Liver fibrosis is closely related to metabolic factors in metabolic associated fatty liver disease with hepatitis B virus infection

This case–control study aimed to identify the clinical characteristics and explore the risk factors for liver fibrosis in metabolic associated fatty liver disease (MAFLD) patients with hepatitis B virus (HBV) infection. The patients were grouped into MAFLD + HBV and MAFLD (without HBV infection). Propensity score matching (PSM) was used to match baseline features between the groups. We included 401 patients with biopsy-proven MAFLD, 179 of whom had HBV infection. A total of 83 pairs were successfully matched via PSM, and steatosis scores and ballooning in the MAFLD + HBV group were lower than those in the MAFLD group, while the inflammation scores and liver fibrosis stages were higher. After adjusted for confounding factors, HBV infection was associated with a higher risk of significant liver fibrosis in patients with MAFLD [odds ratio (OR): 3.140, P = 0.003]. Overall, 43.58% (78/179) of patients in the MAFLD + HBV group had significant liver fibrosis. Further multivariate regression analysis, hypertension (OR: 2.640; P = 0.031), type 2 diabetes (OR: 4.939; P = 0.035), and elevated glutamyl-transferase levels (OR: 3.980; P = 0.001) were risk factors for liver fibrosis in the MAFLD + HBV group. This suggests metabolic rather than viral factors are more closely associated with liver fibrosis in MAFLD patients with HBV infection.


Methods
Study subjects and design. This cross-sectional study included all patients with liver stiffness measurements greater than 7 kPa as determined by Fibroscan prior to liver biopsy between 2011 and 2021 at the Affiliated Hospital of Hangzhou Normal University (Hangzhou, China). Individuals with the following conditions were excluded ( Fig. 1): (1) evidence of chronic liver diseases such as chronic viral hepatitis C, alcoholic liver disease, autoimmune liver disease; (2) history of malignancy or missing data concerning weight, heigh, or etiological markers, including hepatitis B surface antigen (HBsAg), hepatitis B e-Antigen (HBeAg), and hepatitis B virus deoxyribonucleic acid (HBV DNA); (3) use of hypoglycemic or antilipidemic drugs exerting potential effects on liver fibrosis; (4) patients with hepatitis B receiving antiviral therapy. This study conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the Affiliated Hospital of Hangzhou Normal University (Approval Number/ID: 2020 (02)-KS-022). As this was an observational retrospective study, the requirement for informed consent was waived by the Ethics Committee.
Clinical examination, biochemical analyses, and biopsy assessment. Diastolic blood pressure (DBP), systolic blood pressure (SBP), height, and body weight were measured by professional physicians in accordance with standard protocols. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared (kg/m 2 ). Blood samples were obtained after 8 h of fasting, and routine blood and biochemical tests were conducted to assess the following: fasting plasma glucose (FPG), triglycerides (TG), total cholesterol (TC), high/ low-density lipoprotein cholesterol (HDL-c/LDL-c), alanine aminotransferase (ALT), gamma-glutamyl-transferase (GGT), aspartate aminotransferase (AST), albumin (ALB), glycated hemoglobin (HbA1c), and serum uric acid (SUA). The biochemical tests were performed using an automated biochemical analyzer in accordance with the manufacturer's instructions (Model 7180; Hitachi, Tokyo, Japan). Serological markers of HBV infection, including HBsAg, HBcAb, HBeAg, and hepatitis B e-Antibody (anti-HBe), were obtained using commercially available enzyme immunoassays. Serum HBV DNA levels were quantified using a commercially available realtime polymerase chain reaction assay in accordance with the manufacturer's instructions, with a linear dynamic detection range of 3 × 10 1 -10 × 10 9 IU/ml.
All liver biopsies were reassessed by three experienced histopathologists blinded to participant details. The steatosis score (positive if > 5%, according to the Brunt classification), stage of fibrosis (based on a meta-analysis of histological data for viral hepatitis score), ballooning, and degree of inflammation were evaluated [15][16][17] . Fibrosis stage ≥ 2, degree of inflammation ≥ 2, and steatosis score ≥ 2 was defined as significant liver fibrosis, active inflammation, and severe steatosis, respectively.  Consent to participate/consent to publish. As this was an observational retrospective study, the requirement for informed consent was waived by the Ethics Committee.

Results
Establishment of the study. As shown in Fig. 1, a total of 681 patients with biopsy-proven steatosis were included in the data screening. A total of 175 individuals with steatosis were excluded since they did not meet the diagnostic criteria for MAFLD, while 105 individuals were further excluded due to incomplete data. Among the 401 patients diagnosed with MAFLD, 256 patients were overweight/obese, 64 patients had T2DM, and 81 patients with normal or lean weight had metabolic disorders. The average age of the included patients was 43.14 ± 11.31 years; 44.64% (179/401) of patients had MAFLD with HBV infection (MAFLD + HBV group), and 55.36% (222/401) of patients had MAFLD without HBV infection (MAFLD group). The proportion of male patients was higher in the MAFLD + HBV group than in the MAFLD group (88.82% vs. 72.52%, P < 0.001). No significant differences in age, SBP, DBP, BMI, FPG, HDL-c, SUA, or ALB levels were observed between the MAFLD and MAFLD + HBV groups (P > 0.05). However, the MAFLD + HBV group exhibited lower levels of liver enzymes, LDL-c, TG, and TC, as well as lower rates of obesity, hypertension, T2DM, low HDL-c, hypertriglyceridemia, hypercholesterolemia, and hyperuricemia than the MAFLD group (P < 0.05) ( Table 1).

Comparison of histological features in MAFLD patients with and without HBV infection. After
PSM, sex, age, BMI, liver enzymes, and metabolic factors were comparable between patients with and without HBV infection. Inflammation scores and liver fibrosis stages were higher in the MAFLD + HBV group than in the MAFLD group, while steatosis and ballooning scores were lower (P < 0.05) (Fig. 2). In the multivariate analysis, model 1 was adjusted for age and sex, while model 2 was adjusted for model 1 plus metabolic parameters, including BMI, T2DM, low HDL-c, hypertriglyceridemia, hypercholesterolemia, high LDL-c, hypertension, and hyperuricemia. The results indicated that HBV infection was associated with lower hepatic steatosis scores (OR: 0.251, 95% CI: 0.117-0.542, P < 0.001) and ballooning scores (OR: 0.119, 95% CI: 0.049-0.294, P < 0.001) yet higher stages of liver fibrosis (OR: 3.140, 95% CI: 1.479-6.663, P = 0.003) in patients with MAFLD (Table 2). However, no significant differences were observed in inflammation between patients with and without HBV infection after adjusting for confounders. www.nature.com/scientificreports/ (P > 0.05). Among patients in MAFLD + HBV group, those with liver fibrosis had higher levels of GGT and FPG, and a higher percentage of T2DM and obese/overweight status than those without (P < 0.05) ( Table 3). For histological comparison, the proportion of patients with active inflammation (inflammation score ≥ 2) was significantly higher among those with liver fibrosis than those without liver fibrosis (85.90% vs. 37.62%, P < 0.001), whereas the proportions of severe steatosis and ballooning degeneration had no significant difference between the two groups (P > 0.05).

Discussion
MAFLD with HBV infection is a distinct subtype of MAFLD in which metabolic and viral factors co-exist. The current results indicate that the presence of HBV is associated with lower steatosis scores and ballooning grades but a higher liver fibrosis stage in patients with MAFLD. Further risk factor analysis for liver fibrosis revealed that T2DM, hypertension, and elevated GGT levels were independent risk factors for liver fibrosis in MAFLD patients with HBV infection. MAFLD patients with HBV infection exhibit unique histopathological characteristics (Fig. 2). Our findings are in accordance with a recent study showing that, although HBV infection is associated with a lower degree of steatosis and ballooning, it independently increases the risk of liver fibrosis in patients with MAFLD 18,19 . In our study, HBV infection resulted in a threefold increase in the risk of significant liver fibrosis in patients with Table 1. Comparison of clinical characteristics between metabolic dysfunction-associated fatty liver disease with and without hepatitis B virus infection before and after propensity score matching. Data are expressed as mean ± standard deviation or median (interquartile range). MAFLD metabolic dysfunction-associated fatty liver disease, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, SUA serum uric acid, FPG fasting plasma glucose, TG triglycerides, TC total cholesterol, LDL-c low-density lipoprotein cholesterol, HDL-c high-density lipoprotein cholesterol, ALT alanine aminotransferase, AST aspartate aminotransferase, GGT gamma-glutamyl-transferase, T2DM type 2 diabetes mellitus. ‡ P-value calculated using the χ 2 test.

Variables
Before propensity score matching After propensity score matching (1:1) www.nature.com/scientificreports/  www.nature.com/scientificreports/ www.nature.com/scientificreports/ MAFLD (Table 2). Therefore, for MAFLD patients with HBV infection, early screening and intervention for risk factors of liver fibrosis are required, as liver fibrosis has been identified as an accelerator for cirrhosis and hepatocellular carcinoma in chronic liver disease 20 . www.nature.com/scientificreports/ Previous studies have reported that metabolic disorders such as T2DM, hypertension, dyslipidemia, and obesity are closely related to NAFLD liver fibrosis [21][22][23][24][25][26] . HBeAg negativity is associated with more advanced liver fibrosis in patients with chronic hepatitis B 27 . ALT and AST have also been identified as excellent predictors of significant liver fibrosis in patients with CHB 28 . The presence of both viral and metabolic factors may accelerate disease progression in MAFLD patients with HBV infection. Our study indicates that T2DM, hypertension, and elevated GGT levels are independent risk factors for significant liver fibrosis in MAFLD patients with HBV infection even after adjusting for confounding factors (Table 4). While HBeAg positivity and HBV-DNA positivity were not associated with liver fibrosis in MAFLD patients with HBV. It suggested that metabolic factors is more associated with liver fibrosis compared with viral factors in MAFLD patients with HBV infection.

MAFLD MAFLD + HBV P-value MAFLD MAFLD + HBV
This study highlights that T2DM, hypertension, and elevated GGT levels are closely associated with liver fibrosis in MAFLD patients with HBV infection. Liver fibrosis is the result of an excessive production of extracellular matrix (ECM) that is not adequately maintained, resulting in net accumulation. In the liver, hepatic stellate cells (HSCs) constitute the main source of ECM-producing fibroblasts in models of toxic and biliary liver disease and NAFLD 29,30 . Insulin resistance (IR) in T2DM is recognized as an integral component of NAFLD pathogenesis that worsens with disease progression 29,31,32 and the activation of HSC by IR is largely divided into distinct direct and indirect pathways. The renin-angiotensin-aldosterone system is well recognized for its essential role in the physiological regulation of blood volume, blood pressure, and sodium homeostasis 33,34 . Increasing evidence demonstrates that this system is overactive at different stages of liver fibrosis 33,35 , which may explain the association between hypertension and liver fibrosis. As a surface enzyme, GGT can cleave extracellular glutathione (GSH), maintain the balance of GSH in vivo, and play a key role in alleviating the effects of oxidative stress 36 . Previous studies confirmed that elevated GGT was associated with SCRP, low adiponectin, the presence of chronic kidney disease, and hepatic steatosis. It was reported that GGT elevation was associated with hepatic steatosis, and fibrosis in patients with NAFLD 37,38 . In addition to the above factors, the influence of genetic factors such as PNPLA3, TM6SF2, and MBOAT7 on liver fibrosis in MAFLD patients with HBV infection should also be further studied 39 .
The major strength of this study is that, to the best of our knowledge, it is the first to analyze risk factors for liver biopsy-proven significant liver fibrosis in MAFLD patients with HBV infection. However, this study has some limitations, including its retrospective design. Although HOMA-IR and SCRP are mentioned in the diagnostic criteria for lean and normal-weight patients with MAFLD under the new definition, these were absent in our data, which may have caused us to miss some MAFLD cases. Second, given the cross-sectional nature of the study, we were unable to determine the causal relationship between metabolic dysfunction and significant liver fibrosis, highlighting the need for further longitudinal cohort studies to verify the effects of metabolic and viral factors on liver fibrosis in MAFLD patients with HBV infection. Furthermore, due to the gender distribution of MAFLD patients, the number of women included in this study was low; therefore, conclusions may pertain more to male patients, and further research is required to confirm these findings in females.
In conclusion, MAFLD patients with HBV infection have a higher risk of liver fibrosis than patients who have pure MAFLD. Metabolic factors, hypertension and type 2 diabetes, are closely related to liver fibrosis in MAFLD patients with HBV. These results highlight that in addition to traditional antiviral therapy, screening and early intervention of metabolic diseases are required for MAFLD patients with HBV infection. For patients with diabetes and hypertension, blood noninvasive biomarkers or transient elastography should be actively performed to further define the stage of liver fibrosis. If noninvasive screening presents a high risk of liver fibrosis, liver biopsy is recommended.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints.
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