The Role Of ECIRP-STAT3/C/EBPβ Signaling Pathway In The Pathogenesis Of Acute Pancreatitis-induced Lung Injury And The Intervention Of Qingyi Decoction

Objectives: Severe Acute pancreatitis(SAP)is a common clinical acute abdomen,which can lead to acute lung injury(ALI)and even acute respiratory distress syndrome(ARDS).A large number of studies have confirmed that cold-induced RNA-binding protein(e CIRP)excreted after tissue or cell destruction can damage the air-blood barrier of the lung and aggravate lung injury,leading to the increase of neutrophil extracellular traps(NET)into the lung.This study takes the "e CIRP-STAT3/C/EBPβ" signaling pathway as the entry point to explore the mechanism of this signaling pathway in SAPALI,clarify the regulatory mechanism of e CIRP in pulmonary inflammation,and explore new targets for the clinical treatment of SAP-ALI.To further explain the intervention mechanism of Qingyi decoction affecting the changes of SAP-ALI,and to provide new strategies and ideas for the prevention and treatment of SAP-ALI with integrated traditional Chinese and western medicine.Methods: Forty male SD rats were randomly divided into four groups: Sham group(SO),SAP group,Qingyi Decoction group(QYD),and C23 treatment group(CIRP inhibitor).SAP-ALI was induced by retrograde injection of 5% sodium taurocholate(50mg/kg)through the biliopancreatic duct in SAP,QYD and C23 groups.C23 group:C23(8mg/Kg)was injected via the tail vein 2 hours after modeling.QYD group: Qingyi decoction(10.53g/kg)was given by gavage half an hour before surgery and 12 hours after surgery.The rats in the sham group were injected with the same amount of 0.9%sodium chloride injection in the same way,and were given the same volume of QYD pure water by gavage at the same time.After 24 hours of modeling,the rats were anesthetized,and the abdomen was opened under anesthesia.Blood was collected from the abdominal aorta,and pancreatic and lung specimens were collected.Hematoxylineosin(HE)staining was used to reflect the pathological injury,serum Amylase(AMY)activity was detected by amylase detection kit,and the wet/dry weight ratio(W/D)of lung was obtained by vacuum drying method to evaluate the pancreatic and lung injury.The serum CIRP content and proinflammatory factors tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by Enzyme-linked immunosorbent assay(ELISA)to reflect the inflammatory response and degree.The expression of CIRP,STAT3,P-STAT3,C/EBPβ and adhesion molecule-1(ICAM-1)protein in lung tissue was detected by Western-Blot.The expression of CIRP,ICAM-1,STAT3 and C/EBPβmRNA in lung tissue was detected by q RT-PCR.Evans Blue(EB)staining was used to detect the changes in pulmonary vascular permeability.The formation and number of NETs were observed under scanning electron microscope.Results: Compared with healthy rats,the pancreas and lung pathological scores of SAP rats were significantly increased(P<0.001),indicating that the pancreas and lung pathological injuries were serious.The serum amylase level and lung wet to dry weight ratio were significantly increased(P<0.001).The serum CIRP level,TNF-α and IL-6levels in SAP group were significantly increased(P<0.001),indicating that the degree of inflammation was aggravated.The protein levels of CIRP,p-STAT3,C/EBPβ and ICAM-1 in lung tissues were increased(P<0.001).The mrna expression levels of these target factors were also increased in SAP group(P<0.001).EB test results showed that the Evans blue leakage on the surface of lung tissue in SAP group was significantly increased,and the quantitative ratio of leakage was increased(P<0.001).A large number of NETs were observed in the lung tissue of SAP under scanning electron microscope.The results showed that the level of e CIRP and the expression of STAT3/C/EBPβ signaling pathway were significantly increased in the SAP-ALI model.Compared with the SAP group,the QYD treatment group and the C23 inhibitor group had reduced pathological damage,decreased serum amylase and lung wet/dry weight ratio,significantly decreased serum CIRP,TNF-α and IL-6 levels,and significantly improved inflammation.The protein and gene expression levels of CIRP,p-STAT3,C/EBPβ,and ICAM-1 in lung tissue were significantly decreased,the amount of Evans blue exudate was significantly reduced,and the release of NETs was significantly reduced,with statistically significant differences(P<0.05).The results showed that Qingyi Decoction and CIRP inhibitor could inhibit the secretion of CIRP,thereby affecting the activation of STAT3/C/EBPβ signaling pathway,thereby reversing the damage of pancreas and lung and improving the release of downstream inflammatory factors.Conclusions:(1)At 24 hours after retrograde injection of 5% sodium taurocholate solution into the biliopancreatic duct,the pancreas and lung of the rats in the model group were obviously damaged,the serum amylase content was significantly increased,and the wet/dry weight ratio of the lung was significantly increased.The above results proved that the pancreas and lung of the SAP group were obviously damaged,indicating that the SAP-ALI model was successfully prepared.(2)In the lung tissue of SAP-ALI rats,the expression of CIRP at the protein and gene levels was significantly increased,accompanied by an increase in the serum level of CIRP.After QYD and C23 intervention,the expression of CIRP protein and gene in lung tissue decreased,the serum level of CIRP decreased,and the symptoms of ALI were significantly relieved,indicating that CIRP was closely related to the occurrence and development of SAP-ALI.(3)With the increase of CIRP level in rats,the protein and gene expression levels of PSTAT3 and C/EBPβ were significantly increased compared with the sham group.C23 could inhibit the expression of P-STAT3 and C/EBPβ,indicating that CIRP may affect lung injury by regulating STAT3/C/EBPβ signaling pathway.CIRP inhibitor could inhibit e CIRP-STAT3/C/EBPβ signal transduction and alleviate SAP-ALI.(4)Qingyi Decoction intervention can significantly reduce the levels of serum amylase,CIRP,and related inflammatory factors in the SAP group,reduce the pathological damage of pancreas and lung tissue,and improve lung function,indicating that Qingyi decoction has a good therapeutic effect on SAP-ALI.(5)In the SAP group,the air-blood barrier was damaged,the microvascular permeability was increased,and a large number of NETs were flooding into the lungs.Qingyi Decoction and CIRP inhibitor may improve SAP-ALI by regulating the e CIRPSTAT3/C/EBPβ signaling pathway.