Introduction

Methotrexate (MTX) is a chemotherapeutic dihydrofolate reductase inhibitor widely used in the treatment of several types of cancer, such as leukemia, osteosarcoma, lung cancer, and breast cancer (Farber 1966). It is used as an immunosuppressive agent to treat rheumatoid arthritis, psoriasis, and systemic lupus erythematosus. MTX disrupts folate metabolism and inhibits DNA synthesis (Rajagopalan et al. 2002), and its use is therefore associated with multiple adverse effects, including nausea, diarrhea, fever, and vomiting (Khan et al. 2012). In addition, the toxicity profile of MTX has been demonstrated to include nephrotoxicity (Perazella 2009) and hepatotoxicity (Conway and Carey 2017).

Drug-induced liver injury (DILI) is a serious side effect that can occur during the use of MTX (West 1997), thereby limiting its clinical applications. The mechanism of MTX hepatotoxicity is not fully understood; however, studies have highlighted the possible involvement of oxidative damage and mitochondrial dysfunction. MTX triggers cytotoxicity in rat hepatocytes by increasing the production of reactive oxygen species (ROS) and altering mitochondrial membrane potential (MMP) (Al Maruf et al. 2018). In experimental rats, MTX induced hepatic oxidative stress and reduced cellular antioxidants (Mahmoud et al. 2017b, c). In addition to oxidative damage, ROS can activate nuclear factor-kappaB (NF-κB) and the release of pro-inflammatory mediators (Morgan and Liu 2011). Inflammation and redox imbalance have also been associated with the apoptosis of hepatocytes following MTX administration (Mahmoud et al. 2017b, c).

Chicoric acid (CA) is a hydroxycinnamic acid that occurs in at least 63 genera and species, including Cichorium intybus L. and Echinacea purpurea (Lee and Scagel 2013). In plants, CA plays a defensive role to protect against insects and infection (Nishimura and Satoh 2006), and helps in wound healing after mechanical damage (Tomás-Barberán et al. 1997). Recent studies have demonstrated that CA possesses antioxidant and anti-inflammatory efficacies (Ding et al. 2019; Liu et al. 2017; Tsai et al. 2017). CA ameliorated antioxidant defenses in diabetic mice (Zhu et al. 2017), cerebral ischemia/reperfusion (I/R) injury (Jia et al. 2018), and in hepatocytes in vitro (Ma et al. 2018). Despite its potent antioxidant efficacy, nothing has been yet reported on its potential to suppress oxidative stress in MTX-administered rats. Therefore, we investigated the hepatoprotective effects of CA, emphasizing on oxidative stress and the possible involvement of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling in MTX-administered rats. Nrf2 is a redox-sensitive factor conferring protection against oxidative stress by stimulating the expression of antioxidant defenses (Satta et al. 2017). Under normal cellular conditions, Nrf2 is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1). ROS dissociate Nrf2/Keap-1 complex and the liberated Nrf2 translocates into the nucleus and elicits the transcription of multiple genes, including heme oxygenase (HO)-1, superoxide dismutase (SOD), and catalase (CAT) (Itoh et al. 1997). In addition, we evaluated the effect of CA on peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear hormone receptor with central roles in carbohydrate metabolism, adipogenesis, and cell differentiation (Kim et al. 2015). Upon activation, PPARγ forms a heterodimer with retinoid X receptor (RXR) and controls the transcription of several genes through binding to a specific response element (Barish et al. 2006). It promotes the expression of antioxidant defense enzymes and inhibits the release of pro-inflammatory cytokines via NF-κB suppression (Hwang et al. 2005; Remels et al. 2009). Therefore, the activation of Nrf2 and PPARγ can mitigate inflammation, oxidative damage, and liver injury induced by MTX.

Materials and methods

Experimental design

Male Wistar rats (150–160 g), obtained from the National Research Centre (NRC, Giza, Egypt), were housed under standard conditions and given a standard chow diet and water ad libitum.

CA (Chengdu Purify Phytochemicals Ltd., China) was dissolved in 0.5% carboxymethyl cellulose (CMC) and MTX (Shanxi PUDE Pharmaceutical Co., China) was dissolved in physiological saline. After acclimatization for 1 week, the animals were allocated into 4 groups, six rats per group (n = 6), as follows:

  • Group I: received vehicle for 15 days and a single intraperitoneal (i.p.) injection of physiological saline at day 16. This group served as a control.

  • Group II: received vehicle for 15 days and a single injection of MTX (20 mg/kg) at day 16.

  • Group III: received CA (25 mg/kg) orally for 15 days and a single i.p. dose of MTX at day 16.

  • Group IV: received CA (50 mg/kg) orally for 15 days and a single i.p. injection of MTX at day 16.

The doses of CA were selected according to previous studies demonstrating its antioxidant efficacy in vivo (Ding et al. 2019; Jia et al. 2018; Zhu et al. 2017), and MTX-induced liver injury was established as previously described (Mahmoud et al. 2017c).

Collection and preparation of samples

At day 19, overnight fasted rats were sacrificed under thiopental (Eipico, Egypt) anesthesia and blood and liver samples were collected. Pieces from the liver were fixed in 10% neutral buffered formalin (NBF) for 48 h and others were kept at − 80 °C for RNA and protein isolation. Samples from the frozen liver were homogenized in cold PBS (10% w/v) for the determination of glutathione (GSH), SOD, CAT, glutathione peroxidase (GPx), cytochrome c, caspase-3, malondialdehyde (MDA), and nitric oxide (NO). For Western blotting, other frozen samples were homogenized in RIPA buffer containing proteinase inhibitors and protein content was measured by Bradford reagent (Bradford 1976).

Assay of liver function, inflammation, and apoptosis markers

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were assayed using Spinreact kits (Spinreact, Girona, Spain). ELISA kits were used for measuring tumor necrosis factor alpha (TNF-α; R&D systems, USA), cytochrome c (Cusabio, Wuhan, China), and caspase-3 (Cusabio, Wuhan, China).

Assay of oxidative stress and antioxidant markers

ROS (Mahmoud et al. 2017d, e), MDA (Ohkawa et al. 1979), NO (Grisham et al. 1996), GSH (Beutler et al. 1963), SOD (Marklund and Marklund 1974), CAT (Cohen et al. 1970), and GPx (Matkovics et al. 1998) were assayed in the liver homogenates of the control and experimental rats.

Histology and immunohistochemistry

The fixed liver samples were processed for paraffin embedding and 5-μm sections were cut. The sections were stained with hematoxylin and eosin (H&E) for examination using light microscopy. The effect of MTX and CA on the expression of Bcl-2-associated X protein (Bax) was determined using immunohistochemical staining (Mahmoud et al. 2017c). Briefly, the sections were blocked in 3% hydrogen peroxide (H2O2) and then probed with anti-Bax (Santa Cruz Biotechnology, USA; 1:1000 dilution) overnight at 4 °C. After washing, the sections were incubated with biotinylated secondary antibody (Santa Cruz Biotechnology, USA) at 1:1000 dilution and then washed. The slides were incubated with streptavidin/peroxidase conjugate followed by diaminobenzidine, counterstained with hematoxylin, and then examined.

Gene expression

RNA was isolated using TRIzol reagent (Invitrogen, USA), quantitated and reverse transcribed into cDNA which was amplified by SYBR Green master mix and the primers listed in Table 1. The 2−ΔΔCt method (Livak and Schmittgen 2001) was used to analyze obtained amplification data followed by normalization to β-actin.

Table 1 Primers used for qRT-PCR
Full size table

Western blotting

A total of 50 μg protein from each sample was separated using SDS-PAGE followed by transfer to PVDF membranes. After blocking, the membranes were washed and incubated with primary antibodies against Nrf2, PPARγ, and β-actin (Novus Biologicals, USA) overnight at 4 °C. After washing with TBST, secondary antibodies (Novus Biologicals, USA) were added to the membranes for 1 h at room temperature. All antibodies were used at 1:1000 dilution. ImageJ (version 1.32j; NIH, USA) was used to quantify the band intensity and results were normalized to β-actin.

Statistical analysis

The obtained results are expressed as means ± standard error of the mean (SEM). The statistical comparisons were made by one-way ANOVA followed by Tukey’s test using GraphPad Prism 7 (La Jolla, CA, USA). A P value < 0.05 indicated a statistical significance.

Results

CA attenuates MTX-induced liver injury in rats

ALT, AST, and ALP were determined to evaluate the hepatoprotective effect of CA (Fig. 1). MTX-administered rats showed significantly elevated serum ALT, AST, and ALP (P < 0.001). In contrast, both doses of CA alleviated ALT (P < 0.01), AST (P < 0.001), and ALP (P < 0.001).

Fig. 1
figure 1

CA prevents MTX-induced liver injury in. CA alleviated serum levels of a ALT, b AST, and c ALP in MTX-administered rats. Data are expressed as mean ± SEM, n = 6. **P < 0.01 and ***P < 0.001. d Photomicrographs of sections in the liver of control rats showing normal histological structure of the hepatic lobules, hepatocytes, and sinusoids. MTX-induced rats showed degenerative changes of hepatocytes (red arrow), fatty infiltration (black arrow), activated Kupffer cells and inflammatory cells infiltration (green arrow), and hemorrhage (blue arrow). Pre-treatment of the MTX-induced rats with 25 mg/kg and 50 mg/kg CA prevented all histological alterations. (Scale bar 50 μm)

Full size image

The histological findings supported the hepatoprotective efficacy of CA as represented in Fig. 1d. The control group showed normal structure of the hepatic lobules, hepatocytes, and sinusoids. In contrast, multiple histological alterations, including degenerative changes of hepatocytes, cytoplasmic vacuolations, fatty infiltrations, activated Kupffer cells (KCs), inflammatory cells infiltration, hemorrhage, and others were observed in rats received MTX. Both doses of CA prevented the histological alterations in MTX-intoxicated rats.

CA attenuates hepatic oxidative stress in MTX-induced rats

MTX-intoxicated rats exhibited significantly (P < 0.001) elevated hepatic ROS (Fig. 2a), MDA (Fig. 2b), and NO levels (Fig. 2c). CA ameliorated effectively (P < 0.001) hepatic ROS, MDA, and NO. The effect of CA on ROS levels was dose-dependent as represented in Fig. 2a.

Fig. 2
figure 2

CA suppresses a ROS, b MDA, and c NO in the liver of MTX-induced rats. Data are expressed as mean ± SEM, n = 6. P < 0.01 and ***P < 0.001

Full size image

CA alleviates cellular antioxidants in liver of MTX-induced rats

To evaluate the effect of CA on the cellular antioxidants, we determined GSH and activity of the antioxidant enzymes (Fig. 3). MTX administration decreased GSH content (Fig. 3a), SOD (Fig. 3b), CAT (Fig. 3c), and GPx (Fig. 3d) in the liver of rats (P < 0.001). CA boosted antioxidant defenses in MTX-administered rats.

Fig. 3
figure 3

CA enhances a GSH content and activity of b SOD, c CAT, and d GPx in the liver of MTX-induced rats. Data are expressed as mean ± SEM, n = 6. *P < 0.05, P < 0.01, and ***P < 0.001

Full size image

CA suppresses inflammation and apoptosis in MTX-induced rats

To assess the effect of CA pre-treatment on the inflammatory response and apoptosis following MTX administration, we determined serum and liver TNF-α and the expression of pro- and anti-apoptosis markers. MTX injection increased serum and liver TNF-α (P < 0.001; Fig. 4a), an effect that was significantly reversed in 25 and 50 mg/kg CA supplemented animals (P < 0.001). Hepatic Bax mRNA was significantly increased in MTX-administered rats (P < 0.001; Fig. 4b). The upregulation of Bax was confirmed by immunohistochemical staining which showed a noticeable increase in Bax in rats received MTX (Fig. 4c). In addition, Bcl-2 mRNA was decreased (P < 0.01; Fig. 4d), whereas Bax/Bcl-2 ratio (Fig. 4e), cytochrome c (Fig. 4f), and caspase-3 (Fig. 4g) were markedly increased (P < 0.001). CA decreased Bax mRNA abundance (P < 0.01), Bax immunostaining, Bax/Bcl-2 ratio (P < 0.001), cytochrome c (P < 0.001), and caspase-3 (P < 0.001), and up-regulated Bcl-2 (P < 0.01).

Fig. 4
figure 4

CA attenuates inflammation and apoptosis in the liver of MTX-induced rats. Pre-treatment with CA decreased a serum and liver TNF-α levels, down-regulated b Bax mRNA, c Bax protein (arrows), e Bax/Bcl-2 ratio, f cytochrome c, and g caspase-3, and d increased Bcl-2 abundance. Data are expressed as mean ± SEM, n = 6. **P < 0.01 and ***P < 0.001

Full size image

CA upregulates hepatic Nrf2/HO-1 signaling in MTX-intoxicated rats

MTX administration decreased Nrf2 mRNA (P < 0.001; Fig. 5a) and protein (P < 0.01; Fig. 5b) in the liver of rats. MTX-induced suppression of Nrf2 signaling was confirmed by the reduced NQO-1 (Fig. 5c) and HO-1 (Fig. 5d). CA increased the expression of Nrf2, NQO-1, and HO-1 in the liver of MTX-intoxicated rats. The 50 mg/kg dose of CA increased the gene expression levels of Nrf2 significantly (P < 0.01) when compared with the lower dose (Fig. 5a).

Fig. 5
figure 5

CA upregulates a Nrf2 gene, b Nrf2 protein, c NQO1 gene, and d HO-1 gene expression in the liver of MTX-induced rats. Data are expressed as mean ± SEM, n = 6. **P < 0.01 and ***P < 0.001

Full size image

CA increases hepatic PPARγ expression in MTX-induced rats

PPARγ mRNA and protein were reduced significantly in the liver of MTX-intoxicated rats (P < 0.001). Both doses of CA alleviated hepatic PPARγ mRNA and protein in MTX-administered rats as depicted in Fig. 6a, b.

Fig. 6
figure 6

CA upregulates a PPARγ gene and b protein expression in the liver of MTX-induced rats. Data are expressed as mean ± SEM, n = 6. **P < 0.01 and ***P < 0.001

Full size image

Discussion

CA is a hydroxycinnamic acid with promising hepatoprotective efficacy. Previous reports have demonstrated the protective efficacy of CA on oxidative stress induced by H2O2 (Ma et al. 2018) and high glucose (Zhu et al. 2017), and FFA-induced metabolic deregulations (Guo et al. 2018) in hepatocytes in vitro. The ameliorative effect of CA on oxidative stress in vivo has been demonstrated in a murine model of cerebral I/R injury (Jia et al. 2018). In addition, CA suppressed inflammation and acute hepatic steatosis in mice (Landmann et al. 2014). Recent studies have shown the efficacy of CA to prevent MTX-induced acute kidney injury via attenuation of oxidative stress and inflammation (Abd El-Twab et al. 2019). Although CA has demonstrated several beneficial effects, its potential to prevent MTX-induced liver injury has not been investigated yet. Herein, we evaluated the effect of CA supplementation on oxidative stress, inflammation, and apoptosis in the liver of MTX-administered rats, pointing to the role of Nrf2 signaling and PPARγ.

MTX administration triggered liver injury and increased serum ALT, AST, and ALP. Altered circulating levels of these enzymes indicate hepatocyte damage and represent a sensitive marker of liver injury. Accordingly, these liver function markers were elevated in serum of MTX-intoxicated rats (Mahmoud et al. 2017b, c). The histological examination supported the biochemical findings where degenerative changes of hepatocytes, inflammatory cells infiltration, hemorrhage, fatty infiltration, and KCs activation were observed in the MTX-administered rats. Accordingly, similar histological alterations have been reported in MTX-administered rodents (Erdogan et al. 2015; Mahmoud et al. 2017b, c; Moghadam et al. 2015). Oral supplementation of CA ameliorated liver function markers and prevented all histological alterations provoked by MTX, demonstrating a potent hepatoprotective effect. Few studies have shown the in vivo hepatoprotective effect CA. For instance, CA attenuated both alcoholic (Landmann et al. 2014) and non-alcoholic steatohepatitis (Kim et al. 2017) in mice, at least in part, by modulating inflammation. Herein, our results confer new information that CA can prevent MTX-induced liver injury in rats.

Elevated ROS levels play a role in MTX hepato- and nephrotoxicity as previously described (Abd El-Twab et al. 2016; Mahmoud et al. 2018, 2017b, c). Therefore, we investigated the effect of CA on ROS levels, lipid peroxidation (LPO), and antioxidants in rat liver following MTX administration. MTX-induced rats exhibited oxidative stress evidenced by increased ROS and MDA. Increased ROS levels can provoke oxidative damage to different cell components, resulting in cell death (Fink and Cookson 2005). Accordingly, hepatic LPO was increased along with NO levels in MTX-administered rats. NO, generated via inducible NO synthase (iNOS) upregulation (El-Sheikh et al. 2015), can react with superoxide radicals producing peroxynitrite which induces oxidative DNA damage and cell injury (McKim et al. 2003).

The exact mechanisms underlying increased ROS generation triggered by MTX are not fully understood. However, in vitro studies have pointed to the involvement of mitochondrial dysfunction. Rat hepatocytes treated with MTX showed decreased MMP, increased ROS levels, cytochrome c release, and cell death (Al Maruf et al. 2018). Similarly, increased ROS generation and cell death were demonstrated in different lymphocytic cell lines in response to low doses of MTX (Herman et al. 2005). In vivo studies have also added support to the role of MTX in triggering ROS generation. For instance, the kidney of rats received a single dose of MTX-exhibited mitochondrial dysfunction (Heidari et al. 2018), upregulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Arab et al. 2018) and elevated ROS levels (Abd El-Twab et al. 2019). In addition, depletion of the mitochondrial antioxidants machinery has been indirectly implicated in MTX toxicity (Kolli et al. 2014). Accordingly, hepatic GSH, SOD, CAT, and GPx were declined in MTX-administered rats in the present study. Besides oxidative stress, MTX resulted in inflammation as shown by the increased serum and liver TNF-α. These findings supported previous studies showing increased serum pro-inflammatory cytokines in MTX-administered rats (Abd El-Twab et al. 2016; Mahmoud et al. 2018, 2017b, c). Given that ROS can activate NF-κB which elicits the transcription of pro-inflammatory mediators, increased TNF-α in the present study might be a direct consequence of MTX-induced excessive ROS production. In addition, peroxynitrite can activate NF-κB and stimulate KCs leading to the release of pro-inflammatory cytokines (Matata and Galiñanes 2002). Furthermore, MTX has been recently reported to increase ROS generation and activate the NF-κB/NLRP3 inflammasome axis in rat kidney (Abd El-Twab et al. 2019).

Interestingly, CA suppressed ROS generation, LPO, and inflammation, and boosted hepatic antioxidants in MTX-administered rats. The antioxidant and anti-inflammatory effects of CA have been previously demonstrated both in vitro and in vivo. Ma et al. have recently reported the ameliorative effect of CA on oxidative stress induced by H2O2 in hepatocytes (Ma et al. 2018). CA ameliorated oxidative stress in high glucose-induced hepatocytes and inhibited inflammation in diabetic mice (Zhu et al. 2017). In lipopolysaccharide (LPS)-induced KCs, pre-treatment with CA downregulated iNOS and TNF-α gene expression (Landmann et al. 2014). In addition, CA suppressed hepatic iNOS protein expression in a mouse model of acute hepatic steatosis (Landmann et al. 2014). Moreover, CA prevented hepatocyte apoptosis in MTX-administered rats in the present investigation. MTX triggered Bax, cytochrome c, and caspase-3 expression, and suppressed Bcl-2. Bax provokes apoptosis via stimulating mitochondrial cytochrome c release and activation of caspase-3 (Almeida et al. 2000), whereas Bcl-2 inhibits this process (Mahmoud et al. 2014; Yang et al. 1997). Given that the balance between pro- and anti-apoptosis proteins determines the cell fate (Almeida et al. 2000), apoptosis has been demonstrated by the increased Bax/Bcl-2 ratio in MTX-intoxicated rats. CA suppressed hepatic Bax, cytochrome c, and caspase-3, and increased Bcl-2 in MTX-administered rats.

To reveal the underlying mechanism of the antioxidant, anti-inflammatory, and anti-apoptosis efficacies of CA, we evaluated its effect on Nrf2 signaling. CA activated Nrf2/HO-1 signaling in the liver of MTX-administered rats. Although activated by ROS, Nrf2 was suppressed in liver of MTX-administered rats in the present study. This is explained by the prolonged excessive generation of ROS as previously reported in multiple in vivo and in vitro studies (Abd El-Twab et al. 2016; Mahmoud and Al Dera 2015; Mahmoud et al. 2017b, c, d, e). The ability of CA to activate Nrf2 signaling has been recently reported in H2O2-induced hepatocytes where it increased the expression of Nrf2 and HO-1 mRNA (Ma et al. 2018). CA upregulated renal expression of Nrf2, NQO-1, and HO-1 in MTX-administered rats as we reported recently (Abd El-Twab et al. 2019). In animal models of liver injury, Nrf2 activation mitigated ROS generation, NF-κB activation, and pro-inflammatory cytokines (Mahmoud et al. 2017a, e). The suppressed inflammation following Nrf2 activation was supported by the study of Pan et al. who reported NF-κB activation in mouse primary astrocytes lacking Nrf2 (Pan et al. 2012). In addition to Nrf2, CA increased the expression of hepatic PPARγ in MTX-administered rats. PPARγ activation has been reported to induce the expression of antioxidant enzymes (Yu et al. 2014) and suppress NADPH oxidase and NF-κB (Hwang et al. 2005; Remels et al. 2009). Furthermore, the dual activation of PPARγ and Nrf2 has been associated with suppressed oxidative stress and inflammation in rat models of hepatotoxicity, fibrogenesis, and hepatocarcinogenesis (Mahmoud and Al Dera 2015; Mahmoud et al. 2019, 2017b, e).

In conclusion, these results confer new information on the mechanism underlying the protective effect of CA on MTX hepatotoxicity. CA enhanced the antioxidant defenses, prevented MTX liver injury, and mitigated excessive ROS generation, LPO, inflammation, and apoptosis in rats. The dual activation of Nrf2 and PPARγ represents, at least in part, the mechanism underlying the hepatoprotective efficacy of CA as illustrated in Fig. 7. Therefore, CA could be supplemented as an adjuvant treatment to prevent liver injury provoked by MTX, pending further investigations to determine its exact mechanisms of action.

Fig. 7
figure 7

A proposed schematic diagram illustrating the protective efficacy of CA on MTX-hepatotoxicity. ROS, reactive oxygen species; Nrf2, nuclear factor (erythroid-derived 2)-like 2; Keap-1, Kelch like-ECH-associated protein 1; ARE, antioxidant response element; PPARγ, peroxisome proliferator activated receptor gamma; RXR, retinoid X receptor; PPRE, PPAR response element NF-κB, nuclear factor-kappaB; Bax, Bcl-2-associated X protein

Full size image