Cerium oxide nanoparticles improve liver regeneration after acetaminophen-induced liver injury and partial hepatectomy in rats
Background and aims
Cerium oxide nanoparticles are effective scavengers of reactive oxygen species and have been proposed as a treatment for oxidative stress-related diseases. Consequently, we aimed to investigate the effect of these nanoparticles on hepatic regeneration after liver injury by partial hepatectomy and acetaminophen overdose.
All the in vitro experiments were performed in HepG2 cells. For the acetaminophen and partial hepatectomy experimental models, male Wistar rats were divided into three groups: (1) nanoparticles group, which received 0.1 mg/kg cerium nanoparticles i.v. twice a week for 2 weeks before 1 g/kg acetaminophen treatment, (2) N-acetyl-cysteine group, which received 300 mg/kg of N-acetyl-cysteine i.p. 1 h after APAP treatment and (3) partial hepatectomy group, which received the same nanoparticles treatment before partial hepatectomy. Each group was matched with vehicle-controlled rats.
In the partial hepatectomy model, rats treated with cerium oxide nanoparticles showed a significant increase in liver regeneration, compared with control rats. In the acetaminophen experimental model, nanoparticles and N-acetyl-cysteine treatments decreased early liver damage in hepatic tissue. However, only the effect of cerium oxide nanoparticles was associated with a significant increment in hepatocellular proliferation. This treatment also reduced stress markers and increased cell cycle progression in hepatocytes and the activation of the transcription factor NF-κB in vitro and in vivo.
Our results demonstrate that the nanomaterial cerium oxide, besides their known antioxidant capacities, can enhance hepatocellular proliferation in experimental models of liver regeneration and drug-induced hepatotoxicity.
KeywordsLiver regeneration Oxidative stress Cerium oxide nanoparticles Partial hepatectomy Acetaminophen-induced liver injury
cerium oxide nanoparticles
drug-induced liver injury
reactive oxygen species
Dulbecco’s modified Eagle medium
The liver regenerative capability is essential in the success of some treatments for chronic liver diseases, such as tumor resection and donor liver transplantation, which are conditioned by adequate liver regeneration [1, 2]. However, there are some clinical situations in which the liver shows poor regenerative capacity, such as in the case of liver cirrhosis and severe drug-induced liver injury (DILI) [3, 4].
One pathological process that is common to these two clinical conditions is oxidative stress, which is caused by the excessive formation of reactive oxygen species (ROS). The presence of oxidative stress has been described in most of the clinical conditions associated with chronic liver injury (i.e.: nonalcoholic steatohepatitis, hepatitis C viral infection, alcoholic liver cirrhosis) [5, 6]. In addition, recent reports suggest that drug-induced oxidative stress also significantly correlate with increased DILI risk . Concurrently, the association between high levels of oxidative stress and a reduction of antioxidant defenses has also been reported in most of these pathological situations [8, 9]. According to the results obtained by several studies, the imbalance between the production of ROS and antioxidant defense in some of these liver diseases affects liver regeneration [10, 11, 12]. One of the reasons that explain this ROS effect is that ROS modulates the expression of a variety of regulators that play major roles in liver regeneration, including growth factors, transcription factors and cell cycle proteins such as β-catenin , cyclin D , p53 , Nrf2  and JNK/p38 mitogen activated kinases .
Cerium oxide nanoparticles (CeO2NPs) have drawn considerable attention as a potential therapeutic tool in the prevention and treatment of oxidative stress related diseases. This interest relies on the multi-enzyme mimetic properties of CeO2NPs due to their unique electronic structure [18, 19, 20]. At the nanoscale, the oxygen vacancies that appear in the CeO2 nanocrystals modify their electronic structure enabling them to participate as a catalyst in a wide range of reactions (e.g. promoting the simultaneous oxidation of Carbon Monoxide (CO) and hydrocarbons to CO2 and the reduction of Nitrogen Oxides (NOx) to N2 in three way catalytic converters) . Similarly, in biological contexts, it has been reported that cerium oxide nanoparticles can mimic enzymatic antioxidants such as superoxide dismutase  and catalase . The beneficial effects of CeO2NPs have been reported in different clinical conditions associated with excess production of ROS such as neurology [24, 25, 26, 27], diabetes [28, 29], chronic inflammation , cirrhosis  and cancer [32, 33, 34].
Considering the above, we hypothesize that CeO2NPs may improve liver regeneration by scavenging ROS in regenerative livers. To test our hypothesis we studied the hepatic regenerative process using two different rat experimental models of liver regeneration that are commonly associated with ROS production: partial hepatectomy (PHx) [35, 36] and DILI caused by acetaminophen (APAP) overdose. In the last case, the therapeutic effect of CeO2NPs was compared with the gold standard treatment for APAP-induced injury, N-acetylcysteine (NAC) .
Characterization and biodistribution of cerium oxide nanoparticles in rats
Several studies have described that after systemic distribution small inorganic NPs accumulate in the liver and spleen [31, 37]. In agreement, CeO2NPs treated rats showed CeO2NPs retention into the liver and, to a lesser extent, in spleen as early as 90 min following i.v. injection. In these organs and at this time point, CeO2NPs reached concentrations of 160.9 μg and 36.0 μg of CeO2NPs per gram of tissue, respectively (Fig. 1g). Interestingly, cerium was still detected in liver and spleen for over 8 weeks although in lower concentrations. CeO2NPs retention was barely detected in the lungs and the kidneys of the rats at different time points after the intravenous injection (Fig. 1g).
To investigate the antioxidant properties of CeO2NPs, we induced oxidative stress in the hepatocyte cell line HepG2 by H2O2 treatment, as previously reported [38, 39]. ROS were assessed in these cells by using the dichlorofluorescein (DCF) assay . When exposed to H2O2, CeO2NPs-treated HepG2 cells showed a significant reduction in the accumulation of DCF in comparison to that observed in non-treated cells (Fig. 1h).
Rats treated with CeO2NPs showed increased liver regeneration and hepatocellular proliferation after PHx
To further investigate the cause through which CeO2NPs improves liver regeneration, we assessed the expression of the cell proliferation marker Ki67 in liver sections of rats treated with nanoparticles. Rats receiving vehicle or CeO2NPs showed absence of hepatocellular proliferation in resting livers (t = 0 h) (Fig. 2d). However after PHx, the remnant liver from rats treated with CeO2NPs showed a significant increase in Ki67+-hepatocytes at day 1 (*p < 0.05, t = 24 h) that reached a maximum at t = 48 h after partial hepatectomy, compared with vehicle (44.6 ± 4.5% vs. 38.5 ± 6.7% Ki67+ cells, *p < 0.05, t = 48 h); returning to nearly basal levels after 7 days (Fig. 2d). Therefore, improvement of liver regeneration caused by the CeO2NPs treatment is associated with enhanced hepatocyte proliferation.
CeO2NPs treated rats showed decreased liver damage and increased hepatocellular proliferation after acetaminophen-induced liver injury
We performed additional experiments to characterize better the liver injury induced by APAP and the degree of recovery of the different experimental groups. To this end, we quantified the serum concentration of transaminases (ALT, AST), glucose and albumin from CeO2NPs-treated and vehicle-treated rats at t = 0 h, t = 48 h, and t = 96 h. We found that the concentration of transaminases was significantly increased to a similar extent in the vehicle and the CeO2NPs-treated groups 2 days after APAP administration (t = 48 h compared with t = 0 h). However, CeO2NPs treatment was associated with a significant decline in serum ALT, reaching basal levels at 96 h after the APAP injection; compared with the vehicle group (Additional file 1: Figure S2). This recovery phase marker, which is a more specific marker of liver injury than AST, points to a more efficient recovery of the CeO2NPs-treated liver after drug-induced liver damage.
CeO2NPs treatment stimulates cell cycle progression in HepG2 cells and NFκB activation in vitro and in vivo
The NF-κB/Rel family of transcription factors (NFκB) is a central hub of signaling pathways that regulates apoptosis and cell proliferation. In addition, the activity NFκB is modulated by ROS [41, 42, 43]. These confluent evidences let us to assess whether the activity of NF-κB is affected by the CeO2NPs treatment in vitro and in vivo. In HepG2 cells treated with CeO2NPs, the abundance of IκBα (that function as an inhibitor of NFκB) significantly decreased when compared to vehicle-treated HepG2 cells (p < 0.05) (Fig. 4c). Next, the transcriptional activity of NF-κB was assessed by detecting specific p65 DNA binding in nuclear extracts from CeO2NPs treated and non-treated HepG2 cells. As shown in Fig. 4d, CeO2NPs treated cells showed increased p65 transcriptional activity compared with non-treated HepG2 cells (p < 0.05). We next investigated whether the described stimulation of NF-κB activity by CeO2NPs was also detected in regenerative livers in vivo. Before PHx, there were no differences in the expression of IκBα in the livers from vehicle or CeO2NPs treated rats. However, and in agreement with the results obtained in HepG2, the abundance of IκBα was significantly lower in the livers from CeO2NPs treated rats 3 h after PHx, compared with the group of rats receiving vehicle (p < 0.01) (Fig. 4e).
We previously described that CeO2NPs showed hepatoprotective activity in vivo against fibrosis . Another study also demonstrated the beneficial effects of these nanoparticles against hepatic oxidative damage caused by the pyrrolizidine alkaloid monocrotaline, which causes oxidative vascular damage in the liver . Considering this hepatoprotective role of CeO2NPs, we further investigated whether these nanoparticles might also stimulate other hepatoprotective functions, such as liver regeneration. In the present study we describe for the first time that CeO2NPs stimulate liver regeneration in the rat experimental models of partial hepatectomy and DILI caused by acetaminophen overdose. Consistently with this observation, we demonstrated that CeO2NPs treatment decreases liver damage after PHx and APAP-induced liver injury. The treatment with CeO2NPs also leads to the reduction of oxidative stress markers and stimulates the cell cycle progression in hepatocytes and the activation of the transcription factor NF-κB in vitro and in vivo.
It is known that excess of oxidative stress inhibits efficient regeneration after partial hepatectomy or acute liver failure caused by DILI. For example, the genetic deficiency of Nrf2 (a regulator of the cellular antioxidant response) and UCP2 (an uncoupling protein of the electron transport chain) cause enhanced oxidative stress and impaired liver regeneration in partially hepatectomized mice . Moreover, oxidative cellular damage in mitochondria and DNA is associated with deficient liver regeneration after acetaminophen toxicity . Therefore, several strategies have been investigated by others to diminish ROS in the context of liver regeneration in preclinical models. For example, thymoquinone, an essential oil with free radical scavenging capacity, protects rat liver against ischemia/reperfusion injury  and prevents liver injury in hepatectomized mice under I/R by blunting oxidative stress, mitochondrial damage, endoplasmic reticulum stress and apoptosis . Exogenous administration of GSH or resveratrol significantly decreased oxidative stress and protect against APAP-induced liver injury [48, 49]. Similarly, pharmacological inhibition of JNK activity, which enhances mitochondrial-derived ROS production upon APAP toxicity, reduced liver damage . Some drugs approved for its clinical use in different pathologies have also been tested pre-clinically for APAP hepatotoxicity. Both methylene blue and metformin reduced mitochondrial oxidant stress  and protected against APAP liver injury .
Our study has potential limitations regarding the APAP-induced toxicity model in rats. It has been shown that rats are more resistant to acetaminophen damage than mice . Despite this observation, the presence of oxidative stress in the APAP-induced toxicity model in rats has broadly been demonstrated [54, 55, 56], regardless of the severity of the injury between species. Therefore, this experimental model in rats is still valid to test the antioxidant properties of CeO2NPs treatment on liver regeneration after drug-induced injury. Regarding the metabolism of APAP in the rat liver, we found in our study that the concentration of transaminases was significantly increased to a similar extent in the vehicle and the CeO2NPs-treated groups 2 days after APAP administration. This result confirmed that there is significant toxicity induced by APAP overdose in our experimental rat model and that the CeO2NPs pretreatment does not modify the metabolism of APAP in the liver. Nevertheless, the quantification of an intermediary metabolite of APAP (i.e., N-acetyl-p-benzoquinone imine) would have supported more robustly this conclusion.
Our study presents an innovative treatment for diminish ROS in the context of liver regeneration using nanoparticles. Some of the advantages of using CeO2 nanomaterials over the traditional anti-oxidative drugs are: (1) minimal toxicity in cumulative doses [57, 58], (2) the multi-enzyme mimetic activities of CeO2NPs, which targets several sources of ROS generation and (3) the continuous regeneration of the CeO2NPs catalytic activity, which avoids the exhaustion of its anti-oxidative properties . Among these theoretical benefits over traditional drugs, we also observed superior therapeutic performance compared with the gold standard treatment used for acetaminophen toxicity in patients, NAC. In our study, CeO2NPs treatment is equally effective reducing oxidative stress and tissue damage in rats receiving APAP overdose compared with NAC. However, while NAC did not affect hepatocyte proliferation in injured livers, CeO2NPs robustly increased cell proliferation in vivo and in vitro. In addition, we observed a significant decrease in the percentage of CeO2NPs-treated HepG2 cells that underwent apoptosis following 48 h of serum starvation, suggesting that the anti-apoptotic effect associated to the nanoceria treatment may also contribute to enhance liver regeneration.
NF-κB plays a major role in liver homeostasis and liver regeneration. For instance, NF-κB (p65) knockout mice show embryo lethality and display massive hepatocyte apoptosis . In addition, the transduction of rat livers with different forms of IκB, an inhibitor of NF-κB activity, before PHx was associated with impaired liver regeneration . Furthermore, the inactivation of NF-κB in Kupffer cells as well as in hepatocytes impaired the regenerative process after PHx . Here, we demonstrated that the treatment with CeO2NPs lead to the activation of the transcription factor NF-κB in vitro and in vivo. Our results are not sufficient to establish a robust link between NF-κB activation and the beneficial effects of CeO2NPs treatment on liver regeneration yet. However, the agreement between our findings and the studies mentioned above make this relationship plausible.
It has been described that oxidative stress can inhibit the transcriptional activity of NFκB through different mechanisms including direct interaction with NF-κB, inactivation of IKK (an IκB repressor), and alternative inhibitory phosphorylation of IκBα . Our in vitro and in vivo results showed that CeO2NPs are associated with a significant decrease in the intracellular IκBα content, which is translated in the activation of NF-κB. Therefore, our findings suggest that the anti-oxidant property of CeO2NPs is responsible for the increase in NF-κB activation. However, our results are in disagreement with other studies which have shown that CeO2NPs treatment inhibits NF-κB in vivo mostly in the pathological context of sepsis [62, 63, 64]. One of the reasons that may explain the seemingly contradictory findings is differences in the CeO2NPs stability used in these studies, what has been recently recognized as key in order to employ CeO2NPs for medicine . In addition, these studies used higher in vivo doses of CeO2NPs compared with ours (from 0.5 mg/kg to 3.5 mg/kg b.w. compared with 0.1 mg/kg b.w.). Our previous dosage standardization experiments  indicate that at higher doses CeO2NPs shows a hormetic-like dose response characterized by an increasing dose inefficacy in ROS scavenging.
In summary, drug-induced hepatotoxicity and impaired liver regeneration are major concerns in medical practice as they are leading causes of acute liver failure and liver transplantation. Our results demonstrated that CeO2NPs, besides their known antioxidant capacities, can enhance liver regeneration in experimental models that replicate these two clinical scenarios. Hence, CeO2NPs treatment may provide avenues to overcome deficient liver regeneration in patients.
Synthesis and characterization of CeO2NPs
CeO2NPs were synthesized by the chemical precipitation of cerium (III) nitrate hexahydrated (Sigma-Aldrich, St. Louis, MO, USA) in aqueous solution, as reported elsewhere . Controlling the pH of synthesis, small-sized nanoceria can be obtained. Here, we used 4 nm nanoparticles at a concentration of 1 mg/mL. The surface charge of the NPs was characterized by the Z-potential in a Z-sizer (Malvern, Worcestershire, UK) while the crystal size was characterized by High Resolution Transmission Electron Microscopy (HR-TEM) in the Tecnai G2 F20 (FEI, Oregon, USA). Nanocrystalline morphology and composition was analyzed by HR-TEM (Tecnai 200 kV) and XRD (Xpert Pannalytical, MA, USA), and the light interaction by UV–VIS spectroscopy (Shimatzu, Kyoto, Japan). Size distribution was computer analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA). CeO2NPs were stabilized with Tetramethylammonium hydroxide (TMAOH), which was used as vehicle in all the experiments as a control condition, and kept at 4 °C until use. Prior to the animal studies, 15 µL from the nanoparticles solution were dispersed on a copper grid coated with a formvar film. The samples were then let to dry for TEM observation and digital photomicrographs were taken (BioScan Gatan, CA, USA). Free CeO2NPs samples available for evaluation if requested.
Animal experimentation and In vivo CeO2NPs treatment
This study was performed in male Wistar rats (Charles-River, Saint Aubin les Elseuf, France). Rats were fed with standard chow diet and housed on a 12 h light/12 h dark cycle. For each experiment of this study, treated rats were administered 0.1 mg/kg CeO2NPs dispersed in saline solution as a bolus (500 μL), given intravenously through the tail vain twice a week for 2 weeks. Control groups without treatment and treated with vehicle (TMAOH) were also included in the study. At the end of each experiment rats were euthanized by isoflurane anesthesia overdose.
CeO2NPs tissue quantification
For tissue quantification, at the end of the 2 weeks treatment, rats were euthanized at days 1, 21, 42, and 56 (90 min, 3, 6 and 8 weeks respectively) and their livers, spleens, lungs and kidneys dissected and kept at − 80 °C for further analysis. For CeO2NPs quantification, samples were diluted in an aqueous solution of HNO3 2% w/w (Trace Metal Basis; Sigma-Aldrich, St. Louis, MO, USA) and analyzed for cerium concentration by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500; Agilent Technologies, California, USA). The quantitative results were obtained by interpolation in a standard curve obtained from a commercial 1000 ppm Ce standard (Sigma-Aldrich, St. Louis, MO, USA).
Reactive oxygen species quantification
Reactive oxygen species were measured by fluorescence spectrophotometry using 2′,7′-DCF-DA as a probe. For these experiments, HepG2 cells alone or treated with H2O2 in the presence or absence of CeO2NPs were washed with phosphate-buffered saline (PBS) and incubated with 10 μM DCF-DA (Thermo Fisher Scientific, Waltham, MA, USA) in Dulbecco’s modified Eagle medium (DMEM) for 40 min at 37 °C in the dark. The supernatant was collected to measure the extracellular production of ROS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer (FLUOstar Optima; BMG Labtech, Ortenberg, Germany) at 485 nm for excitation and at 520 nm for emission.
In vitro CeO2NPs treatment
The human cell line HepG2 (a suitable in vitro model system for the study of polarized human hepatocytes) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). HepG2 cells were cultured and maintained in DMEM containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Cells were serum-starved and treated with 0.1 mg/mL CeO2NPs, or vehicle as a control condition, for 48 h.
Measurement of NF-κB activation
NF-κB activity in HepG2 cells was measured using a NF-κB Transcription Factor Assay Kit (specific for p65), following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). To this end, nuclear extracts from HepG2 were incubated with double stranded DNA immobilized in a 96-well plate that contained the κB response motif site. The complex p65-DNA was detected by enzyme-linked immunosorbent assay using specific primary antibody directed against p65 and a secondary antibody conjugated to HRP. The results were quantified by measuring absorbance at 450 nm in a FLUOstar Optima (BMG Labtech, Ortenberg, Germany).
Quantitative data were analyzed using GraphPad Prism, version 6 (GraphPad Software, Inc., San Diego, CA, USA) and public libraries from The Comprehensive R Archive Network (CRAN; http://CRAN.R-project.org) rooted in the open source statistical computing environment R, version 3.4 (http://www.R-project.org/). The statistical analysis of the results was performed using unpaired Student’s t-tests and ANOVA models (with Tukey’s post hoc test) with normally distributed data. For other type of data, the Mann–Whitney U-test and the Kruskal–Wallis tests (with Dunn post hoc test) were used. Results are expressed as mean ± sem and considered significant at a p value lower than 0.05.
Additional Materials and Methods are shown in Additional file 1. Free CeO2NPs samples available for evaluation if requested.
We thank Ministerio de Economía y Competitividad, Fundació La Marató de TV3 and CIBERehd (Instituto de Salud Carlos III) for the grants and financial support to this research.
BC-J: experiments, procedures and writing of article; AA-C: experiments, procedures and writing of article; JR: experiments and generation of experimental models; MP: generation of experimental models; DO: experiments, GC: measurement of biochemical parameters, GF-V: measurement of biochemical parameters, EC: synthesis of CeO2NPs and writing of article, VP: synthesis of CeO2NPs and writing of article, WJ: concept, design and writing of article; MM-R: concept, design, experiments and writing of article. All authors read and approved the final manuscript.
This research was supported by grant from the Ministerio de Ciencia, Innovación y Universidades (SAF2016-75358-R to MM-R), co-financed by FEDER, European Union, a way of making Europe; Grant from the Ministerio de Sanidad, Consumo y Bienestar Social (PI15/00777 to GC); Grant from Wuyi University Funding for Hight Talents Introduction (2018TP010 to EC); Grant from Department of Education of Guangdong Province (2017KSYS010 to EC) and Fundació La Marató de TV3 (Marató 120930 to WJ). CIBERehd is financed by Instituto de Salud Carlos III.
Ethics approval and consent to participate
All animals received human care and all procedures were approved by the Investigation and Ethics Committee of Animal Experimentation of the University of Barcelona.
Consent for publication
The authors declare that they have no competing interests.
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