.05).The result of inflammatory factors such as IL-1β,TNF-α,iNOS in lung tissue,total protein amount in BALF,and TUNEL staining to assess apoptosis rate showed similar trends to oxidative stress indicators.The results showed that knockdown of the genes STAT3 or C/EBPβcould improve SAP-ALI-induced oxidative stress and inflammatory responses and inhibited apoptosis.2.Compared with the model group,serum amylase activity(p<0.001),inflammatory factors IL-6,IL-1β,and oxidative stress indicators 4-HNE and MDA were significantly lower(p<0.001,p<0.001,p<0.01 and p<0.05,respectively),and GSH content was significantly higher(p<0.05)in the emodin group.The SO group’s lung tissue mean W/D ratio was 3.44;the SAP group was 5.58,which was considerably higher than the SO group(p<0.001);and the EMO group was 4.31,which was lower than the SAP group(p<0.001).The results indicated that EMO treatment could significantly reduce pulmonary edema.SO group rats had healthy morphology of pancreatic and lung tissues;rats in the SAP group showed severe pancreatic acini liquefaction necrosis and exudative edema,and lung tissues showed pathological changes such as massive inflammatory cell infiltration and edema(p<0.001);the above pancreatic and lung tissue damage was effectively alleviated in the EMO group(p<0.001).Compared with rats in the SO group,PaO2 levels were reduced(p<0.001)and PaCO2 levels were increased(p<0.001)in the SAP group;PaCO2 levels were reduced(p<0.05)and PaO2 levels were increased(p<0.05)after EMO administration.The results showed that EMO treatment significantly improved pulmonary respiratory function.In the model group,STAT3,C/EBPβ,and COX-2 genes and proteins were highly expressed and the apoptosis rate was 40.95%;in the EMO group,STAT3,C/EBPβ,and COX-2 genes and proteins were less expressed(p<0.001)and the apoptosis rate decreased(p<0.001).The data suggest that EMO treatment significantly down-regulated the high expression of STAT3,C/EBPβ and COX-2 genes and proteins in SAP-induced lung tissue and inhibited apoptosis.Conclusions:1.The SAP-ALI rat model was established by injecting 5%sodium taurocholate backward into the biliopancreatic duct.2.The silenced genes STAT3 or C/EBPβ effectively reduced the severity of ALI in SAP rats,and significantly reduced SAP-induced expression of STAT3,C/EBPβ,and COX-2 in rat lungs,as well as reduced oxidative stress,inflammation levels,and apoptosis levels in the SAP-ALI model.3.Emodin could alleviated SAP-ALI by reducing lung tissue pathological damage,inflammatory response and their oxidative stress and apoptosis levels.4.Emodin significantly inhibited SAP-induced expression of STAT3,C/EBPβ,and COX-2 mRNA and protein in the lung of rats,suggesting that it may exert its therapeutic effect on SAP-ALI by regulating the "STAT3/C/EBPβ/COX-2" signaling pathway. |
PDF Full Text Request