The Role Of Cold Inducible Rna-Binding Protein In The Pathogenesis Of Severe Acute Pancreatitis-Associated Lung Injury And The Intervention Of Emodin

Background: With the wide change of life-style in the world,the incidence rate of obesity and gallstones and the incidence of acute pancreatitis(AP)are increasing year by year.AP is a common abdominal emergency in clinic,which is characterized by acute onset and rapid progress.Clinically,acute pancreatitis is often divided into mild acute pancreatitis(MAP)and severe acute pancreatitis(SAP),of which severe acute pancreatitis accounts for about 20%,and about 13-35% of patients with severe acute pancreatitis die due to serious complications.Acute Lung Injury(ALI)is one of the most common complications of SAP,and ALI can further develop into acute respiratory distress syndrome(ARDS).ARDS is one of the main causes of death of SAP patients.At present,the lung damage caused by acute pancreatitis is called acute pancreatitis-associated lung injury(APALI)by scholars at home and abroad.although there are many researches on the pathogenesis and drug intervention of APALI,the therapeutic effect of APALI has not improved significantly and the mortality has not decreased significantly in recent years.Therefore,it is urgent to explore the pathogenesis and effective treatment strategies of APALI.The extracellular cold-induceile RNA-binding protein(eCIRP)is a kind of damage-associated molecular pattern molecules(DAMPs).High levels of serum CIRP can be detected in patients with hemorrhagic shock or sepsis,and the increase degree is positively correlated with mortality.Functionally,eCIRP can activate NLRP3 inflammasome,aggravate inflammatory diseases and lead to tissue damage.NLRP3 inflammasome is a protein complex,which can directly interact with the adaptor apoptosis-associated speck-like protein containing caspase-recruitment domain(ASC)to activate caspase-1,and the activated caspase-1 splits gasdermin D(GSDMD),pro-IL-1? and pro-IL-18,leading to cell pyroptosis,IL-1? and IL-18 secretion.Pyroptosis is a novel form of programmed cell death involved in inflammation to deteriorate the disease process.Lung resident macrophages,such as alveolar macrophages,are the key factors in the pathogenesis of ALI/ARDS.The pyroptosis of alveolar macrophages can aggravate the inflammation in lung by producing inflammatory cytokines,such as IL-1? and IL-18.However,little is known on how CIRP regulates AM pyroptosis and inflammatory cytokine production,which is worth further exploring.Our group works on exploring the pathogenesis and integrative treatment of APALI for 20 years.Our results have confirmed that integrative medicine can significantly shorten disease course and decrease the mortality.Therefore,The application of integrated medicine in the treatment of SAP and APALI will have broad prospects.In many years' clinical practice,the theory of " the lung and the large intestine being interior-exteriorly related " has been fully confirmed,and it has become one of the components of traditional Chinese medicine(TCM)with the development of later physicians.Many studies have shown that the occurrence and development of APALI may be related to the dysfunction of intestinal barrier during SAP,and the enteric bacteria and toxins are absorbed into blood and circulated to the lungs after translocation.The above research results coincide with the theory of " the lung and the large intestine being interior-exteriorly related " in TCM.Rhubarb is commonly used in traditional Chinese medicine to treat AP,and emodin is is a natural ingredient of Rhubarb.In order to explore the possible mechanism of emodin in the treatment of APALI,the present study established rat model of APALI by retrograde injection of 5% STC into rat pancreaticobiliary duct,and selected emodin to intervene,and applied modern biological methods and techniques to explore the potential role of eCIRP in the pathogenesis of APALI and the intervention effect of emodin,so as to further reveal the pathogenesis of APALI and provide evidence for the prevention and treatment of APALI by integrated traditional Chinese and western medicine.Objective: To explore the role of CIRP in the pathogenesis of APALI,the expression changes of CIRP in APALI rat model and the effect of antagonizing CIRP on lung injury in rats with SAP were detected.In addition,the recombinant rat CIRP was used to stimulate rat alveolar macrophages,and the effect of CIRP on the cells was detected.Moreover,emodin was used for intervention therapy to explore its therapeutic targets of APALI,and to provide theoretical basis for the treatment of APALI with emodin.Method:(1)Forty male SD rats were randomly divided into four groups: sham-operated group(CON),severe pancreatitis group(SAP),C23 treatment group(C23)and emodin treatment group(EMO).SAP model of SD rats was induced by retrograde injection of 5% STC through the biliopancreatic duct(1ml/kg,0.1ml/min).Rats in sham-operated group were retrograde injected with the same volume of sterile normal saline through the biliopancreatic duct.In C23 group,C23(8mg/kg)was injected through tail vein 2 hours after modeling.Emodin group was given emodin(40mg/kg)by gavage 2 hours after operation and 12 hours after operation.Twenty-four hours after the injection with STC,rats in each group were anesthetized,and blood was collected through abdominal aorta,and pancreas and lung tissues were collected.Pathological changes of pancreas and lung were observed by HE staining,and the protective effects of C23 and emodin on SAPALI was evaluated by the pathological score of pancreas and lung tissue.Enzyme-linked immunosorbent assay(ELISA)was used to determine CIRP,amylase and IL-1? in serum.Automatic blood gas analyzer was used to analyze arterial blood gas.The expression of CIRP in pancreas and lung tissue and the number of ly6G+ cells in lung tissue were observed by immunohistochemical staining.CIRP mRNA levels in pancreas and lung tissues and the m RNA expression levels of NLRP3,IL-1? and CXCL1 in lung tissues were measured by qRT-PCR.The expressions of CIRP,p-P65,t-P65,p-IKB?,t-IKB?,NLRP3,ASC,Caspase-1,GSDMD,IL-1?,CXCL1 protein were detected by Western blot.The expression of CIRP in islets and NLRP3 and CXCL1 in resident macrophages(F4/80+ cells)in lungs were observed by multiplex fluorescence staining.(2)The in vitro study was divided into two parts.In the first part,NR8383 cells were treated with vehicle as the Control or recombinant rat CIRP(1.5 ?g/ml)for 6 h as the CIRP group.NR8383 cells were pretreated with TAK242(500 ?M)for 24 h and treated with CIRP(1.5 ?g/ml)for 6 h as the CIRP+TAK242 group.Besides,NR8383 cells were pretreated with C23(300 ng/ml)for1 h and treated with CIRP(1.5 ?g/ml)for 6 h as the CIRP + C23 group.Moreover,NR8383 cells were treated with CIRP(1.5 ?g/ml)and emodin(20 ?M)for 6 h as the CIRP + EMO group.The rate of pyroptosis(Caspase-1 and PI double staining)was detected by flow cytometry.The m RNA expression levels of NLRP3,IL-1? and CXCL1 were measured by qRT-PCR.The expression of p-P65,t-P65,p-IKB?,t-IKB?,NLRP3,ASC,Caspase-1,GSDMD,IL-1?,CXCL1 protein in each group was detected by Western blot.In the second part,to examine the role of IL-1? in the CIRP-increased CXCL1 expression in AMs,NR8383 cells were treated with vehicle as the Control or1.5 ?g/ml CIRP for 6 h as the CIRP group.Furthermore,NR8383 cells were transfected with control si RNA or IL-1?-specific si RNA for 48 h and treated with 1.5 ?g/ml CIRP for 6 h as the CIRP+NC or CIRP+Si-IL-1? group,respectively.In addition,NR8383 cells were treated with different concentrations(0,1ng/ml,5ng/ml,10ng/ml)of recombinant rat IL-1? for 24 h and the same concentration of IL-1?(10ng/ml)for different time periods(0,6,12,24h),then the cells were collected,RNA and protein were extracted,and the expression of CXCL1 mRNA and protein were detected by qRT-PCR and Western blot.Results:The results of animal model experiment are as follows:(1)Compared with CON group,SAP group showed obvious pathological injury in pancreas and lung tissue,and the pathological score was significantly higher(P <0.001).In addition,compared with CON group,the levels of CIRP,amylase and IL-1? in SAP group were significantly higher(P < 0.001).The level of PaO₂ in SAP group decreased significantly(P < 0.001),while the level of PaCO₂ in SAP group increased significantly(P < 0.001).Compared with SAP group,the injury of pancreas and lung tissue in C23 group and EMO group was significantly alleviated(P < 0.001),serum CIRP,amylase and IL-1? were significantly decreased(P < 0.001).The level of PaO₂ in arterial blood increased significantly(P < 0.001),while the level of PaCO₂ decreased significantly(P < 0.001)(2)Compared with CON group,the expression of CIRP in pancreas(mainly located in islets)and lung of SAP group was significantly increased(P < 0.001).Compared with CON group,significantly higher levels of NF-?Bp65 and IKB? phosphorylation were detected in the lung of the SAP group of rats(P < 0.001),and the expression of NLRP3,ASC(Monomer,Dimer,Polymer),Caspase-1 p20,GSDMD-N,IL-1?(pro-IL-1? and p17)and CXCL1 in lung were significantly increased in SAP group(P < 0.001).Moreover,the infiltration of neutrophils(ly6g + cells)were significantly increased in SAP group compared with the CON group.(3)Compared with SAP group,CIRP expression in islets and lung of C23 and EMO groups was significantly inhibited.Besides,the expression of p-P65,p-IKB?,NLRP3,ASC(Monomer,Dimer,Polymer),Caspase-1 p20,GSDMD-N,IL-1?(pro-IL-1? and p17),and CXCL1 in lung decreased significantly(P < 0.05),as well as the infiltration of ly6g+ cells in lung were significantly reduced.The results in vitro study were as follows:(1)In vitro study shown that CIRP(1.5?g/ml)stimulation significantly increased the levels of I?B? and NF-?Bp65 phosphorylation(P < 0.001),and also promoted the expression of NLRP3,ASC,caspase-1(p20),GSDMD-N,IL-1?(pro-IL-1? and p17)and CXCL1 protein and the pyroptosis of NR8383 cells(P < 0.001).TAK242,a TLR4 inhibitor,nearly completely abrogated the above cell effects induced by CIRP.The molecular mechanism indicated that CIRP induced the the activation of the NF-?B signal and the NLRP3 inflammasome,thus lead to pyroptosis of NR8383 cells through TLR4.Treatment with emodin,like C23,significantly inhibit the activation of NF-?B,NLRP3 inflammasome formation,pyroptosis and the expression of CXCL1 in NR8383 cells induced by CIRP(p<0.05).(2)We tested whether CIRP could modulate CXCL1 expression and its molecular mechanism.We found that treatment with CIRP significantly up-regulated CXCL1 expression in NR8383 cells(p<0.001).Compared with CIRP group,CXCL1 expression level in CIRP+ Si-IL-1? group decreased significantly(p<0.001),while CXCL1 expression level in CIRP+NC(Negative control)group did not change significantly.It is suggested that IL-1? is the key mediator for CIRP to promote CXCL1 expression in NR8383 cells.Furthermore,treatment with different concentrations(0,1ng/ml,5ng/ml,10ng/ml)of recombinant rat IL-1? for 24 h and the same concentration of IL-1?(10ng/ml)for different time periods(0,6,12,24h)revealed that IL-1? induced CXCL1 expression in NR8383 cells in a dose-and time-dependent manner.Conclusion:(1)24 h after the injection with 5% sodium taurocholate to SD rats,obvious pathological damage of pancreas and lung tissue and significant increase of serum amylase content appeared,meanwhile,arterial blood gas analysis showed obvious respiratory dysfunction in rats,which indicated that APALI model was successfully induced by this method.(2)The expression of CIRP increased in pancreatic islets and lung tissues of APALI rat model,accompanied by the increase of serum CIRP level.With the application of C23,the expression of CIRP was down-regulated in pancreatic islets and lung tissues,and the level of CIRP in serum decreased,which significantly relieved APALI,suggesting that CIRP may be a molecular marker closely related to the occurrence and development of APALI.CIRP antagonist C23 can inhibit the activation of NF-?B,the assembly and activation of NLRP3 inflammasome,the expression of CXCL1 and the infiltration of neutrophils in lung tissue of APALI rats,and alleviate APALI.(3)CIRP may activate NF-?B signal and NLRP3/IL-1?/CXCL1 signal pathway in rat alveolar macrophages through TLR4 receptor,and play an important role in APALI.(4)Emodin can alleviate the pathological damage of pancreas and lung tissue in APALI rats,and also significantly reduce the levels of serum amylase,CIRP and IL-1? in APALI rats,and improve the lung function,showing a good therapeutic effect on APALI.(5)Emodin may reduce neutrophil infiltration into lung by inhibiting NF-?B and NLRP3/IL-1?/CXCL1 signaling pathway mediated by CIRP-TLR4,thus exerting its pharmacological effect on APALI.