Cerium oxide nanoparticles improve liver regeneration after acetaminophen-induced liver injury and partial hepatectomy in rats

Abstract

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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Acetaminophen-induced liver injury; Cerium oxide nanoparticles; Liver regeneration; Oxidative stress; Partial hepatectomy.

Fig. 1

Characterization of cerium oxide nanoparticles. a, b Representative TEM images of CeO₂NPs at different magnifications showing the non-aggregate and spherical shape of the engineered nanoparticles. Inset in b is a High Resolution TEM image of single particle showing pure CeO₂ atomic planes; c UV–Visible absorption spectrum of the as-synthesized CeO₂NPs; d XRD spectrum of the as-synthesized CeO₂NPs after being dried under vacuum. e Z-Potential distribution and f Hydrodynamic diameter measured by DLS of the CeO₂NPs dispersed in the physiological media (saline solution at pH = 5.5); g Cerium concentration in liver, spleen, lung and and kidney from rats treated with CeO₂NPs for 90 min, 3, 6 and 8 weeks (n = 4 for each group). h Oxidative stress was quantified in non-treated and CeO₂NPs-treated HepG2 cells by measuring DCF fluorescence in basal condition and after inducing oxidative stress with 2 mM H₂O₂ added to the culture medium (^#p < 0.0001 vs basal and *p < 0.0001 vs. non-treated, n = 10)

Fig. 2

CeO₂NPs treatment increased liver regeneration and cell proliferation after PHx. a Body weights of control rats without treatment and rats that received vehicle or CeO₂NPs before PHx (n = 8). b Hepatic regenerative index at day 6 after PHx (n = 8, p < 0.05). c Blood levels of ALT (*p < 0.01), AST (*p < 0.05) and LDH (*p < 0.05) in vehicle or CeO₂NPs-treated rats after 3 h post-PHx (n = 8; mean ± SEM). d Representative immunostaining for the Ki-67 antigen in liver histological sections of rats treated with either vehicle or CeO₂NPs at different time points (t = 0 h, 24 h, 48 h, 7 days). Merged images show co-localization of Ki-67 (green) and nuclear DNA (DAPI, blue). Original magnification ×200 (n = 8 for each group and treatment). On the bottom, percentage quantification of positive Ki-67 liver cells for each time point and treatment (n = 8; mean ± SEM; *p < 0.05 compared with vehicle at the same time points)

Fig. 3

CeO₂NPs treatment reduces histological damage and increases cell proliferation after APAP-induced injury. a Hematoxylin-eosin stained liver sections (n = 13). After vehicle or CeO₂NPs treatments, rats received 1 g/kg APAP and were sacrificed after 48 h (vehicle + APAP and CeO₂NPs + APAP, respectively). Another group was treated with 300 mg/kg NAC 1 h after APAP (NAC + APAP). Also, healthy non-treated rats were included as experimental controls (upper left panel), ×100. b Quantification of HNE in liver from vehicle + APAP, NAC + APAP and CeO₂NPs + APAP groups (n = 13; mean ± SEM; *p < 0.05). c Immunostaining for Ki-67 in liver of rats treated with vehicle + APAP, NAC + APAP and CeO₂NPs + APAP. Ki-67 (green) and DAPI (blue), ×200. On the right, quantification of Ki-67 positive cells (n = 13; mean ± SEM; *p < 0.05)

Fig. 4

CeO₂NPs stimulates cell cycle progression and NF-κB activation. a Flow cytometry of HepG2 showing cell cycle profiles from propidium iodide DNA staining after vehicle or CeO₂NPs treatment (n = 5; ^#p < 0.01 and *p < 0.05). b Western blot for activated caspase 3 and cyclin D1 abundance from HepG2 incubated with vehicle or CeO₂NPs. β-actin was used as loading control (mean ± SEM; n = 5; *p < 0.05 versus vehicle in the same experimental condition). O.D.: optical density. c Western blot for IκBα abundance from HepG2 (mean ± SEM; n = 5; *p < 0.05). d Transcription factor immunosorbent assay for NF-κB (p65) activity in HepG2 (n = 5, *p < 0.05). e Western blot for IκBα abundance in the liver vehicle and CeO₂NPs-treated rats before and 3 h post-PHx (mean ± SEM; n = 5; ^#p < 0.01)