Effect of exogenous melatonin on the ultrastructure of hepatocytes in rats with experimental toxic liver injury

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Abstract

BACKGROUND: Prolonged exposure to constant light suppresses melatonin synthesis by the pineal gland and induces desynchronosis, increasing the risk of various pathological conditions, including liver dysfunction. Exogenous melatonin is known to exert a pronounced hepatoprotective effect; however, its role in protecting the liver against carbon tetrachloride (CCl4)-induced toxicity remains insufficiently understood. Moreover, the impact of disrupted circadian rhythmicity under melatonin deficiency on the development of liver pathology, as well as the mechanisms of melatonin’s hepatoprotective action in toxic injury.

AIM: The work aimed to investigate the effects of dark deprivation and exogenous melatonin on the ultrastructure of mitochondria in rat hepatocytes under carbon tetrachloride-induced toxic liver injury.

METHODS: The study involved male Wistar rats (n = 200), aged 6 months, with a body weight of (350 ± 15) g. The animals were divided into five groups: group 1, control group, fixed light–dark cycle; group 2, dark deprivation; group 3, fixed light–dark cycle with intraperitoneal CCl4 (in olive oil, 0.3 mg/kg) every 3 days; group 4, dark deprivation with CCl4 every 3 days; group 5, dark deprivation with CCl4 injections every 3 days, (intraperitoneally) and daily melatonin administration (Sigma-Aldrich, USA; intragastrically, 0.3 mg/kg).

The experiment lasted 3 weeks. The ultrastructure of hepatocytes was evaluated using transmission electron microscopy. The micromorphometric analysis of mitochondria included measurement of organelle area, quantification and length of cristae, and calculation of the concentration of inner mitochondrial membranes. The statistical analysis was performed using GraphPad Prism v8.41 (GraphPad Software, USA).

RESULTS: Dark deprivation caused marked structural changes in hepatocytes, including cytoplasmic swelling, nuclear deformation, ribosomal detachment from the endoplasmic reticulum, reduced mitochondrial number, shortened cristae, and decreased concentration of inner mitochondrial membranes. CCl₄ exposure resulted in more severe damage to hepatocytes, such as cytoplasmic vacuolization, mitochondrial swelling, and necrosis. Under dark deprivation, CCl₄ toxicity was exacerbated: total mitochondrial count decreased with compensatory enlargement, cristae were shortened, and the concentration of inner mitochondrial membranes declined, indicating reduced mitochondrial function. Melatonin has a protective effect, preserving nuclear morphology, reducing lipid vacuole accumulation, and normalizing micromorphometric parameters of mitochondria.

CONCLUSION: Pineal melatonin deficiency under dark deprivation aggravates CCl₄-induced hepatotoxicity due to induction of oxidative stress and mitochondrial dysfunction. Melatonin demonstrates a pronounced hepatoprotective effect by stabilizing hepatocyte ultrastructure and supporting energy metabolism. These findings support the use of melatonin in preventing liver damage under chronic intoxication and circadian rhythmicity disruption.

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About the authors

Sevil A. Grabeklis

Petrovsky National Research Centre of Surgery

Author for correspondence.
Email: grabeklene@gmail.com
ORCID iD: 0009-0002-3290-3768
SPIN-code: 4259-7674
Russian Federation, Moscow

Lyudmila M. Mikhaleva

Petrovsky National Research Centre of Surgery

Email: mikhalevalm@yandex.ru
ORCID iD: 0000-0003-2052-914X
SPIN-code: 2086-7513

Dr. Sci. (Medicine), Professor

Russian Federation, Moscow

Alexandr M. Dygai

Institute Of General Pathology And Pathophysiology

Email: ombn.ramn@mail.ru
ORCID iD: 0000-0001-6286-5315
SPIN-code: 8070-3578

Dr. Sci. (Medicine) Professor, Academician of the Russian Academy of Sciences

Russian Federation, Moscow

Maria A. Kozlova

Petrovsky National Research Centre of Surgery

Email: ma.kozlova2021@outlook.com
ORCID iD: 0000-0001-6251-2560
SPIN-code: 5647-1372

Cand. Sci. (Biology)

Russian Federation, Moscow

Valery P. Chernikov

Petrovsky National Research Centre of Surgery

Email: 1200555@mail.ru
ORCID iD: 0000-0002-3253-6729
SPIN-code: 3125-7837

Cand. Sci. (Medicine)

Russian Federation, Moscow

David A. Areshidze

Petrovsky National Research Centre of Surgery

Email: labcelpat@mail.ru
ORCID iD: 0000-0003-3006-6281
SPIN-code: 4348-6781

Cand. Sci. (Biology)

Russian Federation, Moscow

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2. Fig. 1. Ultrastructure of hepatocytes in rats from the control group (1): G, glycogen; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; M, mitochondrion; Er, erythrocyte; N, nucleus; Nu, nucleolus. Transmission electron microscopy, ×8000 magnification, scale bar 5 µm.

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3. Fig. 2. Ultrastructure of hepatocytes in rats subjected to dark deprivation (group 2): V, vacuole; G, glycogen; RER, rough endoplasmic reticulum; M, mitochondrion; Mf, macrophage; Ed, cytoplasmic edema; R, ribosomes; N, nucleus; Nu, nucleolus. Transmission electron microscopy, ×8000 magnification, scale bar 5 µm.

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4. Fig. 3. Ultrastructure of hepatocytes in rats with toxic liver injury under fixed light–dark conditions (group 3): L, lipids; Ly, lysosome; M, mitochondrion; P, peroxisome; N, nucleus; Nu, nucleolus. Transmission electron microscopy, magnification: a, ×6700; b, ×14000; scale bars: a, 5 µm; b, 2 µm.

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5. Fig. 4. Ultrastructure of hepatocytes in rats with toxic liver injury combined with dark deprivation (group 4): C, collagen fibers; L, lipids; M, mitochondrion; MN, focus of micronecrosis; Er, erythrocyte; N, nucleus, Nu, nucleolus. Transmission electron microscopy, ×6700 magnification, scale bar 5 µm.

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6. Fig. 5. Ultrastructure of hepatocytes in rats treated with melatonin under toxic liver injury and dark deprivation (group 5): M, mitochondrion; P, peroxisome; N, nucleus; Nu, nucleolus. Transmission electron microscopy, ×8000 magnification, scale bar 5 µm.

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