The Role Of “P2X7-NLRP3 Inflammasome” Signal Pathway In The Pathogenesis Of Lung Injury Associated With Severe Acute Pancreatitis And The Therapeutic Effects Of Emodin

Objective:Acute lung injury is one of the most common complications of severe acute pancreatitis.In this experiment,a rat model of acute pancreatitis-induced lung injury was established by retrograde injection of 5%sodium taurocholate into pancreaticobiliary duct.The purpose is to explore the role of"P2X7-NLRP3 inflammasome"signal pathway in the pathogenesis of lung injury induced by severe acute pancreatitis,and to observe the effects of P2X7 receptor inhibitor BBG and emodin,so as to provide reliable experimental and theoretical basis for clinical application of integrated traditional Chinese and western medicine in the treatment of severe acute pancreatitis and its associated lung injury.Methods:Establishment of SAP rat model:forty SD male rats were randomly dividedinto 5 groups?n=8?by random number table:control group?Control,CON?,severe acute pancreatitis?SAP?model group,dexamethasone?DEX?group,P2X7 receptor inhibitor Brilliant Blue G?BBG?group,emodin?EMO?group.Among them,the CON group only turned over the pancreas several times after laparotomy.SAP group established SAP rat model by retrograde injection of 5%sodium taurocholate?50mg/kg?through pancreaticobiliary duct.Rats in DEX group were given dexamethasone injection?dose:10mg/Kg,concentration:5mg/ml?by intraperitoneal injection at the time of anesthesia conscious and 12 hours after the model was established.The BBG group was given intragastric administration?dose:30mg/Kg,concentration:3mg/ml?at the same time.Rats in EMO group were given emodin in the way of intragastric administration?dose:40mg/Kg,concentration:4mg/ml?at the same time.The rats in each group were carried laparotomy under anesthesia 24 hours after the model was established in order to obtain experimental materials.Some lung and pancreas tissues were stained with hematoxylin-eosin?HE?and the pathological injury degree was evaluated.The lung wet/dry weight ratio?W/D?was measured by vacuum drying method.The arterial blood gas indexes of rats in each group were measured by a full-automatic arterial blood gas analyzer:Pa O₂and Pa CO₂.The levels of pro-inflammatory factors TNF-?,IL-1?,IL-18 in serum of rats in each group were detected by ELISA.The content of serum amylase?AMY?was detected by automatic biochemical analyzer.The protein expression level of"P2X7-NLRP3 inflammasome"signal pathway in lung tissue was detected by Western-Blot.The expression of P2X7m RNA and NLRP3m RNA in lung tissue was detected by Real Time-PCR.Results:Compared with CON group,the pathological scores of lung and pancreastissues in SAP group were increased?p<0.001?,which accorded with the pathological features of lung injury induced by severe acute pancreatitis.In SAP group,Pa O2 in arterial blood decreased significantly?p<0.001?while Pa CO2 increased significantly?P<0.001?.The wet/dry weight ratio of lung tissue in SAP group was significantly increased?P<0.001?.In SAP group,the contents of IL-18,IL-1?and TNF-?in arterial serum increased?P<0.001?.The protein expression level of P2X7,NLRP3,ASC,Caspase-1p20in lung tissue increased?p<0.001?.The gene expression levels of P2X7 and NLRP3 in lung tissue of rats with severe acute pancreatitis also increased?p<0.001?.Compared with rats in SAP group,pathological scores of pancreas and lung tissues?p<0.01?,the content of amylase in serum?p<0.001?,wet/dry weight ratio of lung tissues?p<0.01?and blood gas analysis results?p<0.01?in rats after BBG administration have been improved to different degrees.In addition,the generation and release of pro-inflammatory factors IL-18,IL-1?and TNF-??all p<0.001?in lung tissue are reduced,and the protein expressions of P2X7?p<0.001?,NLRP3?p<0.001?,ASC?p<0.05?,Caspase-1p20?p<0.001?are significantly reduced,which indicates that BBG can control inflammatory reaction and reduce lung injury by inhibiting"P2X7-NLRP3 inflammasome"signal pathway.Another group of rats treated with DEX obtained similar experimental results.Interestingly,we observed that compared with SAP group rats,the structural damage and dysfunction of lung tissue of rats treated with EMO were alleviated,and the protein expression of NLRP3?p<0.001?,ASC?p<0.01?,Caspase-1p20?p<0.05?in lung tissue were significantly reduced.The inflammatory factors IL-18?p<0.001?,IL-1??p<0.01?and TNF-??p<0.001?released are significantly reduced,while the expression of P2X7 receptor,the upstream activation signal,has no significant change,which indicates EMO can inhibit the release of downstream active inflammatory factors and alleviate lung injury by effectively inhibiting the activation of NLRP3 inflammasome,but this inhibition does not rely on P2X7 receptor.Conclusion:1.24 hours after the model was established,pancreatic and lung tissues of SAP group rats showed obvious pathological damage,and serum amylase content significantly increased.Blood gas analysis results showed that rats had respiratory dysfunction,and lung wet/dry weight ratio significantly increased.Lung tissues of SAP group rats showed different degrees of damage in structure and function,which proved the successful preparation of acute pancreatitis lung injury model.2.SAP can cause acute lung injury.The specific mechanism may be that DAMPs acts on P2X7 receptor on cell membrane to activate NLRP3 inflammasome during acute pancreatitis,thus generating active caspase-1 fragment,causing cleavage and activation of pro-IL-18 and pro-IL-1?.The release of a large amount of inflammatory factors induces activation of peripheral inflammatory cells to cause inflammatory cascade reaction,resulting in lung injury.3.P2X7 receptor specific inhibitor BBG and dexamethasone can effectively antagonize P2X7 receptor,inhibit expression and activation of NLRP3 inflammasome,thus inhibiting inflammatory reaction and alleviating lung injury.EMO can also effectively reduce the expression of NLRP3,ASC and Caspase-1p20 proteins,thus preventing the assembly and activation of NLRP3 inflammasome in cytoplasm,inhibiting the cleavage,activation and release of a large number of active inflammatory factors,alleviating inflammatory reactions,and playing a protective role in lung injury.It is worth noting that the inhibitory effect of EMO on NLRP3 inflammasome does not rely on P2X7receptor,and the specific mechanism needs to be further explored.