Chronic hepatitis B (CHB) and nonalcoholic fatty liver disease (NAFLD) are two major chronic liver diseases in China. As lifestyle and dietary structure have changed, the number of CHB patients with hepatic steatosis has increased. The incidence varies widely around the world, fluctuating between 27 and 51% [
This prospective cohort study included 245 naive CHB patients who underwent percutaneous liver biopsy from May 2010 to 2016 at the Xiamen Hospital of Traditional Chinese Medicine. All patients underwent percutaneous liver biopsy, and their hepatic tissues were confirmed to exhibit or not exhibit steatosis by liver histological pathological examination. Patients were treated with PEG-IFN
All patients provided informed consent for the liver biopsy procedure. The liver biopsy was performed using 16G biopsy needles guided by ultrasonography. A qualified biopsy specimen was either a minimum of 1.5 cm long or displayed for 6 or more portal tracts. The specimens were fixed, paraffin-embedded, and stained with haematoxylin and eosin (HE staining). Scheuer’s scoring system was used to semiquantify the histological necroinflammation from G0 to G4 and the fibrosis stages from F0 to F4 by two pathologists independently who were blinded to the patients’ biochemical and virologic results. Fibrosis was evaluated in all specimens by subjecting them to Masson Trichrome staining. Biopsied tissue sections were cut into 4 mm samples from prepared representative tissue blocks (lumps) and then placed in a paraffin oven to remove most of the paraffin. To complete deparaffinization, the specimens were then passed through xylene and a series of alcohol dilutions for 5 minutes and underwent microwave treatment using antigen retrieval for 10 minutes. After soaking in the methanol solution with 3% H2O2 for 5 minutes to block endogenous peroxidase, the peroxidase-conjugated Envision Kit (Envision-PO, Envision System; DAKO, Carpinteria, CA, USA) for rabbit primary antibodies was applied to the specimens for immunohistochemical staining of HBcAg in the hepatocytes.
Hepatic steatosis was categorized as follows: no steatosis (steatosis affected less than 5% of hepatocytes, S0), mild steatosis (steatosis affected 5–33% of hepatocytes, S1), moderate steatosis (steatosis affected 34–66% of hepatocytes, S2), and severe steatosis (steatosis affected more than 66% of hepatocytes, S3) (Figure
The distribution of hepatic steatosis was categorized into four types in this study. (a) In the S0 group, less than 5% of hepatocytes were affected by steatosis (HE staining, ×200). (b) In the S1 group, 5–33% of hepatocytes were affected by steatosis (HE staining, ×200). (c) In the S2 group, 34–66% of hepatocytes were affected by steatosis (HE staining, ×200). (d) In the S3 group, more than 66% of hepatocytes were affected by steatosis (HE staining, ×200).
HBcAg expression patterns were classified as follows: no HBcAg expression (HBcAg-negative), cytoplasmic expression (cHBcAg), cytoplasmic dominant expression (cdHBcAg), and cytoplasmic and nuclear mean expression (mHBcAg).
The HBcAg-negative type showed no HBcAg expression in the nucleus or cytoplasm of the hepatocytes, the cHBcAg type showed HBcAg expression only in the cytoplasm but not in the nucleus of the hepatocytes, the cdHBcAg type showed more than 2/3 of HBcAg expression in the cytoplasm, and the mHBcAg type showed 50% HBcAg expression in the nucleus and 50% in the cytoplasm of the hepatocytes (Figure
The distribution of HBcAg in the hepatocytes of CHB patients was classified into four types. (a) The HBcAg-negative type showed no HBcAg expression in either the nucleus or cytoplasm of the hepatocytes (immunohistochemical stain for HBcAg, ×200). (b) The cHBcAg type showed HBcAg expression only in the cytoplasm but not in the nucleus of hepatocytes (immunohistochemical stain for HBcAg, ×200). (c) The cdHBcAg type showed more than 2/3 HBcAg expression in the cytoplasm of hepatocytes (immunohistochemical stain for HBcAg, ×200). (d) The mHBcAg type showed 50% HBcAg expression in the nucleus and 50% HBcAg expression in the cytoplasm of the hepatocytes (immunohistochemical stain for HBcAg, ×200).
HBcAg expression intensity was classified into four types in this study: 0 points, less than 5% hepatitis B core antigen expression; 1 point, 5%-33% hepatitis B core antigen expression; 2 points, 34%-66% hepatitis B core antigen expression; and 3 points, over 66% hepatitis B core antigen expression (Figure
HBcAg expression intensity was classified into four types in this study. (a) The 0 point group showed HBcAg expression in less than 5% of hepatocytes (immunohistochemical stain for HBcAg, ×200). (b) The 1 point group showed HBcAg expression in 5%-33% of hepatocytes (immunohistochemical stain for HBcAg, ×200). (c) The 2 point group showed HBcAg expression in 34%-66% of hepatocytes (immunohistochemical stain for HBcAg, ×200). (d) The 3 point group showed HBcAg expression in over 66% of hepatocytes (immunohistochemical stain for HBcAg, ×200).
The HBsAg expression intensity was classified into three types in this study: 1 point, 5%-33% hepatitis B surface antigen expression; 2 points, 34%-66% hepatitis B surface antigen expression; and 3 points, over 66% hepatitis B surface antigen expression (Figure
HBsAg expression intensity was classified into three types in this study. (a) The 1 point group showed HBsAg expression in 5%-33% of hepatocytes (immunohistochemical stain for HBsAg, ×200). (b) The 2 point group showed HBsAg expression in 34%-66% of hepatocytes (immunohistochemical stain for HBsAg, ×200). (c) The 3 point group showed HBsAg expression in over 66% of hepatocytes (immunohistochemical stain for HBsAg, ×200).
All patients were treated with 180
All statistical analyses were performed using SPSS 22.0 software (IBM Co., Armonk, NY, USA). Continuous variables are presented as the
The baseline characteristics are listed in Table
Baseline characteristics of 226 chronic hepatitis B patients.
Variable | Degrees of hepatic steatosis | |||||
---|---|---|---|---|---|---|
S0 ( | S1 ( | S2 ( | S3 ( | |||
Male ( | 82 (68.91%) | 65 (85.53%) | 21 (95.46%) | 9 (100%) | 14.828 | 0.002 |
Age | 2.063 | 0.106 | ||||
Age >40 ( | 6 (5.04%) | 15 (19.74%) | 1 (4.55%) | 0 (0%) | 13.28 | 0.004 |
Course of disease | 0.327 | 0.806 | ||||
TC | 5.703 | 0.001 | ||||
TG | 13.180 | 0.001 | ||||
UA | 4.271 | 0.006 | ||||
Hyperuricaemia ( | 18 (15.13%) | 17 (22.37%) | 9 (40.91%) | 6 (66.67%) | 18.255 | 0.001 |
Metabolic syndrome ( | 1 (0.84%) | 3 (3.95%) | 1 (4.55%) | 1 (11.11%) | 4.537 | 2.063 |
GLU | 9.404 | 0.001 | ||||
ALT | 4.347 | 0.005 | ||||
AST | 7.560 | 0.001 | ||||
HBVlog10 (IU/mL) DNA | 1.224 | 0.302 | ||||
HBsAg (log IU/mL) | 1.273 | 0.283 | ||||
HBV genotype | 1.00 | 0.616 | ||||
B | 35 (29.41%) | 23 (30.26%) | 7 (31.83%) | 3 (33.33%) | ||
C | 78(65.55%) | 50 (65.79%) | 14(63.64%) | 6 (66.67%) | ||
Mix | 6(5.04%) | 3 (3.95%) | 1 (4.55%) | 0 | ||
BMI | 36.996 | 0.001 | ||||
14 (11.76%) | 54 (71.05%) | 14 (63.64%) | 7 (77.77%) | 80.932 | 0.001 |
Continuous variables are expressed as the
The baseline hepatic histological necroinflammation and hepatic steatosis stages were negatively correlated. The more severe the hepatic steatosis was, the less severe the liver inflammation activity (Table
The correlation between baseline hepatic histological necroinflammation and baseline hepatic steatosis in each group.
Feature | Total cases | G1 | G2 | ≥G3 | Spearman’s rank correlation analysis | Coefficient of contingency |
---|---|---|---|---|---|---|
S0 | 119 | 2 | 74 | 43 | 0.036 | -0.140 |
S1 | 76 | 1 | 55 | 20 | ||
S2 | 22 | 0 | 16 | 6 | ||
S3 | 9 | 0 | 9 | 0 |
The correlation between baseline hepatic fibrosis stage and baseline hepatic steatosis degree.
Feature | Total cases | F1 | F2 | ≥F3 | Spearman’s rank correlation analysis | Coefficient of contingency |
---|---|---|---|---|---|---|
S0 | 119 | 69 | 40 | 10 | 0.202 | -0.085 |
S1 | 76 | 50 | 20 | 6 | ||
S2 | 22 | 2 | 6 | 14 | ||
S3 | 9 | 7 | 2 | 0 |
The categorical variables are described by counts and proportions. The Pearson chi-square test, Spearman’s rank correlation analysis, and coefficient of contingency were used for the statistical analysis.
The categorical variables are described by counts and proportions. The Pearson chi-square test, Spearman’s rank correlation analysis, and coefficient of contingency were used for the statistical analysis.
Clinical parameters (sex, age, disease course, baseline TC, baseline TG, metabolic syndrome, baseline ALT, baseline AST, baseline HBV DNA,
Multivariate analysis of clinical parameters independently associated with significant histological abnormalities.
Patient characteristics | Clinical parameters | OR (95% CI) | |
---|---|---|---|
Presence of hepatic steatosis | 5.481 (2.992-10.041) | 0.001 | |
Presence of significant hepatic steatosis (≥S2) | Baseline AST | 0.978 (0.959-0.998) | 0.029 |
Baseline UA | 1.006 (1.000-1.012) | 0.040 | |
Presence of significant fibrosis (≥F2) | Baseline AST | 1.006 (1.000-1.012) | 0.038 |
The VR, BR, and CR rates were observed in the four groups after 48 weeks of treatment. The rates of VR, BR, and CR differed significantly between the four groups (
The virological response, biochemical response, and complete response in the four groups after 48 weeks of treatment.
Groups | Cases | The rate of VR | The rate of BR | The rate of CR |
---|---|---|---|---|
CHB with S0 | 119 | 42.9% | 75.6% | 28.6% |
CHB with S1 | 76 | 30.3% | 40.9% | 14.5% |
CHB with S2 | 22 | 18.2% | 31.8% | 9.1% |
CHB with S3 | 9 | 0% | 22.2% | 0% |
11.777 | 32.228 | 9.552 | ||
0.007 | 0.001 | 0.018 |
Categorical variables are described by counts and proportions. The Pearson chi-square test was used for the statistical analysis.
During 24 weeks follow-up, there were thirteen patients who had an elevation of ALT levels >10 times ULN discontinued follow-up and switch to NAs therapy and seven patients intended to NA therapy so drop out continue the follow-up. 206 patients finished the follow-up.
The VR, BR, and CR rates were observed in the four groups after 24 weeks of follow-up. The rates of VR, BR, and CR differed significantly between the four groups (
The virological response, biochemical response, and complete response in the four groups after 24 weeks of follow-up.
Groups | Cases | The rate of VR | The rate of BR | The rate of CR |
---|---|---|---|---|
CHB with S0 | 110 | 40.0% | 68.2% | 34.5% |
CHB with S1 | 72 | 26.3% | 36.1% | 20.8% |
CHB with S2 | 18 | 16.7% | 27.8% | 11.1% |
CHB with S3 | 6 | 0% | 16.7% | 0% |
8.505 | 25.988 | 8.390 | ||
0.029 | 0.001 | 0.032 |
Categorical variables are described by counts and proportions. The Pearson chi-square test was used for the statistical analysis.
The four degrees of hepatic steatosis have different HBcAg expression patterns. The rate of HBcAg-negative and c-HBcAg was 43.70% in the S0 group, whereas it was 31.58%, 31.82%, and 33.33% in the S1, S2, and S3 groups, respectively (
The difference in HBcAg expression patterns in different degrees of steatosis.
Degrees of steatosis | Total cases | HBcAg-negative group | c-HBcAg group | cd-HBcAg group | m-HBcAg group | Pearson’s chi-square test |
---|---|---|---|---|---|---|
S0 | 119 | 5 | 47 | 63 | 4 | 0.015 |
S1 | 76 | 10 | 14 | 48 | 4 | |
S2 | 22 | 3 | 4 | 13 | 2 | |
S3 | 9 | 0 | 3 | 5 | 1 |
The difference in HBsAg expression levels in different degrees of steatosis.
Degrees of steatosis | Total cases | 1 | 2 | 3 | Rank test |
---|---|---|---|---|---|
S0 | 119 | 8 | 10 | 101 | 0.085 |
S1 | 76 | 3 | 13 | 60 | |
S2 | 22 | 1 | 5 | 16 | |
S3 | 9 | 0 | 1 | 8 |
The difference in HBcAg expression levels in different degrees of steatosis.
Degrees of steatosis | Total cases | 0 | 1 | 2 | 3 | Rank test |
---|---|---|---|---|---|---|
S0 | 119 | 8 | 28 | 20 | 63 | 0.257 |
S1 | 76 | 10 | 13 | 17 | 36 | |
S2 | 22 | 3 | 4 | 5 | 10 | |
S3 | 9 | 0 | 1 | 3 | 5 |
In this study, we compared the baseline clinical data of CHB patients with different degrees of hepatic steatosis. CHB patients with hepatic steatosis were more likely to have higher levels of TC, TG, GLU, UA, and BMI; be of themale sex; be aged >40 years; have hyperuricaemia and a
The multivariate logistic regression analysis showed that a
In this study, we present our findings that a higher degree of hepatic steatosis is associated with a lower level of inflammation in the liver tissue; moreover, there was no correlation between the degree of liver fibrosis and the degree of hepatic steatosis. One domestic study [
However, our study investigated the difference in expression levels of HBsAg and HBcAg in different groups and found that there was no significant difference in the expression levels of HBsAg and HBcAg between groups with different severities of hepatic steatosis. Furthermore, we demonstrated that HBcAg expression patterns were related to hepatic steatosis. In the group without steatosis, 43.70% of patients were HBcAg-negative and c-HBcAg, whereas 31.77% of patients with steatosis were HBcAg-negative and c-HBcAg. One study [
In conclusion, ALT and AST levels and the inflammatory activity of liver tissue in CHB patients with steatosis were significantly lower than those in patients without steatosis; this result may be due to the low expression of the HBcAg-negative and c-HBcAg type in liver tissue that leads to a poor immune response, eventually causing a poor antiviral effect. Antiviral therapy with PEG-IFN
There are several limitations of the present study. We evaluated the efficacy of PEG-IFN
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
This study was supported by the National Natural Science Foundation of China (No. 81673660, No. 81873242), the National Science and technology major special project (No: 2017zx10202201), the Fujian Province Health and Family Planning Medical Innovation Research Talent Training Project (No. 2018-CXB-28; No. 2018-2-68), the Xiamen Science and Technology Program Grant (No. 3502Z20174028), and the joint research of Xiamen Important and Major Disease Project (No. 3502Z20179047), the sixth group of experts in traditional Chinese medicine in China (2017).