The Role Of NLRP3/Caspase-1 Signaling Pathway In The Pathogenesis Of Severe Acute Pancreatitis Associated Lung Injury And The Study Of Emodin Intervention

Objective: The incidence rate and mortality of acute lung injury(ALI)caused by severe acute pancreatitis(SAP)are high,but the mechanism is unclear.In this study,the activation of NLRP3 / Caspase-1 inflammatory pathway in acute lung injury model was studied to explore the pathogenesis of severe acute pancreatitis associated lung injury(SAP-ALI).By studying the effect of emodin on the key molecules of NLRP3 / Caspase-1 signaling pathway,the mechanism of emodin in inhibiting lung injury in acute pancreatitis was discussed,which provides a new idea for the prevention and treatment of lung injury in acute pancreatitis by combining traditional Chinese and Western medicine.Methods: Experiment 1: Rats were randomly divided into Sham operation group(Sham group,n = 10),disease model group(SAP group,n = 10),emodin group(EMO group,n = 10)and dexamethasone group(DEX group,n = 10).4% phenobarbital(30-40 mg / kg)was used for anesthesia.Rats in SAP,EMO and DEX group were retrogradely perfused with 5% sodium taurocholate(1ml / kg)through pancreaticobiliary duct to establish acute lung injury model;and the pancreas was gently turned over and closed three times in the Sham operation group.In the EMO group,emodin suspension was administered to rats at a dose of 40 mg / kg,2 h and 12 h after modeling.In the DEX group,dexamethasone was injected intraperitoneally to the rats at a dose of 10 mg / kg,2 h after modeling.Detected respectively in 24 h after operation,the wet/dry ratio of pancreatic tissue and lung tissue;the serum amylase level;the rats were observed by H & E staining pathological changes of pancreas and lung tissue;flow cytometry to detect the number of apoptotic cells in the lung;westetn blot to detect Caspase3? Bax and Bcl2 in the lung;the expression distribution of Caspase3 in lung of rats were detected by immunohistochemistry.Experiment 2: Rats were randomly divided into Sham operation group(Sham group,n = 10),disease model group(SAP group,n = 10),emodin group(EMO group,n = 10)and dexamethasone group(DEX group,n = 10).Animals were treated in the same way as Experiment 1.Westetn blot was used to detect the expression of NLRP3 / Caspase-1 signaling pathway proteins NLRP3,ASC and Caspase-1;detection of IL-1 ? and IL-18 in serum of rats in each group by ELISA;detection of expression and distribution of Ly6 G in lung of rats in each group by immunohistochemistry;extraction of neutrophils in blood by magnetic bead method,the expression of i CAM and IL-8 protein of neutrophils in blood was detected by westetn blot.Experiment 3: 5ml of blood was collected from abdominal aorta of 15 normal SD rats,neutrophils were isolated by magnetic bead method,a few neutrophils were used for purity detection(flow cytometry),the rest were used for in vitro culture.10% FBS DMEM medium was used to culture neutrophils in 37 degree incubator;the neutrophils in vitro were divided into five groups: normal control group,LPS,LPS + MCC950,LPS + DEX,LPS + emodin.One cell sample was used for RNA extraction,and RT-PCR was used to detect the m RNA expression level of TNF – ??NLRP3?ASC?Caspase-1 and IL-1?.The other was used for westetn blot of egg white to detect TNF – ??NLRP3?ASC?Caspase-1 and IL-1?.The remaining two were frozen in-80 refrigerator.The rest of the neutrophils were cultured for repeated experiments.Result: 1.The results showed that in SAP group,the intestine became dark purple,the pancreas was necrotic,the lung was colorless and blood stasis.Compared with the Sham operation group,the wet/dry ratio of pancreas and lung tissue was significantly higher(P < 0.05)? the serum amylase level of SAP group was significantly higher(P < 0.01)?the pathological score of pancreas and lung tissue was significantly higher(P < 0.01)?the apoptosis ratio of lung tissue was significantly higher(P < 0.001)?the Caspase3 of apoptosis related protein in lung tissue was significantly higher?the expression level of Bax was significantly higher(P < 0.001)?the expression of Bcl2 was significantly lower(P < 0.01),and the expression distribution of Caspase3 in lung tissue was increased(P < 0.01).After the treatment with emodin and dexamethasone,the above indexes were improved compared with SAP group(P < 0.05).It should be noted that there was a significant difference between emodin group and dexamethasone group(P < 0.05)in the detection of serum amylase level?the proportion of apoptosis in lung tissue?the expression of Caspase3 and Bax in lung tissue.2.The expression of NLRP3 / Caspase-1 pathway related proteins NLRP3,ASC and Caspase-1 in lung tissue of SAP group was significantly higher than that of Sham operation group(P < 0.01)?the expression of IL-1 ? and IL-18 increased significantly(P < 0.001)? the number of neutrophil specific antibody Ly6 G increased significantly(P < 0.001),and the expression of neutrophil migration protein i CAM and IL-8 in peripheral blood increased significantly(P < 0.05).After adding dexamethasone and emodin,the expression of the above indexes decreased significantly(P < 0.05).It should be noted that,compared with dexamethasone group,emodin group had more significant intervention effect on the above indicators,and the difference was statistically significant(P < 0.05).3.Compared with the control group,the expression levels of TNF-? and NLRP3 /Caspase-1 pathway related genes NLRP3?ASC?Caspase-1?IL-1 ? m RNA and protein were significantly higher after LPS stimulation(P < 0.05).After the intervention of NLRP3 inhibitor MCC950 and emodin,the expression levels of the above genes m RNA and protein were significantly lower than that of LPS group(P < 0.05).The protein expression of the above indicators in the dexamethasone group was significantly reduced(P <0.05).The intervention effect of emodin and MCC950 was better than that of dexamethasone(P < 0.05).Conclusion: 1.In SAP group,the injury of pancreas and lung was obvious,and the apoptosis of lung tissue was aggravated.2.NLRP3 / Caspase-1 pathway was activated in the lung injury model of severe acute pancreatitis,and promoted the migration of neutrophils and infiltration in the lung tissue,thus aggravating the lung injury.3.Inhibiting the activation of NLRP3 inflammatory bodies in neutrophils can reduce the release of inflammatory factors.4.Emodin can alleviate lung injury in severe acute pancreatitis by inhibiting the activation of NLRP3 / Caspase 1 pathway.