Establishment A Nomogram Based On Pathological Scores To Diagnosis Of Liver Pathological Damage In Hepatitis B Patients With ALT≤2ULN And Preliminary Exploration Of Its Pathogenesis

BackgroundHepatitis B virus(HBV)infection is a major public health problem worldwide[1].In 2015,the United Nations adopted the 2030 agenda for sustainable development,in which viral hepatitis has become one of the goals of the United Nations’ global health work[2].Subsequently,the World Health Organization(WHO)formulated a global hepatitis strategy to completely eliminate viral hepatitis by 2030,reduce the incidence of new chronic hepatitis B and hepatitis C by 90%,reduce the mortality of hepatitis B and hepatitis C to 65%,and treat 80%of patients with chronic hepatitis B and hepatitis C[3].The 194 member countries of the World Health Organization have committed themselves to eliminating viral hepatitis as a public health threat by 2030,with special attention to hepatitis B and hepatitis C virus infection[4].This "2030 goal"may have become the "No.1 topic" in the field of liver disease prevention and treatment in the world.According to the current situation of our country,our country’s hepatology experts updated the guideline of chronic hepatitis B,which pointed out that HBV DNA is positive,ALT>ULN,other causes are excluded,and antiviral therapy is given.Compared with our 2015 CHB Guideline[6],the broadest antiviral standard ever proposed is due to the research progress on the immune pathogenesis of chronic hepatitis B.It is found that the HBV infection stage of ALT<ULN and ALT 1-2ULN no longer represents the stable stage of the disease,and the risk of HBV DNA activation and the risk of disease progress and HCC development cannot be ignored[7,8].A Dutch[9]study of 2991 patients found significant liver fibrosis in 7.2%of HBV-infected patients with ALT<ULN,and in HBV-infected patients with ALT≤12ULN,significant hepatic fibrosis accounted for 25%.In addition,a German retrospective study showed that in patients with untreated ALT<ULN or 1-2ULN,the rates of fibrosis and cirrhosis were 35.9%and 17.9%,respectively[10].These data suggest that patients with chronic HBV infection with ALT≤2ULN may also develop significant liver fibrosis or cirrhosis and HCC.While antiviral therapy for ALT≤2ULN in chronic HBV-infected individuals,the guidelines state that assessment of liver histopathology damage should not be ignored.In order to correctly identify the degree of liver damage in patients infected with ALT≤2ULN,to select the appropriate population,to carry out early anti-virus intervention therapy,and to use medical resources reasonably as far as possible,to avoid the risk of non-treatment and the waste of medical resources due to over-treatment,we conducted the first part of the study,aiming to establish a non-invasive diagnostic model,to predict the pathological damage of liver in patients with chronic hepatitis B virus(HBV)infection by ALT≤2ULN,and to provide a scientific diagnostic evaluation tool for realizing the "2030 target" as soon as possible.In recent years,our research group has been devoted to the study of the immunopathogenesis of HBV infection.The preliminary results of Wang W[11],Chen W[12]and Xie X[13]showed that the immunopathogenesis of HBV infection is not related to the immune function,HBeAg with strong immunogenicity plays a key role in the activation of macrophages.So is the immunological mechanism of liver pathological injury in patients with ALT≤2ULN related to HBeAg and macrophages?To this end,we carry out the second and third part of the study.In order to achieve the following objectives through the subject of a number of experiments.Objectives:1.From the aspect of clinical research,the nomogram was constructed according to the pathological score as the gold standard.The Nomogram was used to diagnosethe probability of significant pathological changes of liver injury(SHCHI)in HBV infected individuals with ALT≤2ULN,guiding the evaluation of clinical antiviral initiation and treatment effectiveness.The SHCHI was defined as a Scheuer score of≥ G2 and/or≥S2.2.From the perspective of basic research,to explore the possible immunopathogenesis of liver histopathological damage in HBV infected individuals with ALT≤2ULN.Methods:1.The study included 384 patients with chronic HBV infection who underwent liver puncture with ALT≤2ULN and were hospitalized in a hospital from January 2017 to December 2021.They were randomly assigned to a training cohort(n=270)and a validation cohort(n=114),with a ratio of 7:3.Single factor and multivariate logistic regression analysis were used to screen the risk factors for SHCHI,and the rms software package R4.1.0(http://www.r-project.org/)was used to draw a Nomogram,and internal validation was conducted.The receiver operating characteristic curve(ROC)was used to compare the diagnostic performance of Nomogram with APRI and FIB-4.2.The study included 42 patients with chronic hepatitis B with ALT≤2ULN who were hospitalized or followed up in an outpatient department of a hospital from January 2019 to December 2021 and underwent liver biopsy and pathological testing.The study population was divided into healthy control group,HBeAg positive group,and HBeAg negative group.The levels of cytokines IL-17A,TNF-α,IL-6,IL-1β,and IL-10 in each group were detected by enzyme linked immunosorbent assay(ELISA).Flow cytometry was used to detect the expression of CD 14+macrophages,CD14+CD86+M1 macrophages,and CD14+CD163+M2 macrophages in each group.The study population was further divided into healthy control group,light-moderate CHB group,severe CHB group,and liver cirrhosis group according to the severity of the disease.Liver pathological specimens were evaluated for the degree of inflammation and fibrosis of the liver pathological tissue using HE staining and reticular fiber argyrophilic staining,and the ratio of CD4+IL-17A+Th-17 cells in each group was detected by flow cytometry.Pairwise comparisons of three or more groups of variables were conducted using one-way ANOVA.The correlation between the two variables was studied using Spearman correlation analysis.Results:1.The probability of SHCHI among patients with chronic HBV infection with ALT≤2ULN was 74.44%in the modeling group and 76.32%in the validation group.Logistic regression analysis showed that HBeAg,AST,GGT,and PT were independent risk factors for predicting SHCHI(P<0.05).The nomogram established using the above four indicators can diagnose the probability of significant liver pathological damage with chronic HBV with ALT≤2ULN.2.Nomogram had good discrimination and calibration,with AUROC of 0.80 and 0.75 for the model group and validation group,and Brier scaled scores of 0.147 and 0.150,respectively.3.The AUROC for diagnosis of SHCHI by Nomogram was 0.80(95%CI,0.74-0.86),which was higher than the Aspartate Transaminase Platelet Ratio Index(APRI)score model 0.78(95%CI,0.71-0.84),and the Fibrosis 4(FIB-4)score model 0.76(95%CI,0.69-0.82).The cutoff value of the Nomogram score is 119,and patients with a score below 119 have a lower risk of SHCHI.4.The number of CD14+macrophages in the peripheral blood of HBV infected patients with ALT≤2ULN significantly increased,especially in the HBeAg positive group.After being stimulated by HBeAg,CD 14+macrophages mainly differentiated into M1 type.5.M1 macrophages could promote the differentiation of CD4+T cells into Th-17 cells,and the IL-17A secreted by Th-17 cells was positively correlated with the degree of liver inflammation and fibrosis,as well as the severity of liver diseases.Conclusion:1.The nomogram constructed with four indicators,HBeAg,AST,GGT,and PT,has good efficacy in diagnosing the risk of SHCHI in patients with chronic hepatitis B virus infection with ALT≤2ULN.2.HBV infected individuals with ALT≤2ULN have a higher incidence of SHCHI.Patients with a score of 119 or more are high-risk groups with SHCHI(positive predictive value is 87.84%).This group of people should be closely monitored,dynamically observing the pathological damage of liver tissue based on the nomograph score,and initiating antiviral treatment in a timely manner.3.HBeAg positive or negative patients with ALT≤2ULN have different pathogenesis.HBeAg-M1 macrophage-Th-17-IL-17A Axis may be the mechanism of liver immune injury in HBeAg-positive patients.Control of Th-17 cell differentiation and anti-IL17A treatment may provide a new theoretical basis for the treatment of Hepatitis B virus infection.