Anti-fibrotic impact of Carvedilol in a CCl-4 model of liver fibrosis via serum microRNA-200a/SMAD7 enhancement to bridle TGF-β1/EMT track

Circulating microRNAs (miRNAs) play a role in modulating the prevalence of fibrosis and have been a target of the cardiac anti-fibrotic effect of Carvedilol. However, the impact of miRNAs on the hepatoprotective effect of this non-selective β-blocker has not been yet elucidated. Hence, the current goal is to evaluate the potential role of circulating miR-200a in the hepatic anti-fibrotic pathway of Carvedilol. Male Wistar rats were randomized into normal, CCl4 (2 ml/kg, i.p, twice weekly for 8 weeks), and CCl4 + Carvedilol (10 mg/kg, p.o, daily). Carvedilol over-expressed the circulating miR-200a to modulate epithelial mesenchymal transition (EMT) markers (vimentin, E-Cadherin). In turn, Carvedilol increased SMAD7 gene expression and protein content to attenuate the pro-fibrogenic marker transforming growth factor β1 (TGF-β1) and the inflammatory markers (p-38 MAPK and p-S536-NF-κB p65). The anti-fibrotic potential was reflected on the decreased expression of the mesenchymal product and EMT marker α-SMA, besides the improved histopathological examination, and the fibrosis scores/collagen quantification to enhance liver functions (AST, ALT, ALP, and AST/platelet ratio index; APRI). In conclusion, circulating miR-200a/SMAD7/TGF-β1/EMT/MAPK axis is crucial in the hepatic anti-fibrotic mechanism of Carvedilol.


Results
Effect of Carvedilol on miR-200a and SMAD7 in CCl 4 -induced liver fibrosis. As depicted in Fig. 1, CCl 4 intoxicated group showed a marked downregulation of (A) serum miR-200a gene expression, as compared to the control group. This effect entailed its downstream SMAD7, where the insult decreased its hepatic (B) gene expression and (C) protein content. On the other hand, Carvedilol upregulated serum miR-200a and hepatic SMAD7 by 5 and 2.5 fold, respectively, this effect was reflected also on the protein content of SMAD7 (2.1 folds).  Fig. 2, CCl 4 reduced the hepatic content of (A) E-Cadherin (668.7+/− 84.67 vs normal 979.1+/−106.6), while increased that of (B) vimentin to reach 2.7 folds, as compared to the vehicle group. Carvedilol, however, reverted the CCl 4 effects to raise the MET marker E-Cadherin significantly and to reduce the EMT marker vimentin.

Effect of Carvedilol on hepatic content of inflammatory markers in CCl 4 -induced liver fibrosis.
The insulted group (Fig. 3) showed a significant increase in the hepatic contents of (A) p38 MAPK and (B) the p(S536) NF-κB p65 by 2.7 and 2.1 folds, respectively as compared to the vehicle group. However, Carvedilol counteracted these increments by reducing their levels, respectively by 28% and 26%, as compared to the insulted group. Figure 4 confirmed the CCl 4 -induced liver injury, where in the intoxicated group, the serum levels of (A) ALT, (B) AST, and (C) ALP were elevated by 2.5, 2.7, 3.1 folds, respectively, as compared to the vehicle group, while treatment with Carvedilol has reduced these values significantly. Moreover, (D) the AST to platelet ratio index (APRI) has increased in the insulted group by 3.9 folds relative to the vehicle group, whereas Carvedilol reduced it by 69%, as compared to the CCl 4 intoxicated group. . Statistical analysis was carried out using one-way ANOVA followed by Tukey's post hoc test, P < 0.05. As compared to (*) control group or (#) CCl 4 intoxicated group. Carved treatment (10 mg/kg, p.o, daily) started 2 weeks post CCl 4 (2 ml/kg, i.p) initiation until week 8. Carved: Carvedilol; CCl 4 : carbon tetrachloride. . Statistical analysis was carried out using one way ANOVA followed by Tukey's post hoc test, P < 0.05. As compared to (*) control group or (#) CCl 4 intoxicated group. Carved treatment (10 mg/kg, p.o, daily) started 2 weeks post CCl 4 (2 ml/kg, i.p) initiation until week 8. Carved: Carvedilol; CCl 4 : carbon tetrachloride. Effect of Carvedilol on immune-histochemical examination of pro-fibrogenic marker/HSC activation markers. As depicted in Fig. 5a, immune-histochemical staining analysis showed that Carvedilol has reduced hepatic expression of the pro-fibrogenic marker TGF-β1 and the marker of HSC activation α-SMA showing minimal brown staining, as compared to the CCl 4 intoxicated group. These results were further confirmed by the percentage area expression of TGF-β1 and α-SMA, as shown in Fig. 5b and c, respectively. A sum-up of the Carvedilol hepatic anti-fibrotic mechanisms. To recapitulate the current tackled mechanisms/pathways by Carvedilol, Fig. 8 shows that CCl 4 abated miR200-a/SMAD7, as well as E-Cadherin, while enhanced the fibrotic-related markers (vimentin, TGF-β1, α-SMA), as well as the inflammatory markers (p38MAPK, p-NF-κBp65) to further augment liver injury. Administration of Carved, however, proved its beneficial effect, as it modulated these pathways significantly in favor of reducing fibrosis.

Discussion
Liver fibrosis is described as a response to chronic injury that can progress to organ failure upon extended damage 22 . This ailment is an endpoint of several factors that assimilate to activate the fibrogenic process. Among these factors is the activity of the sympathetic nervous system, which regulates hepatic fibrogenesis by exerting an effect on hepatic stellate cells 23 . Moreover, Nuamnaichati et al. recently described the mechanism of β-blockers in fibrosis revealing that continuous stimulation of the β-adrenergic receptors induces the synthesis and secretion of growth factors in cardiac myocytes that activate cardiac fibroblast, thus, imposing a correlation between these receptors and the activation of fibroblast 24 .
Despite this ailment is a historic disease, yet anti-fibrotic therapies are powerless to restrain liver fibrosis in an efficient approach 25 . Nevertheless, Carvedilol, a non-selective β-blocker approved for the treatment of hypertension, has been proven for its efficient hepatic anti-fibrotic properties that are mainly ascribed to its antioxidant and anti-inflammatory properties 16,18,21 . However, in our study, we have evaluated the impact of circulating miR-200a/SMAD7/TGF-β1 axis and the suppression of EMT on the anti-fibrotic effect of Carvedilol.   In this study, Carvedilol significantly upregulated the serum miR-200a gene expression, as compared to the CCl 4 intoxicated group, an effect that is partly responsible for the decreased hepatic fibrosis. In support to our findings, previous studies reported that the expression of miR-200a is downregulated in case of liver fibrosis 26 .
Moreover, growing evidence showed that miRNAs participated in hepatic fibrosis through targeting SMAD proteins in liver 27 . SMADs are a group of proteins that mediate the TGF-β signaling and they are targeted by miRNAs 11 . TGF-β is considered as the most powerful commanding pro-fibrogenic cytokine that triggers fibrosis through the enhancement of SMAD-based pathways 28 . This association was further reinforced by a research article revealing that the over expression of miR-200a inhibited SMAD3 activity and attenuated TGF-β1-induced fibrosis 29 ; however, the correlation between miR-200a and SMAD7 in liver has not been studied before.
In the current study and in parallel to the miR-200a results, hepatic level of SMAD7 was upregulated in the Carvedilol treated group, as compared to the CCl 4 intoxicated one. This effect was associated with a decrease in the protein expression of TGF-β1, as documented by the current immune-histochemical examination. Our results concur with an earlier study in a model of renal fibrosis 9 , where Xiong et al. established a correlation between SMAD7 and miR-200 family and they stated that activation of SMAD7 leads finally to hindrance of the TGF-β1-mediated miR-200 downregualtion. Furthermore, a recent study conducted on cardiac fibrosis model, established in human aortic endothelial cells, reported that increased TGF-β1 is responsible for the downregulated expression of miR-200a 30 .
A further confirmation for the Carvedilol anti-fibrotic capacity, immunostaining of liver sections with α-SMA antibody divulged a minimal staining of liver tissues in Carvedilol treated group indicating a decline in collagen content, as compared to the CCl 4 intoxicated group, while the latter group showed a brown staining as compared to the vehicle treated group. Similarly, an earlier study confirmed the α-SMA immune-histochemical results 16 . The increased protein expression of α-SMA can be owed to the enhanced TGF-β1, in the current model to match previous findings; Li et al. 31 have reported that increased TGF-β1 triggers a rise in α-SMA in a model of renal fibrosis.
EMT is a basic part of liver fibrosis pathology 32 ; E-Cadherin and vimentin are classified as type 2 epithelial mesenchymal transition (EMT) markers and this type is responsible for organ fibrosis, wound healing, and tissue regeneration in the liver 33 . In tissue fibrosis, the alteration in E-Cadherin during EMT process is the archetype epithelial marker 34,35 . However, vimentin is a mesenchymal product recognized as type III intermediate filament protein, expressed at the site of injury during the repair phase and declines subsequent to injury resolution 5 . Indeed, E-Cadherin downregulation is mediated through vimentin upregulation to hinder E-cadherin trafficking to the cell 36   miR-200 family obstructed EMT and maintained a high level of E-Cadherin 9 . Additionally, a recent study further supports our results; the authors mentioned in a model of pancreatic fibrosis that TGF-β1 promotes EMT, while miR-200a inhibits it via a negative regulation of TGF-β1-induced pancreatic stellate cells activation 38 . It is noteworthy to mention that Gong et al. also proved in a recent model of proximal tubules EMT that miR-200a impeded TGF-β1-induced EMT 39 . The reduction of vimentin and the increase of E-Cadherin were enhanced by the upregulation of SMAD7 and the inhibition of TGF-β1. CCl 4 is a toxin recognized for the induction of liver damage, partially via the production of inflammatory cytokines; these cytokines are associated with the activation of mitogen-activated protein kinases (MAPKs) and NF-κB 40 . In normal cells, NF-κB dimer interact with IκBα and produce an inactive complex localized in the cytoplasm. Under stressful conditions, the hepatic cells are activated and this leads to the phosphorylation/degradation of IκBα, which frees activated NF-κB to be translocated into the nucleus 41 . Liver fibrosis pathogenesis is linked to p38 MAPK that phosphorylates and activates the nuclear kinase mitogen-and stress-activated protein kinase (MSK1). In return, activation of MSK1 plays a central role in the activation of NF-κB 42 . Activated p38MAPK can be the missing loop between fibrosis and inflammation, where apart from its fibrogenic role, increased TGF-β1 extends its effect to activate p38MAPK, which in turn stimulates the transcription factor NF-κB. In 2008, Sorrentino et al. correlated p38MAPK with the fibrogenic cytokine TGF-β1 after its binding to the TBR1 receptor in a SMAD-independent pathway 43 . This fact should not rule out the role of SMAD7, which when downregulated activates NF-κB p65 through inhibiting IκBα proteins expression to endorse its degradation by phosphorylation 11,44 . These evidences are in agreement with our results, where the hepatic levels of p38MAPK and the active form NF-κB p65 (pSer 536) were significantly raised in CCl 4 intoxicated group, as compared to the normal control group, whereas treatment with Carvedilol significantly diminished them compared to the intoxicated group. We may conclude that the anti-inflammatory capacity of Carvedilol relies on both the inhibition of TGF-β1, as well as the overexpression of SMAD7 to coincide with the results of Wang et al. in a renal fibrosis model 44 .
The Carvedilol anti-fibrotic effect was mirrored on the improved liver function; as a consequence to the CCl 4 -induced hepatic injury, liver enzymes; viz., AST, ALT and ALP, were markedly elevated, whereas treatment with Carvedilol leveled them off to signify its hepato-protective effect and to match with a previous study 16 . The ratio of AST to platelet count (APRI) is one of the non-invasive methods used in the assessment and diagnosis of liver fibrosis 45 . In our study, CCl 4 intoxicated group revealed a rise in APRI, however the Carvedilol treated group showed a significant decline in APRI, as compared to the CCl 4 group. Additionally, our results were associated by an improvement in the histopathological examination using H&E and MTC stain, besides the morphometrical collagen quantification test. These examinations revealed collagen deposition and extensive fibrosis detected as karyomegaly of hepatic nuclei and hyperplasia of bile duct in the CCl 4 intoxicated group and this was in agreement with Li et al. 46 . On the other hand, Carvedilol improved liver histology and hindered collagen deposition triggered by CCl 4 . The significant differences between the intoxicated and Carvedilol groups in fibrosis scoring confirmed the results of the histopathology and morphometry, as Carvedilol has reduced the semi-quantitative fibrosis score in the liver specimens 47 . Our histopathological and morphometrical analysis were in parallel with previous literature 16 .
Our results established a recent approach associating the anti-fibrotic mechanism of the non-selective β-blocker Carvedilol with the circulating miR-200a, the newly diagnostic non-invasive biomarkers of liver fibrosis, SMAD7, and through the suppression of EMT.

Materials and Methods
Animals. Thirty adult male Wistar rats (180-200 g) were purchased from National Research Center (Cairo, Egypt). Animals were kept under controlled environmental conditions at a constant temperature (23 ± 2 °C), humidity (60 ± 10%) and a light/dark (12/12 h) cycle at the animal facility of Faculty of Pharmacy, Misr International University (MIU). Food (standard diet pellets; EL-Nasr Co., Abu Zaabal, Egypt) and water were available ad libitum until the beginning of the experiment. Animal handling and experimental protocols comply with the Guide for the Care and Use of Laboratory Animals 48 , and were approved (PT:1772) by the Research Ethical Committee of the Faculty of Pharmacy, Cairo University (Cairo, Egypt).

Induction of fibrosis and experimental design.
Rats were randomly distributed into three groups (10 rats each); animals in group1 received the vehicle dimethyl sulphoxide (1% DMSO), whereas those in group 2 received CCl 4 in olive oil (1:1 v/v) (El Gomhorya Co, Cairo, Egypt; 2 ml/kg; i.p) twice weekly for 8 weeks 49 . Rats in group 3, received CCl 4 as in group 2 and were gavaged Carvedilol (Carvid ® MAP S.A.E, Cairo, Egypt) in a dose of 10 mg/kg. Two weeks after initiation of CCl 4 , Carvedilol (suspended in 1% DMSO) was administered daily till week 8 16 .
Following the termination of experimental protocol, rats were anesthetized with an overdose of thiopental and blood samples were withdrawn from the femoral vein. One aliquot was immediately analyzed manually for platelet count and then confirmed using hemocytometer to be used later for calculating the AST platelet ratio index. Sera were separated from another aliquot and were used for the assessment of liver function tests and circulatory miR-200a. The animals were sacrificed and the liver was harvested and divided into 3 portions. One portion was homogenized in phosphate buffered saline (PBS; 10% w/v), divided into aliquots, and frozen at −80 °C until analysis. Histopathological and immune-histochemical examination. Specimens from 3 representative animals/group were fixed in 10% buffered formaldehyde, embedded in paraffin, and sliced with a microtome into 5μm sections. These sections were used for the histopathological and immuno-histochemical examinations. Liver sections were deparaffinised in xylene, washed with ethanol, and placed on glass slides to be stained with Hematoxylin and Eosin (H&E) and Masson trichome (MTC) for histopathological examination of structural changes.
The remaining slices were then immuno-stained with primary antibody anti-TGF-β1 or anti-α-SMA antibody (Santa-Cruz Biotechnology, CA, USA) at a concentration of 1 μg/ml containing 5% BSA (bovine serum albumin) in buffered saline, then incubated with goat anti-rabbit secondary antibody. The percentage expression area of TGF-β1 and α-SMA, as well as liver sections were examined under a light microscope using Leica Quin Plus version 3 (magnification power were x100 x400).
Morphometric analysis and fibrosis scoring. Morphometric analysis of fibrosis was quantified as the mean of 3 consecutive sections/rat/group as a percentage of the total area that was positive for MTC stain in the digital photomicrographs using a computerized image analysis system (Leica Quin plus version 3 software/magnification power x400). The extent of liver fibrosis was evaluated blindly by a pathologist based on METAVIR liver fibrosis scoring system 47 . The scale ranges from 0-4; F0: No fibrosis, F1: Portal fibrosis without septa, F2: Portal fibrosis with few septa, F3: Numerous septa without cirrhosis, and F4: Cirrhosis.
Quantitative real-time PCR for hepatic SMAD7 and serum miR-200a. Total RNA was extracted and purified from serum samples or homogenized liver tissue samples using using miRneasy mini kit including QIAzol Lysis Reagent according to the manufacturer's instructions (Qiagen; Hilden, Germany). For the serum samples, this protocol allows the purification of separate fractions enriched in miR and other small RNA species; for miR recovery a RNeasy MinElute clean up kit was used according to the manufacturer's instructions. SMAD7 and MiR200a gene expression were carried out using gene-specific oligonucleotide primers (Invitrogen, Thermo Fisher Scientific, Inc; CA, USA). Primers sequence for both genes and the housekeeping gene β-actin are presented in Table 1. Total RNA was reverse transcribed to cDNA and amplified in one tube using one-step RT-PCR with SYBR ® Green iScript PCR kit (BioRad, CA, USA). Complete reaction mix was incubated in a real time thermal detection cycler as follows: cDNA synthesis at 50 °C for 10 min, reverse transcriptase inactivation 5 min at 95 °C followed by 30 to 45 cycles of amplification 10 s at 95 °C and 30 s at 55 °C to 60 °C. SMAD7 gene expression was carried out using gene-specific oligonucleotide primers (Invitrogen, Thermo Fisher Scientific, Inc; CA, USA). Mean Ct values were used to calculate the relative expression levels of the target gene for the experimental groups, relative to those in the control group. The gene expression data were normalized relative to the housekeeping gene β-actin using the 2 −ΔΔCt formula. Statistical analysis. Parametric data are presented as mean ± S.D (n = 7), all statistical analyses were performed using one-way analysis of variance (ANOVA; p < 0.05) followed by Tukey's multiple comparison post hoc test. Non-parametric data are presented as box and whiskers with median (max-min) and analysed by Mann-Whitney U test between model and treatment (GraphPad (Prism) software, version 5 ® ).