Short hairpin RNA attenuates liver fibrosis by regulating the peroxisome proliferator-activated receptor-γ and nuclear factor-κB pathways in hepatitis B virus-induced liver fibrosis in mice

Background: Progressive liver fibrosis, caused by chronic viral infection and metabolic disorders, results in the development of cirrhosis and hepatocellular carcinoma. However, no antifibrotic therapies have been approved to date. In our previous study, adeno-associated virus (AAV) short hairpin RNAs (shRNAs) targeting hepatitis B virus (HBV) and transforming growth factor (TGF)-β administration could persistently inhibit HBV replication and concomitantly prevent liver fibrosis. However, the differentially expressed proteins and critical regulatory networks of AAVshRNA treatment remain unclear. Accordingly, in this study, our major goal was we aimed to analyze differentially expressed proteins in the liver of AAV-shRNAs-treated mice with HBV infection and liver fibrosis using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics and to elucidate the underlying antifibrotic mechanisms. Results: In total 2743 proteins were recognized by iTRAQ-based quantitative proteomics analysis. Gene ontology analysis suggested that the differentially expressed proteins were mostly participated in peptide metabolism in the biological process category, cytosolic ribosomes in the cell component category, and structural constituents of ribosomes in the molecular function category. Kyoto Encyclopedia of Genes and Genomespathway analysis indicated that oxidative stress and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were actived after treatment. Verification studies showed that AAVshRNAs inhibited hepatic stellate cell activation and inflammation by suppressing nuclear factor-κB p65 phosphorylation and α-smooth muscle actin expression via upregulation of PPAR-γ. Hepatocytes steatosis was also decreased by activating PPAR signaling pathway and improving lipid metabolism. TGF-β level was decreased owning to increase PPAR-γ expression and directly inhibition using AAVshRNAs targeting TGF-β. TGF-β-induced oxidative stress was suppressed by increasing glutathione S-transferase Pi 1 and reducing

peroxiredoxin 1. Conclusions: Our results indicated that AAV-shRNAs were effective for modulating liver fibrosis by reducing oxidative stress, inflammation and activating PPAR signaling pathway. Background Hepatitis B virus (HBV) infection is a major health problem, which cause acute and chronic hepatitis, and progress to cirrhosis, and hepatocellular carcinoma (HCC) [1][2][3][4]. More than 780,000 people die from HBV infection or related complications each year [5].
Liver fibrosis is a common wound healing process response to chronic liver injury via excessive production and deposition of extracellular matrix (ECM) [9]. Hepatic stellate cells (HSCs) are major producers of matrix components and play pivotal roles in regulating the production and secretion of the ECM [10]. Typically, HSCs remain in a quiescent state and function in the storage of vitamin A. Upon liver injury, HSCs may undergo transdifferentiation, transform into highly proliferative myofibroblast-like cells, and acquire fibrogenic properties, including expression of α-smooth muscle actin (α-SMA), type I collagen, and type III collagen, which are vital components of the ECM [11]. Although inhibiting of HSC activation has been proposed as therapeutic strategy in anti-fibrosis treatment of fibrosis [12], novel approaches for uncovering the mechanisms of liver fibrosis and for development of antifibrotic treatments are still challenging.
TGF-β has also been shown to inhibit the antioxidant system and hence induce oxidative stress or redox imbalance [18][19][20]. Such a redox imbalance dedicates importantly to TGF-β 4 related pathophysiologic effects containing fibrosis [16]. Therapeutics targeting TGF-βinduced ROS-dependent cellular signaling may be a new therapeutic method in the treatment of fibrotic disorders. However, the mechanisms underlying of liver fibrosis associated with redox-sensitive targets remain unclear.
Peroxisome proliferator-activated receptors (PPARs) including PPAR-α, PPAR-β/δ, and PPAR-γ, are ligand-activated transcription factors belonging to the nuclear hormone receptor family [21][22][23][24][25]. Previous studies have shown that PPAR-γ is predominantly present in adipose tissue, and plays an important role in many biological processes, such as adipogenesis, cell differentiation, cell growth regulation and inflammatory reactions [26,27]. PPAR-γ has also been shown to be a driver of liver fibrosis in hepatic tissues, and its activation promotes insulin sensitivity and inhibits the transformation of HSCs from a quiescent to activated state [28][29][30][31]. Previous studies have indicated that the activation of PPAR-γ can reduce connective tissue growth factor expression induced by TGF-β1 in HSCs [32] and that the PPAR-γ agonist rosiglitazone can enhance PPAR-γ expression in activated HSCs, leading to reduced oxidative stress and decreased expression of α-SMA and In this study, we analyzed liver proteins in shRNA-treated mice using iTRAQ-based quantitative proteomics in order to identify differentially expressed proteins and to elucidate the therapeutic mechanisms of liver fibrosis.

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.
6 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.    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-7 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    Table S4).
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.

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. 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]. PPARα 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.

Discussion 11
Chronic HBV infection is a major health problem in developing countries, including China, and up to one-third of chronically HBV-infected individuals will progress to fibrosis, cirrhosis, and even HCC [55][56][57]. Liver fibrosis involves inflammation induced by a vicious circle of hepatic damage, driving HSC activation and worsening liver damage [9,10]. Liver fibrosis is a reversible process that represents the pivotal early stage of hepatic cirrhosis [58], and few therapies for liver fibrosis have been developed. Thus, it is necessary to elucidate the mechanisms of hepatic fibrosis and develop new medicines for blocking and reversing hepatic fibrosis. Our previous study showed that AAVshRNA had anti-hepatic fibrosis effects in HBV-replicated mice with liver fibrosis [44]. Moreover, fibrotic markers, including α-SMA, collagen I, and collagen III, were significantly reduced. However,the mechanisms mediating the antifibrotic effects of AAVshRNA are still unclear. In this study, ITRAQ-based quantitative proteomics was used to elucidate the antifibrotic mechanism of AAVshRNA. Through a comprehensive analysis comparing the treatment group and HBV(+) mice, we found that ribosomal proteins, downstream proteins of the PPAR signaling pathway, and inflammation-and oxidative stress-related proteins were significantly enriched in the AAVshRNA-treated group. In order to elucidate the mechanisms of liver fibrosis, we investigated the involvement of oxidative stress, the PPAR signaling pathway, and inflammation, which are closely associated with liver fibrosis.
In this study, reduction of TGF-β expression was observed following treatment withAAVshRNA-TGF-β by direct inhibition of TGF-β mRNA at the transcript level, resulting in upregulation of PPAR-γ. The findings indicated that AAVshRNA treatment alleviated oxidative stress by reducing TGF-β expression. PRDXs, as redox-regulating proteins, function to eliminate various ROS and maintain cellular redox homeostasis [66]. PRDX1 can be easily over oxidized on its catalytically active cysteine upon stimulation with various stimuli [60]. PRDX1 was significantly upregulated in HBV(+) mice compared with that in HBV(-) mice and was downregulated after treatment, indicating that oxidative stress was reduced. As an important phase II enzyme, GSTP1 can protect cells from oxidative stress in human cancers [59,67]. In accordance with a previous study, we found that GSTP1 was elevated to alleviate oxidative stress and played a critical role in antioxidant defense after AAVshRNA treatment. Collectively, these findings showed that AAVshRNA treatment could prevent oxidative stress through suppressing the oxidative stress inducers TGF-β and PRDX1 and enhancing GSTP1 expression.
In the PPI network, proteins up-or downstream of the PPAR signaling pathway (including ACAA1, ME1, and FABP1) were found to be regulated, suggesting activation of the PPAR signaling pathway. Notably, FABP1 and ME1 were downregulated in the PPAR signaling pathway, as demonstrated by KEGG analysis. These proteins also played pivotal roles in fatty acid synthesis and transport. ACAA1 is broadly expressed in humans and animals and can catalyze free cholesterol and long-chain fatty acids to synthesize esterified cholesterol [68]. ACAA1 is also a marker of beta-oxidation [69,70]. ME1 is the cytoplasmic component of the NADPH pool and is used by fatty acid synthase as a primary lipogenic enzyme. ME1 is also dysregulated in many types of cancers and is involved in tumorigenesis and metastasis [71,72]. . Therefore, inhibition of the NF-κB pathway may have therapeutic effects on liver fibrosis. In this study, we also found that NF-κB p65 phosphorylation was inhibited in treated mice compared with that in HBV(+) mice and in cells transfected with AAVshRNA. Overall, these data suggested that AAVshRNA inhibited liver fibrosis by blocking the NF-κB pathway.
Based on these findings, we proposed an antifibrotic model for AAVshRNA (Figure.

Protein preparation and iTRAQ labeling
Total protein extraction was performed using a kit (FOCUS-Mammalian Proteome; G-Biosciences, USA) in accordance with the manufacturer's instructions. Protein samples were stored at −80°C for proteomic analysis and western blotting. The iTRAQ method was described previously [52]. Briefly, total protein concentrations were determined using an EZQ Protein Quantitation Kit (Invitrogen, USA), and protein samples from treated mice, HBV(+) mice, and HBV(-) mice were reconstituted in dissolution buffer, denatured, reduced, and trypsinzed. Next, tryptic digests of the samples were labeled with iTRAQ reagents (Table S1). All samples were balanced, mixed, and preseparated for liquid chromatography (LC)-mass spectrometry (MS)/MS analysis.

Nano-LC−MS/MS analysis
LC-MS/MS analysis was performed with an Easy-nLC1000 (Thermo Fisher Scientific, USA) and Q ExactiveMS (Thermo Fisher Scientific). A reversed-phase ReproSil-PurC18-AQ column (column: 3 μm, 120 Å, 100 μm×10 cm) was used to separate the peptides at a flow rate of 600 nL/min. The LC linear gradient elution was performed from 6% to 9% B for 15 min, 9% to 14% B for 20 min, 14% to 30% B for 60 min, 30% to 40% B for 15 min, and 40% to 95% B for 3 min, followed by elution with 95% B for 7 min. A precursor scan was performed using an Orbitrap instrument by scanning from m/z 300--650 for detection with Q ExactiveMS. The MS resolution was 60000 at 400 m/z. The parameters of MS/MS settings were as follows: the product ion scan range started atm/z 100; the activation type was CID; the minimum signal required was 1500; the isolation width was 3; the normalized collision energy was 40; the default charge state was 6; the activation Q was 0.25; the activation time was 30 s; and the data dependent MS/MS was up to the top 5 most intense peptide ions from the preview scan in the Orbitrap.

Functional Analysis of differentially expressed proteins
In order to reduce false positives of differentially expressed proteins, an additional cut off of fold change greater than 1.30 or less than 0.77 (1/1.3) was exploited for all iTRAQ ratios [53]. Proteins with iTRAQ ratios greater than 1.30 or less than 0.77 were considered upregulated or downregulated, respectively. Gene ontology (GO) annotations, pathway enrichment, and protein-protein interaction (PPI) networks for all the identified proteins and differentially expressed proteins were evaluated with Omicsbean (http://www.omicsbean.cn). GO annotations were classified into three major categories, including biological processes (BPs), cell components (CCs), and molecular functions (MFs). Pathway enrichment analysis was performed with Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping. PPI networks were applied to obtain key nodes, such as degree centrality, betweenness, closeness, and cluster coefficient.

Immunoblotting analysis
For immunoblotting, 10 μg protein from liver tissue was separated by sodium dodecyl The membranes were then incubated with goat anti-rabbit secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Finally, the signal was visualized using an electrochemiluminescent reagent kit (Millipore Corporation, Billerica, MT, USA), and blots were imaged using X-ray film.

Cell lines
LX-2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C in a humidified incubator with 5% CO2. LX-2 cells were seeded into 6-well plates at 4×105cells/well and cultured. pSSV9-HBV1.2 was transfected into LX-2 cells using Lipofectamine 2000(Thermo Fisher Scientific) with or without pAAV-shRNAs (3μg) according to the manufacturer's instructions. Lipofectamine was used as a negative control. The supernatants and transfected cells were collected 72 h after transfection and subjected to protein extraction with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific).The concentration of protein was determined with a Thermo Scientific Pierce BCA Protein Assay Kit.

Statistical analysis
The data are reported as means ± standard deviations. One-way analysis of variance (GraphPad Prism 5.0) was used to determine statistically significant differences between groups. Differences with P values of less than 0.05 were considered statistically significant.  Table 1. Baseline characteristics of mice used in this study.

Supplementary Files
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