Effect Of Autophagy And Macrophage Polarization Mediated By STING Pathway Activation On The Pathogenesis Of HBV-Associated Chronic Acute Liver Failure And The Establishment Of Clinical Prognostic Model

Background Hepatitis B virus-related acute-on-chronic liver failure(HBV-ACLF)is a severe acute liver injury that occurs on the basis of chronic hepatitis B(with or without cirrhosis),accompanied by a complex series of intrahepatic and extrahepatic syndromes,with a high short-term mortality rate.The specific pathogenesis of this condition is not fully understood.Stimulator of interferon gene(STING)is an essential protein anchored in the endoplasmic reticulum,regulating inflammation and immune responses in various diseases.Previous studies have indicated its role in liver injury,but there is limited research on the impact of STING on the progression and related mechanisms of HBV-ACLF.Although various experimental models of acuteon-chronic liver failure have been developed for mechanistic studies,there is a lack of research on models specifically based on chronic hepatitis B as the foundation.Many clinical research teams have developed multiple models for assessing the prognosis of HBV-ACLF patients,which have high accuracy but are often complex and not readily applicable in clinical settings.Therefore,developing a simple and practical clinical predictive model remains important.Objectives1.To establish an experimental mouse model of HBV-related acute-on-chronic liver failure based on chronic hepatitis B,and to study the effect and mechanism of STINGrelated pathways on the disease process.2.To develop a simple and easy-to-use short-term prognostic model for HBV-ACLF based on research results and retrospective population cohorts.MethodsPart Ⅰ: Establishment of an Experimental Model of HBV-Related Acute-onChronic Liver Failure and Study on the Role of STING Molecules1.Establishment of the HBV-ACLF mice model: Wild-type C57BL/6 mice were used,and a chronic HBV replication mouse model was established by hydrodynamic injection of the p AAV/HBV1.2 plasmid.Acute liver injury was induced using acetaminophen(APAP)on the foundation of the chronic HBV replication mouse model.2.Model mice were pretreated with STING agonist(di ABZI,1.5mg/kg)or STING inhibitor(C-176,20mg/Kg).Liver injury was analyzed by tissue staining and liver biochemical parameters;macrophage polarization was analyzed by flow cytometry;STING pathway activation,autophagy and inflammation-related molecular expressions were analyzed by western blotting or RT-PCR to study the role of STING and its related mechanisms.Part Ⅱ: Development of a Prognostic Model for HBV-ACLF Patients Based on PrealbuminA cross-sectional cohort(n = 291)and a retrospective cohort(n = 185)were included in the study.Laboratory and clinical data were collected,and univariate and multivariate logistic regression analyses were used to determine independent predictors of 30-day mortality.Based on these findings,a new prognostic score(HIAPP)for HBV-ACLF was developed and its performance was evaluated using receiver operating characteristic(ROC)curves.ResultsPart Ⅰ: Establishment of an Experimental Model of HBV-Related Acute-onChronic Liver Failure and Study on the Role of STING Molecules1.A mouse model of HBV-ACLF was successfully established based on chronic hepatitis B.This model showed liver inflammation,lymphocyte infiltration,and the persistence of HBV viremia for at least 8 weeks,which were similar to the clinical characteristics of chronic hepatitis B(CHB)and could be used to mimic chronic HBV infection in human.Acute liver failure was induced using APAP on this CHB model,creating a suitable mouse model for HBV-ACLF.2.6 hours later,the HBV-ACLF group showed upregulated expression of STING pathway molecules p-TBK1 and p-IRF3,thus,STING pathway was activated;increased autophagy marker LC3 B levels,decreased P62 levels,and enhanced autophagic flux compared to the chronic HBV control group.24 hours later,CD11b+F4/80+macrophages in the HBV-ACLF group exhibited significantly increased CD86 expression and decreased CD206 expression,indicating M1 polarization of macrophage,which contributed to disease progression.3.At 6 h after STING agonist treatment to HBV-ACLF mice,p-TBK1 and p-IRF3 protein expression in non-parenchymal cells(NPCs)were increased and STING pathway was activated.Additionally,LC3 B expression was increased;P62 expression was decreased and autophagic flux was enhanced both in NPCs and in parenchymal cells.Compared to untreated control group,H&E staining showed that intrahepatic inflammatory infiltration and liver injury was significantly reduced in STING agonisttreated group;and the levels of liver injury markers in serum were also significantly decreased.In contrast,pretreatment with a covalent STING inhibitor reduced autophagy in NPCs,increased P62 expression,and reduced autophagic flux.H&E and TUNEL staining of liver tissues indicated increased inflammatory infiltration and damage in the STING inhibitor-treated group compared to the untreated control group.4.At 24 h of STING agonist treatment to HBV-ACLF mice,CD86 expression on CD11b+ F4/80+ macrophages in the STING agonist-treated group were significantly increased,indicating M1 polarization of macrophage,which was associated with increased liver inflammatory infiltration and injury.At 48 h of STING agonist treatment to HBV-ACLF mice,the STING agonist-treated group showed worsened intrahepatic inflammatory infiltration and injury compared to the untreated control group,indicating that STING activation exacerbated liver injury.In contrast,in the STING covalent inhibitor-treated group,M2 polarization of macrophage was observed at 24 hours,and liver injury was significantly reduced.Part Ⅱ: Development of a Prognostic Model for HBV-ACLF Patients Based on Prealbumin1.Prealbumin(PAB)levels at admission were a strong independent predictor of 30-day mortality in HBV-ACLF patients,with an AUROC of 0.760.2.Based on five independent prognostic variables,including PAB,platelet count(PLT),international normalized ratio(INR),occurrence of hepatic encephalopathy(HE),and age,a prognostic model called HIAPP score was constructed.The HIAPP score in the survival group was significantly lower than in the death group.Furthermore,the HIAPP score was positively correlated with the Model for End-Stage Liver Disease(MELD)score.3.In predicting 30-day short-term mortality in HBV-ACLF,the HIAPP score had an AUROC value of 0.899,which outperformed the MELD score(AUROC = 0.795),CLIF-C ACLF score(AUC = 0.781),and COSSH-ACLF II score(AUC = 0.825).The score was validated in a separate cohort.Conclusion1.The study revealed that STING-induced autophagy plays a protective role in early liver injury,while persistent STING activation may exacerbate liver injury in the later stages by inducing M1 polarization of hepatic macrophages.2.In HBV-ACLF patients,prealbumin levels at admission were identified as a simple and effective predictor of 30-day mortality,and the HIAPP score was an easy-to-use and practical prognostic score.