Melatonin Attenuates Ferritinophagy/Ferroptosis by Acting on Autophagy in the Liver of an Autistic Mouse Model BTBR T+Itpr3tf/J

Abstract

Autism spectrum disorders (ASDs) are a pool of neurodevelopment disorders in which social impairment is the main symptom. Presently, there are no definitive medications to cure the symptoms but the therapeutic strategies that are taken ameliorate them. The purpose of this study was to investigate the effects of melatonin (MLT) in treating ASDs using an autistic mouse model BTBR T⁺Itpr3^tf/J (BTBR). We evaluated the hepatic cytoarchitecture and some markers of autophagy, ferritinophagy/ferroptosis, in BTBR mice treated and not-treated with MLT. The hepatic morphology and the autophagy and ferritinophagy/ferroptosis pathways were analyzed by histological, immunohistochemical, and Western blotting techniques. We studied p62 and microtubule-associated protein 1 light chain 3 B (LC3B) for evaluating the autophagy; nuclear receptor co-activator 4 (NCOA4) and long-chain-coenzyme synthase (ACSL4) for monitoring ferritinophagy/ferroptosis. The liver of BTBR mice revealed that the hepatocytes showed many cytoplasmic inclusions recognized as Mallory-Denk bodies (MDBs); the expression and levels of p62 and LC3B were downregulated, whereas ACSL4 and NCOA4 were upregulated, as compared to control animals. MLT administration to BTBR mice ameliorated liver damage and reduced the impairment of autophagy and ferritinophagy/ferroptosis. In conclusion, we observed that MLT alleviates liver damage in BTBR mice by improving the degradation of intracellular MDBs, promoting autophagy, and suppressing ferritinophagy/ferroptosis.

Keywords: Mallory–Denk bodies; autism spectrum disorder; autophagy; ferritinophagy; ferroptosis; liver injury; melatonin.

Figure 1

Morphopathological evaluation of hepatocytes containing MDBs. Representative photomicrographs of hematoxylin–eosin staining of (a) BTBR mice, (b) MLT-treated BTBR mice, and (c) CTR mice. Original magnification: 200×; insert: 400×; bars = 50 µm. (d) Quantitative analysis of MDB-containing hepatocytes. * p < 0.05 vs. BTBR mice; # p < 0.05 vs. MLT-BTBR mice.

Figure 2

Hepatic p62 and LC3B immunohistochemical evaluations. Representative photomicrographs of liver p62 (a–c) and LC3B (d–f); immunostainings of (a,d) BTBR mice, (b,e) BTBR mice treated with MLT, and (c,f) CTR mice. Black arrows indicate positivity in MDBs. Original magnification: 400×; insert: 1000× (b); bars = 20 µm.

Figure 3

Hepatic p62 and LC3B Western blotting. Representative immunoblots of p62, LC3B I, and LC3B II of total liver samples from BTBR mice, BTBR mice treated with MLT, and CTR mice. β-actin was used as loading control. Relative expression quantification of Western blotting for p62 and LC3B. * p < 0.05 vs. BTBR mice; # p < 0.05 vs. MLT-BTBR mice.

Figure 4

Hepatic NCOA4 and ACSL4 immunohistochemical evaluations. Representative photomicrographs of liver NCOA4 (a–c) and ACSL4 (d–f) immunostaining of (a,d) BTBR mice, (b,e) BTBR mice treated with MLT, and (c,f) CTR mice. Original magnification: 400×; bars = 20 µm.

Figure 5

Schematic drawing of the experiment plan. (A) indicates the five mice per group transcardically perfused, whereas (B) indicates the other five mice per group which were euthanized by cervical dislocation. The black arrows indicate adequate liver samples collection; the blue arrows indicate the experimental treatment duration expressed in number of days (1 to 8); the white box indicates light exposition of mice and the black box indicates the dark exposition of mice. B-test: behavioral test; CTR: control; h: hour; MLT: melatonin; MLTV: melatonin vehicle.