Transcriptome And Proteome Analysis Of The Pathogenesis Of Acute Pancreatitis-associated Acute Lung Injury And The Intervention Of Emodin

Background: Acute pancreatitis（AP）,the most common acute abdominal diseases in clinic,which is characterized by sudden onset and rapid development。15-20% AP patients will develop severe acute pancreatitis（SAP）with SIRS/MODS,and caused 10%-30% SAP-associated death.Acute lung injury（ALI）is the most common remote organ complications induced by severe acute pancreatitis.It is reported that ALI also occur in about 10-25% of all AP cases,and accounts for 60-70% SAP-associated deaths within the first seven days.In recent year,the damages induced by AP were defined as acute pancreatitis associated lung injury（APALI）.Althoug many studies about the pathogenesis and treatment of APALI were prefromed,the mechanisms involved in APALI remain incompletely understood and effective drug for APALI is still limited.Early aggressive surgical treatment not only cannot alleviate AP,often aggravating the disease due to surgical stress and trauma.Therefore,making further research to explore mechanisms and potential strategies for SAP-ALI is very necessary.Our team works on exploring the pathogenesis and integrative treatment of APALI for 20 years.Our results suggested that integrative medicine can significantly shorten disease course and decrease the mortality.Therefore,combination of traditional medical theories and modern medical knowledge and methods will be promising in the treatment of SAP and APALI.It is believed that the intestinal barrier dysfunction is the pathological basis of APALI.This is consistent with the theory of Traditional Chinese Medicine“exterior-interior relation between the lung and the large intestine”。Yao shukun,a famous scholar of integrative Chinese and western medicine,suggested that systems biology is the bridge of integrative Chinese and western medicine,and that substance is the carrier of biological information and the basis of biological function.The combination of multi-omics have greatly help to explore the the treatment and mechanism of disease.In the present study,the SAP model was induced by administering fresh 5.0% sodium taurocholate（0.1?m L/100?g body weight）into the biliopancreatic duct by standard retrograde infusion.RNA-seq,proteomics,Western Blot,Real-time PCR,Immunohistochemistry etc.were used to reveal the expression level of genes and proteins,and the mechanism of EMO in the treatment of APALI.This study provides a new found of the pathogenesis of APALI and the prevention and treatment of APALI by combining traditional Chinese and western medicine,and lays a theoretical and experimental basis for the reduction of the death rate of severe acute pancreatitis.Part One: Study on the treatment of acute pancreatitis associated lung injury with emodin by regulating the expression network of lnc RNA-m RNAObjective: To explore the role of lncRNA-mRNA in the pathogenesis of APALI and the effect of EMO and DEX.Method: SAP model was induced by administering fresh 5.0% sodium taurocholate into the biliopancreatic duct by standard retrograde infusion.Sixty male SD rats were randomly divided into 5 following groups（n = 12/group）: control group,SAP model group（SAP）,sham-operated group with pancreas marginally rotated（Sham）,positive drug group with DEX treatment（SAP-DEX）,and administration group with EMO treatment（SAP-EMO）.Meanwhile,each group was further divided into 6 h,24 h subgroups（n = 6 / subgroup）.Pathologic changes of pancreas and lungs were evaluated by hematoxylin & eosin（HE）staining.Ly6G+ cells were determined by immuno-histochemical straining.The level of amylase,TNF-α,IL-6 were detected using ELISA kit.The blood gas analysis was measured by an automatic blood gas analyzer.Ultrastructure of alveolar II epithelial cells were carried out by the transmission electron microscope.RNA-seq was used to reveal the expression rule of lnc RNAs and m RNAs.q RT-PCR was used to verify the expression level of lnc RNAs and m RNAs。Result:Contrasting with the Sham and Control groups,tissues of SAP-ALI rats presented severe edema,large amount of inflammatory cell infiltrations,extensive hemorrhage,multiple pancreatic acinar necrosis cells,ultrastructure of the nuclei of alveolar type II epithelial cells were irregular or constricted,the lamellar cortices were significantly reduced,the microvilli were widely detached and disappeared,the basement membrane was obviously vesiculated,and up-regulated serum AMY level,indicating the successfully established SAP-ALI rat model.After DEX or EMO treatment,the pathological damage of both pancreatitis and lung tissues were greatly alleviated in both 6 and 24 h treatment time.The pancreatitis and lung tissue pathological scores as well as serum AMY level,which were increased sharply in the SAP group,were also significantly recovered,indicating that both EMO and the positive drug DEX,could effectively alleviated the pancreatic and lung injuries induced by SAP.Then,the level pro-inflammatory cytokine TNF-α of rat blood samples was detected by ELISA assays,and the levels of blood Pa O2 and Pa CO2 were determined by blood gas analyses.Obviously,the SAP-ALI significantly up-regulated the pro-inflammatory cytokine TNF-α and the Pa CO2,and decreased the Pa O2 in the blood.Interestingly,EMO and DEX could significantly reverse the dysregulation of pro-inflammatory cytokine TNF-α and the levels of Pa O2 and Pa CO2,especially when the rats were in a severe ALI stage（SAP-24 h groups）.The neutrophil cells infiltration in the lung tissue of 24 h groups were further determined by immunohistochemical straining of Ly6G+.A large number of neutrophils infiltrated into the lung tissue were observed in SAP groups,whereas DEX and EMO treatment groups showed much fewer neutrophils infiltration.The results showed that both emodin and DEX had therapeutic effects on SAP-ALI.RNA-seq showed that m RNA and lnc RNA expression of SAP group changed significantly compared to controls and were further affected by both drug treatments.The correlation of gene expression profiles between emodin and control groups is higher than that between DEX and control groups.Comparing control to SAP group,the numbers of upregulated m RNAs and lnc RNAs were more than downregulated ones in emodin group,but reverse for DEX group.Function analysis showed that both the upregulated m RNA genes and co-expressed genes with upregulated lcn RNAs were enriched in pathways including inflammatory and immune response,response to LPS,reduction of oxidative stress.our study showed that emodin associated lnc RNA-m RNA co-expression network were different from DEX,which indicated difference in potential therapeutically mechanisms for SAP-ALI.EMO may suppress the immune response and regulate cell differentiation,promote the repair and inhibit apoptosis in the lung tissue of SAP-ALI rats model through Lnc RNA-Nrp1 and Lnc RNA-Tbx2-Cdkn-1a pathway,respectively,but not DEX.Conclusion:1)EMO can inhibit the level of TNF-α,IL-6,protect ultrastructure of alveolar type II epithelial cells,improve the respiratory function of SAP and inhibit the infiltration of PMN in the SAP rat model.2)EMO regulated m RNA genes and co-expressed genes with upregulated lcn RNAs were enriched in pathways including inflammatory and immune response,response to LPS,reduction of oxidative stress.3)emodin associated lnc RNA-m RNA co-expression network were different from DEX,which indicated difference in potential therapeutically mechanisms for SAP-ALI.EMO may suppress the immune response and regulate cell differentiation,promote the repair and inhibit apoptosis in the lung tissue of SAP-ALI rats model through Lnc RNA-Nrp1 and Lnc RNA-Tbx2-Cdkn-1a pathway,respectively,but not DEX.Part Two: proteome analysis of the mechanism of emodin for the treatment of acute pancreatitis-associated acute lung injuryObjective: To investigate the molecular mechanism of EMO in the treatment of acute pancreatitis associated lung injury at the protein level.Method:SAP model was induced by administering fresh 5.0% sodium taurocholate into the biliopancreatic duct by standard retrograde infusion.Sixty male SD rats were randomly divided into 5 following groups（n = 12/group）: control group,SAP model group（SAP）,sham-operated group with pancreas marginally rotated（Sham）,positive drug group with DEX treatment（SAP-DEX）,and administration group with EMO treatment（SAP-EMO）.Meanwhile,each group was further divided into 6 h,24 h subgroups（n = 6 / subgroup）.LC-MS/MS based quantitative proteomic analyses were applied to systematically elucidate the differential protein expression levels in SAP-ALI lung tissues with or without treatment.Systematical analysis of protiens changes were performed to reveal the mechanism of EMO.Moreover,EMO-GO function-proteins network was established.The expression level of Lamc2,Serpin-b1,Serpin-a1 were verified bu western blot.Result:14973 unique peptides and 2219 distinct proteins were successfully quantified,in which 1130 proteins were quantified at least in two rats of each group and were used for further analysis.Compared with control groups,22 and 15 proteins were up-regulated,and 20 and 78 proteins were down-regulated in the lung tissues of SAP₆h and SAP₂4h groups,respectively.3 and 7 up-regulated as well as 6 and 22 down-regulated proteins in SAP-ALI rats were greatly recovered at 6 h and 24 h EMO treatment,respectively.Besides,8 and 10 up-regulated as well as 8 and 32 down-regulated proteins in SAP-ALI rats were greatly recovered at 6 h and 24 h DEX treatment,respectively.Among these recovered proteins,2 and 6 up-regulated as well as 4 and 14 down-regulated proteins were recovered by both EMO and DEX at 6 h and 24 h.Among the dysregulated proteins,Sftpa,Spy（down-regulated at 6h）,Ckm（down-regulated at 6h and 24h）,Serpin-b1（up-regulated at 6h and 24h）and AQP5,Sftpb,Sec14l3（down-regulated at 24h）indicated lung injury were induced by SAP.Partial of these markers,AQP5 and Sftpb were significantly recovered by EMO treatment,proven its therapeutic effect in APALI again.Subsequently,GO analysis suggested that EMO and DEX can both involve in regulation of endopeptidase and peptidase activity as well as negative degradation of collagen-containing extracellular matrix and laminin complex.Lamc2 and Serpin-b1 were up-regulated as well as Serpin-a1 was down-regulated in lung tissues of APALI.Importantly,EMO can recover the expression level of Lamc2,Serpin-b1 and Serpin-a1.PPI network suggested that EMO may provide protective effect by negatively regulated NPs activity via up-regulated Serpin-a1,and block the altered loop of neutrophils/NPs/Lamc2 in APALI.Conclusion:1)High expression levels of serpin-b1 in the lung tissues of SAP-ALI for the first time and maybe correlated with high neutrophils and monocytes recruited to the lungs,which suggested it might be a novel biomarker of severity degree.2)neutrophils/ NPs,Lamc2,may form a loop in pathogenesis of APALI.3)EMO may provide protective effect by negatively regulated NPs activity via up-regulated Serpin-a1,and block the altered loop of neutrophils/NPs/Lamc2 in APALI.