Chronic HBV infection exhibited beneficial effects on BMI, hepatocyte steatosis and related impairments
In the present study, demographic, clinical and pathological indices were compared between NAFLD patients with (NAFLD-HBV group) or without chronic HBV infection (NAFLD group). Interestingly, the NAFLD-HBV group presented the BMI much lower than that of NAFLD group (NAFLD group vs NAFLD-HBV group: 27.4 ± 3.2 vs 25.7 ± 2.6, P = 0.023) (Table 1). Significant amelioration of NAFLD-specific pathological characteristics, including hepatocyte steatosis (NAFLD group vs NAFLD-HBV group: 2.32 ± 0.65 vs 1.33 ± 0.49, P < 0.001) and ballooning (NAFLD group vs NAFLD-HBV group: 1.84 ± 0.37 vs 1.13 ± 0.52, P < 0.001), was documented in the NAFLD-HBV instead of NAFLD group (Table 1). In contrast to the comparability in most biochemical indices, there was a statistically decreased ALT activity in the NAFLD-HBV group in comparison to that of the NAFLD group (NAFLD group vs NAFLD-HBV group: 64.1 IU/L (39.5 IU/L, 110.3 IU/L) vs 53 IU/L (23.4 IU/L, 69.5 IU/L), P = 0.025) (Table 1). Thus, the beneficial impact of chronic HBV infection, especially on the lipid metabolism and related hepatic injury, was indicated in the NAFLD patients.
Table 1
Demographic, clinical and pathological data of all patients
Variable
|
NAFLD (n = 41)
|
NAFLD-HBV (n = 21)
|
P
|
Male, n(%)
|
26 (63.4)
|
15 (71.4)
|
0.528
|
Age (years)
|
40 ± 14
|
37 ± 14
|
0.439
|
Body mass index (kg/m2)
|
27.4 ± 3.2
|
25.7 ± 2.6
|
0.023 *
|
Waist-to-hip ratio
|
0.93 ± 0.05
|
0.92 ± 0.07
|
0.569
|
Total bilirubin (µmol/L)
|
17.3 ± 15.6
|
14.2 ± 4.3
|
0.379
|
Alkaline phosphatase (IU/L)
|
84.6 (62.3, 100.7)
|
78 (63, 96.4)
|
0.693
|
Alanine aminotransferase (IU/L)
|
64.1 (39.5, 110.3)
|
53 (23.4, 69.5)
|
0.025 *
|
Aspartate aminotransferase (IU/L)
|
37.7 (25, 65.4)
|
28.6(20.3, 45.8)
|
0.14
|
Gamma-glutamyl transferase (IU/L)
|
60.6 (38, 91.1)
|
31 (22.2, 60)
|
0.058
|
Fasting glucose (mmol/L)
|
5.4 (4.6, 6.5)
|
4.8 (4.7, 5.5)
|
0.797
|
Total cholesterol (mmol/L)
|
4.5 (4.3, 5.2)
|
5.2 (4.1, 5.7)
|
0.322
|
HDL-cholesterol (mmol/L)
|
1.2 (1.1, 1.3)
|
1.2 (1, 1.6)
|
0.451
|
LDL-cholesterol (mmol/L)
|
2.9 (2.6, 3.2)
|
2.8 (2, 3.5)
|
0.7
|
Total triglycerides (mmol/L)
|
1.6 (1.1, 2.4)
|
1.2 (1, 2.2)
|
0.612
|
Hepatocyte steatosis
|
2.32 ± 0.65
|
1.33 ± 0.49
|
< 0.001 **
|
Lobular inflammation
|
0.65 ± 0.66
|
0.67 ± 0.62
|
0.915
|
Ballooning
|
1.84 ± 0.37
|
1.13 ± 0.52
|
< 0.001 **
|
Liver fibrosis
|
1.68 ± 1.14
|
1.13 ± 1.13
|
0.134
|
The continuous variables were presented as mean ± SD or median (interquartile range). HDL, high density lipoprotein; LDL, low density lipoprotein. * P < 0.05, ** P < 0.01. |
Serum lipidomics differentiated NAFLD patients with or without chronic HBV infection
Multivariate analyses were employed in our study to take an overview of the serum lipidomics between NAFLD and NAFLD-CHB groups. Dramatically, 3D PCA score plot of serum lipidomics distinctly differentiated the NAFLD patients with or without chronic HBV infection (Fig. 1A). Similar group discrimination was also obtained by the OPLS-DA score plot (Fig. 1B).
To reveal the role of chronic HBV infection in lipid metabolism, a total of 239 serum lipids was exposed to UPLC-MS/MS in both NAFLD and NAFLD-HBV groups. In result, 64 lipids among these ones (26.78%) were filtered to be statistically different by unpaired Student’s t-test. Detailedly, the profile of differential serum lipids comprised 17 free fatty acid (FFAs), 8 lysophosphatidylcholine (LPCs), 3 lysophosphatidylcholine plasmalogen (LPC-Os), 1 lysophosphatidylethanolamine (LPE), 2 lysophosphatidylethanolamine plasmalogen (LPE-Os), 2 lysophosphatidylinositol (LPIs), 3 phosphatidylcholine (PCs), 18 cholineplasmalogen (PC-Os), 5 phosphatidylethanolamine (PEs), 2 ethanolamine plasmalogen (PE-Os), 1 phosphatidylinositol (PI) and 2 sphingomyelin (SMs) (Table 2). On the other hand, S-plot put forward 31 differential serum lipids with VIP > 1.0, including 7 free fatty acid (FFAs), 3 lysophosphatidylcholine (LPCs), 10 phosphatidylcholine (PCs), 8 cholineplasmalogen (PC-Os), 1 sphingomyelin (SM) and 2 triacylglycerol (TGs) (Fig. 2).
Table 2
Differential serum lipids between NAFLD patients with or without chronic HBV infection by unpaired Student’s t test
Lipids
|
NAFLD-HBV/NAFLD
|
P
|
Lipids
|
NAFLD-HBV/NAFLD
|
P
|
Lipids
|
NAFLD-HBV/NAFLD
|
P
|
FFA 12:0
|
0.81
|
0.021
|
LPC 20:3
|
0.67
|
0.016
|
PC-O 36:5
|
1.33
|
0.002
|
FFA 14:0
|
0.70
|
0.002
|
LPC 22:6
|
0.63
|
0.001
|
PC-O 38:4
|
1.31
|
< 0.001
|
FFA 14:1
|
0.59
|
0.009
|
LPC 24:0
|
1.35
|
0.014
|
PC-O 38:5
|
1.35
|
0.001
|
FFA 16:0
|
0.75
|
0.001
|
LPC-O 16:0
|
0.68
|
0.008
|
PC-O 40:4
|
1.40
|
< 0.001
|
FFA16:1
|
0.61
|
0.001
|
LPC-O 16:1
|
0.70
|
0.008
|
PC-O 40:5
|
1.37
|
0.001
|
FFA 18:1
|
0.67
|
< 0.001
|
LPC-O 18:1
|
0.68
|
0.024
|
PC-O 42:4
|
1.38
|
< 0.001
|
FFA18:2
|
0.61
|
< 0.001
|
LPE 22:6
|
0.83
|
0.047
|
PC-O 42:5
|
1.45
|
< 0.001
|
FFA 18:3
|
0.58
|
< 0.001
|
LPE-O 16:1
|
0.56
|
0.006
|
PC-O 42:6
|
1.42
|
0.001
|
FFA 20:1
|
0.66
|
0.003
|
LPE-O 18:1
|
0.62
|
0.019
|
PC-O 44:5
|
1.42
|
0.001
|
FFA 20:2
|
0.59
|
< 0.001
|
LPI 18:0
|
0.30
|
0.004
|
PC-O 44:6
|
1.48
|
0.001
|
FFA 20:3
|
0.44
|
0.001
|
LPI 20:4
|
0.66
|
< 0.001
|
PE 34:1
|
1.47
|
0.016
|
FFA20:4
|
0.44
|
0.002
|
PC 34:2
|
1.16
|
0.049
|
PE 34:2
|
1.43
|
0.012
|
FFA20:5
|
0.20
|
0.012
|
PC 34:3
|
1.25
|
0.043
|
PE 36:2
|
1.33
|
0.044
|
FFA 22:2
|
0.59
|
0.043
|
PC 36:2
|
1.25
|
0.029
|
PE 36:3
|
1.49
|
0.009
|
FFA 22:4
|
0.63
|
0.004
|
PC-O 32:0
|
1.28
|
0.001
|
PE 38:5
|
1.28
|
0.021
|
FFA 22:5
|
0.50
|
< 0.001
|
PC-O 34:0
|
1.25
|
0.003
|
PE-O 34:3
|
1.28
|
0.017
|
FFA 22:6
|
0.41
|
0.001
|
PC-O 34:1
|
1.28
|
0.001
|
PE-O 36:3
|
1.29
|
0.017
|
LPC 16:0
|
0.72
|
0.004
|
PC-O 34:2
|
1.29
|
0.031
|
PI 34:1
|
1.30
|
0.035
|
LPC 17:0
|
0.68
|
0.007
|
PC-O 34:3
|
1.34
|
0.003
|
SM 34:0:3
|
1.26
|
0.014
|
LPC 18:0
|
0.73
|
0.018
|
PC-O 36:2
|
1.27
|
0.003
|
SM 36:0:2
|
0.66
|
0.005
|
LPC 18:3
|
0.70
|
0.003
|
PC-O 36:3
|
1.28
|
0.012
|
|
|
|
LPC 20:2
|
0.64
|
0.008
|
PC-O 36:4
|
1.31
|
0.012
|
|
|
|
The serum lipids are expressed in a pattern of name carbon numbers: double bond numbers. FFA, free fatty acids; LPC, lysophosphatidylcholine; LPC-O, lysophosphatidylcholine plasmalogen; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; PC, phosphatidylcholine; PC-O, choline plasmalogen; PE, phosphatidylethanolamine; PI, phosphatidylinositol; SM, sphingomyelin. |
FFAs, LPCs, PCs and PC-Os alteration characterized the effects of chronic HBV infection on serum lipidomics
Integrating unpaired Student’s t-test and S-plot, 17 differential serum lipids with P < 0.05 and VIP > 1.0 were identified to characterize the lipidomics of NAFLD patients upon chronic HBV infection. They were classified into FFAs (FFA 16:0, FFA 16:1, FFA 18:1, FFA 18:2, FFA 20:4, FFA 22:6), LPCs (LPC 16:0, LPC 18:0, LPC 18:3), PCs (PC 34:2, PC 36:2), and PC-Os (PC-O 34:2, PC-O 34:3, PC-O 36:4, PC-O 36:5, PC-O 38:4, PC-O 38:5), respectively (Table 3). When compared to those of the NAFLD group, serum PCs (NAFLD-HBV/NAFLD: 1.21–1.25) and PC-Os levels (NAFLD-HBV/NAFLD: 1.29–1.35) in the NAFLD-HBV group exhibited significant upregulation (Table 3, Fig. 3). Contrastively, serum levels of FFAs (NAFLD-HBV/NAFLD: 0.41–0.75) and LPCs (NAFLD-HBV/NAFLD: 0.70–0.73) experienced statistical downregulation in the NAFLD patients with concurrent chronic HBV infection (Table 3, Fig. 3). These characteristics convinced the dominating role of FFAs, LPCs, PCs and PC-Os in differential lipid profile.
Table 3
Differential serum lipids between NAFLD and NAFLD-HBV groups (P < 0.05, VIP > 1)
lipids
|
Ratio of concentration (NAFLD-HBV/NAFLD)
|
|
Fold
|
Trend
|
P
|
VIP
|
FFA16:0
|
0.75
|
↓
|
0.010
|
2.85
|
FFA16:1
|
0.61
|
↓
|
0.005
|
1.18
|
FFA18:1
|
0.67
|
↓
|
0.001
|
2.98
|
FFA18:2
|
0.61
|
↓
|
0.001
|
2.93
|
FFA20:4
|
0.44
|
↓
|
0.022
|
1.14
|
FFA22:6
|
0.41
|
↓
|
0.015
|
1.10
|
LPC16:0
|
0.72
|
↓
|
0.014
|
3.80
|
LPC18:0
|
0.73
|
↓
|
0.018
|
1.85
|
LPC18:3
|
0.70
|
↓
|
0.013
|
1.47
|
PC34:2
|
1.21
|
↑
|
0.049
|
7.76
|
PC36:2
|
1.25
|
↑
|
0.029
|
7.37
|
PC-O34:2
|
1.29
|
↑
|
0.009
|
1.23
|
PC-O34:3
|
1.34
|
↑
|
0.001
|
1.32
|
PC-O36:4
|
1.31
|
↑
|
0.002
|
1.97
|
PC-O36:5
|
1.33
|
↑
|
0.002
|
1.41
|
PC-O38:4
|
1.31
|
↑
|
< 0.001
|
1.15
|
PC-O38:5
|
1.35
|
↑
|
< 0.001
|
1.74
|
Low-level FFAs upon chronic HBV infection associated with improvements in hepatocyte steatosis and related impairments
Correlation between differential serum lipids (FFAs, LPCs, PCs and PC-Os) and biochemical indices (TBIL, DBIL, ALP, γ-GT, ALT and AST), together with NAFLD-specific pathological characteristics (hepatocyte steatosis, ballooning, lobular inflammation, fibrosis), was subjected to assessment in this study. Noticeably, low-level FFAs among these lipids showed close association with lessened hepatocyte steatosis (FFA16: 1, r = 0.39, P = 0.01; FFA18:1, r = 0.39, P = 0.015; FFA18:2, r = 0.33, P = 0.036) and ballooning (FFA16:0, r = 0.31, P = 0.037; FFA16:1, r = 0.45, P < 0.001; FFA18:1, r = 0.37, P = 0.005; FFA18:2, r = 0.30, P = 0.013) (Fig. 4). Furthermore, the reduction of ALT (FFA16: 0, r = 0.16, P = 0.043; FFA16: 1, r = 0.05, P = 0.02; FFA18:1, r = 0.16, P = 0.004; FFA18:2, r = 0.13, P = 0.017) and AST activities (FFA16: 0, r = 0.34, P = 0.003; FFA16: 1, r = 0.37, P < 0.001; FFA18:1, r = 0.33, P < 0.001; FFA18:2, r = 0.42, P < 0.001) in patients with low-level FFAs reflected an attenuation of steatosis-related hepatic impairments (Fig. 4). The down-regulatory effect of chronic HBV infection on FFAs, and the association of low-level FFAs and steatosis improvements, resultantly convinced a beneficial role of chronic HBV infection in the lipid profile and related NAFLD.