Ameliorative effects of sildenafil against carbon tetrachloride induced hepatic fibrosis in rat model through downregulation of osteopontin gene expression

The liver carries out many essential tasks, such as synthesising cholesterol, controlling the body’s storage of glycogen, and detoxifying metabolites, in addition to performing, and regulating homeostasis. Hepatic fibrosis is a pathological state characterized by over accumulation of extracellular matrix (ECM) including collagen fibers. Sildenafil (a selective inhibitor of type 5 phosphodiesterase) has anti-inflammatory, antioxidant and anti-apoptotic properties. It is commonly used to treat erectile dysfunction in male. The purpose of the current investigation was to evaluate sildenafil’s hepatoprotective potential against liver fibrosis in rats that was caused by carbon tetrachloride (CCl4). Liver enzymes and oxidative markers as well as profibrotic genes were determined. The findings showed that sildenafil alleviates the hepatic dysfunctions caused by CCl4 by restoring normal levels of ALT, AST, and GGT as well as by restoring the antioxidant status demonstrated by increased glutathione (GSH), and catalase. In addition, a significantly down-regulated the mRNA expressions of profibrotic genes [collagen-1α, IL-1β, osteopontin (OPN), and transforming growth factor-β (TGF-β)]. Additionally, sildenafil lessens the periportal fibrosis between hepatic lobules, congestion and dilatation in the central vein, and the inflammatory cell infiltrations. As a result, it is hypothesized that sildenafil may be helpful in the management of hepatotoxicity brought on by CCl4 through suppressing OPN.


Experimental design
Four equal groups of ten rats each were randomly assigned to the experimental rats.The group 1 (G1) was preserved as a normal control, and the group 2 (G2) was administered orally sildenafil dissolved in saline by stomach tube at a dose of 20 mg/kg b.wt. 13.daily for 4 weeks.The group 3 (G3) (intoxicated group) was administered intraperitoneal (I/P) CCl 4 (30% in olive oil) at a dose of 3 mg/kg b.wt 14 .twice a week for 4 weeks.Group 4 (G4), which received co-treatment, received sildenafil via stomach tube at a dose of 20 mg/kg b.wt.daily and CCl 4 I/P at a dose of 3 mg/kg b.wt.twice a week for 4 weeks.Throughout the experiment, all groups' clinical symptoms were noted.At the end of the experiment, the experimental rats were euthanized using isoflurane inhalation.Blood for serum samples was collected from the abdominal aorta, and liver specimens were collected from all rats in each group at the end of the fourth week of the experiment for evaluation of liver function, oxidative markers, gene expression, histopathological changes, and immunohistochemically markers.

Preparation of liver homogenate
The liver tissue homogenate was made in compliance with Farid and Hegazy 15 .The total protein and the oxidative indicators [catalase (CAT), reduced glutathione (GSH), and lipid peroxidation by-products (MDA) level] were measured in the obtained supernatant.

Oxidative markers in liver homogenate
The levels of MDA 18 , GSH 19 , CAT 20 , and the total proteins content 21 were performed in the liver homogenates.

Hepatic mRNA gene expression
The mRNA expression of hepatic collagen-1α, interleukin-1β (IL-1β), osteopontin (OPN), and TGF-β genes were analyzed using a 7300 real-time PCR system (PCR) (Applied Biosystems, Foster City, CA, USA).The expression of hepatic collagen-1α, IL-1β, OPN, and TGF-β cytokines and β-actin (as a housekeeping gene) were analyzed with sense and antisense primers according to a previously published method 22 used the primers sets (Table 1).The changes in gene expression were estimated from the obtained cycle threshold (Ct) values provided by the real-time PCR equipment using the comparative Ct method to a reference (β-actin, housekeeping gene) 23 .
www.nature.com/scientificreports/Histopathological examinations Tissue specimens from liver of each rat were collected immediately after euthanized, rinsed with isotonic saline, and fixed in 10% neutral buffered formalin and dehydrated by serial ethanol solutions (70, 95, and 100%, respectively).Paraffin-embedded tissue sections (~ 5 μm thickness), and stained with hematoxylin and eosin (H&E) and with Masson's trichrome 24 .For a histopathological assessment, the slides were inspected using a light microscope (Olympus BX50, Japan).

Immunohistochemistry staining
The liver slices (5 µm) were adhered to glass microscope slides with positive charge.Sections deparaffinized and then rehydrated with decreasing alcohol concentration.For thirty minutes, a microwave was used to heatinduced antigen retrieval using a citrate buffer solution.After allowing the sections to cool at room temperature, nonspecific binding was stopped for 20 min at that temperature using regular goat serum.Incubation with the diluted primary antibodies [alpha-smooth muscle actin (α-SMA) Monoclonal Antibody, Catalog # 14-9760-82, Thermo Fisher Scientific, Manor Park, United Kingdom] at a dilution rate of 1:200 for 60 min at 37 ℃.After that, sections were exposed to a peroxidase suppressor for 15 min at room temperature in order to inhibit the activity of endogenous peroxidase.Subsequently, the DAB chromogen mixture was applied to each slide after they had been incubated for one hour at room temperature with the secondary antibody (goat anti-rat peroxidase conjugated streptavidin).PBS was used for washing and drying after each step.To show the nuclei, sections were counter-stained with Meyer's hematoxylin.A dark brown response indicated good immuno-expression.Except for adding the primary antibodies, a negative control underwent the previously described procedures 25 .

Image analysis
Using an Olympus CX51 microscope, the percentage of the area (area %) covered by the blue color in the Masson's trichrome staining or immunostaining area for α-SMA was examined, and photos were captured with an Olympus digital camera (E-620, United States).Digital image analysis was used to evaluate and quantify the images using the computer programme Scion Image Beta 4.03 (Scion Corporation, USA).

Statistical analysis
The information was shown as mean ± standard deviation (SD).ANOVA was used to determine the significance of mean differences, and Duncan's multiple range test was then performed using SPSS for Windows (version 20.0; SPSS Inc., Chicago, Ill.).The difference of means at P < 0.05 is considered significant.

Consent to participate
Acceptance of participation, each individual participant in this study provided both oral and written informed permission.

Results
No any symptoms or rat died in any of the experimental groups over the course of the experiment.

Liver function tests
After the fourth week of treatment, the CCl 4 -treated group's levels of ALT, AST, and GGT considerably increased in comparison to the other experimental groups.However, following the fourth week of the trial, ALT, AST, and GGT levels were mitigated in the group that received sildenafil and CCl 4 (Table 2).

Changes in the liver oxidative markers
When compared to the other experimental groups, the CCl 4 -treated group's liver tissue exhibited considerably lower GSH contents and lower CAT activities.However, following the fourth week of the trial, the group that received both sildenafil and CCl 4 co-treatment exhibited alleviation of these activities.When compared to the other experimental groups, the CCl 4 -treated group's levels of MDA, a marker of LPO in liver tissue, were also significantly higher; however, after the fourth week of the experiment, the group that received both sildenafil and CCl 4 co-treatment showed mitigation of MDA levels (Table 3).

Histopathological assessment of liver tissue
Examination of liver sections stained with H and E revealed that CCl 4 treated rats showed sever degenerative changes with distorted histological architecture, vacuolization (circle), sever congested central vein (CV), congested portal tract vasculature (PT), large inflammatory cells infiltrates (asterisk), some congested sinusoids (arrow), and other dilated sinusoids (angled arrow) were seen (Fig. 2B, C).Treatment of rats with sildenafil and CCl 4 showed improvement of the hepatic architecture but mild congestions between the hepatocytes were still noticed (Fig. 2D).The hepatic tissues of rats that administered sildenafil appeared to be normal (Fig. 2A).

Masson's trichrome stain examination
Examination of liver sections stained with Masson's trichrome showed that rats received CCl 4 displayed a significant increase in the amount of collagen fiber deposition in the portal area and around the central veins (Fig. 3B,  C).The Masson's trichrome area (%) was markedly increased in the CCl 4 treated group (G3) after 4 weeks of experiment (Fig. 3E).On the other hand, treatment of rats with sildenafil and CCl 4 showed reduced collagen fiber deposition (Fig. 3D).The Masson's trichrome area (%) was markedly decreased when rats treated with sildenafil and CCl 4 after 4 weeks of experiment (Fig. 3E).The minimal collagen expression just around the central veins were detected in the control, and sildenafil-treated groups (Fig. 3A).

Immunohistochemical assessment of liver tissue
Α-smooth muscle actin (α-SMA) immunohistochemical staining sections of liver tissues observed that rats exposed to CCl 4 revealed that α-SMA protein is highly expressed in the area of central vein, portal tract, and between hepatocytes indicating fibrotic changes (Fig. 4C).The α-SMA area (%) was markedly increased in the CCl 4 treated group (G3) after 4 weeks of experiment (Fig. 4E).While rats treated with sildenafil and CCl 4 showed mild α-SMA-expression, and the level of positivity is lower than in the CCl 4 treated rats (Fig. 4D).The α-SMA area (%) was markedly decreased in the sildenafil and CCl 4 treated group (G4) after 4 weeks of experiment (Fig. 4E).The α-SMA immunohistochemical staining sections of liver tissues of normal control, and sildenafiltreated rats showed a minimal reaction in the vascular wall (Fig. 4A, B).

Discussion
The purpose of this study was to investigate if sildenafil could adequately support liver function in Wistar rats given CCl 4 to induce liver fibrosis.Chronic liver diseases causes health problem with high morbidity and mortality rates all over the world 26 .Liver fibrosis, which is defined by an imbalance between excessive matrix synthesis and matrix breakdown, is the result of persistent tissue injury-induced defective wound healing 27 .It has been determined that HSCs are the primary cells that produce matrix along the course of liver fibrosis 28 .Multiple cell types of hepatic progenitor cells are involved in the process of liver repair; the HSCs or liver pericytes, and liver progenitor cells (LPCs) or oval cells are the most important cells involved in this process 29 .Chronic liver injury results in sustained activation of quiescent HSCs (qHPCs) which is transformed into active HPCs (aHPCs) or myofibroblasts, which secrete collagen into the ECM 30 .Furthermore, LPCs are reprogrammed into fiber secreting cells (epithelial to mesenchymal transition) and recruited immune cells (effector-profibrogenic) amplify the process of fibrogenic deposition 31 .
The current study revealed that the activity of ALT, AST and GGT markedly increased that indicated damage of the hepatic tissue of CCl 4 -treated group after 4 weeks administration.This could be the result of inflammation and hepatocellular damage, which raises the permeability of the cell membrane and releases transaminases into the blood stream.A similar results were recorded in rats treated with CCl 4

32
. GGT activity increase is linked to all types of primary and secondary hepatobiliary diseases; higher blood levels of GGT are associated with cholestasis brought on by intrahepatic or extrahepatic biliary blockage 33 .Marked decreases in serum ALT, AST, and GGT levels in rats co-treated with sildenafil + CCl 4 were recorded.The hepatoprotective activity may be attributed to www.nature.com/scientificreports/an anti-inflammatory property of sildenafil.These findings coincided with Molehin et al. 34 who found that the sildenafil moderately reduced hepatotoxicity of CCl 4 .
In this work, rats treated with CCl 4 showed decreased GSH contents and CAT activity after the fourth week of therapy, and an increase in LPO as evidenced by an elevation in MDA levels.This effect may have resulted from damage to cellular components and peroxidation of membrane lipids 35 .The primary mechanism of CCl 4 -induced liver damage is oxidative stress, whereby hepatic cytochrome P450 metabolizes CCl 4 to produce the trichloromethyl radical; promote hepatocyte necrosis, impair liver functioning, and upset hepatic architecture as a result of free radicals produced by CCl 4 induction 36 .The reactive oxygen species (ROS) which deplete endogenous antioxidants and interact with cellular lipids leading to lipid-peroxidation 36 .Rats co-treated with sildenafil and CCl 4 showed decreased LPO (MDA) levels and increased GSH content and CAT activity.These findings corroborate those of Abd El Motteleb et al. 37 , who discovered that sildenafil protects against oxidative stress and liver impairment brought on by bile duct ligation.Sildenafil depresses the generation of hydrogen peroxide by inducing the activity of antioxidants and preventing ROS generation.Thus, sildenafil improved the antioxidants concentrations and reduced oxidative stress.The GSH is the most important cellular antioxidant that plays an important role in scavenging of free radicals 38 .
In the present study, there was statistically significant upregulation of hepatic collagen-1α, IL-1β, OPN, and TGF-β mRNA expression when rats treated with CCl 4 after the fourth week of the experiment.Collagen-1α, IL-1β, OPN, and TGF-β mRNA expression were all shown to be downregulated in rats given sildenafil and CCl 4 .These findings agree with Kaleta et al. 39 .Sildenafil caused suppressive effects on many pro-inflammatory cytokines through its effect on oxidative and inflammatory pathways and the overexpression of pro-inflammatory cytokines, such as IL-1β, was reduced by sildenafil; these cytokines have synergistic effects that stimulate immune and non-immune cells, as well as other cytokines and adhesion molecules 40 .
The CCl 4 -treated rats significantly increased the expression of OPN.These findings corroborate those of Kaleta et al. 41 .Hepatic OPN expression correlated with inflammatory cell infiltration in the portal area; during CCl 4 intoxication, OPN facilitated the accumulation of macrophages at sites of injury and consequently enhance hepatic inflammation, hepatic stellate cells, and fibrogenesis 42 .Phosphodiesterase inhibitors elevate cGMP level, which inhibits fibroblast-to-myofibroblast transition that represent an important source of osteopontin 43 .Moreover, Kaleta et al. 39 demonstrated that sildenafil downregulates OPN gene expression in human peripheral mononuclear cells; that secret OPN during chronic liver injury as patients with primary biliary cirrhosis, autoimmune hepatitis, hepatic cirrhosis, and primary sclerosing cholangitis have all been shown to have elevated hepatic OPN levels.Sildenafil downregulates OPN gene expression, this may refer to the immunomodulatory and antiinflammatory effects of sildenafil on human immune system cells 44 .Numerous cell types express OPN during the hepatic fibrotic healing process.Hepatocytes and immune cells (macrophages, Kupffer cells) express more OPN mRNA, and OPN also attracts more neutrophils and macrophages to the area.Moreover, hepatic progenitor cells like HSC express OPN significantly while transitioned from Q-HSC to become MF-HSC then OPN stimulate the migration of hepatic progenitor cells towards the site of necrosis in the liver.It has been demonstrated that OPN stimulates HSC migration, proliferation, and epithelial-mesenchymal transition; it also produces factors that activate nearby progenitor cells and cells that produce ECM.It contributes to liver tissue scarring in both an autocrine and paracrine manner 45 .
The CCl 4 -treated rats significantly increased the expression of hepatic collagen-1α and TGF-β.While cotreated with sildenafil + CCl 4 showed down regulation of these genes.These findings corroborate those of Abd El Motteleb et al. 37 .A profibrogenic reaction was set off by damaged liver cells, which increased TGF-β expression and transformed latent TGF-β into active form, which activated HSCs.Higher hepatic expression of collagen-1α indicates upregulation of TGF-β gene content linked to HSC activation.TGF-β1 phosphorylates Smad2/3 proteins via activating its own receptors on HSCs.Increased production of collagen-1α is caused by phosphorylated Smad2/3 protein translocating into the nucleus and forming a complex with Smad4.One important cytokine that has been identified as mediating liver fibrosis is TGF-β 46 .Activated HSCs promote the migration and proliferation of myogenic markers, such as α-SMA, transforming them into the primary cells that produce collagen-1α; the primary constituent of the ECM that accumulates and plays a crucial role in the onset and progression of liver fibrosis 47 .The group that received sildenafil and CCl 4 had lower levels of hepatic TGF-β and collagen-1α gene expression; these findings are consistent with those reported by Xiang et al. 48.The potential reason for sildenafil's antifibrotic impact could be its ability to inactivate HSC through the inhibition of TGF-β production 49 .
The improvement in α-SMA immunohistochemistry characteristics of the liver of rats treated with CCl 4 was corroborated by these data; revealed that α-SMA protein is highly expressed in the area of central vein, portal tract, and between hepatocytes indicating fibrotic changes 50 .While rats treated with sildenafil and CCl 4 showed mild α-SMA-expression.α-SMA is frequently employed as a diagnostic tool for the initial phases of fibrosis 51 .
These findings were consistent with the improvement in the liver's histological characteristics of sildenafiltreated rats compared with the liver of CCl 4 -treated rats, which showed sever degenerative changes, vacuolization, sever congested central vein, congested portal tract vasculature, large inflammatory cells infiltrates, in addition to congested and dilated sinusoids.The outcomes aligned with the results of an earlier investigation 37 that used bile duct ligation to cause liver fibrosis and sildenafil co-treatment.These findings were also corroborated by those of the Masson's trichrome stain examination.It revealed the improvement of collagen fiber deposition in the portal area and around the central veins in rats treated with sildenafil and CCl 4 .These results aligned with those of an earlier investigation 52 that induced liver fibrosis in rats by CCl 4 and co-treated with Zataria multiflora Boiss essential oil.

Conclusion
Oxidative stress and profibrotic genes (OPN and TGF-β) played pivotal role in chronic liver diseases.Modulation of oxidative stress and suppression of profibrotic genes by sildenafil decreased hepatic progenitor response and protected the liver against fibrosis.Thus, sildenafil is a potentially attractive anti-fibrotic strategy in the liver fibrosis.
Figure1shows the expression of mRNA genes for liver collagen-1α, IL-1β, OPN, and TGF-β in the control group and groups treated with sildenafil, CCl 4 , and sildenafil+CCl 4 after four weeks of treatment.The liver mRNA of the rats treated with CCl 4 showed a significant elevation of collagen-1α, IL-1β, OPN, and TGF-β.Conversely, there was a noticeable downregulation of these genes in the group that received sildenafil and CCl 4 together.

Figure 1 .
Figure 1. mRNA expression of hepatic collagen-1α, IL-1β, OPN, and TGF-β.Total RNA was prepared from hepatic tissues of rats treated with sildenafil, CCl 4 , sildenafil against CCl 4 , and control on 4 weeks after treatments.Real-time PCR was evaluated the expression levels.P ˂ 0.05 compared with control values.Bars represent means ± SDM. (n = 10).

Figure 2 .
Figure 2. Liver sections of rats from different experimental groups.(A) The control group (G1) and (G2) received sildenafil and showed a normal histological features of the liver with no abnormalities.Cords of hepatocytes (H) radiating from the central vein (CV).(B,C) show liver of G3 rats that received CCl 4 .(B) Shows sever degenerative changes with distorted histological architecture, vacuolization (circle), congested portal tract vasculature (PT), large inflammatory cells infiltrates (asterisk), and congested sinusoids (arrow).(C) Shows Sever congested central vein (cv), dilated sinusoids (angled arrow), and large inflammatory cells infiltrates (asterisk).(D) G4 received sildenafil and CCl 4 and showed improvement of hepatic architecture with congestions between some hepatocytes (H&E ×100).

Figure 3 .
Figure 3. Liver sections of rats from different experimental groups stained with Masson's trichrome.(A) The control group (G1) and (G2) received sildenafil and showed minimal collagen expression just around the central veins.(B,C) Show liver of G3 rats that received CCl 4 ; showed increased collagen fibers deposition in the portal area and around the central veins.(D) G4 received sildenafil and CCl 4 and showed reduced collagen deposition compared to G3 group.(Masson's trichrome ×100).(E) Show digital morphometric study of area (%) of Masson's trichrome of liver of experimental rats.Data were used to estimate the degree of Masson's trichrome stain.P < 0.05 compared with different experimental groups.Bars represent mean ± SDM. (n = 10).

Figure 4 .
Figure 4. Immunohistochemical expression of α-smooth muscle actin (α-SMA) in liver tissue from different experimental groups.The brown color indicating positivity.(A) The control group (G1) showed slight amount of α-SMA-positivity merely seen in vascular wall.(B) G2 received sildenafil showed no difference compared with control.(C) G3 rats that received CCl 4 showed the α-SMA protein is highly expressed in the area of central vein, portal tract, and between hepatocytes.(D) G4 received sildenafil and CCl 4 and showed mild α-SMA-expression; however, the level of positivity is lower than in the G3.(Anti-α-SMA immunostaining ×100).(E) Digital morphometric study of area (%) of α-SMA immunohistochemical staining sections of liver of experimental rats.Data were used to estimate the degree of α-SMA immunohistochemical staining.P < 0.05 compared with different experimental groups.Bars represent mean ± SDM. (n = 10). https://doi.org/10.1038/s41598-024-67305-1www.nature.com/scientificreports/

Table 1 .
The primer sets of the assessed genes.

Table 2 .
Effect of sildenafil against carbon tetrachloride (CCl 4 ) on liver function test in various gatherings of the experiment after 4 weeks of treatment (mean ± standard deviation), (n = 10).Means with different superscripts in the same row are significantly different at P < 0.05.

Table 3 .
Effect of sildenafil against carbon tetrachloride (CCl 4 ) on oxidative markers in the liver homogenates of various gatherings of the experiment after 4 weeks of treatment (mean ± standard deviation), (n = 10).Means with different superscripts in the same row are significantly different at P < 0.05.