Direct-acting antivirals sofosbuvir and daclatasvir attenuate carbon tetrachloride-induced liver fibrosis in mice

Abstract

Background and aim

Advanced liver fibrosis is a major risk for developing hepatocellular carcinoma (HCC) in chronic hepatitis C virus (HCV) patients. Direct-acting antivirals (DAAs) which are used for treating HCV infection, produce more than 90% cure rate but do not seem to diminish the rate of occurrence or recurrence of HCC. This study aimed to investigate the effect of DAAs sofosbuvir (SOF) and daclatasvir (DAC) on carbon tetrachloride (CCl₄)-induced fibrotic changes in mice.

Methods

Eighty adult male Swiss albino mice were randomly allocated into 8 groups (10 mice/group): normal control group, SOF group (receiving SOF 80 mg/kg body weight (BW), oral gavage, daily), DAC group (receiving DAC 30 mg/kg BW, oral gavage, daily), SOF + DAC group (receiving a combination of both, daily), CCl4 model group (receiving CCl₄ 2 mL/kg BW, intraperitoneal twice weekly) and three CCl₄-intoxicated groups receiving either SOF or DAC or their combination. All CCl₄ groups received CCl₄ for 12 weeks followed by DAAs for another 12 weeks.

Results

CCl₄-induced a significant elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and produced histopathological evidence of fibrosis and liver degeneration along with a significant increase (P ≤ 0.001) of the proliferation markers (proliferating cell nuclear antigen (PCNA) and Ki-67), hepatic stellate cells (HSCs) activation markers (alpha-smooth muscle actin (α-SMA) and glial fibrillary acidic protein (GFAP)), fibrosis marker (matrix metalloproteinase-9 (MMP-9)) and pro-inflammatory cytokine (tumor necrosis factor-alpha (TNF-α)). CCl₄-intoxicated mice treated with SOF, DAC, or their combination revealed a significant amelioration (P ≤ 0.001) of CCl₄-induced elevation of liver enzymes, fibrotic changes, and liver degeneration along with a significant attenuation (P ≤ 0.001) of CCl₄-induced upregulation of all tested markers. The effects of SOF, DAC, and their combination on liver enzymes were comparable while the effect of SOF + DAC combination on mitigating CCl₄-induced upregulation of the proliferation and HSCs activation markers was significantly stronger than either SOF or DAC alone. As for MMP-9 and TNF-α, the effects of DAC and SOF + DAC combination were comparable and both were more significant than that of SOF alone.

Conclusions

SOF and DAC may possess an antifibrotic effect that is independent of their role as antiviral agents against CCl₄-induced liver injury. This might exclude the role of DAAs in early occurrence or accelerated recurrence of HCC through the progression of the HCV patients' pre-existing fibrosis. However, HCC patients treated with DAAs should be closely monitored with continuous HCC surveillance during and post-therapy.

1. Introduction

Hepatocellular carcinoma (HCC) is ranked the sixth most prevalent cancer around the world and one of the leading causes of cancer-related mortality. It is also the most common primary liver cancer.¹ Chronic hepatitis C virus (HCV) infection is considered a major risk factor for developing HCC, especially in cirrhotic patients. This risk is due to the induction of chronic inflammation and most importantly fibrosis.² Chronic HCV infection leads to liver fibrosis which is a wound healing process characterized by the activation of hepatic stellate cells (HSCs) which normally store vitamin A and express glial fibrillary acidic protein (GFAP), into the extracellular matrix (ECM)-producing myofibroblasts. The activated HSCs are proliferating and characterized by high production of alpha-smooth muscle actin (α-SMA). They promote ECM accumulation by collagen type I and III which leads to the formation of a fibrotic scar.^3,4 In response to liver injury, Kupffer cells and macrophages produce pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) which is believed to have a role in inflammation, proliferation, and fibrosis. By activating the nuclear factor kappa B (NF-κB) pathway, TNF-α induces the production of matrix metalloproteinase-9 (MMP-9), a key player in ECM remodeling. MMP-9 promotes hepatocyte proliferation in addition to HSC activation through the induction of the profibrogenic factor transforming growth factor-beta (TGF-β).^5,6

It has been suggested that chronic liver injury like HCV infection, could lead to hepatocyte death which subsequently initiates inflammation, liver fibrosis, excessive production of reactive oxygen species (ROS), and DNA damage. It also triggers compensatory hepatocyte proliferation which comes as a consequence of the necrosis produced by the liver injury. Repetitive cycles of these destructing-regenerating mechanisms are suggested to launch replication-related mutations in the hepatocytes which ultimately lead to HCC.⁴ Liver fibrosis is strongly correlated to HCC as it has been found in up to 90% of HCC patients and indirect measures of fibrosis like liver stiffness and high fibrosis index were found to be positively correlated with the risk of developing HCC.7, 8, 9 Liver fibrosis has also been linked to the recurrence of HCC after curative procedures like resection or ablation.^10,11

The combination of pegylated interferon (PegIFN) and ribavirin used to be standard therapy for HCV patients before the development of the direct-acting antivirals (DAAs) which provided an all-oral, non-interferon treatment of HCV infection with a much higher cure rate, shorter period of treatment, and better tolerability.¹² The DAAs regimens are based on combining molecules targeting different non-structural (NS) proteins that are crucial for the RNA replication process of HCV. DAAs are divided into three classes that have both different antiviral potencies and barriers to resistance: the protease inhibitors (anti-NS3/4A) such as simeprevir, the RNA-dependent polymerase inhibitors (anti-NS5B) such as sofosbuvir (SOF), and anti-NS5A such as daclatasvir (DAC). SOF and DAC are both pangenotypic, which means they can be used to produce sustained virological response (SVR) in almost all HCV genotypes. The combination of SOF and DAC is one of the widely endorsed combinations worldwide for the treatment of several genotypes of HCV as it produces extremely high SVR rates in almost all HCV genotypes regardless of the disease state of the liver.¹³

There has been evidence that the eradication of HCV by antiviral therapy reduces the risk of developing HCC.¹⁴ Before DAAs, the PegIFN and ribavirin-based therapy led to an average cure rate of 50% in the treated patients and SVR showed a correlation with the diminished risk of developing HCC.^15,16 When DAAs were introduced, they produced SVR in more than 90% of the treated patients even in those with advanced fibrosis.¹⁷ This has raised the expectations that successful DAAs treatment would lead to a decline in the rate of development of HCV-related HCC due to the improvement of the chronic inflammation related to the viral infection.¹⁸

Several studies investigated the effect of the DAAs therapy on the development of HCC in HCV-related cirrhotic patients expecting to observe a drastic decline in the rate of HCC occurrence and even a decline in its recurrence in patients who used to have HCC but underwent curative procedures. However, the results were surprising as it was found in one study that 3.16% of the patients with no history of HCC developed it after the DAA treatment and in another study, about 27%–29% of the patients who had a history of HCC but underwent curative procedures, had recurrence post-treatment.^18,19

Since liver fibrosis has a strong positive correlation with the development of HCC in HCV patients, this study was undertaken to investigate the effect of DAAs, represented by a combination of SOF and DAC, on carbon tetrachloride (CCl₄)-induced liver fibrosis in mice. Liver enzymes were assayed and fibrotic changes were assessed by histopathological and immunohistochemical examination where the latter estimated expression of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67, HSCs activation markers α-SMA and GFAP, fibrotic marker MMP-9 in addition to pro-inflammatory cytokine TNF-α.

2. Materials and methods

2.1. Ethical approval

All animal protocols were conducted according to the National Institute of Health guidelines for the care and use of laboratory animals and approved by the Ethical Committee of the Faculty of Pharmacy, Tanta University, Tanta, Egypt.

2.2. Animals

Adult male Swiss albino mice (Animal Unit Facility, Faculty of Pharmacy, Mansoura University, Dakahliya, Egypt) aging 6–10 weeks and weighing 20–25 g were given access to standard mice chow and water ad libitum. The mice were placed in plastic cages (5 mice per cage) under the controlled laboratory conditions of temperature (25 ± 2 °C) and 12 h light/dark cycles. After a 7-day acclimatization period, the animals were randomly divided into 8 groups (see below).

2.3. Induction of liver fibrosis

Fibrosis was induced using CCl₄ where 2 mL/kg of CCl₄ 50% (v/v) solution in paraffin oil (Al Gomhouria Co., Cairo, Egypt), was administered to mice via intraperitoneal (i.p.) injections twice weekly (Sunday and Thursday) for 12 weeks. The mice were treated with phenobarbital (0.3 g/L in drinking water) to enhance CCl₄ hepatotoxicity.²⁰

2.4. Administration of DAAs

SOF (Eva Pharma Co., Cairo, Egypt) and DAC (Eva Pharma Co., Cairo, Egypt) were freshly prepared immediately before use by suspending in distilled water and were orally administered by gavage daily for 12 weeks at 80 mg/kg body weight (BW) and 30 mg/kg BW for SOF and DAC, respectively.21, 22, 23

2.5. Design of the study

A total of 80 mice were randomly divided into 8 groups (10 mice for each group): normal control group; SOF group: mice daily received SOF (oral gavage, 80 mg/kg BW) for 12 weeks; DAC group: mice daily received DAC (oral gavage, 30 mg/kg BW) for 12 weeks; SOF + DAC group: mice daily received both SOF (80 mg/kg BW) and DAC (30 mg/kg BW) by oral gavage for 12 weeks; CCl₄ model group: mice were administered CCl₄ (i.p., 2 mL/kg BW) twice a week for 12 weeks; CCl₄+SOF group: mice were i.p. injected CCl₄ (2 mL/kg BW) twice a week for 12 weeks, then orally administered SOF (80 mg/kg BW) by gavage once a day for another 12 weeks; CCl₄+DAC group: mice were i.p. injected CCl₄ (2 mL/kg BW) twice a week for 12 weeks, then orally administered DAC (30 mg/kg BW) by gavage once a day for another 12 weeks; CCl₄+SOF + DAC group: mice were i.p. injected CCl₄ (2 mL/kg BW) twice a week for 12 weeks then orally administered both SOF (80 mg/kg BW) and DAC (30 mg/kg BW) by gavage for another 12 weeks, completing 24 weeks from the beginning of the experiment.

2.6. Preparation of blood samples and liver specimens

At 24 h from the last treatment dose, the mice were anesthetized with diethyl ether, and blood samples were collected using direct cardiac puncture. The collected blood was allowed to clot at room temperature, then centrifuged at 3000×g for 10 min, for separation and collection of sera, which were then stored at −20 °C for biochemical analysis. Afterwards, 5 mice from each group were sacrificed under anesthesia, and the livers were immediately excised. Liver specimens were fixed in 10% neutral buffered formalin and embedded in paraffin for histopathological and immunohistochemical studies.

2.7. Biochemical analysis

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in the collected sera according to the manufacturer's instructions and previously reported method of determination,²⁴ using assay kits obtained from Biodiagnostics Company (Cairo, Egypt).

2.8. Histopathology

Liver sections (4 μm thick) were cut, processed, and stained with either hematoxylin and eosin (H&E) for studying histopathological changes or Masson's trichrome stain for determination of the area-based percentage of reactive collagen fibers. Data were obtained using a full HD microscopic imaging system (Leica Microsystems GmbH, Germany) and operated by Leica Application software for tissue sections' analysis.

2.9. Immunohistochemistry (IHC)

IHC staining for PCNA, Ki-67, α-SMA, GFAP, MMP-9, and TNF-α was done for all animal groups. All histochemical staining was conducted according to the manufacturer's protocol. Deparaffinized sections were immersed in citrate buffer for antigen retrieval and then treated with 0.3% H₂O₂ to block endogenous peroxidase. After that, they were incubated overnight at 4 °C with the primary antibodies anti-PCNA (Cat.# PA5-32541, Invitrogen, USA; dilution 1/100), anti-Ki-67 (Cat.#M7248, Dako, USA; dilution 1/100), anti-α-SMA (Cat.# PA5-16697, Invitrogen, USA; dilution 1/100), anti-GFAP (Cat.# MA5-11757, Invitrogen, USA; dilution 1/200), anti-MMP-9 (Cat.# PA5-13199, Thermofisher, USA; dilution 1/100) and anti-TNF-α (Cat.# NBP1-19532, Novus Bio. Co., USA; dilution 1/100). Afterwards, they were rinsed with phosphate-buffered saline (PBS) and incubated with the secondary antibody horseradish peroxidase (HRP) Envision kit (DAKO) for 30 min at room temperature. Slides were visualized with a diaminobenzidine (DAB) kit and eventually stained with Mayer's haematoxylin as a counterstain. The labeling indexes of PCNA and Ki-67 were assessed and presented as the percent of positive cells per 1000 cells. Staining intensity for GFAP and α-SMA were determined using ImageJ analysis software (NIH, USA) and data were expressed as the percent of positive area. Data for MMP-9 and TNF-α were expressed as the area percentage of immunohistochemical expression, obtained using a Full HD microscopic imaging system (Leica Microsystems GmbH, Germany) and operated by Leica Application software for tissue sections analysis.

2.10. Statistical analysis

Descriptive statistics are presented in the form of means and standard deviations of the percentage of positive cells in each group. Testing for the effect of the different drugs, the effect of the disease, and the interaction of the disease and drugs was done using two-way analysis of variance (ANOVA). Due to the presence of interaction, the analysis of the simple main effect of the drugs and the simple main effect of the disease is presented with statistical significance by receiving a Bonferroni adjustment. IBM SPSS statistics 26.0 (IBM Corp, NY, USA) for Windows software was used for the analysis. P < 0.05 is considered statistically significant.

3. Results

3.1. SOF and DAC ameliorate CCl₄-induced elevation of liver injury biomarkers in mice

As described in Table 1, a significant increase (P ≤ 0.001) in the activities of ALT and AST were observed in the CCl₄ model group, as compared to the normal control group. CCl₄-intoxicated mice treated with SOF showed a significant decline (P ≤ 0.01) of ALT mean level by 36.4% while mean AST level was significantly reduced (P ≤ 0.001) by 40.7%, as compared to the CCl₄ model group. CCl₄+DAC and CCl₄+SOF + DAC groups revealed a significant reduction (P ≤ 0.001) of 51.6% and 57.5% for ALT, and 50.3% and 55.0% for AST mean levels, as compared to the CCl₄ model group, respectively. There were no significant differences between the CCl₄-intoxicated groups treated with SOF, DAC or their combination for either ALT or AST levels neither were between the normal control group nor the other treated control groups.

Table 1.

Effects of SOF and DAC on levels of serum ALT and AST of mice.

Group	ALT (U/L)	AST (U/L)
Normal control	31.35 ± 4.09	46.51 ± 3.25
SOF	33.64 ± 3.27	43.32 ± 2.80
DAC	31.72 ± 3.77	44.83 ± 3.88
SOF + DAC	36.57 ± 3.30	49.95 ± 3.57
CCl4	292.99 ± 42.46 ∗∗∗	474.38 ± 36.06 ∗∗∗
CCl4+SOF	186.37 ± 21.85 ##	281.48 ± 22.14 ###
CCl4+DAC	141.78 ± 14.88 ###	236.72 ± 12.42 ###
CCl4+SOF + DAC	124.45 ± 17.62 ###	213.40 ± 19.55 ###

Data are represented as the mean ± SEM (n = 5 per group). ∗∗∗P ≤ 0.001 vs. normal control group; ^##P ≤ 0.01, ^###P ≤ 0.001 vs. CCl₄ model group.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCl₄, carbon tetrachloride; DAC, daclatasvir; SOF, sofosbuvir.

3.2. SOF and DAC attenuate CCl₄-induced liver fibrosis in mice on a histopathological level

3.2.1. SOF and DAC alleviate CCl₄-induced histopathological manifestation of liver damage and fibrosis in mice

The liver sections of mice in the normal control group and both groups receiving either SOF or DAC showed normal hepatic architecture with normal hepatocytes arranged in cords radiating from the central vein. There were also normal hepatic sinusoids between hepatocytes plates lined by endothelial cells (Fig. 1a, b, and c) while mild hepatic degeneration characterized by granular hepatic cells was observed in sections of mice receiving a combination of SOF and DAC (Fig. 1d).

Fig. 1.

Histopathological examination of mice livers stained with H&E. Normal control group (a): showing normal hepatocytes arranged in cords around the central vein (arrow), which is similar to control groups treated with SOF or DAC (arrow) (b and c, respectively). Control group treated with SOF + DAC combination (d): showing mild hepatic degeneration (arrow indicates granular hepatic cells). CCl₄ model group: showing perivascular fibrosis (arrow) and necrotic changes (e) in addition to periportal both hepatic necrosis (arrowheads) and fibroblastic cell proliferation (arrows) (i). CCl₄+SOF group is showing moderate attenuation of CCl₄-induced fibrosis (arrow, f) in addition to a mild degree of periportal mononuclear inflammatory cells infiltration (arrow) and decrease of the hepatic vacuolation (arrowhead) (j). CCl₄+DAC group is showing moderate fibrosis (arrows) (g) as well as a mild degree of hepatic vacuolation (arrows) (k). CCl₄+SOF + DAC group is showing a marked decrease of interstitial and perivascular fibrosis (arrow) (h) and of hepatic degeneration unless little hepatic vacuolation (arrow) (l), as compared to the CCl₄ model group (e and i). Original magnification, ×200. Abbreviations: CCl₄, carbon tetrachloride; DAC, daclatasvir; H&E, hematoxylin and eosin; SOF, sofosbuvir.

In contrast to normal control group, liver sections of CCl₄-intoxicated groups revealed varying degrees of fibrosis and hepatic degeneration. Mice that only received CCl₄, showed perivascular fibrosis, necrotic changes (Fig. 1e) in addition to periportal hepatic necrosis, fibroblastic cell proliferation (Fig. 1i), mononuclear inflammatory cell infiltration, and hepatic vacuolation. Compared to mice only receiving CCl₄, those which received either SOF or DAC in addition to CCl₄ showed a moderate decrease in both fibrosis (Fig. 1f and g) and necrotic changes except for a mild degree of periportal mononuclear inflammatory cells infiltration. They also showed a decrease in hepatic vacuolation (Fig. 1j and k). Sections of CCl₄-intoxicated mice which received a combination of SOF and DAC showed a marked decrease in fibrosis with only mild hepatic vacuolation (Fig. 1h and l).

3.2.2. SOF and DAC attenuate CCl₄-induced collagen deposition in mice

Liver sections from normal control mice as well as other control mice treated with SOF, DAC, or their combination, showed normal distribution of collagen fibers (Fig. 2A) and there were no significant differences between all these four groups (Fig. 2B). On the other hand, mice in the CCl₄ model group revealed extensive collagen deposition (Fig. 2A) that was significantly higher (P ≤ 0.001) than that of the normal control group (Fig. 2B) suggesting liver fibrosis. Collagen deposition was significantly diminished (P ≤ 0.001) in the CCl₄-intoxicated mice treated with SOF, DAC, or their combination (Fig. 2A), as compared to the CCl₄ model group (Fig. 2B). There was no significant difference in collagen deposition between CCl₄-intoxicated mice treated with either SOF or DAC, but those treated with SOF + DAC combination showed significantly (P ≤ 0.001) less fibrotic areas than mice treated with either of them alone (Fig. 2B).

Fig. 2.

Effect of SOF and DAC on collagen deposition in liver tissues of mice.(A) Representative images of liver sections stained by Masson's trichome stain. Original magnification, ×400. (B) Quantification of collagen deposition in liver tissue sections. Data are expressed as the means ± SEM (n = 6 per group). ∗∗∗P < 0.001 vs. normal control group. ^###P < 0.001 vs. CCl₄ model group. ^$$$P < 0.001 vs. CCl₄-intoxicated group treated with SOF + DAC combination. NS vs. normal control group. Abbreviations: CCl₄, carbon tetrachloride; DAC, daclatasvir; H&E, hematoxylin and eosin; NC, normal control; NS, not significant; SEM, stand error of the mean; SOF, sofosbuvir.

3.3. Immunohistochemical expression of several-associated biomarkers

3.3.1. SOF and DAC diminish CCl₄-induced hepatic cell proliferation in mice

Immunohistochemical staining images revealed that the number of positive cells of PCNA and Ki-67 was increased in the CCl₄ model group and decreased by SOF, DAC, and combination treatment (Fig. 3A and C). The mean expression was the highest in the CCl₄ model group, at 67.7% and 47.7% for PCNA and Ki-67, respectively, which was significantly higher (P ≤ 0.001) than that of the normal control group (Fig. 3B and D). PCNA expression levels in hepatocytes demonstrated a significant reduction (P ≤ 0.001) in CCl₄-intoxicated groups that received SOF, DAC, and combination by 31.4%, 33.0%, and 57.6% as compared to the CCl₄ model group, respectively (Fig. 3B). Mean Ki-67 immune expression was also significantly reduced (P ≤ 0.001) in the CCl₄ groups that received either SOF, DAC, or combination reaching a 31.4%, 38.2%, and 60.0% reduction in comparison with the CCl₄ model group, respectively (Fig. 3D). The increase in proliferating hepatocytes characterized by increased PCNA and Ki-67 nuclear expressions in the CCl₄ model group was mainly periportal.

Fig. 3.

An anti-proliferative effect of SOF and DAC is proven by PCNA and Ki-67 downregulation.(A) Representative immunohistochemical staining of liver PCNA staining images are shown. Original magnification, × 200. (B) Quantification of PCNA-positive cells. (C) Representative immunohistochemical staining of liver Ki-67 staining images are shown. Original magnification, × 200. (D) Quantification of Ki-67-positive cells. In all statistical plots, data are expressed as the mean ± SEM (n = 5 per group). ∗∗∗P ≤ 0.001 vs. normal control group. ^###P ≤ 0.001 vs. CCl₄ model group. ^$$$P ≤ 0.001 vs. CCl₄-intoxicated group treated with SOF + DAC combination. NS vs. normal control group. Abbreviations: CCl₄, carbon tetrachloride; DAC, daclatasvir; NC, normal control; NS, not significant; PCNA, proliferating cell nuclear antigen; SEM, stand error of the mean; SOF, sofosbuvir.

3.3.2. SOF and DAC mitigate CCl₄-induced activation of HSCs in mice

Immunohistochemical staining images revealed that the number of α-SMA-positive cells and GFAP-positive cells was increased in the CCl₄ model group and decreased by SOF, DAC, and combination treatment (Fig. 4A and C). Both α-SMA and GFAP showed similar staining patterns where the highest level was detected in the CCl₄ model group at 41.6% and 76.7% for α-SMA and GFAP, respectively, which was significantly higher (P ≤ 0.001) than that of the normal control group (Fig. 4B and D). Immune expression of α-SMA which was mainly periportal (Fig. 4A) was significantly reduced (P ≤ 0.001) in CCl₄-intoxicated groups which received SOF, DAC, and combination by 45.8%, 46.3%, and 76.3% respectively as compared to the CCl₄ model group (Fig. 4B). Similarly, GFAP expression was significantly reduced (P ≤ 0.001) in the same groups by 51.7%, 50.7%, and 75.9% for CCl₄-intoxicated groups treated with SOF, DAC, and combination, respectively (Fig. 4D).

Fig. 4.

Inhibitory effect of SOF and DAC on HSCs activation is proven by α-SMA and GFAP downregulation.(A) Representative immunohistochemical staining of liver α-SMA staining images are shown. Original magnification, × 200. (B) Quantification of α-SMA-positive cells. (C) Representative immunohistochemical staining of liver GFAP staining images are shown. Original magnification, × 200. (D) Quantification of GFAP-positive cells. In all statistical plots, data are expressed as the mean ± SEM (n = 5 per group). ∗∗∗P ≤ 0.001 vs. normal control group. ^###P ≤ 0.001 vs. CCl₄ model group. ^$$$P ≤ 0.001 vs. CCl₄-intoxicated group treated with SOF + DAC combination. NS vs. normal control group. Abbreviations: α-SMA, alpha-smooth muscle actin; CCl₄, carbon tetrachloride; DAC, daclatasvir; GFAP, glial fibrillary acidic protein; HSCs, hepatic stellate cells; NS, not significant; SEM, stand error of the mean; SOF, sofosbuvir.

3.3.3. SOF and DAC attenuate CCl₄-induced fibrosis in mice

Immunohistochemical staining revealed the highest expression of MMP-9 to be in the CCl₄ model group (Fig. 5A) which was significantly higher (P ≤ 0.001) than that of the normal control group (Fig. 5B) that presented with minimal expression of MMP-9 along with other control groups treated with SOF, DAC, or their combination. There was no significant difference in MMP-9 expression among all four control groups (Fig. 5B). CCl₄-intoxicated mice treated with SOF, DAC, or their combination revealed a significantly mitigated (P ≤ 0.001) MMP-9 expression by 24.6%, 52.3%, and 59.0%, as compared to the CCl₄ model group, respectively (Fig. 5B). No significant difference was found between CCl₄-intoxicated mice groups treated with either DAC or SOF + DAC combination, however, both groups showed significantly lower (P ≤ 0.001) MMP-9 expression than the CCl₄-intoxicated group treated with SOF (Fig. 5B).

Fig. 5.

Effect of SOF and DAC on the fibrosis marker MMP-9.(A) Representative immunohistochemical staining of liver MMP-9 staining images are shown. Original magnification, × 200. (B) Quantification of MMP-9 expression. Data are expressed as the mean ± SEM (n = 6 per group). ∗∗∗P < 0.001 vs. normal control group. ^###P < 0.001 vs. CCl₄ model group. ^$$$P < 0.001 vs. CCl₄-intoxicated group treated with SOF + DAC combination. NS vs. CCl₄-intoxicated group treated with SOF + DAC combination. Abbreviations: CCl₄, carbon tetrachloride; DAC, daclatasvir; MMP-9, matrix metalloproteinase-9; NS, not significant; SEM, stand error of the mean; SOF, sofosbuvir.

3.3.4. SOF and DAC lower CCl₄-induced inflammation in mice

The expression of TNF-α was increased in the CCl₄ model group (Fig. 6A), which was significantly higher (P ≤ 0.001) than that of the normal control group (Fig. 6B). SOF, DAC, and their combination significantly alleviated the CCl₄-induced TNF-α upregulation, by 43.3%, 62.3%, and 72.0% as compared to the CCl₄ model group, respectively (Fig. 6B). Similar to the results of MMP-9 expression, there was no significant difference between the CCl₄-intoxicated groups treated with DAC or SOF + DAC combination, but both groups revealed significantly lower (P ≤ 0.001) expression of TNF-α as compared to the CCl₄-intoxicated group treated with SOF (Fig. 6B).

Fig. 6.

Effect of SOF and DAC on TNF-α.(A) Representative immunohistochemical staining of liver TNF-α staining images are shown. Original magnification, × 200. (B) Quantitative analysis for TNF-α expression. Data are expressed as the mean ± SEM (n = 6 per group). ∗∗∗P < 0.001 vs. normal control group. ^###P < 0.001 vs. CCl₄ model group. ^$$$P < 0.001 vs. CCl₄-intoxicated group treated with SOF + DAC combination. NS vs. CCl₄-intoxicated group treated with SOF + DAC combination. Abbreviations: CCl₄, carbon tetrachloride; DAC, daclatasvir; NS, not significant; SEM, stand error of the mean; SOF, sofosbuvir; TNF-α, tumor necrosis factor-alpha.

4. Discussion

DAAs are known to produce SVR in more than 90% of HCV patients.¹⁷ However, unlike expectations, several studies found that DAAs did not seem to diminish the rate of development of HCC or its recurrence in patients who underwent curative procedures.^18,19 Since fibrosis is a major risk factor for HCC, this study investigated the effect of SOF, DAC, and their combination on the fibrotic changes induced in mice by CCl₄. SOF and DAC were found to possess an antifibrotic effect against CCl₄-induced fibrosis which was evident by a decline in HSCs activation markers α-SMA and GFAP, proliferation markers PCNA and Ki-67, profibrogenic marker MMP-9, and pro-inflammatory cytokine TNF-α and confirmed by histopathological changes. The antifibrotic effect was found when the drugs were used either separately or in combination showing an augmented effect in the latter (Fig. 7).

Fig. 7.

Brief summary of the effects of SOF and DAC on CCl₄-induced liver fibrosis. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; α-SMA, alpha-smooth muscle actin; CCl₄, carbon tetrachloride; DAC, daclatasvir; GFAP, glial fibrillary acidic protein; H&E, hematoxylin and eosin; MMP-9, matrix metalloproteinase-9; NC, normal control; PCNA, proliferating cell nuclear antigen; SOF, sofosbuvir; TNF-α, tumor necrosis factor-alpha. This figure was created with Biorender.com.

CCl₄ model was employed for induction of liver fibrosis in mice due its high reproducibility, fast induction, and ability to closely mimic the inflammatory process of chronic liver disease in humans.^4,20,25 CCl₄ initiate an inflammatory process through the stimulation of Kupffer cells leading to the production of several ROS and pro-inflammatory mediators like cyclooxygenase-2 (Cox-2) and interleukins which eventually results in HSCs activation and thus liver fibrosis.^4,26,27

In the current study, the application of CCl₄ to mice produced clear signs of fibrosis and hepatic degeneration which initially manifested as a marked increase in liver enzymes: ALT and AST. Histopathologically, CCl₄-intoxicated mice showed extensive deposition of collagen fibers in liver tissues stained with Masson's trichrome stain. With H&E stain, they presented with perivascular fibrosis, periportal hepatic necrosis, fibroblastic cell proliferation, mononuclear inflammatory cell infiltration, and hepatic vacuolation which is in accordance with the results produced by Rocha et al.²⁸ and Chhimwal et al.²⁹ CCl₄ intoxication also induced the activation of HSCs, being the central event of fibrosis, which manifested as a marked increase of α-SMA and GFAP levels which coincides with the findings of previous studies.29, 30, 31 Similar to the other finding,³² a marked increase in hepatocyte proliferation was recorded in the form of severe elevation of PCNA and Ki-67-positive hepatocytes with nuclear expression, mainly in the periportal area. CCl₄ also induced upregulation of both the fibrogenic marker MMP-9 and its regulator, the pro-inflammatory cytokine TNF-α, which is in agreement with several studies that showed significant elevation in the levels of both markers in fibrosis whether in humans or experimentally induced in animal studies.33, 34, 35

The present findings illustrated that both SOF and DAC exhibited remarkable hepatoprotective and antifibrotic effects which are independent of their role as antiviral agents. The antifibrotic effect was evident when either of them was used separately and found to be augmented when used in combination. Mice received oral doses of either SOF or DAC of 30 and 80 mg/kg BW, respectively, which was used based on several previous studies of SOF and DAC in mice.^21,22 In a similar pattern, both SOF and DAC also showed a moderate reduction of proliferation markers PCNA and Ki-67 when used separately and significantly greater reduction when used in combination. SOF and DAC also mitigated CCl₄-induced upregulation of the profibrogenic marker MMP-9 and the pro-inflammatory cytokine TNF-α with a significantly more profound effect experienced with DAC and SOF + DAC combination than SOF by itself which is in agreement with a previous study that examined the effect of SOF and DAC on both markers.³⁵

In support of the current findings, several human studies found SOF-based therapy whether used in combination with DAC or other DAAs could induce significant improvement of fibrosis in chronic HCV patients of different genotypes.36, 37, 38 Furthermore, several experimental models of fibrosis in rats identified the antifibrotic effect of SOF, DAC and their combination against liver damage by CCl₄ as well as other hepatotoxic agents like thioacetamide (TAA) regardless of their role as antiviral agents.^35,39 Ibrahim et al.³⁸ attributed the antifibrotic effect of SOF+ DAC to their anti-oxidant and anti-inflammatory effects by arguing that liver injury including HCV infection is usually accompanied by secretion of pro-inflammatory and profibrogenic mediators such as TNF-α and interleukin-1β from several types of cells like Kupffer cells, natural killer (NK) cells, lymphocytes among others. These mediators along with ROS produced by the liver injury lead to HSC activation, the main event of fibrosis. The findings of Ibrahim et al.³⁸ stated that SOF and DAC whether used separately or in combination, significantly reduced the inflammatory foci and fibrotic changes in TAA-intoxicated mice and their hepatoprotective effect was accompanied by amelioration of the fibrotic marker tissue inhibitors of metalloproteinase-1 (TIMP-1), the pro-inflammatory cytokine TNF-α and the oxidative stress parameters (malondialdehyde content, and superoxide dismutase and catalase activities) which suggests that SOF and DAC may possess hepatoprotective and antifibrotic effects which are carried out by anti-inflammatory and anti-oxidant mechanisms independent of their role as antiviral agents.³⁸ It's worth mentioning that unlike the findings by the current study, Ibrahim et al.³⁸ did not find giving SOF at a dose of 80 mg/kg for 4 weeks to improve the TAA-induced hepatotoxicity in rats. The current study found that using SOF at the mentioned dose for 12 weeks, showed a hepatoprotective effect that was augmented when used in combination with DAC in CCl₄-intoxicated mice. Future studies with different liver injury models, SOF doses, and duration of treatments are needed to confirm either find.

Despite being known to closely mimic the inflammatory process of chronic liver disease in humans,^4,20,25 the CCl₄ fibrosis model in mice is not without limitations. Most importantly, there are several species-specific differences between mice and humans in their gene regulation, immune response, and metabolic, pharmacological, and tissue responses.^39,40 That's why it is recommended that future studies in both humans and humanized mice models of HCV-induced fibrosis are carried out to further explore the effect of DAAs on fibrosis and HCC. Other study limitation is unexplained mild hepatic damage in control mice that received SOF + DAC combination, reflected only in histopathological examination with H&E stain with no other sign of damage in all tested markers and liver enzymes. Several case reports mentioned the possibility of drug-induced hepatotoxicity with different DAAs including SOF and DAC whether used together or either of them used with other DAAs.41, 42, 43, 44 Future investigations of the underlying mechanism of DAA-induced liver injury should be carried out. Besides, DAAs are known to have an inhibitory effect on the NK cells, the most prevalent innate immune cells in the liver, which then facilitates the progression of the neoplastic clone.¹⁹ It has also been proven that DAAs cause a rapid decline in the activation of NK cells, normalization of their cytotoxic effector functions in both the blood and the liver, and a decline in the serum level of the cytokines that activate them. This may result from the rapid decline of viremia following DAAs therapy.⁴⁵ Future studies are needed to investigate the potential role of DAAs in the disruption of immunosurveillance and its effect on the development of HCC in HCV patients.

In conclusion, the present study suggests that DAAs represented by SOF and DAC may have an antifibrotic effect independent of their role as antiviral agents. This might exclude the role of DAAs in early occurrence or accelerated recurrence of HCC through the progression of the existing fibrosis of HCV patients. However, HCC patients treated with DAAs should be closely monitored with continuous HCC surveillance during and post-therapy.

Authors’ contributions

M. M. Abdelsalam collected data, performed analyses, and also interpreted and drafted the manuscript. N. EI-Mahdy and S. Abou-Saif revised the manuscript and provided direct supervision and guidance. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.