The Impact Of Non-alcoholic Fatty Liver Disease On The Liver Inflammation Activity,clinical Efficacy Of Antiviral Agents And Fibrosis Progression In Patients With Chronic Hepatitis B

Although population of chronic hepatitis B virus（HBV）infection（CHB）has decreased significantly with the popularization of vaccine and prevention of mother-to-child transmission.CHB is still a major global public health challenge,especially in low/middle income countries,with an estimated 292million cases worldwide.It is the main cause of liver cirrhosis and hepatocellular carcinoma（HCC）,causing 0.5-1.2 million deaths per year with nearly 60%were HCC.As a result,investigations into influential factors of liver fibrosis and HCC progression for timely interventions are vital.Non-alcoholic fatty liver disease（NAFLD）is another major cause of chronic liver disease worldwide due to increasing incidences of obesity and metabolic syndrome（Met S）,with a global prevalence of 25.24%.As a difficult and hot issues in the field of liver disease,concurrent of CHB and NAFLD is common with a prevalence of 25%-30%.To improve their outcomes,it is imperative to clarify the influence of NAFLD on the liver inflammation activity,therapeutic response and liver fibrosis progression in CHB,especially among biopsy-proven patients.At present,long-term antiviral therapy is a most efficient intervention to decelerate the CHB progression,though only a small proportion of patients can achieve function cure with it.The necessity of initiating antiviral therapy depends on the histological grade of liver inflammation and stage of fibrosis in patients with detectable HBV DNA in those without other indications.Liver biopsy is gold standard for grading the liver inflammation,but is limited in practice as being an invasive operation.Serum alanine aminotransferase（ALT）levels remain being a most common laboratorial indicator of liver inflammation.Whereas,many studies pointed out the inconsistency between them.CHB patients with detectable HBV DNA and normal ALT levels may also develop moderate or serious liver inflammation.NAFLD related fat accumulation and oxidative stress may increase the risk of developing liver inflammation in CHB,with higher risk of liver fibrosis progression and HCC pathogenesis.Nonetheless,the influence of NAFLD on the incidence of significant inflammation in treatment na?ve CHB patients with detective HBV DNA and normal ALT levels has not been investigated and deserves detailed assessment.Therapeutic response is vital and requires being monitored along with the management of CHB.NAFLD can regulate the host immunity to influence the HBV replication.Some research has indicated the negative relationship between liver steatosis degrees and HBV DNA levels.Whereas,no such relationship was found in other researches which based on liver biopsy.Besides,there is controversy about the influence of NAFLD on the incidence rates of complete virological response（CVR）and liver enzymes normalization in CHB patients after the treatment of nucleoside analogue（NA）.Low-level viremia（LLV）and HBV pre-genomic RNA（HBV pg RNA）are novel important indicators of virological response,and have been used in assessing low HBV replication in patients with detectable HBV DNA or the underlying HBV activity in patients with undetectable HBV DNA.They are significantly associated with the development of HCC.Liver cirrhosis and HCC may still occur even after the antiviral treatment.Studies have proposed that concurrent NAFLD was associated with stage of liver fibrosis and incidence of adverse outcomes.However,there is no study for investigating the dynamic changes of liver fibrosis in multiple time-points in patients with CHB and NAFLD.Such investigations can provide meaningful recommendation for timely monitoring or intervention along with the management of CHB.Our study was designed to clarify above issues in three parts.As begin,we enrolled biopsy-proven patients with CHB alone or CHB with NAFLD.They had normal ALT levels,detectable baseline HBV DNA and were not being on-treatment.We analysis the incidences of significant liver inflammation in the overall cohort and different subgroups classified by the age,status of hepatitis B e antigen（HBe Ag）and HBV DNA levels,aiming to identify associated risk factors of significant liver inflammation.Secondly,histologically-proven patients with initial first antiviral agents were selected to analyze the differences of virological,serological and biochemical response,LLV incidences and HBV pg RNA levels at multiple time-points between the two groups.Multivariable regression analysis was also conducted to identify associated factors of CVR.At last,on-treatment patients were followed-up to find important time-point of liver fibrosis regression or progression by observing the long-term changes of liver fibrosis,which was measured with non-invasive serological models（FIB-4,APRI and GPRI）.Part One The impact of non-alcoholic fatty liver disease on the incidence of significant liver inflammation in treatment naive patients with chronic hepatitis B and normal alanine aminotransferase levelsObjective:To clarify the impact of non-alcoholic fatty liver disease（NAFLD）on the incidence of significant liver inflammation in treatment naive patients with chronic hepatitis B（CHB）and normal alanine aminotransferase（ALT）levelsMethods:Liver biopsy-proven CHB with or without concurrent NAFLD between May 2007 and October 2021 from Third Hospital of Hebei Medical University.Their demographic,anthropometric,laboratory tests and histopathological data were collected.Significant liver inflammation was defined as Grade≥2 evaluated by Scheuer scoring system.The incidences of significant inflammation in overall and different subgroups were analyzed and compared between patients with and without concurrent NAFLD by chi-square test,and its associated factors were identified by Logistic regression analysis（stepwise forward:LR）.Results:We enrolled 660 patients,445 with CHB alone and 215 with concurrent NAFLD.1.Basic characteristic of the study populationAs a whole,their ages were 38.0（31.0,47.0）years,male accounted for a percentage of 59.4%,and the incidence of significant liver inflammation was52.1%.Along with above three variables,body mass index（BMI）,levels of ALT and GGT,and incidence of metabolic syndrome（Met S）were significantly higher in patients with concurrent NAFLD when compared with patients with CHB alone（significant inflammation:58.6%vs 49.0%,P=0.024）.2.The incidence of significant liver inflammation in different subgroupsIncidences of significant liver inflammation in patients with age<50 and≥50years were 49.8%and 61.0%,respectively.And,concurrent NAFLD significantly increased the incidence in patients with age≥50 years（from53.6%to 73.1%,P=0.023）.Besides,the incidence of significant inflammation increased with HBV DNA levels in HBe Ag negative patients and was 73.1%in high viral load（VL）(HBV DNA≥5.0 log₁₀IU/m L),and were 81.0%when superimposed NAFLD.The incidence was 72.2%in patients with significant liver fibrosis.What’s more,compared with patients with CHB alone,the incidences in patients with concurrent NAFLD were significantly higher in the subgroups of HBe Ag negative（60.7%vs 48.1%,P=0.025）,low VL(HBV DNA<3.3 log₁₀IU/m L)（55.7%vs 40.3%,P=0.033）,and HBe Ag negative with low VL（55.6%vs 39.6%,P=0.035）.3.The differences between patients with significant liver inflammation and no/mild inflammationCompared with patients with no/mild liver inflammation,the age,platelet counts,levels of ALT,AST and GGT,and scores of fibrosis associated serological models were all higher in patients with significant inflammation（P<0.05）.And,the percentage of male was lower in them（54.9%vs 64.2%,P=0.015）.In consistent with the differences of serological models between the two groups,the incidences of significant and advanced liver fibrosis were also higher in patients with significant inflammation（77.0%vs 32.3%and 27.9%vs 6.0%,both P<0.001）.4.Regression analysis to identify significant liver inflammation related factors in the overall populationMultivariable analysis revealed that female（adjusted OR 1.889,P=0.001）,platelet counts（a OR 0.994,P=0.001）,AST（a OR 1.040,P=0.001）,GGT（a OR1.015,P=0.010）and concurrent NAFLD（a OR 1.537,P=0.042）were independent associated factors of liver inflammation.5.Regression analysis to identify significant liver inflammation related factors in different subgroupsThe multivariable analysis showed that NAFLD was independent risk factors of significant liver inflammation in the subgroups of age≥50 years,female,HBe Ag negative,low VL and high VL.Conclusions:Concurrent NAFLD was independent risk factors of significant liver inflammation in CHB patients with normal ALT levels and without being on-treatment.The incidences of significant liver inflammation were high in CHB patients with age≥50 years or with negative HBe Ag and high VL,and were even higher when superimposed with NAFLD.In the subgroups of age≥50 years,female,HBe Ag negative,low VL and high VL,NAFLD was an independent risk factor of significant liver inflammation.Part Two Adverse Effect of Nonalcoholic Fatty Liver Disease on the Therapeutic Response in Patients with Chronic Hepatitis BObjective:The impact of nonalcoholic fatty liver disease（NAFLD）on the treatment outcome of chronic hepatitis B（CHB）is undefined and deserves an in-depth investigation.Methods:Histologically-proven CHB receiving first-line antiviral regimens as initial therapy was enrolled and grouped by the concurrence of NAFLD,and followed-up at 6-monthly intervals.Therapeutic response related data were recorded and compared at multiple time-points.Kaplan-Meier and Cox regression analyses were utilized to estimate the impact of NAFLD on complete virological response（CVR）.Results:We enrolled 267 patients（CHB:164;CHB with NAFLD:103）with comparable follow-up durations.They were also comparable in baseline HBV DNA levels and HBe Ag positivity.Patients with concomitant NAFLD showed less significant decline in HBV DNA,q HBs Ag,pg RNA,and liver enzyme levels over time;moreover,their accumulative incidences of CVR were significantly lower and that of low-level viremia（LLV）were significantly higher at 6,12,18,24 months.First CVR was delayed with the presence NAFLD（11.0 vs 7.0 months,P<0.001）and further prolonged with higher grade of liver steatosis（Grade 2-3 vs 1:13.0 vs 9.0 months）in CHB patients.On multivariate analysis,HBe Ag positivity（HR:0.650,P=0.036）,grade of steatosis（G2:HR 0.447,P=0.004;G3:HR 0.085,P=0.002）and HBV DNA(log₁₀IU/m L)（HR:0.687,P<0.001）were significantly associated with delayed CVR,whereas grade of necroinflammation（HR:1.758,P<0.001）accelerated the CVR.Conclusions:In CHB patients receiving initial antiviral therapy,NAFLD was associated with higher levels of HBV DNA,pg RNA,and liver enzymes,and higher incidence of LLV and delayed CVR.Part Three Characteristics of the dynamic changes of liver fibrosis in patients with chronic hepatitis B and nonalcoholic fatty liver diseaseObjective:Clarify the characteristics of the long-term dynamic changes of liver fibrosis in patients with chronic hepatitis B（CHB）and nonalcoholic fatty liver disease（NAFLD）through evaluating the non-invasive serological models.Methods:Histologically-proven CHB was enrolled and grouped by the concurrence of NAFLD.Data during follow-up were collected and used to calculate scores of APRI、GPRI and FIB-4.Their dynamic changes were observed and compared between the two groups.Results:We enrolled 690 patients（CHB alone:486;CHB with NAFLD:204）.Their durations of follow-up were 43.0 and 35.0 months,respectively.Significant and advanced liver fibrosis accounted for 57.8%and 31.7%in patients with CHB alone at baseline,with 54.4%and 23.5%in CHB patients with concurrent NAFLD.When compared to patients with CHB alone,the baseline FIB-4（1.13 vs 0.93,P=0.002）and APRI（0.61 vs 0.46,P=0.001）values were significantly higher in patients with concurrent NAFLD with comparable GPRI values（0.15 vs 0.17,P=0.349）at baseline.Their values decreased in the first 12 months in both groups.But,their values in patients with NAFLD decreased less significantly,reaching comparable or even higher levels between 6 and 12 months（FIB-4 at 6 months:0.89 vs 1.08,P=0.195;APRI at 6 months:0.37 vs 0.39,P=0.470;GPRI at 12 months:0.16 vs 0.12,P<0.001）.The values in patients with CHB alone further decreased with prolonged therapy in the 36 months,and did not decrease,even increased after that time-point,especially after 64 months（FIB-4:0.92 at 60 months and 0.99at 84 months）.In contrast,values in patients with CHB and NAFLD did not decrease after 12 months,even increased after 36 months（FIB-4:0.99 at 36months and 1.18 at 84 months）.Conclusions:Liver regression occurred in the first 12 months in both groups.Liver fibrosis in CHB patients with NAFLD may progress after 12 months.In patients with CHB alone,fibrosis did not further regress after 36 months,and even progressed after 60 months.NAFLD can significantly promote the liver fibrosis progression in CHB patients.