| Background Acute-on-chronic liver failure(ACLF)is a complex clinical syndrome characterized by jaundice,coagulation dysfunction,hepatorenal syndrome,hepatic encephalopathy and ascites,of which short-term mortality can reach 50%-90%.In China,hepatitis B virus(HBV)is the most common cause of ACLF.Early evaluation of the disease and judgement for prognosis is necessary to guide the clinical work because of acute onset,rapid progress and limited treatment of HBV-ACLF.In patients with HBV-ACLF,here is a severe immune disorder that inflammatory reaction and compensatory antiinflammatory reaction coexist.Therefore,immune and inflammation play important roles in the occurrence and development of the disease.Artificial liver support system(ALSS)is an effective treatment to improve clinical symptoms and reduce mortality.Exploring the characteristics of transcriptomics changes of peripheral blood immune cells can help us to study the characteristic immune changes of BV-ACLF and Li’ artificial liver system(Li-ALS)treatment,in order to screen the prognostic markers,which is of great significance to the prognosis evaluation of the disease.Aim1.To describe the clinical characteristics and outcome of patients with HBV-ACLF,find out the independent risk factors of short-term adverse prognosis,and evaluate the predictive roles of several prognostic models.2.To explore the prognosis related miRNA-mRNA regulatory network in peripheral blood immune cells of patients with HBV-ACLF,and screen for the key pathways and biomarkers that affect the severity of the disease and poor prognosis.3.To explore the characteristics of gene expression profile of peripheral blood immune cells in patients with HBV-ACLF after Li-ALS treatment,and screen for the key genes related to treatment.Methods1.We retrospectively collected the clinical data and prognosis information of patients with HBV-ACLF who met the CLSSH criteria in our hospital from January 2017 to November 2020.Then we analyzed the disease characteristics and clinical outcomes.The risk factors that may lead to poor prognosis of patients with HBV-ACLF were explored through COX regression analysis.The receiver operating curve(ROC)was used to evaluate the predictive effect of each prognostic score model in short-term prognosis of these patient.2.We explored the changes of prognosis related miRNA and mRNA expression profiles in peripheral blood immune cells of patients with HBV-ACLF in the discovery cohort through transcriptomics and constructed the miRNA-mRNA co regulatory network by a series of bioinformatics analysis.Quantitative real-time polymerase chain reaction(q RT-PCR)and ROC curve analysis were performed in the validation cohort to screen for prognosis related biomarkers.Then we determined the targeting effect of related miRNA and mRNA using luciferase reporter assay.In addition,the levels of plasma cytokines were measured by enzyme-linked immunosorbent assay to judge the differences in different prognosis groups.3.We explored the gene expression profile changes of peripheral blood immune cells in patients with HBV-ACLF(different prognosis)after Li-ALS treatment through transcriptomics.Subsequently we analyzed the enriched biological functions and signaling pathways of differential expressed genes(DEGs).Protein protein interaction(PPI)network was constructed by STRING and Cytoscape and then screen for key genes according to the dergee value.Results1.In this study,323 patients with HBV-ACLF were analyzed for clinical characteristics and outcomes.The results showed that age,liver cirrhosis,international normalized ratio(INR)were independent risk factors for 28-day poor prognosis while age,liver cirrhosis,creatinine and INR were independent risk factors for 90-day poor prognosis.The au ROC(95% CI)of COSSH-ACLFs,chronic liver failure consortium acute on chronic liver failure score(CLIF-C ACLFs),chronic liver failure consortium organ failure assessment score(CLIF-C OFs)and the model for end stage liver disease(MELD)score in predicting 28 day and 90 day mortality were 0.797(0.746-0.842),0.762(0.708-0.809),0.794(0.742-0.839),0.735(0.680-0.785)and 0.782(0.730-0.829),0.748(0.694-0.797),0.727(0.671-0.777),0.706(0.650-0.758),respectively.2.Eight patients in 28-day survival group and eight patients in 28-day death group were included in the discovery group.And then 39 differentially expressed miRNAs and1032 differentially expressed mRNA were screened out by miRNA and mRNA expression profiles.We construct a prognosis related co regulatory network composed of 38 miRNAs and 313 mRNAs,which was mainly enriched immune and inflammation related signaling pathways like cytokine-cytokine receptor interaction,Th17 cell differentiation.The results of q RT-PCR and ROC curve analysis showed that mi R-6861-3p(au ROC=0.729)and mi R-6840-3p(au ROC=0.680)were significantly different among different prognosis groups,which could be used as short-term prognostic markers.There was a direct targeting effect between mi R-6840-3p and JADE2.At the same time,the levels of IL-6,IL-8 and PDGF-BB in the28-day death group were significantly higher than those in the survival group,and the distribution of IL-6 level in different 90-day prognosis groups was significantly different.3.This study included 10 patients with HBV-ACLF(5 patients with 28-day survival and 5 patients with 28-day death)who were treated with Li-ALS for the first time in our hospital.And 601 differential expressed genes were screened out by gene expression microarray,including 443 up-regulated genes and 158 down-regulated genes.Bioinformatics analysis showed that the differential expressed genes were mainly enriched in inflammatory,infection related signaling pathways and platelet activity.The PPI network was constructed using common genes from three different gene sets.We screen out 10 key genes related to Li-ALS treatment including 8 upregulated genes(PF4,SELP,ITGA2 B,EGF,PPBP,GNG11,P2RY12 and ADORA3)and 2 down regulated genes(CXCL10 and CX3CR1).Interestingly,half of these genes were involved in the cytokine-cytokine receptor interaction pathway.Conclusion In patients with HBV-ACLF meeting COSSH criteria,age,liver cirrhosis and INR were independent risk factors for 28-day and 90-day poor prognosis.COSSH-ACLFs was a superior short-term prognostic model.mir-6840-3p-JADE2 may promote the progression of ACLF and lead to poor prognosis.Meanwhile,mi R-6840-3p and mi R-6861-3p could be used as markers of short-term prognosis.Finally,Li-ALS treatment was not only the blood material exchange of patients,but also changed part of the immune state of patients,in which cytokine-cytokine receptor interaction may play an important role that influence the therapeutic effect. |
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