Fasudil Ameliorates Methotrexate-Induced Hepatotoxicity by Modulation of Redox-Sensitive Signals

Methotrexate (MTX) is one of the most widely used cytotoxic chemotherapeutic agents, and it is used in the treatment of different autoimmune disorders. However, the clinical applications of MTX are limited by its hepatic toxicity. Hence, the present study was conducted to evaluate the efficacy of fasudil (Rho-Kinase inhibitor) in the amelioration of MTX hepatotoxicity and the possible underlying mechanisms. Experimentally, 32 male Sprague Dawley rats were used and divided into four groups: control, MTX (20 mg/kg, i.p., single dose), fasudil (10 mg/kg/day i.p.) for one week, and fasudil plus MTX. It was found that MTX significantly induced hepatitis and hepatocellular damage, as shown by abnormal histological findings and liver dysfunction (ALT and AST), with up-regulation of the inflammatory mediators NF-κB-p65 and IL-1β. Moreover, MTX remarkably disrupted oxidant/antioxidant status, as evidenced by malondialdehyde (MDA) up-regulation associated with the depletion of superoxide dismutase (SOD), catalase, and reduced glutathione (GSH) levels. Moreover, MTX reduced the hepatic expression of B-cell lymphoma 2 (Bcl-2). On the contrary, the i.p. administration of fasudil significantly ameliorated MTX hepatotoxicity by histopathological improvement, restoring oxidant/antioxidant balance, preventing hepatic inflammation, and improving the hepatic anti-apoptotic capability. Furthermore, fasudil hepatic concentration was determined for the first time using the validated RP-HPLC method. In conclusion, the present study revealed that fasudil has a reliable hepatoprotective effect against MTX hepatotoxicity with underlying antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. It also introduced a new method for the determination of fasudil hepatic tissue concentration using the RP-HPLC technique.


Introduction
Methotrexate (MTX) acts as an antifolate and antimetabolite, which is used extensively in the treatment of different cancerous diseases [1]. MTX, used in different autoimmune and inflammatory diseases, has shown a potent immunomodulatory effect [2]. MTX acts by blocking dihydrofolate reductase, which inhibits folate metabolism and the synthesis of purines and pyrimidines, resulting in decreased RNA and DNA synthesis. It has been reported that MTX administration leads to serum elevation of aminotransferase and has been linked to the induction of liver diseases such as liver fibrosis and cirrhosis [3].
Different regimens using MTX in humans are classified as high-dose, intermediate, or low-dose according to the type of cancer and route of drug administration [4]. MTX

Chromatographic Method Validation for Fasudil Tissue Concentration
To create a sensitive and accurate HPLC method for the quantitation of fasudil, the mobile phase parameters were enhanced to produce symmetric and sharp peaks. After several attempts, an optimum mobile phase was established, which was composed of 15% methanol/85% water containing 10 mM SDS and 20 mM ammonium acetate, and the column temperature was set at 35 • C. The chromatogram is shown in Figure 1, where the retention time of fasudil was obtained at 2.12 min. The developed method was validated concerning ICH guidelines regarding linearity, accuracy, precision, the limit of detection (LOD), and the limit of quantitation (LOQ) (Tables 1-3). The system suitability criteria for the developed method such as the tailing factor, asymmetry factor, number of theoretical plates, and the height equivalent to a theoretical plate (HETP) were evaluated (Table 4).   36 11.964 99.71 SD = standard deviation; RSD = relative standard deviation.    In the present study, the fasudil concentration in the hepatic tissue homogenate was found at 5.32 ± 1.5 µg/g proteins in the fasudil group, while the MTX + fasudil group showed a higher concentration of fasudil as it was found at 6.91 ± 1.33 µg/g proteins. These results suggested that the co-administration of MTX with fasudil significantly (p > 0.05) increased the concentration of fasudil in hepatocytes against the administration of fasudil alone.

Effect of Fasudil on Hepatic Enzymes and Lipid Peroxidation after MTX Challenge
The i.p. administration of MTX significantly (p < 0.05) induced hepatic injury characterized by marked elevation of the hepatic MDA concentrations (2.95 nmol/mg protein) accompanied by an elevation of the hepatic enzymes ALT (74 U/L) and AST (95.6 U/L) serum concentrations compared to the control group (1.57 nmol/mg protein) for MDA, (21.5 U/L) for ALT, and (28.75 U/L) for AST ( Figure 2). However, the concomitant administration of fasudil with MTX significantly (p < 0.05) attenuated the MTX hepatic tissue-damaging effect with 2.25 nmol/mg protein for MDA, 40 U/L for ALT, and 57.25 U/L for AST, though the i.p. administration of fasudil for seven consecutive days did not produce significant changes in the formerly mentioned parameters compared to the control group ( Figure 2).

Effect of Fasudil on Hepatic Aberrations Induced by MTX Injection
The hepatic tissue of the control and fasudil groups showed normal cellular architecture with hepatocytes in normal arrangement. On the other hand, the hepatic tissue of the MTX group showed different histopathological changes, including hepatic lobular changes with hepatocyte degeneration and congestion with inflammatory cell infiltration. However, the histopathological lesions in MTX + fasudil groups were notably ameliorated compared to the MTX group with mild inflammatory cell infiltration and congestion ( Figure 3). Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX control group (p < 0.05). **** p < 0.0001. NS, non-significant.

Effect of Fasudil on Hepatic Aberrations Induced by MTX Injection
The hepatic tissue of the control and fasudil groups showed normal cellular architecture with hepatocytes in normal arrangement. On the other hand, the hepatic tissue of the MTX group showed different histopathological changes, including hepatic lobular changes with hepatocyte degeneration and congestion with inflammatory cell infiltration. However, the histopathological lesions in MTX + fasudil groups were notably ameliorated compared to the MTX group with mild inflammatory cell infiltration and congestion (

Effect of Fasudil on the Hepatic GSH Content as well as Antioxidant Enzyme Activity
The i.p. administration of MTX by a 20 mg/kg dose significantly (p < 0.05) decreased the GSH content of the hepatic tissue (7.3 ± 0.45 μmol/mg protein) compared to the control group (14.23 ± 1.33 μmol/mg protein). On the other hand, the i.p. administration of fasudil could restore GSH hepatic contents (10.87 ± 0.98 μmol/mg protein) and ameliorate MTX

Effect of Fasudil on the Hepatic GSH Content as well as Antioxidant Enzyme Activity
The i.p. administration of MTX by a 20 mg/kg dose significantly (p < 0.05) decreased the GSH content of the hepatic tissue (7.3 ± 0.45 µmol/mg protein) compared to the control group (14.23 ± 1.33 µmol/mg protein). On the other hand, the i.p. administration of fasudil could restore GSH hepatic contents (10.87 ± 0.98 µmol/mg protein) and ameliorate MTX depleting effects, while the i.p. administration of fasudil to control animals did not produce any significant changes in the hepatic GSH concentrations ( Figure 4).  Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). **** p < 0.0001. NS, non-significant.

Effect of Fasudil on IL-1β Expression
The i.p. administration of MTX produced a significant elevation of the inflammatory mediator IL-1β up to 191.12 pg/mg protein compared to 94.87 pg/mg protein for the control group, which was significantly attenuated by i.p. administration of fasudil when combined with MTX (127.87 pg/mg protein), while the i.p. administration of fasudil did not produce any significant change in the IL-1β compared to the control group ( Figure 5).  Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). **** p < 0.0001. NS, non-significant.
In the present study, the hepatic tissue concentrations of SOD and catalase in the control group were found (28 U/mg protein and 1.57 U/mg protein, respectively), whereas the i.p. administration of MTX produced a significant (p < 0.05) reduction of both enzymes to 19.12 U/mg protein and 0.73 U/mg protein, respectively. However, the i.p. administration of fasudil in combination with MTX could significantly attenuate the MTX depleting effect on these antioxidant enzymes concentrations, namely, 19.12 U/mg protein for SOD and 0.73 U/mg protein for catalase ( Figure 4).

Effect of Fasudil on IL-1β Expression
The i.p. administration of MTX produced a significant elevation of the inflammatory mediator IL-1β up to 191.12 pg/mg protein compared to 94.87 pg/mg protein for the control group, which was significantly attenuated by i.p. administration of fasudil when combined with MTX (127.87 pg/mg protein), while the i.p. administration of fasudil did not produce any significant change in the IL-1β compared to the control group ( Figure 5).

Effect of Fasudil on IL-1β Expression
The i.p. administration of MTX produced a significant elevation of the inflammatory mediator IL-1β up to 191.12 pg/mg protein compared to 94.87 pg/mg protein for the control group, which was significantly attenuated by i.p. administration of fasudil when combined with MTX (127.87 pg/mg protein), while the i.p. administration of fasudil did not produce any significant change in the IL-1β compared to the control group ( Figure 5).  Results are presented as mean ± SEM (n = 8); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). **** p < 0.0001. NS, non-significant.

Effect of Fasudil on NF-κB-p65 and Bcl-2 Expressions
As shown in Figure 6, the distribution of NF-κB-p65 inside hepatic tissue using the immunostaining technique showed that MTX significantly up-regulated NF-κB-p65 expression compared to the control group, whereas fasudil i.p. administration significantly reduced NF-κB-p65 tissue up-regulation induced by MTX. On the other hand, fasudil administration didn't produce any change in the NF-κB-p65 expression compared to the control group. Moreover, hepatic tissue immunostaining for the detection of Bcl-2 distribution showed that MTX significantly down-regulated Bcl-2 distribution and expression compared to the control group. On the other hand, fasudil treatment significantly ameliorated the MTX-depleting effect on Bcl-2, and treated animals showed up-regulation of Bcl-2 compared to the MTX-treated group (Figure 7).

Effect of Fasudil on NF-κB-p65 and Bcl-2 Expressions
As shown in Figure 6, the distribution of NF-κB-p65 inside hepatic tissue using the immunostaining technique showed that MTX significantly up-regulated NF-κB-p65 expression compared to the control group, whereas fasudil i.p. administration significantly reduced NF-κB-p65 tissue up-regulation induced by MTX. On the other hand, fasudil administration didn't produce any change in the NF-κB-p65 expression compared to the control group. Moreover, hepatic tissue immunostaining for the detection of Bcl-2 distribution showed that MTX significantly down-regulated Bcl-2 distribution and expression compared to the control group. On the other hand, fasudil treatment significantly ameliorated the MTX-depleting effect on Bcl-2, and treated animals showed up-regulation of Bcl-2 compared to the MTX-treated group (Figure 7).   The bar graph represents the quantitative determination of Bcl-2 protein expression in different groups (n = 6); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). ** p < 0.05. *** p < 0.001. **** p < 0.0001. NS, non-significant.

Discussion
Methotrexate (MTX) is a folate antagonist with immunomodulatory effects, which has been used extensively in the treatment of different autoimmune and cancerous diseases [2], but due to its severe toxicity (mainly hepatotoxicity), its clinical applications are limited. Therefore, the present study examined Rho-associated kinase (ROCK) pathway The bar graph represents the quantitative determination of Bcl-2 protein expression in different groups (n = 6); a: significant difference from the control group (p < 0.05); b: significant difference from the MTX group (p < 0.05). ** p < 0.05. **** p < 0.0001. NS, non-significant.

Discussion
Methotrexate (MTX) is a folate antagonist with immunomodulatory effects, which has been used extensively in the treatment of different autoimmune and cancerous diseases [2], but due to its severe toxicity (mainly hepatotoxicity), its clinical applications are limited. Therefore, the present study examined Rho-associated kinase (ROCK) pathway inhibition using fasudil as a new modality to attenuate MTX hepatotoxicity, which could increase MTX clinical applications and enhance patient tolerance.
MTX in low doses can induce changes in the histology of the liver, and in the long run it can produce a different type of hepatotoxicity. On the other hand, high-dose administration, as in leukemia, can result in severe hepatic tissue damage and elevation of the hepatic enzymes with progressive fibrosis and cirrhosis [10,30]. In the present study MTX prominently induced histopathological changes and hepatic tissue injury as found by histopathological examination. This was in agreement with preceding studies [8], which found that MTX can induce hepatic lesions, such as focal fibrosis, congestion, and dilatation of sinusoids with fatty vacuolation, and portal vein inflammation [31], which could be recognized, as the liver is the major site of MTX metabolism into the toxic agent 7-hydroxy-MTX, and the MTX can be stored inside hepatocytes in polyglutamated form, leading to hepatotoxicity [32,33].
On the other hand, in the present study, it was found that the i.p. administration of fasudil could significantly ameliorate the MTX injurious effect on the hepatic tissue, which could be attributed to ROCKs inhibition. Hence, ROCKs are considered the major regulator of tissue responses to injury, and fasudil, a ROCK inhibitor, and its metabolite hydroxyfasudil selectively inhibit ROCKs by competing with ATP for binding to the kinase, with minimal effects on other intracellular signaling pathways [34] in addition to antioxidant and anti-inflammatory properties [35].
The mechanism of MTX-inducing hepatotoxicity is not fully elucidated, and it can somewhat be attributed to oxidative stress generation [36]. Hence, different reports stated that MTX administration results in antioxidant enzyme depletion and free radical generation [12,37], which was in agreement with our findings. In the present study, we found that the i.p. administration of MTX significantly decreased the antioxidant enzymes SOD and catalase concentrations with depletion of hepatocellular GSH stores compared to the normal group. Hence, we used fasudil to ameliorate these effects, and we found that fasudil noticeably ameliorates the depleting effect of MTX on SOD and catalase enzymes and protected the hepatic GSH stores from diminution, assuming that Rho-ROCK pathway inhibition using fasudil has a hepatoprotective effect by amelioration of the oxidative stress inside hepatic tissue. This was in agreement with former studies, which found that the abnormal activation of the Rho/ROCK pathway was involved in different metabolic disorders, including oxidative stress, and the inhibition of Rho-ROCK pathway can diminish the oxidative stress [38].
Studies have reported that MTX administration could inhibit the cytosolic nicotinamide adenosine diphosphate (NADP)-dependent dehydrogenases and other cellular antioxidants [39,40]. NADPH is required to maintain the reduced state of cellular glutathione and protects against free radical generation, which is involved in the damage of biomolecules, such as lipids, leading to MDA upregulation [41]. In the present study, MTX significantly upregulated MDA hepatic tissue concentrations compare to the normal group, whereas the i.p. administration of fasudil remarkably attenuated the MDA upregulation by MTX, preserving the hepatocellular structure integrity, which confirms the hepatoprotective properties of fasudil. Furthermore, we found that the i.p. administration of fasudil ameliorated MTX hepatic enzyme (ALT and AST) upregulation, which was consistent with other studies' findings [42].
RhoA can modulate several cellular functions including monocyte/macrophage chemotaxis, adhesion, and proliferation [43]. RhoA can activate the NF-κB-p65 inflammatory pathway, indicating its potential role in the inflammatory process [44]. In the present study, we examined the fasudil attenuation effect on the inflammatory process induced by MTX as a proposed hepatoprotective mechanism, and we found that fasudil could significantly attenuate the inflammatory mediators NF-κB-p65 and IL-1B upregulation produced by MTX administration, as presented by immunohistochemistry and biochemical analysis, respectively.
Researchers have found that the inhibition of the Rho/ROCK signal pathway can increase anti-apoptotic family members' tissue concentrations [45], and the inhibition of ROCK by Y-27632 resulted in the upregulation of the anti-apoptotic protein (Bcl-2) and downregulation of the pro-apoptotic proteins [46], and it was reported that the inhibition of the ROCK pathway using fasudil decreased the number of apoptotic cells in cases of myocardial ischemic reperfusion injury [47], which agreed with our findings. Thus, we found that fasudil could significantly ameliorate Bcl 2 protein depletion by MTX, indicating the ability of fasudil to protect hepatocytes against apoptosis and degradation.
Furthermore, the present study introduced a new validated method for the detection and determination of fasudil concentration inside hepatic tissue, and we found that the MTX combined administration with fasudil significantly increased concentrations of the hepatic tissue content of fasudil compared to the fasudil only treated group, which could be attributed to decreased metabolic capacities of the liver due to MTX hepatotoxicity, leading to fasudil accumulation, increasing the fasudil protective effect by upregulating its hepatic concentrations.

Drugs, Reagents, and Chemicals
Fasudil and MTX were purchased from Sigma-Aldrich, St. Louis, Missouri, USA. Carbon tetrachloride (CCl4) and thiobarbituric acid (TBA) were purchased from Merck, Darmstadt, Germany. Methanol and sodium dodecyl sulfate were purchased from Thermo Fisher Scientific, Waltham, Massachusetts, USA, and Milli-Q water used to prepare buffer solutions was obtained by a Millipore ® purification system (Merck, Bedford, MA, USA). Other chemicals and reagents were of high analytical grade and certified sources.

Animals
Thirty-two adult male Sprague Dawley rats (130-150 g, 12-15 weeks) were used. Animals were kept under standard laboratory conditions of 12 h light/12 h dark cycle and 25 ± 2 • C (2 animals per cage), and they were fed standard rat chow (not less than 19% protein, 6% fiber, 3.5% fat, and 6.5% ash; EL Nasr Chemical Company, Abou-Zaabal, Cairo, Egypt) and water ad libitum. Animals were obtained from the Helwan animal breeding house, Cairo, Egypt. All animal treatments and procedures were conducted according to the ethical committee of the Faculty of Pharmacy at South Valley University (P.S.V.U 012/21), which complies with the ARRIVE guidelines and the EU Directive 2010/63/EU for animal experiments.

Study Design
Experimentally, rats were randomly divided into 4 groups (8 rats/group) as follows: Group I, control animals, received 0.5 mL saline/rat i.p. for seven consecutive days. Group II rats received an i.p. injection of fasudil (10 mg/kg/day) for seven consecutive days [48,49]. Group III rats received an i.p. injection of MTX (20 mg/kg) as a single dose [50,51]. Group IV rats received MTX (20 mg/kg, i.p.) as a single dose followed by fasudil (10 mg/kg/day, i.p.) for seven consecutive days.
After 24 h fasting from the last dose, rats were sacrificed (9:00 a.m.) under ketamine anesthesia (50 mg/kg, i.p.), and blood samples were collected from the inferior vena cava and cooled and centrifuged at 4000 rpm for 15 min. Sera were collected and refrigerated at −20 • C for biochemical analysis. The animals' livers were rapidly dissected from each animal and washed using ice-cold saline, and then a portion of the hepatic tissue was taken from all animals and homogenized using 100 mmol KH 2 PO 4 buffer solution, cooled, and centrifuged at 3000 rpm for 15 min to produce 10% homogenate. At the end of centrifugation, the supernatants were kept at −80 • C for further analysis. The remained portions of the hepatic tissues were stored in 10% formalin-buffered saline for histopathological and immunohistochemical examination. The chromatographic system consisted of a 1260 Infinity LC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a binary pump and a DA detector.

Preparation of Standard Solutions
A methanolic stock solution of fasudil was prepared to obtain a concentration of 25.0 mg/mL. Working standard solutions were freshly prepared by further dilution of the aliquots of the stock solution with the mobile phase to obtain concentrations of 1.0-12.0 µg/mL for fasudil.

Chromatographic Conditions
The chromatographic separation of fasudil was performed using a Symmetry C18 column (150 mm × 4.6 mm, 5 µm particle size). The mobile phase consisted of a mixture of methanol/water containing 20 mM of ammonium acetate and 10 mM SDS in a 15/85 v/v ratio. Fasudil was eluted by an isocratic technique at a flow rate of 0.5 mL min −1 ; the column temperature was 35 • C, the detection wavelength was measured at λ = 275 nm, and the injection volume was 20 µL. Several chromatographic conditions were examined, optimized, and validated following ICH guidelines [52].

Calibration Curve of Fasudil in Hepatic Tissue Homogenate
Appropriate volumes of working standard solution of fasudil were added to an aliquot of hepatic tissue homogenates (free from fasudil, obtained from the control animal group). The mixtures were vortex-mixed (for 3 min) and centrifuged at 6000 rpm (for 5 min) at room temperature. From the clear supernatant, 20 µL was injected into the HPLC system for analysis.

Histopathological and Immunohistochemical Examination
At room temperature, hepatic tissue snaps were successfully fixed in 10% formalinbuffered saline for 24 h. Liver snaps were terminated, washed, treated with different grades of alcohol, and sectioned with a thickness of 5 µm using a microtome. Then, the sections were deparaffinized using xylene and routinely stained with hematoxylin and eosin (H&E) following the standard procedures [53]. In addition, immunopositive slide sections were conducted for the immune detection of NF-κB-p65 [54] and Bcl-2 [55] according to the standard immunohistochemical procedure. Quantification of immunopositivity expression of NF-κB-p65 and Bcl-2 was conducted using ImageJ ® software (National Institutes of Health, Bethesda, USA) according to the previously reported investigation [56].

Liver Enzymes
Rat sera were used for the estimation of ALT and AST concentrations using the available commercial kits (Biodiagnostics, Cairo, Egypt) following the manufacturer's instructions [58]. The hepatic activity of SOD was measured kinetic spectrophotometrically using the SOD activity kit (Biodiagnostics, Cairo, Egypt) following the manufacturer's protocol. Absorbance was measured at 440-460 nm using the ELISA reader.

Catalase Activity
Samples of hepatic tissue homogenate were used for catalase activity determination using the catalase activity kit purchased from Biodiagnostics, Cairo, Egypt, and following the manufacturer's protocol. Samples were treated using triton X-100 for solubilization of the enzyme before the assay, and the enzyme concentration was assayed using a spectrophotometric procedure based on the disappearance of hydrogen peroxide, whereas the activity of the enzyme was expressed as units/mg protein [59].

Malondialdehyde (MDA) Content
Lipid peroxidation was determined in the hepatic tissue by measuring the MDA content. The method was a spectrophotometric assay based on the reaction between MDA and thiobarbituric acid at 95 • C in an acidic medium, producing a pink color, and its absorbance was determined at 532 nm [60]. 4.6.6. Proinflammatory Marker, IL-1β The concentration of IL-1β was quantified in the collected serum using an IL-1β ELISA kit specific for rats following the manufacturer's protocols [61].

Statistical Analysis
GraphPad Prism software (version 9.2.0) was used for the statistical analysis of the present results. Results were expressed as means ± standard errors of the means (SE) of 8 variables. One-way analysis of variance (ANOVA) was used for statistical analysis between different groups followed by the Tukey-Kramer test to compare the mean of each group. The results were considered statistically significant when p < 0.1.

Conclusions
In conclusion, the present study introduced fasudil as a potent hepatoprotective agent against hepatic injury produced by MTX, with underlying mechanisms as an effective anti-inflammatory (IL-1β and NF-κβ inhibition), antioxidant (SOD, catalase, and GSH upregulation), and anti-apoptotic (Bcl-2 up-regulation) drug, which may widen the clinical applications and decrease the toxicity to one of the most potent immune suppressants and anticancer drugs (MTX), increasing its therapeutic usefulness.