The c-Met inhibitor capmatinib alleviates acetaminophen-induced hepatotoxicity
Kareem M. Saada, Mohamed E. Shakera,b,⁎, Ahmed A. Shaabana,c, Rehab S. Abdelrahmana,d,
Eman Saida
a Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
b Department of Pharmacology, Faculty of Pharmacy, Jouf University, Sakaka 2014, Saudi Arabia
c Department of Clinical Pharmacy and Pharmacy Practice, Faculty of Pharmacy, Aqaba University of Technology, Aqaba 77110, Jordan
d Department of Pharmacology and Toxicology, Faculty of Pharmacy, Taibah University, Al Madinah Al-Munawwarah 30001, Saudi Arabia
Abstract
Acetaminophen (APAP)-induced hepatotoxicity comes among the most frequent humans’ toxicities caused by drugs. So far, therapeutic interventions for such type of drug-induced toxicity are still limited. In the current study, we examined the influence of capmatinib (Cap), a novel c-Met inhibitor, on APAP-induced hepatotoxicity in mice when administered 2 h prior, 2 h post and 4 h post APAP-challenge. The results revealed that Cap administration significantly attenuated APAP-induced liver injury when administered only 2 h prior and post APAP-administration. Cap hepatoprotective effect was mediated by lowering the excessive formation of lipid peroxidation and nitrosative stress products caused by APAP. Besides, Cap attenuated APAP-induced over- production and release of proinflammatory mediators like TNF-α, IL-1β, IL-17A, IL-6, and MCP-1. Cap treatment also led to avoidance of APAP-subsequent repair by abating APAP-induced elevation of hepatic IL-22 and PCNA expressions. In conclusion, c-Met receptor inhibition may be a potential strategy for alleviating APAP-hepato- toxicity, especially when administered in the early phase of intoxication.
1. Introduction
The mesenchymal-epithelial transition (c-Met), a member of tyr- osine kinase receptors, is responsible for cellular proliferation, motility and migration [1]. This cell surface localized receptor is activated conventionally by binding with hepatocyte growth factor (HGF) [2]. Otherwise, this receptor can also be activated upon its hetero- dimerization with other surface molecules, such as the semaphorin receptor [3], CD-44 [4], FAS receptor [5] and integrins [6]. The HGF/c- Met axis is well-known for its major role in organogenesis during em- bryo development [7]. This axis has also a protective role in adulthood as it promotes the cellular survival under stress, healing and re- generation alongside suppressing the chronic inflammation and fibrosis [8]. Despite its beneficial role, aberrant c-Met receptor activation triggered cell migration, proliferation, invasion and angiogenesis in several malignancies [9–12]. Otherwise, selective deletion of c-Met
receptors in liver hepatocytes impaired the repair and survival of he- patocytes [13].
Acetaminophen (APAP) is a widely used analgesic and antipyretic due to its safety and efficacy at the normal prescribed doses. However,
exposure to an overdose of APAP can lead to massive hepatocellular necrosis that may end up in severe hepatic failure and the need for hepatic transplantation [14]. Thus, it not surprising that APAP-poi- soning has a high rate of mortality around the world [15].After APAP administration, the major part of the dose is excreted from the kidney as sulfate and glucuronide conjugates, while a minor part is oxidized to the reactive N-acetyl-p-benzoquinone imine (NAPQI) metabolite [16]. In normal doses, NAPQI is efficiently neutralized by the non-enzymatic antioxidant glutathione (GSH). In high doses, how- ever, NAPQI can deplete and bypass GSH detoxification to form adducts with many cellular proteins. These abnormalities initiate the first phase of oxidative stress-induced hepatocellular injury and death, which eventually becomes worse upon release of danger-associated molecular patterns (DAMPs), the drivers of the second phase of the sterile in- flammatory injury [17,18]. To resolve APAP-damage, a third phase of hepatic regeneration is initiated and orchestrated by certain cytokines, mitogenic pathways and other factors [19]. For instance, cytokines like IL-6 and IL-22 are released to activate the signal transducer and acti- vator of transcription 3 (STAT3)-dependent survival and compensatory hepatocellular proliferation pathways for recovery from APAP-intoxication [20]. Consequently, proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase δ, is elevated to mediate hepatocellular proliferation and DNA repair from APAP-over- dose [21,22].
c-Met inhibitors are a new subset of tyrosine kinase inhibitors that have been recently introduced for the treatment of diverse malignancies [23]. Of these inhibitors, capmatinib (Cap) is a selective and potent ATP-competitive inhibitor of the c-Met receptor that has a favorable safety profile [24,25]. Because the activity of c-Met receptor is linked to inflammation and adaptive cellular responses, we sought to examine the impact of switching off the activation of this receptor on APAP- induced hepatotoxicity by Cap administration in mice at time intervals related to the clinical application.
2. Materials and methods
2.1. Drugs and chemicals
Cap was provided as a gift from Novartis, Switzerland, while APAP was purchased from Sigma, USA.
2.2. Animals
Male BALB/c mice (12–14 week-old, 30–35 g) had free access to food and water throughout the acclimatization and experimentation. The experimental procedures were concordant with those of the National Institutes of Health guidelines and the Research Ethics Committee for Care of Laboratory Animals at Faculty of Pharmacy, Mansoura University, Egypt.
2.3. Experimental design and groups
Both Cap and APAP were dissolved in physiological normal saline and were administered intraperitoneally. Cap-dose (10 mg/kg/10 mL) was chosen to achieve a substantial c-Met inhibition with no apparent adverse effects based on the guidance from the supplier, preliminary trials and the previous study [26]. APAP-dose (500 mg/kg/20 mL) was selected to provide eminent hepatotoxicity with minimal mortality based on the previous study [27]. The mice were randomized into 5 groups (Fig. 1):
(1) Normal Control: received the vehicle without Cap, and after 2 h, the vehicle without APAP;
(2) APAP: received the vehicle without Cap and after 2 h, APAP;
(3) Cap 2 h prior APAP: received Cap and after 2 h, APAP;
(4) Cap 2 h post APAP: received first APAP and after 2 h, Cap;
(5) Cap 4 h post APAP: received first APAP and after 4 h, Cap.
An additional group was included, which comprised mice that were post treated with the standard antidote N-acetyl cysteine (NAC) (100 mg/kg/10 mL, oral) 2 h after APAP-administration. Twenty-four hours after receiving APAP, the mice were euthanized by thiopental (120 mg/kg/10 mL). Also, 2 groups of mice were administered saline or Cap 2 h prior APAP and euthanized after 1 h from the intoxication. Thereafter, blood samples were withdrawn from the heart and cen- trifuged at 1000g for 10 min at 4 °C for isolating sera, which were stored at −80 °C till analysis. Thereafter, a portion of the liver was stored at −80 °C for oxidative stress/antioxidant assays and the enzyme-linked immunosorbent assay (ELISA). Another portion of the liver was also fixed in 10% (v/v) neutral-buffered formalin solutions for 48 h prior processing into paraffin blocks for histopathological and im- munohistochemical evaluations.
2.4. Serum biochemical indices of hepatic injury
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were evaluated as indicators of hepatic injury by kinetic kits (Spectrum-diagnostics, Egypt).
2.5. Hepatic histopathological and immunohistochemical evaluations
For hepatic histopathological evaluation, pre-fixed liver slices were processed into paraffin blocks, followed by preparing 5 μm sections mounted on slides and staining with hematoxylin-eosin (HE). Histopathological scoring for the lesion severity (necrosis, lobular and portal inflammation) was as follows: 0, absent; 1, mild; 2, moderate; 3, severe. For the immunohistochemical evaluation of PCNA, 5 μm sections were transferred to coated glass slides, followed by antigen unmasking, blockade of endogenous peroxidases and non-specific pro- tein binding. Thereafter, the sections mounted on slides were pre-in- cubated with PCNA primary antibody (BioLegend, USA), followed by the secondary antibody and chromogen. Image J software (USA) was used for PCNA-immunohistochemical quantification.
Fig. 1. Schematic presentation of the experimental design.
2.6. Cytokines assessments
Cytokines assessments were done by the aid of ELISA kits supplied by BioLegend (San Diego, USA). Tumor necrosis factor-α (TNF-α), in- terleukin (IL)-6 and IL-17A were estimated in liver lysates, whereas IL- 1β and monocyte chemoattractant protein-1 (MCP-1) were quantified in serum. Moreover, IL-22 was estimated in both liver lysates and serum. Liver lysates were prepared by homogenizing liver tissue (10% w/v) in a cooled lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 2 mM AEBSF). Subsequent to centrifugation at 4000g for 10 min at 4 °C, the supernatants of liver lysates alongside pre-diluted sera (2:1 in phosphate-buffered saline) were loaded on the pre-coated 96 well plates for the ELISA according to the manufacturer protocol. Protein concentrations were quantified in lysates with the Bradford reagent [28].
2.7. Quantification of hepatic oxidative stress and antioxidant parameters
Liver tissue (10% w/v) was homogenized in an ice-cold buffer (20 mM Tris-HCl, 1 mM EDTA, pH 7.4) and then, centrifuged at 2000g for 15 min at 4 °C. Protein concentrations were quantified in super- natants according to the referred method [29].
2.7.1. Quantification of hepatic reduced glutathione (GSH)
Reduced glutathione (GSH) was estimated as an index of the non- enzymatic antioxidants in the liver according to the referred assay with minor modifications [30]. For protein precipitation in samples, 0.025 mL of 50% (w/v) trichloroacetic acid was mixed with liver homogenate (0.225 mL) samples, which were centrifuged at 3000g for 10 min at 4 °C. Thereafter, 0.125 mL of the clear supernatant was mixed with 1 mL of assay buffer (0.2 M Tris-HCl, 1 mM EDTA, pH 8.9) and 0.05 mL methanol containing 10 mM 5,5′-dithiobis(2-nitrobenzoic acid) (Sigma-Aldrich, USA). The generated yellow color was measured and quantified from a standard calibration curve of GSH (Acros Or- ganics, USA) spectrophotometrically at a wavelength of 412 nm.
2.7.2. Quantification of hepatic malondialdehyde (MDA) and 4- hydroxynonenal (4-HNE)
Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were analyzed as indices of lipid peroxidation in the liver [31]. Aliquots of liver homogenate (0.4 mL) were mixed with 0.65 mL of the chromogen [acetonitrile containing 10.3 mM N-methyl-2-phenylindole and me- thanol containing 32 μM FeCl3 in a ratio of 3 to 1] and 0.15 mL of 15.4 M methanesulfonic acid (Merck, Germany). The mixtures were
incubated at 45 °C for 40 min and centrifuged at 3000g for 10 min at 4 °C afterward. The absorbances of supernatants were measured spec- trophotometrically at a wavelength of 586 nm against sample blanks, followed by estimation of the total MDA and 4-HNE concentration from a standard calibration curve of 1,1,3,3-tetramethoxypropane (Sigma- Aldrich, USA).
2.7.3. Quantification of hepatic total nitrate/nitrite (NOx)
The total nitrate/nitrite (NOx) was determined as an indicator of nitrosative oxidative stress in the liver according to the referred method [32]. Fifty microliters of 25% (w/v) sulfosalicylic acid were mixed with liver homogenate aliquots (0.45 mL), which were centrifuged at 2000g afterward for 10 min at 4 °C for deproteination. Aliquots of clear su- pernatants (0.3 mL) were mixed with 0.3 mL 1 M HCl containing 0.8% (w/v) vanadium trichloride (Sigma-Aldrich, USA). Thereafter, the re- action mixtures were incubated for 45 min at 37 °C subsequent to adding 0.4 mL of the chromogen (2% (w/v) sulfanilamide in 5% (v/v) HCl premixed 0.1% (w/v) N-(1-naphthyl) ethylenediamine dihy- drochloride in ratio 1 to 1). The produced violet color was measured spectrophotometrically at a wavelength of 540 nm, followed by an es- timation of the total NOx concentration from a standard calibration curve of sodium nitrate.
2.8. Statistical analysis
Parametric data (means ± SE) were statistically analyzed by one- way ANOVA, ensued by the Tukey-Kramer multiple comparison test. The histopathological scores were analyzed non-parametrically by the Kruskal-Wallis test with Dunn’s multiple comparison post test. GraphPad Prism 7 software (USA) was used for statistical analysis. The cutoff of statistical significance was at P < 0.05. 3. Results 3.1. Cap abrogates APAP-instigated hepatic injury and necrosis Administration of APAP caused a significant (P < 0.001) elevation of serum ALT, AST, and LDH activities (Fig. 2A–C), compared to the control group. Treatment with cap 2 h prior and post APAP-challenge led to a marked decrease in these parameters of hepatocellular injury in comparison to the untreated APAP-group. However, Cap treatment 4 h post APAP caused mediocre or no decrease in the aforementioned parameters of hepatocellular injury, compared to the untreated APAP-group. HE-stained staining for liver sections showed that APAP-over- dose induced severe hepatic necrosis and hydropic degeneration, cen- trilobular and portal inflammation (Fig. 3A–D). Meanwhile, Cap re- duced the extent of these hepatic abnormalities, when used 2 h before or after APAP-administration, rather than the 4 h time point. Note-worthy, the protection of Cap, when administered 2 h post APAP-in- toxication, was almost equivalent to that of the standard antidote NAC. Fig. 2. The influence of capmatinib (Cap) treatments on acetaminophen (APAP)-elicited changes in serum alanine aminotransaminase (ALT, A), aspartate amino- transaminase (AST, B) and lactate dehydrogenase (LDH, C). Statistical significances of data (means ± S.E., n = 6/group) were denoted as **P < 0.01 and ***P < 0.001 from the control group, while ##P < 0.01 and ###P < 0.001 from the APAP group. Fig. 3. The influence of capmatinib (Cap) treatments on acetaminophen (APAP)-elicited changes in scores of hepatocellular necrosis (A), centrilobular inflammation (B), portal inflammation (C) and hepatic histopathology (hematoxylin-eosin staining, 100×, D). Statistical significances of data (means ± S.E., n = 6/group) were denoted as **P < 0.01 and ***P < 0.001 from the control group, while #P < 0.05 and ##P < 0.01 from the APAP group. 3.2. Cap does not affect APAP-induced depletion of GSH but counteracts APAP-induced oxidative and nitrosative stresses Overproduction of NAPQI following APAP-overdose leads to the consumption of hepatic GSH pool and an increase of oxidative and nitrosative stresses within the hepatocytes. Accordingly, we sought to investigate whether the protection exerted by Cap against APAP-he- patotoxicity is related to the oxidative/antioxidant balance. In the liver, APAP-insult caused a severe reduction of GSH concentration after 24 h, and Cap did not reverse that reduction at the 3-time points (Fig. 4A). To ascertain whether Cap can affect APAP-metabolism, we compared GSH depletion within 1 h after APAP with or without Cap (when given 2 h prior APAP). The depletion of GSH after 1 h of APAP-insult was almost similar to that of 24 h, and Cap administration to APAP mice did not show a significant difference from the APAP-mice without Cap at 1 h from the intoxication (Fig. 4B). Noteworthy, APAP caused elevation of lipid peroxidation (MDA + 4-HNE) and nitrosative stress (NOx) pro- ducts in the mice livers (Fig. 4C-D). However, Cap significantly sup- pressed APAP-induced elevation of lipid peroxidation and NOx con- centrations in the liver, when used 2 h prior and post, but not 4 h post, APAP-administration. 3.3. Cap attenuates APAP-induced overproduction and release of proinflammatory mediators Following the initial phase of APAP-oxidative injury, several DAMPs are extracellularly liberated from necrotic hepatocytes and stimulate various immune cells recruited to the liver, which release more in- flammatory mediators that amplify the initial phase of injury elicited by APAP-oxidative injury. As speculated, APAP-intoxication markedly in- creased serum MCP-1 and IL-1β concentrations, compared to the normal control mice (Fig. 5A, B). Administration of Cap 2 h post APAP achieved the most optimal therapeutic timing for lowering APAP-in- duced rise of serum MCP-1 and IL-1β concentrations. In the same context, the results elucidated that mice intoxicated with APAP had a higher hepatic expression of proinflammatory cytokines like TNF-α, IL- 6 and IL-17A, compared to the normal counterparts (Fig. 5C–E). Meanwhile, using Cap 2 h prior and post APAP-intoxication abated the higher hepatic expression of these proinflammatory cytokines. Fig. 4. The influence of capmatinib (Cap) treatments on acetaminophen (APAP)-elicited changes in hepatic concentrations of reduced glutathione (GSH, A after 24 h and B after 1 h of insult), total nitrate/nitrite (NOx, C) and total malondialdehyde + 4-hydroxynoneal (MDA + 4-HNE, D). Statistical significances of data (means ± S.E., n = 6/group) were denoted as ***P < 0.001 from the control group and ##P < 0.01 from the APAP group. 3.4. Cap reverses dysregulation of IL-22 and bypasses the proliferation of hepatocytes induced by APAP In comparison to the normal control group, APAP-challenge raised IL-22 concentration in the liver, while decreased the serum con- centration of IL-22 (Fig. 6A, B). In the liver, Cap treatment (2 h prior and 2 h post, but not 4 h post, APAP-insult) significantly (P < 0.05) lowered hepatic IL-22 concentration in comparison to the untreated APAP-group. In the serum, Cap treatment (2 h prior, 2 h post and 4 h post APAP-insult) insignificantly raised serum IL-22 concentration, compared to APAP-group. Hepatocyte damage caused by APAP-in- toxication can initiate the proliferation of hepatocyte as an attempt for hepatocyte regeneration. To address the impact of Cap on this phe- nomenon, nuclear PCNA immunohistochemical was evaluated in the liver sections (Fig. 6C, D). APAP elicited the elevation of hepatic PCNA expression, while the administration of Cap 2 h post APAP achieved the most optimal therapeutic timing for lowering this to an extent similar to that exhibited by NAC. 4. Discussion APAP-overdose is one of the leading causes of drug-induced hepa- totoxicity in humans. Although experimental studies have introduced several prophylactic approaches for this kind of injury, clinically re- lated studies that dealt with treatment subsequent to APAP-exposure are still scarce. Accordingly, the pharmacological inhibition of c-Met by Cap at different time intervals (2 h prior, 2 h post and 4 h post insult) of APAP-hepatotoxicity in mice was investigated in the current study. The biochemical results indicated that Cap provided protection 2 h prior and post APAP-insult with an efficacy comparable to that of NAC as indicated by combating the elevation in serum ALT, AST and LDH ac- tivities. Histopathologically, the administration of Cap 2 h prior and post insult also repressed APAP-induced hepatocellular necrosis and inflammation alongside maintaining the normal architecture of the liver. The results of oxidative stress and antioxidant status indicated that APAP markedly reduced hepatic GSH content and elevated hepatic NOx and MDA + 4-HNE contents. One of the major factors implicated in APAP-overdose hepatotoxicity is the induction of oxidative stress, which arises from cytochrome P (CYP) 450-mediated metabolism, fol- lowed by mitochondria [33]. During APAP-phase I metabolism medi- ated by CYP2E1 and 3A4, the noxious generated NAPQI binds with GSH alongside mitochondrial vicinal thiols [34]. Due to excessive generation of NAPQI, the former reaction leads to GSH depletion, while the latter causes mitochondrial permeability transition alongside overproduction of superoxide anion radical that simultaneously reacts with nitric oxide radical [35]. As a consequence, the reactive peroxynitrite radical is formed and nitrates tyrosine-protein residues or decomposes to the highly reactive hydroxyl radical [36]. The latter radical eventually at- tacks cell membrane lipids, leading to elevation of lipid peroxidation products and cell membrane disruption. The current data revealed that Cap (2 h prior or post APAP) reversed APAP-induced abnormalities in hepatic contents of NOx and MDA + 4-HNE, but not GSH. Thereby, Cap did not interfere with the initial phase of APAP-depletion of GSH, but inhibited the later phase of mitochondrial oxidative stress that is linked with protein nitration and lipid peroxidation. Fig. 5. The influence of capmatinib (Cap) treatments on acetaminophen (APAP)-elicited changes in concentrations of serum monocyte-chemoattractant protein-1 (MCP-1, A), serum interleukin-1β (IL-1β, B), hepatic tumor necrosis factor-α (TNF-α, C), hepatic interleukin-17A (IL-17A, D) and hepatic interleukin-6 (IL-6, E). Statistical significances of data (means ± S.E., n = 6/group) were denoted as *P < 0.05 from the control group, while #P < 0.05, ##P < 0.01 and ###P < 0.001 from the APAP group. Sterile inflammation is another major contributor to liver damage caused by APAP-overdose secondary to oxidative stress [37]. Following the initial phase of hepatocellular necrosis due to mitochondrial oxidative stress, diverse DAMPs like high mobility group box 1, DNA and ATP are released and recognized by TLRs 4/9 and the purinergic P2X7 receptor localized in Kupffer cells, neutrophils and dendritic cells [18,38]. The nuclear transcription factor NF-κB subsequently becomes activated and translocates to the nucleus, leading to upregulation of proinflammatory cytokines like TNF-α, IL-1β and IL-6 [39]. Upon extracellular secretion, these cytokines can upregulate themselves and other proinflammatory cytokines subsequent to stimulation of their receptors localized in liver immune cells, ending up in aggravation of the inflammatory response and worsening of liver damage [40]. Ac- cordingly, the impact of Cap treatments on APAP-mediated production of TNF-α, IL-1β and IL-6 was investigated. The results showed that Cap when administered 2 h, but not 4 h, post APAP-administration attenuated APAP-induced elevation of hepatic TNF-α concentration. Be- sides, administration of Cap (2 h prior, 2 h post and 4 h post insult) adequately hindered APAP-induced rise of hepatic IL-6 expression. Fig. 6. The influence of capmatinib (Cap) treatments on acetaminophen (APAP)-elicited changes in concentrations of hepatic and serum interleukin-22 (IL-22, A and B) and hepatic proliferating cell nuclear antigen (PCNA) quantification for immunohistochemical expression (400×, C and D). Statistical significances of data (means ± S.E., n = 6/group) were denoted as *P < 0.05, **P < 0.01 and ***P < 0.001 from the control group, while #P < 0.05 and ##P < 0.01 from the APAP group. The attenuation of APAP-induced elevation of TNF-α by Cap is considered an important mechanism in limiting hepatic injury, because exogenous exposure to recombinant TNF-α led to more than 50% rise in serum ALT by APAP, while that of IL-1β or IL6 showed merely 20% rise in ALT [41]. Furthermore, TNF-α neutralization with specific anti- bodies or genetic deletion abolished the hepatotoxic effects of APAP [42,43]. The released TNF-α can also activate c-Jun-N-terminal kinase (JNK) in the liver hepatocytes, leading to apoptosis. Protection against APAP by JNK inhibitors was confirmed by several investigators [44,45]. The activation of NLRP3-inflammasome has been also implicated in APAP-hepatotoxicity, especially in the second phase of injury [37]. In recruited inflammatory cells, IL-1β needs to be proteolytically cleaved prior cellular release via cleaved caspase 1, which is activated by binding with ASC and NLRP3 through activation of the P2X7 receptor by ATP and NAD released from adjacent dying hepatocytes [46]. Also, NLPR3-assembly can occur by other agents involved in potassium ion efflux like uric acid and nigericin, which were shown to trigger ne- crosis-mediated passive IL-1β secretion [47]. Moreover, mice lacking components needed for assembly of the NLRP3-inflammasome and processing of IL-1β were having less hepatocellular injury and inflammation when subjected to APAP-overdose [46,48,49]. Thus, the decrease in IL-1β release by Cap-treatment, especially at 2 h post APAP- insult, is another mechanism mediating Cap-hepatoprotective effect. T-helper (Th)-17 cells, a subdivision of CD4+ Th cells secreting IL- 17A and IL-22, have been also reported to be elevated in the early context of APAP-liver failure in humans [50]. The proinflammatory cytokine IL-17A is released after IL-23 stimulation for Th-17 cells sub- sequent to TLR4-stimulation and is implicated in driving neutrophil infiltration into sites of injury after APAP-overdose [51]. Meanwhile, IL-22 is a member of the IL-10 cytokine family that is produced also by Th-22 and natural killer cells alongside Th-17 [52]. Accordingly, the impact of Cap on IL-17A and IL-22 changes occurring in APAP-toxicity was assessed. APAP caused a mediocre rise in hepatic IL-17A con- centration, but this was normalized by Cap 2 h prior APAP. APAP- overdose markedly elevated IL-22 concentration in the liver, with a reduction in its serum levels. Hence, it can be inferred that the hepatic overexpression of IL-22 acts as a compensation mechanism for the de- creased secretion of IL-22 in the systemic blood concentration. Fig. 7. Schematic illustration of the protective mechanism of capmatinib (Cap) against APAP-hepatotoxicity. The precise function of IL-22 in the context of hepatic inflammation is still not fully understood and controversial. For instance, the binding of IL-22 to its heterodimeric receptor IL-22R1/IL-10R2 leads to the activation of STAT3 for mediating cell survival and repair mechanisms [53]. Moreover, the brief exposure to exogenous IL-22 protected mice against APAP-hepatic injury through STAT3 activation, while the chronic constitutive overexpression of IL-22 aggravates APAP-hepatic injury by raising the levels of CYP2E1 and APAP-toxic metabolites [54]. Thus, it can be speculated that Cap treatment spared the hepatocytes from damage, leading to avoidance of subsequent repair by abating APAP-induced dysregulation of IL-22 in the liver and systemic circu- lation. This speculation was consistent with the elevation of PCNA nuclear expression by APAP as an index of repair after damage and the decrease of PCNA expression by Cap treatment that attenuated the hepatocellular injury caused by APAP. Take these findings together, inhibition of c-Met activation by Cap markedly conferred liver protection when given 2 h prior or post ex- posure to APAP-overdose, but the hepatoprotective effect was lost upon administration of Cap 4 h post APAP. Overall, the c-Met receptor is one of the potential receptors involved in driving APAP-hepatotoxicity, especially in the early phases and its inhibition by Cap may be an effective approach for attenuation of this drug-induced toxicity en- countered in humans’ livers as suggested (Fig. 7). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.intimp.2020.106292. References [1] Y. Zhang, M. Xia, K. Jin, S. Wang, H. Wei, C. Fan, Y. Wu, X. Li, X. Li, G. Li, Z. Zeng, W. 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