Dibenzazepine combats acute liver injury in rats via amendments of Notch signaling and activation of autophagy
Lamiaa A. Ahmed 1 & Rana H. Abd El-Rhman 2 & Amany M. Gad 2 & Sherifa K. Hassaneen 2 & Mohamad F. El-Yamany 1
Received: 15 April 2020 /Accepted: 13 September 2020
# Springer-Verlag GmbH Germany, part of Springer Nature 2020
Paracetamol is a commonly used over-the-counter analgesic and antipyretic drug. Nevertheless, an overdose of paracetamol leads to hepatic necrosis that can be lethal. This study aimed to assess the potential hepatoprotective effects of dibenzazepine, a Notch inhibitor, against acute liver injury in rats via interfering with oxidative stress, inflammation, apoptosis, autophagy, and Notch signaling. Silymarin (200 mg/kg, p.o.) or dibenzazepine (2 mg/kg, i.p.) were administered to rats for 5 days before a single hepatotoxic dose of paracetamol (800 mg/kg, i.p.). Pretreatment with silymarin and dibenzazepine significantly mitigated oxidative stress, inflammatory and apoptotic markers induced by paracetamol hepatotoxicity where dibenzazepine showed greater repression of inflammation. Furthermore, dibenzazepine was found to be significantly more efficacious than silymarin in inhibiting Notch signaling as represented by expression of Notch-1 and Hes-1. A significantly greater response was also demonstrated with dibenzazepine pretreatment with regard to the expression of autophagic proteins, Beclin-1 and LC-3. The aforementioned biochemical results were confirmed by histopathological examination. Autophagy and Notch signaling seem to play a significant role in protection provided by dibenzazepine for paracetamol-induced hepatotoxicity in rats, which could explain its superior results relative to silymarin.
Keywords Autophagy . Dibenzazepine . Hepatotoxicity . Notch . Paracetamol
Paracetamol is a commonly used antipyretic and analgesic drug with weak antiinflammatory activity (James et al. 2003). It is considered nontoxic at therapeutic doses (10–15 mg/kg) (Rumack 2004; Larson 2007; Jaeschke 2015). However, paracetamol overdose causes lethal hepatotoxicity that leads to liver injury both in experimental animals (Galal
et al. 2012; Prabu et al. 2017; Papackova et al. 2018) and in humans (McGill et al. 2012; Jaeschke 2015).
Cytochrome P450–mediated metabolism of paracetamol generates reactive oxygen species (ROS), leading to subse- quent liver injury. Cell death is caused by the toxic paraceta- mol metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), that activates Kupffer cells to produce inflammatory cytokines such as interleukin-12 (IL-12), IL-18, and tumor necrosis
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00210-020-01977-0) contains supplementary material, which is available to authorized users.
* Lamiaa A. Ahmed [email protected]
Mohamad F. El-Yamany [email protected]
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Department of Pharmacology, Egyptian Drug Authority formerly
Sherifa K. Hassaneen [email protected]
National Organization for Drug Control and Research, Giza, Egypt
factor alpha (TNF-α) which in turn stimulate natural killer (NK) cells (Yoon et al. 2016). Inflammatory mediators and chemokines recruit and cause accumulation of neutrophils in the liver exacerbating its damage (James et al. 2005; Cover et al. 2006).
Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) (Flora et al. 1998) and used as a standard hepatoprotective agent in various liver diseases (Lien et al. 2016; Wang et al. 2018; Gillessen and Schmidt 2020). Several studies have examined hepatoprotection provided by silymarin against paracetamol toxicity (Kiruthiga et al. 2007; Alam et al. 2017; Papackova et al. 2018). In parallel with its antioxidant activity, silymarin has strong antiinflammatory properties that might be correlated to its ability to hinder tran- scription factor, nuclear factor kappa B (NFκB), which stim- ulates the production of proinflammatory mediators (Deep and Agarwal 2007; Ramasamy and Agarwal 2008). Indeed, searching for other pathways that may modulate the pathogen- esis of paracetamol liver injury is required for discovering new strategies against its deleterious effect.
Overexpression of Beclin-1 and LC-3 observed in chronic hepatitis and cirrhosis suggests a central role for autophagy as a stress-mediated process that may limit liver injury (Kotsafti et al. 2012). Furthermore, Ni et al. (2012) showed that autoph- agy protected against paracetamol-induced hepatotoxicity. This protection could be provided through the removal of abnormal mitochondria and therefore limiting a major intra- cellular source of ROS production (Levine and Kroemer 2008). Autophagy involves a cascade of morphological pro- cesses, with the activation of several signaling transduction pathways, including apoptosis and the Notch pathway (Shi et al. 2017).
Notch signaling plays a crucial role in cell–cell communi- cation from embryogenesis to adulthood (Bray 2006; Ables et al. 2011). Activation of Notch signaling induces the Notch intracellular domain (NICD) to translocate from the cytoplasm to the nucleus where it binds to C protein binding factor-1 (CSL) and converts the complex from a repressor to an acti- vator of Notch target genes. These genes include the hairy and enhancer of split-1 (Hes-1) and hairy/enhancer of split related with YRPW motif protein-1(Hey-1) (Struhl 1998). Hes-1 in- duces NF-κB gene transcription, which links Notch and in- flammatory signaling pathways (Oswald et al. 1998). Notch signaling also seems to play an important role in the patho- genesis of paracetamol-induced hepatotoxicity (Jiang et al. 2017).
Dibenzazepine (DBZ) is a gamma-secretase inhibitor (GSI) that interferes with Notch signaling and effectively prevents the activation of all Notch receptors by inhibiting gamma- secretase complex (Kopan and Ilagan 2004). Particularly, GSIs showed both antiinflammatory and antiproliferative properties in experimental models of abdominal aortic aneu- rysm and angiotensin II (Ang II)–induced cardiac
inflammation and fibrosis (Hans et al. 2012; Pan et al. 2012). Moreover, Fiorotto et al. (2013) revealed that inhibition of Notch with DBZ ameliorated experimentally-induced biliary damage in mice. Thus, Notch signaling demon- strates an important role in liver damage and its associated fibrosis.
Accordingly, exploring the potential protective effect of DBZ compared with a standard hepatoprotective agent (silymarin) in paracetamol-induced hepatotoxicity would be of significance based on the promising finding of Notch inhi- bition by GSI in another model of liver injury.
Materials and methods
Adult male rats of Sprague Dawley strains (150–200 g) were accommodated at the animal house of the National Organization for Drug Control and Research (NODCAR, Giza, Egypt). Animals had access to food and water ad libitum. Rats were maintained at controlled temperatures (21–24 °C) and relative humidity (40–60%) with a 12-h light-dark cycle. Animals were allowed to acclimatize for 1 week before used in the study. Experimental procedures were conducted following the Guide for Care and Use of Laboratory Animals provided by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and were accepted by the Ethics Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University (Permit Number: PT 2385). All efforts were made to reduce animal suffering or pain during experimentation.
Paracetamol was provided by El-Nasr Company for pharma- ceutical Chemicals (Cairo, Egypt) while silymarin was sup- plied by Sedico Pharmaceutical Company (Cairo, Egypt). DBZ was purchased from Sigma-Aldrich Chemical Co (St Louis, MO, USA). Paracetamol, DBZ, and silymarin were prepared in DMSO and corn oil mixture in a ratio of 1:9, where paracetamol and DBZ were injected intraperitoneally (i.p.) using a 23-gauge needle. Silymarin was administered orally using a curved feeding needle with a round tip (16- gauge; tip diameter, 3 mm; length, 75 mm). Other chemicals not otherwise mentioned were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Animals were divided into five groups, 10 animals per group. Group I included control animals that received DMSO/corn oil mixture (1:9) by i.p. injection, daily for seven consecutive
days. Group II served as DBZ control; animals were injected with DBZ (2 mg/kg, i.p) for seven consecutive days (Fiorotto et al. 2013). Group III served as a positive control; animals received vehicle only for 5 days, and on the 5th day, a single dose of paracetamol (800 mg/kg, i.p.) (Acharya and Lau-Cam 2010). Group IV and V rats were given silymarin daily (200 mg/kg/p.o.) (Fakurazi et al. 2008) as a standard hepatoprotec- tive agent and DBZ (2 mg/kg, i.p), respectively, for 5 days followed by a single injection of paracetamol (800 mg/kg, i.p.) on the 5th day. This treatment was followed by oral adminis- tration of silymarin or i.p. injection of DBZ daily till the 7th day, respectively.
At the end of the experiment, rats were anesthetized with thiopental (50 mg/kg, i.p.). Blood samples were collected from retroorbital sinuses for serum separation. Animals were then weighed and sacrificed by cervical dislocation under an- esthesia, and liver tissues were dissected, washed with ice- cold saline, dried, and weighed. Liver tissues were homoge- nized in 50 mM phosphate buffer (pH 7.4) to prepare 10% homogenates. Homogenates were centrifuged at 5000 rpm for 10 min at 4 °C using a cooling centrifuge (Hermile Labortechnik, Germany) to remove cell debris. Tissue super- natants were used for biochemical estimations. Protein content in tissue supernatants was determined as described by Lowry et al. (1951). Other liver tissues were used for PCR, Western blotting, and histopathology. For each group, two sets of ex- periments were conducted: one (n = 6) for biochemical mea- surement and the other (n = 4) for histological examination.
Liver samples were cleared in xylene and embedded in paraf- fin at 56 °C in a hot air oven for 24 h. Paraffin bees wax tissue blocks were prepared and cut into 4-micron sections with a sledge microtome. Tissue sections were collected on glass slides, deparaffinized, stained with hematoxylin and eosin (H&E) for examination using the light microscope (Banchroft et al. 1996). Ten randomly selected fields from each section (five sections for each liver) were evaluated blindly by a pathologist unaware of treatments. Images were captured and processed using Adobe Photoshop version 8. Lesions were scored as 0 (none), 1 (mild), 2 (moderate), and 3 (severe) as previously described (Ackerman et al. 2010).
Determination of serum liver function tests
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatases (ALP), and total bilirubin levels were assessed using Biodiagnostic kits (Egypt). All proce- dures were performed following the manufacturer’s
instructions. Results were expressed as mg/dl for total biliru- bin and U/l for the other previously mentioned parameters.
Determination of oxidative stress biomarkers
Liver reduced glutathione (GSH), malondialdehyde (MDA), total nitrate/nitrite (NOx), and total antioxidant capacity (TAC) were assessed using Biodiagnostic kits (Egypt). All procedures were performed following manufacturer’s instruc- tions, and results were expressed as mg/mg protein for GSH, nmol/mg protein for MDA and TAC, and μmol/mg protein for NOx.
Determination of inflammatory biomarkers
Involvement of inflammation in paracetamol-induced hepato- toxicity was assessed by measuring TNF-α, IL-6, and NF-κB. Hepatic TNF-α and IL-6 were measured using rat specific ELISA kits purchased from SinoGeneClon Biotech CO., Ltd, Hangzhou, China (Cat. # SG-20127; SG-20267 respec- tively). Likewise, NF-κB level was quantified with a rat spe- cific ELISA kit from Bioassay Technology CO., Shanghai, China (Cat. # E0287Ra). All procedures were performed fol- lowing the manufacturer’s instructions, and results were expressed as ng/mg protein.
Determination of apoptotic biomarkers
Active caspase-3 content was detected in liver homogenates using a rat specific ELISA kit purchased from SinoGeneClon Biotech CO., Ltd, Hangzhou, China (Cat. # SG-20396). All procedures were performed following the manufacturer’s in- structions, and results were expressed as ng/mg protein.
Quantitative RT-PCR analysis Notch-1 receptor and Hes-1
Total RNA was purified from liver tissues using an RNeasy Kit (Qiagen, Valencia, CA) following the manufacturer’s in- struction. The purity (A260/A280 ratio) and concentration of RNA were assessed using a spectrophotometer (Beckman, USA).
Quantitative real-time PCR
In brief, first-strand cDNA synthesis was performed with the SuperScript Choice System (Life Technologies, Breda, Netherlands) following the manufacturer’s protocol. In real- time PCR, detection methods are based on changes in fluores- cence that are proportional to increases in target genes. Monitoring during each PCR cycle provides an amplification
plot and allows determination of the initial amount of target gene expression with great precision. All procedures were com- pleted following the manufacturer’s instructions (Qiagen, USA). Sequences of the used primers are listed in Table 1.
Western blot analysis of Notch-1, Hes-1, Beclin-1, and LC-3 Part of each liver was homogenized in lysis buffer and quan-
tified for protein levels using a Bicinchoninic acid protein assay (BCA) kit (Thermo Fisher Scientific Inc., USA). Protein expression was assessed as previously described (Kandil et al. 2018) using primary antibodies against Notch- 1, Hes-1, Beclin-1, and LC-3 (Thermo Fisher Scientific Inc., USA) in addition to β-actin (Santa Cruz Biotechnology, CA, USA). Protein levels were assessed using a scanning laser densitometer by densitometric analysis of autoradiograms. Results were expressed as arbitrary units after being normal- ized for β-actin protein.
Results were expressed as mean ± standard error (SEM). Multiple comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. Histological scores were expressed as median (min–max) and were analyzed using nonparametric Kruskal–Wallis tests (one-way ANOVA) follow- ed by Dunn’s multiple comparison test. p < 0.05 was the crite- rion for significance. Statistical analysis was performed using Graph Pad Prism software version 5 (San Diego, CA, USA). Correlation coefficients were determined by linear regression analysis. Graphs were drawn using Graph Pad Prism program.
Histopathological examination of liver sections of control rats showed normal histological structure (Fig. 1a). Administration of DBZ only caused no histopathological alteration (Fig. 1b). In contrast, paracetamol-induced hepatotoxicity induced conges- tion in the portal vein, fibroblastic cell proliferation, hyalenosis,
and inflammatory cells infiltration with newly formed bile duct. Diffuse Kupffer cell proliferation in the parenchyma was also observed (Fig. 1c) with a histological score 3 (2–3). In contrast, rats pretreated with silymarin showed marked improvement of the liver histology with only mild dilatation and congestion of the portal veins associated with few inflammatory cells infiltra- tion in the portal area (Fig. 1d) with a histological score 0 (0–1). Similarly, rats pretreated with DBZ showed focal areas of in- flammatory cells infiltration in the portal vein accompanied by mild dilation and congestion of parenchyma in the portal area (Fig. 1e) with a histological score 0 (0–1).
Liver function tests
Paracetamol treatment caused marked deterioration of liver function as shown by elevated serum levels of ALT, AST, ALP, and total bilirubin by 187.5%, 95.8%, 163.1%, and 166.6%, respectively, compared with control group. Notably, these levels were significantly suppressed in silymarin and DBZ pretreated groups compared with paracetamol group. Moreover, these levels remained unchanged in rats treated with DBZ only compared with control animals (Table 2).
Oxidative stress biomarkers
GSH and TAC contents were significantly reduced in paracetamol-injected rats by 64.9% and 69.69%, respectively, while MDA and NOx contents were markedly increased by 110.5% and 197.1%, respectively compared with control values. Rats pretreated with silymarin or DBZ revealed signif- icant increase in GSH and TAC contents, whereas MDA and NOx contents were significantly decreased compared with para- cetamol group. Besides, animals administered DBZ only dem- onstrated no significant changes in these markers compared with control group (Table 3).
Liver contents of TNF-α, IL-6, and NF-κB were increased by 467.2%, 235.5%, and 159.2%, respectively, after administration
Table 1 The sequence of the
primers used in the quantitative real time-polymerase chain
Target gene Primers sequence Gene bank accession number
Notch-1 (rat) F: 5′-CACCCATGACCACTACCCAGTT-3′ R:5′-CCTCGGACCAATCAGAGATGTT-3′
F: 5′-GGTCGGTGTGAACGGATTTGG-3′ R: 5′-ATGTAGGCCATGAGGTCCACC-3′
Fig. 1 Effect of pretreatment with silymarin and dibenzazepine on paracetamol-induced histological changes in liver tissues (magnification × 400). a–f H & E staining. a Control group and b DBZ only group showing normal histological structures of the portal vein (pv), bile duct (bd) and surrounding hepatocytes (h). c Paracetamol group showing congestion in the portal vein (pv), fibroblastic cells proliferation (f), hyalenosis (hy) and inflammatory cells infiltration (arrow), with newly formed bile duct (bd) and diffuse Kupffer cells (k). d Silymarin pretreatment and e DBZ pretreatment show mild dilation and congestion in the portal vein (pv)
with infiltration of few inflammatory cells in the portal area (arrow). f Quantitative image analysis for histological examination expressed as a histological score. Data are presented as medians (min–max) from four rats. Statistical analysis was carried out using nonparametric Kruskal– Wallis test (One-way ANOVA) followed by Dunn’s multiple comparison test. *p < 0.05 vs. control, #p < 0.05 vs. paracetamol. DBZ: dibenzazepine. Representative histological images of different scores of hepatic damage are presented in supplementary figure 1
of paracetamol. Pretreatment with silymarin and DBZ significant- ly attenuated elevation of these parameters compared with the paracetamol group. Pretreatment with DBZ showed greater re- pression of IL-6 and NF-κB than silymarin pretreatment. On the other hand, group pretreated with DBZ only revealed more or less similar values to control group (Fig. 2).
Paracetamol caused a significant elevation of hepatic active caspase-3 (251.6%) as compared with control group. On the other side, silymarin and DBZ administration succeeded to significantly reduce active caspase-3 compared with
paracetamol group with no significant effect in rats pretreated with DBZ only compared with control group (Fig. 2).
Notch signaling pathway
As an evidence of liver damage, treatment with paracetamol enhanced gene expression of Notch-1 and Hes-1 6- and 5- fold, respectively as well as protein expression of Notch-1 and Hes-1 4- and 5-fold, respectively, as compared with con- trol group. In contrast, pre-treatment with silymarin and DBZ significantly downregulated gene and protein expression of Notch-1 and Hes-1 compared to paracetamol-treated group.
Table 2 Effect of pretreatment
with silymarin and dibenzazepine on paracetamol-induced changes of liver function tests in rats
Groups ALT (U/L) AST (U/L) ALP (U/L) Total bilirubin (mg/dl)
Control 19.37 ± 0.22# 46.09 ± 0.62# 102.1 ± 1.21# 1.08 ± 0.05#
DBZ only 21.33 ± 0.65# 49.97 ± 1.21# 110.8 ± 2.73# 1.34 ± 0.07#
Paracetamol 55.69 ± 0.42* 90.26 ± 1.02* 268.6 ± 4.26* 2.88 ± 0.12*
Silymarin pretreatment 26.08 ± 0.61*,# 53.46 ± 1.55*,# 123.8 ± 4.84*,# 1.59 ± 0.11*,#
DBZ pretreatment 25.93 ± 0.51*,# 53.87 ± 1.96*,# 124.8 ± 2.19*,# 1.58 ± 0.09*,#
Data are represented as the mean ± SEM of 6 observations. Statistical analysis was done using one way ANOVA followed by Tukey’s post-hoc test. *p< 0.05 vs. control, # p< 0.05 vs. paracetamol. DBZ, dibenzazepine; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase
Table 3 Effects of pretreatment
with silymarin and dibenzazepine on paracetamol-induced changes of oxidative stress markers in rats
Groups GSH (mg/mg protein)
MDA (nmol/mg protein)
NOx (μmol/mg protein)
TAC (nmol/mg protein)
Control 94.03 ± 0.89# 178.6 ± 2.76# 19.50 ± 0.28# 0.33 ± 0.01#
DBZ only 87.93 ± 1.94# 183.2 ± 3.62# 21.20 ± 0.35# 0.28 ± 0.02#
Paracetamol 32.95 ± 1.24* 375.9 ± 8.35* 57.93 ± 0.61* 0.10 ± 0.01*
79.64 ± 1.79*,#
199.4 ± 6.02#
25.58 ± 0.76*,#
0.23 ± 0.01*,#
DBZ pretreatment 78.41 ± 1.84*,# 195.2 ± 2.08# 24.25 ± 0.77*,# 0.23 ± 0.01*,#
Data are represented as the mean ± SEM of 6 observations. Statistical analysis was done using one way ANOVA followed by Tukey’s post hoc test. *p< 0.05 vs. control, # p< 0.05 vs. paracetamol. DBZ, dibenzazepine; GSH, reduced glutathione; MDA, malonadialdehyde; NOx, total nitrate/nitrite; TAC, total antioxidant capicity
Interestingly, DBZ pretreatment showed significant attenua- tion of Notch-1 and Hes-1 expression compared with the silymarin group. Compared with control group, rats adminis- tered with DBZ only did not show any significant change in both gene and protein expression of Notch-1 and Hes-1 (Figs. 3 and 4).
The elevation of oxidative stress and inflammatory markers as well as the stimulation of Notch signaling in paracetamol group was accompanied by a significant reduction of Beclin- 1 and LC-3 protein expression by 86.6% and 88.6%,
respectively compared with control values. However, protein expression of Beclin-1 and LC-3 was significantly elevated by silymarin pretreatment compared with paracetamol-treated rats. A significantly greater response was demonstrated using DBZ pretreatment. Notably, Beclin-1 and LC-3 protein ex- pression remained at normal levels in rats treated with DBZ only (Fig. 5).
Estimation of ALT level as a specific marker of hepatic dam- age was found to be strongly correlated with levels of GSH, MDA, NF-κB, caspase-3, Notch-1, Hes-1, Beclin-1, and LC-
Fig. 2 Effect of pretreatment with silymarin and dibenzazepine on the hepatic contents of a TNF-α, b IL-6, c NF-κB, and d caspase-3 in liver tissues of paracetamol- injected rats. Data are presented as mean ± SEM (n = 6 rats per group). Statistical analysis was done using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. control, #p < 0.05 vs. paracetamol, @p < 0.05 vs. silymarin. DBZ: dibenzazepine
Fig. 3 Effect of pretreatment with silymarin and dibenzazepine on the mRNA levels of a Notch-1 and b Hes-1 in liver tissues of paracetamol-injected rats. Data are presented as mean ± SEM (n = 6 rats per group). Statistical analysis was done using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. control, #p < 0.05 vs. paracetamol, @p < 0.05 vs. silymarin. DBZ: dibenzazepine
3 (r = - 0.9801, r = 0.9786, r = 0.8683, r = 0.9714, r = 0.9821, r = 0.9334, r = - 0.9197, r = - 0.9690), respectively, (p < 0.0001) (Fig. 6).
Drug-induced hepatotoxicity is a known cause of liver failure and accounts for about half of acute liver injury cases
(Kaplowitz 2004). The present investigation was the first to explore hepatoprotective effects of DBZ, a Notch inhibitor, compared with a standard hepatoprotective agent (silymarin) in paracetamol-induced hepatotoxicity in rats. Additionally, probable mechanisms underlying this hepatoprotective effect were explored.
Paracetamol-treated rats showed a marked elevation in levels of serum ALT, AST, ALP, and total bilirubin with respect to their control values. Similar results were reported
Fig. 4 Effect of pretreatment with silymarin and dibenzazepine on protein expression of a Notch-1 and b Hes-1 in liver tissues of paracetamol-injected rats. Data are presented as mean ± SEM (n = 6 rats per group). Statistical analysis was done using one-way ANOVA
followed by Tukey’s post hoc test. *p < 0.05 vs. control, #p < 0.05 vs. paracetamol, @p < 0.05 vs. silymarin. DBZ: dibenzazepine. The western blots were cropped; whole blots are presented in supplementary figure 2
Fig. 5 Effect of pretreatment with silymarin and dibenzazepine on protein expression of a Beclin-1 and b LC-3 in liver tissues of paracetamol-injected rats. Data are presented as mean ± SEM (n = 6 rats per group). Statistical analysis was done using one-way ANOVA
followed by Tukey’s post hoc test. *p < 0.05 vs. control, #p < 0.05 vs. paracetamol, @p < 0.05 vs. silymarin. DBZ: dibenzazepine. The western blots were cropped; whole blots are presented in supplementary figure 3
by other investigators who showed that treatment of rats with single doses of paracetamol revealed a significant increase in serum levels of liver enzymes such as ALT, AST, and ALP
(Yilmaz et al. 2015; Okokon et al. 2017; Chakradhar and Sarath Babu 2018) and caused a remarkable increase in serum levels of total bilirubin (Okokon et al. 2017; Rao et al. 2018).
Fig. 6 Correlation analysis. Correlation coefficients between serum ALT levels and levels of GSH (A), MDA (B), NF-κB (C), caspase-3 (D), Notch-1 (E), Hes-1 (F), Beclin-1 (G) and LC-3 (H) in liver tissues of
paracetamol-injected rats. Statistical analysis was done by linear regression analysis ( < 0.0001)
These results reflect liver damage and subsequent release in- tracellular liver enzymes into circulation (Sallie et al. 1991; Gutiérrez and Solís 2009). In the current study, results showed that administration of silymarin and DBZ before paracetamol markedly attenuated paracetamol-induced changes in these markers of hepatic damage in blood.
The findings of this study denoted reduction of liver GSH and TAC contents as well as elevation of MDA and NOx contents affirming that oxidative stress is triggered by para- cetamol toxicity. Immensely, these results are consistent with a previous investigation (Tai et al. 2015). This study demon- strated elevated oxidative stress markers in the livers of para- cetamol intoxicated rats. These results might be attributed to the excessive production of NAPQI associated with a reduc- tion of levels of free GSH. Covalent binding of NAPQI to vital hepatic proteins may cause cytotoxicity (Cohen and Khairallah 1997). Aberrant mitochondrial structure also hin- ders mitochondrial respiration and consequently, triggers en- ergy depletion, ROS formation, and lipid peroxidation (Reid et al. 2005; Jaeschke and Bajt 2006; Jaeschke et al. 2012). Furthermore, enhanced lipid peroxidation observed in the present study is consistent with several earlier studies (Abirami et al. 2015; Hamza and Al-Harbi 2015; El Faras and El Sawaf 2017). This lipid peroxidation reflects the inabil- ity of antioxidant defense mechanisms in the liver to prevent the formation of excess amounts of free radicals (Niki 2009; Farghaly and Hussein 2010; Du et al. 2016). Notably, pretreat- ment of paracetamol-injected rats with silymarin or DBZ ame- liorated the oxidative stress by maintaining GSH and TAC levels and lowering MDA and NOx levels. These findings are in agreement with previous studies showing that agents with antioxidant properties revealed protective effects against paracetamol-induced hepatotoxicity (Palabiyik et al. 2016).
The antioxidant activity of DBZ could be attributed to its function as a Notch inhibitor. Notch-1 signaling plays a sig- nificant role in the regulation of lipid peroxidation. A previous clinical study of Alzheimer disease found a significant eleva- tion in levels of intracellular and secreted amyloid β-peptide (Aβ) by hydrogen peroxide. This finding was attributed to c- Jun N-terminal kinase (JNK)-dependent activation of gamma- secretase and upregulation of Notch-1 (Shen et al. 2008). Moreover, Notch-1 may regulate lipid oxidation indirectly by affecting transcriptional activators or repressors that, in turn, regulate the expression of oxidative genes (Song et al. 2016). Consequently, inhibition of Notch-1 signaling could affect lipid peroxidation and oxidative stress status as ob- served herein.
In addition to oxidative stress, inflammation plays an es- sential role in the pathology of paracetamol-induced hepatic injury. In this study, paracetamol-treated rats showed an ap- preciable increase in inflammatory cytokines; TNF-α, IL-6, and NF-κB. These results were confirmed by histopathologi- cal findings showing necrosis and inflammatory cells
infiltration in hepatocytes, together with congestion in portal vein and fibrosis. These data are in agreement with previous studies (Galal et al. 2012; Okokon et al. 2017).
NF-κB plays an important role in several biological pro- cesses, including immune responses, inflammation, and regu- lation of cell differentiation, proliferation, and survival (Gerondakis et al. 1999; Pasparakis et al. 2006). In addition, NF-κB stimulates the production of proinflammatory cyto- kines such as TNF-α (Beutler 2000; Kerfoot and Kubes 2005). A previous study showed enhanced expression of in- flammatory cytokines in the liver after paracetamol injury (Zhang et al. 2016). Excessive production of the paracetamol metabolite, NAPQI, causes primary hepatic damage accom- panied by elevation of inflammatory mediators resulting in tissue necrosis (Blazka et al. 1995), and triggering of an innate immune response with overproduction of TNF-α (Jaeschkea and Bajta 2010). TNF-α has the ability to stimulate an inflam- matory cascade that induces other proinflammatory cytokines, such as IL-1β, IL-6, IFN-γ, and cell adhesion molecules in association with hepatocyte damage (Tiegs et al. 1989; Lee et al. 2010; Oztay et al. 2010).
Particularly, GSI possesses both antiproliferative and antiinflammatory properties (Piggott et al. 2011; Hans et al. 2012; Pan et al. 2012). GSI actions include inhibition of mac- rophage activation, T cell infiltration, and cytokine expression (Piggott et al. 2011; Hans et al. 2012). Interestingly, the Notch pathway plays a significant role in hepatic fibrosis by modu- lating inflammatory responses and macrophage function (Geisler and Strazzabosco 2015). Pharmacologically, inhibi- tion of Notch reduced fibrosis in an experimental model of carbon tetrachloride (CCl4)–induced liver injury (Outtz et al. 2010). Mechanistically, TNF-α induces phosphorylation of histone H3 via the Hes-1 promoter (Maniati et al. 2011). Additionally, Hes-1 induces NF-κB gene transcription, pro- moting further stimulation of proinflammatory cytokines which links Notch and inflammatory signaling pathways (Oswald et al. 1998). Being a GSI, DBZ pretreatment signif- icantly attenuated increases, at the translational level, of the Notch pathway molecules and inflammatory markers. Moreover, the elevation of inflammatory mediators was ac- companied by upregulation of protein expression of Beclin-1 and LC-3 in the current study. A previous study reported that the increase in TNF-α activated expression of autophagy genes (LC-3 and Beclin-1) through stimulation of JNK signal- ing and impeding protein kinase B (Akt) activation (Jia et al. 2006). TNF-α also induces autophagy through extracellular signal-regulated kinases (ERK1/2) pathway (Sivaprasad and Basu 2008; Venkatesan et al. 2016).
Apoptosis is also a key factor in paracetamol-induced liver injury. The present results show that paracetamol overdose triggered apoptotic cell death as measured by significant ele- vation of the active form of caspase-3 in liver tissues. Moreover, effects of paracetamol on levels of caspase-3
enzyme were notably reduced by silymarin and DBZ pretreatments. This result is consistent with Bajt et al. (2006) and Jaeschke et al. (2011), who noted that paracetamol over- dose induced release of cytochrome c from the mitochondrial intermembrane space and consequently stimulated apoptosis.
Autophagy plays a significant role in cell death and can protect cells by avoiding programmed apoptotic cell death. Mechanistically, sequestration of damaged mitochondria pre- vents the release of cytochrome c and the formation of acti- vated apoptosomes in the cytoplasm. Autophagy also protects cells from caspase-independent death. This activity was clar- ified in a previous experimental study of rapamycin pretreat- ment (Ravikumar et al. 2006). Furthermore, stimulation of autophagy enhances autophagosome formation and inhibits Notch signaling, including Notch-1, NICD, and Hes-1. These interactions clarify the link between autophagy and Notch signaling (Wu et al. 2016). Interestingly, in the present study, pretreatment with DBZ was more efficacious than the standard hepatoprotective agent (silymarin) regarding modu- lation of parameters of inflammation (IL-6, NF-κB), autoph- agy (Beclin-1, LC-3), and Notch signaling (Notch-1, Hes-1).
Finally, the present data show a strong correlation between serum ALT level and oxidative stress, inflammation, apopto- sis, autophagy, and Notch signaling. Serum ALT activity has been used as a specific biomarker for hepatic damage in pre- clinical studies and humans (Yang et al. 2014). The present findings are consistent with previous studies showing that ALT activity was negatively correlated with hepatic GSH concentration in patients with acute viral hepatitis in addition to its positive correlation with MDA in women with pre- eclampsia (Atiba et al. 2016; Swietek and Juszczyk 1997). Likewise, a correlation was reported between hepatic damage and up-regulation of IL-6 and NF-κB in acute liver injury (Borkham- Kamphorst et al. 2013). In our study, ALT activity was positively correlated with hepatic NF-κB concentration, which supports a relationship between liver damage and in- flammation. Furthermore, a previous study showed a strong correlation between ALT activity and caspase-3 activity in serum from patients with acute hepatitis C viral (HCV) infec- tion (Choi et al. 2016). These data are in agreement with the results of our present investigation. Interestingly, the current data demonstrate for the first time a strong positive correlation between ALT activity and Notch signaling (i.e., Notch-1 and Hes-1 gene and protein expression) in addition to a negative correlation with autophagy (i.e., Beclin-1 and LC-3 protein expression). Thus, these data confirm the correlation between the degree of hepatic damage and oxidative stress, inflamma- tion, apoptosis, autophagy, and Notch signaling.
In summary, our results suggest that DBZ is a promising drug for protection against paracetamol-induced hepatotoxic- ity. Beneficial activity of DBZ was confirmed by significant improvement of liver function and reduction in markers of oxidative stress, inflammation, and apoptosis. Interestingly,
DBZ was more efficacious than silymarin (a well-known he- patoprotective agent), which could be attributed to the greater effect of DBZ on autophagy and Notch signaling. These re- sults pave the way for further mechanistic investigations of the Notch signaling in experimental models of hepatotoxicity.
Acknowledgments The authors would like to thank Prof. Dr. A. Bakear (Pathology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt) for his assistance in the histopathological examinations.
Authors’ contributions LAA, RHA, SKH, and MFE conceived and de- signed research. RHA, AMG, and SKH conducted experiments. LAA, RHA, AMG, and MFE analyzed data. RHA and LAA wrote the manu- script. All authors read and approved the final manuscript. All data were generated at National Organization for Drug Control and Research (NODCAR, Giza, Egypt), and no paper mill was used.
Data availability All data generated or analyzed during this study are included in this published article.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethics approval All procedures performed in this study were conducted in accordance with the regulations approved by the Ethics Committee at Faculty of Pharmacy, Cairo University (permit number: 2385). The in- vestigation complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011).
Consent to participate Not applicable. Consent to publish Not applicable.
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