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Abstract

Methotrexate (MTX) is a commonly used anticancer and immunosuppressive agent. However, MTX can induce hepatotoxicity due to oxidative stress, inflammation, and apoptosis. Hydrogen sulfide (H₂S), the endogenous gaseous molecule, has antioxidant, anti-inflammatory, and anti-apoptotic effects. The present work explored the probable protective effect of H₂S against MTX hepatotoxicity in rats and also the possible mechanisms underlying this effect. MTX was given at a single intraperitoneal (i.p.) dose of 20 mg/kg. Sodium H₂S (56 µmol /kg/day, i.p.), as H₂S donor, was given for 10 days, starting 6 days before MTX administration. H₂S significantly reduced serum alanine aminotransferase, hepatic malondialdehyde, interleukin 6, nuclear factor κB p65, cytosolic cytochrome c, phosphorylated signal transducer and activator of transcription 3, and Bax/Bcl-2 ratio and significantly increased hepatic total antioxidant capacity and endothelial nitric oxide synthase (eNOS) in rats received MTX. In addition, H₂S minimized the histopathological injury and significantly decreased the expression of STAT3 in liver tissue of MTX-challenged rats. The effects of H₂S were significantly antagonized by administration of glibenclamide as K_ATP channel blocker, Nω-nitro-l-arginine, as eNOS inhibitor, or ruthenium red, as transient receptor potential vanilloid 1 (TRPV1) antagonist. It was concluded that H₂S provided significant hepatoprotection in MTX-challenged rats through its antioxidant, anti-inflammatory, anti-apoptotic effects. These effects are most probably mediated by the ability of H₂S to act as IL-6/STAT3 pathway modulator, K_ATP channel opener, eNOS activator, and TRPV1 agonist.

Keywords

Methotrexate IL-6/STAT3 eNOS TRPV1

Introduction

Methotrexate (MTX) is an antimetabolite folic acid antagonist widely used as a chemotherapeutic agent in the treatment of solid and hematological malignancies, and as immunosuppressive in the treatment of rheumatoid arthritis, psoriasis, and inflammatory bowel diseases. Despite clinical effectiveness of MTX therapy, liver injury is a dose-limiting adverse effect. Elevated serum aminotransferases were observed in nearly 20% of patients treated with high dose of MTX, and approximately 3% of patients develop histopathological abnormalities, including liver fibrosis and cirrhosis.^1,2 The exact pathophysiological mechanisms underlying MTX hepatotoxicity are not fully elucidated. However, the mechanisms seem to play a crucial role include increased generation of reactive oxygen species (ROS), exhaustion of endogenous antioxidants, and augmented lipid peroxidation leading to increased production of malondialdehyde (MDA), the end product of lipid peroxidation of biomembranes.³ Moreover, oxidative stress enhances the inflammatory responses increasing the production of inflammatory cytokines, as interleukin 6 (IL-6).⁴ The signal transducer and activator of transcription 3 (STAT3) is accumulated and phosphorylated on Tyr705 in response to pro-inflammatory cytokines, particularly IL-6. Then, nuclear translocation of phosphorylated STAT3 (pSTAT3) activates gene transcription of inflammatory and apoptotic factors.⁵ Together, all these operators eventually lead to hepatocellular necrosis and apoptosis, and liver dysfunction.⁶ In agreement with these mechanisms, it was reported in the literature that antioxidants, anti-inflammatory, and anti-apoptotic agents significantly protected against MTX hepatotoxicity.^7
–9

Hydrogen sulfide (H₂S) is an endogenous gaseous molecule produced by the metabolic pathway of the thiol-containing amino acid, cysteine. It is generated in most mammalian tissues, including the liver. It has many important bioactivities, including cytoprotective, antioxidant, anti-inflammatory, antiapoptotic, and smooth muscle relaxing effects.^10,11 These effects were attributed to the ability of H₂S to act as K_ATP channel opener,¹² endothelial nitric oxide synthase (eNOS) activator,¹³ and transient receptor potential vanilloid-1 (TRPV1) agonist.¹⁴ It is well-known that K_ATP channel openers can reduce oxidative stress and lipid peroxidation, maintain endogenous antioxidant defense, and mitigate inflammation.¹⁵ In addition, it was reported that eNOS activators maintain microcirculation and decrease oxidative stress and apoptotic biomarkers.¹⁶ Moreover, it was demonstrated that activation of TRPV1 can preserve antioxidant status and attenuate inflammation.¹⁷ Previous reports indicated that exogenous H₂S significantly protected against liver injury induced by ischemia–reperfusion, uranium, carbon tetrachloride, and acetaminophen.^18

–21 Thereby, H₂S has the potential to defend against MTX hepatotoxicity, and therefore, the present work was performed to investigate the possible hepatoprotective effect of H₂S in rats challenged with MTX. In addition, the possible underlying mechanisms of the protective effect of H₂S were investigated using glibenclamide (GBD) as K_ATP channel blocker, Nω-nitro-l-arginine (LNNA) as eNOS inhibitor, and ruthenium red as TRPV1 antagonist.

Materials and methods

Drugs and chemicals

Sodium hydrogen sulfide (NaHS), as H₂S donor, MTX, GBD, LNNA, and ruthenium red (RR) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). NaHS, MTX, RR, and LNNA were dissolved in physiological saline, while GBD was prepared in carboxymethylcellulose (CMC) 0.5% solution. The doses of all agents used in the present work were chosen based on previous investigations.^19,22

–25

Laboratory animals

Forty-two male Sprague-Dawley rats, weighing 220–250 g, were obtained from the National Research Centre (Giza, Egypt). Rats were kept at standard housing facilities (24°C, 45% humidity, and 12 h light/12 h dark cycle), provided with the ordinary chew and tap water ad libitum, and left to acclimatize for 7 days. The research was approved by the Research Ethics Committee, Faculty of Medicine, Minia University (approval number: 144:12019). International guidelines for care and use of laboratory animals were implemented in this study.

Study plan

The rats were randomly divided into six equal groups (n = 7, each) as follows:

Group 1: served as control and received an intraperitoneal (i.p.) injection of physiological saline daily for 10 days.

Group 2: received a single injection of MTX (20 mg/kg, i.p.), and i.p. physiological saline daily for 10 days, starting 6 days prior to MTX administration.

Group 3: received a single injection of MTX (20 mg/kg, i.p.), and NaHS (56 µmol /kg/day, i.p.) for 10 days, starting 6 days prior to MTX administration.

Group 4: received a single injection of MTX (20 mg/kg, i.p.). In addition, GBD (as K_ATP channel blocker) at a dose of 1 mg/kg/day, i.p., and NaHS (56 µmol/kg/day, i.p.) were given for 10 days, starting 6 days prior to MTX administration.

Group 5: received a single injection of MTX (20 mg/kg, i.p.). In addition, LNNA (as eNOS inhibitor) at a dose of 20 mg/kg/day, i.p., and NaHS (56 µmol/kg/day, i.p.) were given for 10 days, starting 6 days prior to MTX administration.

Group 6: received a single injection of MTX (20 mg/kg, i.p.). In addition, RR (as TRPV1 blocker) at a dose of 3 mg/kg/day, s.c., and NaHS (56 µmol/kg/day, i.p.) were given for 10 days, starting 6 days prior to MTX administration.

In the current study, NaHS was given for 6 days prior to and 4 days after MTX injection to study the effect of a combination of prophylactic and therapeutic regimens of H₂S in the current model.

Sampling and biochemical studies

At the end of experiments, rats were euthanized by urethane (1 g/kg, i.p.). Blood was aspirated through left ventricular puncture, and subsequently serum alanine aminotransferase (ALT) was measured by a colorimetric kit (Biodiagnostic, Egypt).

The liver was dissected and homogenized in cold potassium phosphate buffer (pH 7.4, 0.05 M). The homogenates were centrifuged at 4000 r/min for 10 min at 4°C. The supernatant was used to assess MDA, and total antioxidant capacity (TAC) by colorimetric kits (Biodiagnostic). ELISA kits were used to measure IL-6 (R&D Systems, Minneapolis, Minnesota, USA), eNOS (MyBioSource, San Diego, California, USA), and Bax, and Bcl-2 (LifeSpan Biosciences, Seattle, Washington, USA).

Additionally, an ELISA kit was used to determine the pSTAT3 in liver homogenates as indicated by the manufacturer (RayBiotech, Norcross, Georgia, USA). In brief, STAT3 was at first bound to an immobilized antibody. Then, rabbit anti-phospho-STAT3 (Tyr705) antibody was utilized to identify pSTAT3. Thereafter, horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G, and a substrate solution (3,3,5,5′-tetramethylbenzidine) were added. The developed color was proportional to the amount of bound pSTAT-3 (Tyr705). Finally, a stop solution was added to change the color from blue to yellow, and the color intensity was measured at 450 nm. The absorbance obtained with different groups was compared to the control group.

Moreover, a portion of the liver homogenate was recentrifuged at 15,000 r/min for 30 min at 4°C. The cytosolic fraction (supernatant) was used to measure cytochrome C by an ELISA kit (R&D Systems), and nuclear fraction (pellet) was used to measure NF-κB p65 unit by an ELISA kit (Novus Biologicals, Centennial, Colorado, USA).

Histopathology studies

Parts of liver tissue were fixed in formalin 10% solution and embedded in paraffin wax. Sections were cut at 5 µm, stained with hematoxylin and eosin, and examined under light microscope by a specialist unaware of the slide identity. In addition, a semiquantitative score was used to assess the percent of hepatocellular necrosis; in which 0: no necrosis; 1: <6%; 2: 6–25%; 3: 26–50%; and 4: >50%.²⁶

Immunohistochemistry studies

The sections were deparaffinized, rehydrated, and hydrogen peroxide (H₂O₂) (3%) in methanol was used to inhibit endogenous peroxidase. Sections were pretreated in 10 mM of citrate buffer (pH 6.0) in a microwave and incubated with rabbit polycolonal antibody against rat STAT3 (1:50; Thermo Scientific, Fremont, California, USA). Sections were incubated with biotinylated goat anti-polyvalent, streptavidin peroxidase, and lastly with 3,3′-diaminobenzidine (DAB) as chromogen. Counterstaining by hematoxylin was done, and immunostaining was detected by light microscope using a digital imaging software program (cellSens; Olympus Corporation, Miami, Florida, USA) to evaluate the immunostained area (μm²) in five different microscopic fields. The mean ± standard error of mean (SEM) of each group was calculated.

Statistical analysis

GraphPad Prism Software Program (version 6.01) was used for data analysis by applying one-way analysis of variance test, followed by Tukey test for post hoc comparisons. Results are shown as mean ± SEM, and significance was at p < 0.05.

Results

Biochemical results

Administration of a single MTX dose (20 mg/kg, i.p.) significantly elevated (p < 0.05) serum ALT, liver MDA, and IL-6, and significantly reduced (p < 0.05) liver TAC and eNOS activity, as compared to the control values (Figure 1(a) to (e)). On the other hand, rats treated with H₂S showed significant decrements (p < 0.05) of serum ALT, liver MDA, and IL-6 and significant increments (p < 0.05) of liver TAC and eNOS activity, as compared to rats challenged with MTX and non-treated with H₂S (Figure 1(a) to (e)). Treatment with GBD, LNNA, or RR significantly antagonized (p < 0.05) the effects observed with H₂S on serum ALT, liver MDA, TAC, IL-6, and eNOS in rats received MTX (Figure 1(a) to (e)).

Figure 1.

Influence of H₂S and different pretreatments (GBD, LNNA, and RR) on: (a) serum ALT; (b) liver TAC; (c) MDA; (d) IL-6; (e) eNOS in rats challenged with MTX. Results are mean ± SEM, *p < 0.05 versus control, ^·p < 0.05 versus MTX, ^≠p < 0.05 versus H₂S + MTX. H₂S: hydrogen sulfide; GBD: glibenclamide; LNNA: Nω-nitro-l-arginine; RR: ruthenium red; ALT: alanine aminotransferase; TAC: total antioxidant capacity; MDA: malondialdehyde; IL-6: interleukin 6; eNOS: endothelial nitric oxide synthase; MTX: methotrexate; SEM: standard error of mean.

In addition, rats received MTX injection demonstrated significant increases (p < 0.05) in hepatic NF-κB p65, cytosolic cytochrome C, pSTAT3, and Bax/Bcl-2 ratio, as compared to the control rats (Figure 2(a) and (b)). Contrarily, H₂S-treated rats displayed significant reductions (p < 0.05) of hepatic NF-κB p65, cytosolic cytochrome c, pSTAT3, and Bax/Bcl-2 ratio, as compared to MTX-challenged rats without H₂S treatment (Figure 2(a) and (b)). Moreover, GBD, LNNA, and RR treatments significantly inhibited (p < 0.05) the effects of H₂S on hepatic NF-κB p65, cytosolic cytochrome C, pSTAT3, and Bax/Bcl-2 ratio in rats challenged with MTX (Figure 2(a) and (b)).

Figure 2.

Influence of H₂S and different pretreatments (GBD, LNNA, and RR) on: (a) cytosolic cytochrome C and NF-κB p65; (b) pSTAT3 and Bax/Bcl-2 ratio in liver of rats challenged with methotrexate (MTX). Results are mean ± SEM, *p < 0.05 versus control, ^·p < 0.05 versus MTX, ^≠p < 0.05 versus H₂S + MTX. H₂S: hydrogen sulfide; GBD: glibenclamide; LNNA: Nω-nitro-l-arginine; RR: ruthenium red; NF-κB p65: nuclear factor-κB p65; pSTAT3: phosphorylated signal transducer and activator of transcription 3; MTX: methotrexate; SEM: standard error of mean.

Histopathological results

Administration of MTX caused a widespread distortion of the liver architecture, hepatocellular necrosis, ballooning degeneration, and cytoplasmic vacuolization of the hepatocytes, sinusoidal dilatation and congestion, and inflammatory cell infiltration (Figure 3). Treatment with H₂S markedly minimized the liver tissue injury, preserved the normal liver histology, and significantly decreased (p < 0.05) the hepatocellular necrosis score in MTX-challenged rats (Figure 3). On the contrary, the hepatoprotective effect of H₂S was significantly antagonized by administration of GBD, LNNA, or RR (Figure 3).

Figure 3.

H&E (×100) of rat liver of: (a) control manifesting normal histology; ((b) and (c)) MTX group displaying marked distortion of hepatic architecture, hepatocellular necrosis, ballooning degeneration, and cytoplasmic vacuolization of the hepatocytes (black arrows), sinusoidal dilatation and congestion (white arrows), and inflammatory cell infiltration (black heads); (d) H₂S + MTX showing that normal liver histology was preserved; ((e), (f), and (g)) GBD + H₂S + MTX, LNNA + H₂S + MTX, and RR + H₂S + MTX, respectively, revealing that the protective effect of H₂S was diminished; (h) hepatocellular necrosis score. Results are mean ± SEM, *p < 0.05 versus control, ^·p < 0.05 versus MTX, ^≠p < 0.05 versus H₂S + MTX. H&E: hematoxylin and eosin; H₂S: hydrogen sulfide; GBD: glibenclamide; LNNA: Nω-nitro-l-arginine; RR: ruthenium red; ALT: alanine aminotransferase; MTX: methotrexate; SEM: standard error of mean.

Immunohistochemical results

The expression of STAT3 was significantly increased (p < 0.05) in the liver of rats that received MTX, as compared to the control group. Treatment with H₂S significantly decreased (p < 0.05) STAT3 expression in MTX-challenged rats, as compared to rats non-treated with H₂S (Figure 4). Additionally, GBD, LNNA, and RR significantly suppressed (p < 0.05) the effect of H₂S on hepatic STAT3 expression in rats received MTX (Figure 4).

Figure 4.

STAT3 immunohistochemistry (×100) in rat liver of: (a) control group exhibiting minor staining; (b) MTX group manifesting a significant increment of STAT3 immunostaining in brown color; (c) H₂S + MTX revealing a significant decrement in STAT3 reactivity; ((d), (e), and (f)) GBD + H₂S + MTX, LNNA + H₂S + MTX, and RR + H₂S + MTX, respectively, showing significant increments in STAT3 reactivity; (g) immunopositive area (µm²). Results are mean ± SEM, *p < 0.05 versus control, ^·p < 0.05 versus MTX, ^≠p < 0.05 versus H₂S + MTX. STAT3: signal transducer and activator of transcription 3; H₂S: hydrogen sulfide; GBD: glibenclamide; LNNA: Nω-nitro-l-arginine; RR: ruthenium red; MTX: methotrexate; SEM: standard error of mean.

Discussion

Oxidative stress, inflammation, and apoptosis are the main factors incriminated in the pathogenesis of liver injury and dysfunction induced by MTX.³ This was evidenced in many previous studies and also in agreement with the present work which showed that MTX insult caused a significant increase in lipid peroxidation of biomembranes and exhaustion of endogenous antioxidant defenses. Additionally, in the same line with previous reports, MTX augmented the generation of pro-inflammatory cytokines, particularly IL-6.^7
–9,27 Besides, similar to the present work, MTX-induced oxidative stress caused degradation of IκB proteins and released NF-κB p65, the active subunit of NF-κB, in the cytoplasm. Translocation of NF-κB p65 to the nucleus promotes gene transcription of inflammatory cytokines.⁶

In addition, STAT3 which is a member of STAT protein family plays a pivotal role in many pathophysiologic conditions, including cell differentiation, proliferation, inflammation, and apoptosis. Binding of IL-6 to its cell membrane receptor, which belongs to JAK-STAT receptor family, induces accumulation and phosphorylation of STAT3 on Tyr705. pSTAT3 plays a key role in the pathogenesis of acute-phase response in cases of liver injury.⁵ Then, pSTAT3 translocates to the nucleus and acts as a transcriptional factor, which enhances the expression of genes involved in inflammatory and apoptotic signaling.^5,28 Moreover, it was demonstrated that non-phosphorylated STAT3 accumulated in response to IL-6 and activated cytokine-dependent signaling through a mechanism completely different from that of pSTAT3. Non-phosphorylated STAT3 can bind NF-κB, and this complex migrates to the nucleus and stimulates transcription of NF-κB-responsive genes of inflammation and apoptosis.²⁹

The present work showed that MTX caused significant increases in IL-6, STAT3, and pSTAT3 in liver tissue. Upregulation of IL-6/STAT3 signaling pathway is associated with augmented inflammatory responses and cell apoptosis. This was evidenced by activation of the mitochondrial pathway of cell apoptosis. In line with the current study, previous investigations showed that MTX insult increased Bax, the proapoptotic protein, and decreased Bcl-2, the antiapoptotic protein, in liver tissue. The imbalance between Bax and Bcl-2 disrupts mitochondrial membrane permeability with subsequent release of cytochrome c into the cytoplasm. As a result, caspase family of proteases is activated leading to apoptotic cell death.^30,31

The endogenous gaseous molecule, H₂S, is produced mainly by the enzymes, cystathionine β-synthase, and cystathionine γ-lyase involved in cysteine metabolic pathway. It has many physiological activities, including cytoprotective, vasodilator, antioxidant, anti-inflammatory, and antiapoptotic effects.^10,11 In addition, previous investigations revealed that exogenous supplementation of H₂S effectively protected against oxidative stress, inflammatory injury, and cell apoptosis in different models of liver injury.^18

–21 There are several proposed mechanisms for the beneficial effects of H₂S most notably its action as K_ATP channel opener, eNOS activator, and TRPV1 agonist.^12
–14 It was reported in the literature that K_ATP channel openers attenuate oxidative stress and lipid peroxidation, maintain endogenous antioxidant capacity, and alleviate inflammation.¹⁵ Additionally, eNOS activators maintained the microcirculation and produced antioxidant and antiapoptotic effects.¹⁶ Activation of TRPV1 was also shown to keep the antioxidant status and attenuate inflammation.¹⁷ Therefore, through these mechanisms H₂S scavenges ROS, induces endogenous antioxidant defenses, inhibits membrane lipid peroxidation, maintains vascular homeostasis, prevents microcirculatory dysfunction, suppresses NF-κB-dependent inflammatory responses, and blocks apoptotic pathways.^10,11

Previous studies showed that H₂S effectively scavenged ROS, such as H₂O₂, superoxide anion, and hydroxyl radical, and also inhibited NADPH oxidase, which is a major source of ROS overproduction.^32,33 This could explain the effect of H₂S in the current investigation, in which H₂S significantly protected against oxidative stress as indicated by significant reduction of hepatic MDA, the end product of lipid peroxidation, and preservation of hepatic TAC. In addition, H₂S significantly mitigated inflammation, and apoptosis of liver tissue in rats challenged with MTX. Treatment with H₂S also prevented the MTX-induced elevation of serum ALT, which indicated that the liver function was preserved. In addition, the present work showed that GBD (K_ATP channel blocker), LNNA (eNOS inhibitor), and RR (TRPV1 blocker) significantly antagonized H₂S effects indicating that these mechanisms are involved in this model.

The current investigation also revealed that H₂S significantly downregulated IL-6/STAT3 signaling pathway which was induced by MTX in rat liver. To the best of our knowledge, no previous investigations demonstrated the role of this pathway in MTX-induced hepatotoxicity. Previous studies, in line with the current one, showed that H₂S supplementation inhibited lipopolysaccharide-induced inflammation by suppressing IL-6/STAT3 pathway.^34,35 Previous studies also revealed that K_ATP channel activators produced their antioxidant, anti-inflammation, antiapoptotic effects by causing significant decreases in IL-6 production.^36,37 Other studies showed that IL-6 inhibited eNOS expression in acute-phase inflammatory response either directly or through activation of IL-6/STAT3 pathway.^38
–40 Besides, prior investigations showed that activation of TRPV1 by capsaicin resulted in significant inhibition of constitutive and IL-6-induced activation of STAT3.^41,42 Therefore, it could be postulated that the cross-talking between these different mechanisms contributes, at least in part, to the protective effects afforded by H₂S.

From the present results, it can be concluded that H₂S significantly protected against MTX hepatotoxicity in rats by blocking oxidative stress, and inhibiting inflammatory and apoptotic responses. The present study also indicated that suppression of IL-6/STAT3 signaling pathway, opening K_ATP channels, stimulation of eNOS enzyme, and activation of TRPV1 are the mechanisms underlying the hepatoprotective effect of H₂S in this model. However, in the present work, NaHS was given for 6 days before and 4 days following MTX administration to investigate the effect of a combination of prophylactic and therapeutic regimens of H₂S in the current model. In addition, the effect of NaHS was studied using only one dose regimen. Therefore, further investigations are recommended to study the dose–response effect of NaHS and to compare the hepatoprotective effects of prophylactic and therapeutic treatments in MTX-challenged rats.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

AA Fouad

HM Hafez

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Hydrogen sulfide modulates IL-6/STAT3 pathway and inhibits oxidative stress,inflammation,and apoptosis in rat model of methotrexate hepatotoxicity

Abstract

Keywords

Introduction

Materials and methods

Drugs and chemicals

Laboratory animals

Study plan

Sampling and biochemical studies

Histopathology studies

Immunohistochemistry studies

Statistical analysis

Results

Biochemical results

Histopathological results

Immunohistochemical results

Discussion

Footnotes

Declaration of conflicting interests

Funding

ORCID iDs

References