Baseline characteristics of the study mice
Our previous study demonstrated that co-administration of shRNAs targeting TGF-β and HBV decreased HBV antigens, HBV DNA, and liver fibrosis markers in the serum and livers of HBV-replicated mice [44]. In order to explore the mechanisms underlying the antiviral and antifibrotic effects, AAVdual-shRNA and AAVshRNA-TGF-β co-injection was evaluated. HBV(+) and HBV(-) mice were used as positive and negative controls, respectively. All three AAVshRNA-treated mice showed lower HBsAg and HBV DNA levels in the serum compared with that in untreated mice (Table 1). HBsAg and HBcAg levels were significantly decreased in the livers of AAVshRNA-treated mice, as demonstrated by IHC staining (Figure. 1).
Collagen levels were significantly decreased in the livers of treated mice compared with those in HBV(+) mice (Figure. 2a, Table 1).Total collagen was also quantitatively assessed using hydroxyproline assays; lower collagen levels were observed in the livers of treated mice and HBV(-) mice than in those of HBV(+) mice (Table 1). Masson staining and Sirus Red staining showed that the percentages of collagen deposition in hepatocytes were decreased by approximately 67.71% and 80.01%, respectively, after treatment. Collagen I and collagen III levels in serum were also significantly reduced in the treated group compared with that in HBV(+) mice (Table 1).
Next, we detected the expression of α-SMA, a marker of fibrosis,in the liver by IHC staining (Figure. 2a) and western blotting (Figure. 2d). As indicated by IHC staining, α-SMA expression was markedly reduced in the treated group compared with that in the HBV(+) group (Figure. 2a). The percentage of α-SMA expression was decreased by over 45% in the livers of treated mice compared with that in HBV(+) mice, as demonstrated by western blotting (Figure. 2d). Taken together, these data indicated that the mouse model in this study was appropriate.
Proteomic analysis of shRNA-treated HBV-replicated mice by iTRAQ-based quantitative proteomics
Next, we investigated differentially expressed proteins and potential pathways for attenuating liver fibrosis using iTRAQ-based quantitative proteomics by comparing these three groups of mice in order to elucidate the potential antifibrotic mechanisms. AAVdual-shRNA-and AAVshRNA-TGF-β-treated groups were analyzed by iTRAQ-based quantitative proteomics, as shown in the flowchart in Figure. 3a. HBV(+) mice were used as a positive control, and HBV(-) mice were used as a negative control. In total, 2743 proteins were identified in all groups (Figure. 3b, c). Notably, 7 6 upregulated and 122 downregulated proteins were found in the treated group compared with that in the HBV(+) group (Additional file 1: Table S2). Sixty-one proteins were upregulated, and 134 proteins were downregulated in HBV(+) mice compared with that in HBV(-) mice (Additional file 1: Table S3). We also evaluated the differentially expressed proteins in all three groups using Venn-Euler diagrams (Figure. 3d) and found 41 upregulated and 15 downregulated proteins in the treated group versus the HBV(+) group compared with the HBV(+) group versus the HBV(-) group. Two proteins were upregulated in both comparisons, and two proteins were downregulated in both comparisons (Figure. 3d; Additional file 1: Table S4).
In order to obtain an overall functional view of the differentially expressed proteins, we used GO functional annotations and KEGG metabolic pathway analyses. Comparison of the treated group and HBV(+) group revealed enrichment of 2185 BPs; 321 of these BPs were significant according to analysis of P values. Additionally, 89 CCs were significantly altered among 337 enriched CCs, and 484 MFs were enriched, among which 144 MFs were significant. Seventeen KEGG terms among 99 enriched KEGG terms were significant (Figure. 4a). In order clarify the functions and features of the identified proteins, we annotated protein functions and features based on GO and KEGG analyses. An overview of the GO analysis is shown in Figure 4b. There were 10 distinctly enriched categories of BPs, CCs, and MFs. The top proteins enriched in BPs were involved in organ-nitrogen compound metabolic process (45%), and some proteins enriched in BPs were related to liver fibrosis, e.g., lipid metabolic process (11%), oxidation-reduction (11%), response to oxidative stress (5%), negative regulation of cell adhesion (4%), and cellular oxidant detoxification (2%; Additional file 1: Figure S1 a). The main MF category of enriched proteins was cytoplasm (85%). Proteins involved in hepatic fibrosis and oxidative stress were also observed in MFs, including adherent junction (7%), endoplasmic reticulum membrane (7%), complex of collagen trimers (2%), and the TRAF2-GSTP1 complex (1%; Additional file 1: Figure S1 c). Proteins enriched in CCs were involved in nucleic acid binding (40%), hydrolase activity (8%), oxidoreductase activity (4%), organic acid binding (3%), transferase activity (3%), vitamin binding (2%), and vitamin B6 binding (2%; Additional file 1: Figure S1 c).
These proteins were also mapped to KEGG pathways based on their KEGG gene IDs. There were eight KEGG pathways presented, including metabolic pathways (14%), ribosome (13%), PPAR signaling pathway (6%), chemical carcinogenesis (3%), protein digestion and absorption (2%), protein export (1%), tryptophan metabolism (2%), and valine, leucine, and isoleucine degradation (2%; Figure. 4c). The significant (P < 0.01) pathways were ribosome, PPAR signaling pathway, and chemical carcinogenesis (Additional file 1: Figure S2 a).
To clarify the functional relationships of the identified proteins, a PPI network was created using Omicsbean. In the PPI network, GSTP1, which participated in glutathione metabolism, chemical carcinogenesis, and metabolism of xenobiotics by cytochrome P450, and ribosomal proteins, including Rpl13, Rpl37, and Rpl27a, were upregulated. Additionally, FABP1, ME1, and ACAA1,which were relevant to the PPAR signaling pathway and metabolic pathways, were downregulated in the treatment group compared with that in HBV(+) mice (Figure. 4d and Additional file 1: Figure S2 b).
Verification of proteins associated with oxidative stress and PPAR signaling pathway by western blotting
In order to identify the therapeutic mechanisms of liver fibrosis by AAVshRNA treatment, we next focused on differentially expressed proteins related to oxidative stress, the PPAR signaling pathway, lipid metabolism, and inflammation, which are involved in hepatic fibrosis.Indeed, in our previous study, oxidative stress was found to play an important role in liver fibrosis [45]. Additionally,differentially expressed proteins related to oxidative stress, including GSTP1 and PRDX1, were identified by iTRAQ-based quantitative proteomics. Thus, in order to verify changes in oxidative stress after treatment, we evaluated the expression of GSTP1, PRDX1, and TGF-β, which are involved in oxidative stress and the redox imbalance, by western blotting (Figure. 5). GSTP1 (Figure. 5a) was significantly upregulated in the treated group compared with that in the HBV(+) group (increased 1.57-fold) and was significantly downregulated in HBV(+) mice compared with that in HBV(-) mice (decreased 0.54-fold). PRDX1 (Figure. 5b) and TGF-β (Figure. 5c) were significantly downregulated in the treated group compared with that in the HBV(+) group (decreased 0.35-and 0.74-fold, respectively) and were upregulated in the HBV(+) group compared with that in the HBV(-) group (increased 3.31-and 4.20-fold, respectively). Changes in the expression levels of GSTP1 and PRDX1 verified by western blotting were consistent with the alterations found by iTRAQ-based quantitative proteomics analysis.
Bioinformatics analysis showed that the PPAR signaling pathway was activated in the treated group, as demonstrated by downregulation of ACAA1, ME1, and FABP1. Therefore, we next investigated the differential expression of these proteins regulated by PPAR signaling pathway in the liver by western blotting. The three proteins were significantly downregulated to 11.90% (ACAA1; Figure. 5d), 42.50% (ME1; Figure. 5e), and 47.10% (FABP1; Figure. 5f) in the treated group compared with that in the HBV(+) group. In a comparison of the HBV(+) with HBV(-) groups, we found that the expression levels of ACAA1 and FABP1 were not significantly altered, whereas ME1 was significantly upregulated (increased 1.43-fold; Figure. 5e).
PPAR-γ played key roles in activating PPAR-signaling pathway
There are three different isoforms of PPARs, i.e., PPAR-α, PPAR-β/δ, and PPAR-γ[54]. PPAR-α is mainly expressed in the liver, and PPAR-γ is expressed in adipose and liver tissues. Therefore, we evaluated PPAR-α and PPAR-γ expression by western blotting. The results showed that PPAR-α expression was not altered in all three experimental groups. PPAR-γ was significantly upregulated by 3.20-fold in the livers of treated mice compared with those of HBV(+) mice; however, no significant changes were observed in the livers of HBV(+) and HBV(-) groups (Figure. 6). These findings suggest that PPAR-γ may play an important role inactivating the PPAR signaling pathway following AAVshRNAs treatment and that PPAR-α may not have an important a role as PPAR-γ in activating the PPARsignaling pathway.
AAVshRNA attenuated NF-κB P65 phosphorylation in the liver and decreased IL-6 secretion into the serum.
H&E staining was used to investigate the pathological process of liver fibrosis. Although most hepatocytes appeared histologically normal in all three groups, some hepatic necrosis was observed at 6 months in HBV(+) mice (Figure7A). NF-κB is a key mediator of inflammatory signaling and plays important roles in liver fibrogenesis[38]. Thus, we then examined the levels of NF-κB p65 and phosphorylated NF-κB p65 in vivo and in vitro using western blotting. The levels of phosphorylated NF-κB p65/NF-κB p65 were markedly reduced in the treatment group compared with that in the HBV(-) group in livers and in LX-2 cells after transfection with AAVshRNA;90.5% and 69% decreases were observed in vivo and in vitro after treatment, respectively (Figure. 7b). The expression of the inflammatory factor IL-6 was also measured by ELISA in serum. IL-6 levels in the serum were decreased by over 92% after treatment (Figure. 7c).