Morphofunctional State of the Liver Under Conditions of Three-Month Dark Deprivation: The Influence of Circadian Disruptions and Melatonin

The modern urban environment is characterized by an unprecedented level of light pollution. According to the International Dark-Sky Association, over 80% of the world’s population lives under skies where natural day–night cycles are substantially distorted by artificial lighting [1,2]. Night shift work, the use of light-emitting screens (smartphones, tablets, computers) during evening and nighttime hours, and the 24/7 illumination of megacities have rendered chronic desynchronosis an integral attribute of the modern lifestyle. As early as 2007, the World Health Organization classified night shift work as a probable carcinogenic factor (Group 2A) [3,4]. However, over the past years, compelling evidence has accumulated that the spectrum of pathological consequences of desynchronosis is considerably broader, encompassing obesity, type 2 diabetes mellitus, cardiovascular diseases, neurodegenerative disorders, and accelerated organismal aging [2,5].

While the core principles of circadian biology—endogenous, entrainable oscillations with a period of approximately 24 h—are conserved across kingdoms, the underlying molecular mechanisms exhibit fascinating diversity. In plants, the circadian system is decentralized, with most cells containing an autonomous oscillator that is primarily light-entrained, coordinating essential processes like photosynthesis, flowering, and stomatal opening [6,7]. In contrast, animals have evolved a hierarchically organized system, with a master pacemaker in the brain (the suprachiasmatic nuclei) that synchronizes peripheral oscillators in organs like the liver, heart, and kidney through neural and hormonal signals, most notably melatonin. Despite these architectural differences, both kingdoms share conserved molecular feedback loops involving transcription-translation, demonstrating the fundamental evolutionary importance of anticipating daily environmental changes [8,9].

The mammalian circadian system represents a hierarchically organized structure. The central oscillator is localized in the suprachiasmatic nuclei (SCN) of the hypothalamus and is synchronized by external light signals via the retinohypothalamic tract [10,11]. Peripheral oscillators are present in virtually all cells of the body, including hepatocytes, cardiomyocytes, adipocytes, and pancreatic cells. At the molecular level, clock function is ensured by autonomous transcription–translation feedback loops. The heterodimer of transcription factors BMAL1 (ARNTL) and CLOCK binds to E-box elements in the promoters of Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes, activating their expression [12,13,14]. PER and CRY proteins, accumulating in the cytoplasm, form complexes, translocate to the nucleus, and inhibit BMAL1/CLOCK activity, thereby suppressing their own transcription. The cycle is completed by PER/CRY degradation and resumption of transcription. Additional feedback loops involving nuclear receptors REV-ERBα/β and RORα modulate the amplitude and period of oscillations [15,16].

The key link converting the light signal into an endocrine output is the pineal gland. In darkness, under the influence of norepinephrine released from postganglionic sympathetic fibers, the enzyme aralkylamine N-acetyltransferase (AANAT) is activated in pinealocytes, converting serotonin into N-acetylserotonin, which is further metabolized into melatonin (N-acetyl-5-methoxytryptamine) by hydroxyindole-O-methyltransferase (HIOMT) [17]. Light inhibits AANAT activity, leading to a rapid decrease in hormone levels. Melatonin enters the bloodstream and cerebrospinal fluid, delivering photoperiodic information to all body cells. Beyond its chronobiotic function, melatonin is a potent endogenous antioxidant, immunomodulator, apoptosis inhibitor, and geroprotector. Its receptors (MT1 and MT2) belong to the G-protein-coupled receptor family and are abundantly expressed in the liver, kidneys, heart, and blood vessels [18].

The liver occupies a special position among peripheral oscillators. Up to 80–90% of genes encoding proteins of intermediary metabolism in hepatocytes are subject to circadian regulation [19,20,21]. Moreover, the hepatic parenchyma exhibits pronounced daily fluctuations not only at the molecular but also at the tissue level: organ volume, xenobiotic detoxification rate, synthesis of albumin, cholesterol, and bile acids, and the activity of glycolysis and gluconeogenesis enzymes vary with time of day [22,23]. BMAL1 regulates the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), controlling gluconeogenesis. Mice with liver-specific Bmal1 knockout lose the ability to maintain normoglycemia during the rest phase. The clock controls the activity of sterol regulatory element-binding protein 1 (SREBP1), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN). Deletion of Cry genes results in hypertriglyceridemia and steatosis. The expression of many cytochrome P450 isoforms (CYP2E1, CYP3A4, CYP7A1) is under circadian control, which explains the chronopharmacological effects of drugs [24,25,26,27,28,29,30,31,32,33,34,35,36].

Thus, the liver represents an ideal model for studying the impact of desynchronosis on metabolic health. The most adequate experimental model for investigating the consequences of chronic light pollution is housing laboratory animals under constant light conditions (24 h light, 0 h darkness), also termed “dark deprivation”. This regimen leads to elimination of daily rhythmicity in behavior and physiological functions; profound and sustained suppression of endogenous melatonin synthesis (by 80–90% of baseline); desynchronization of molecular clocks in the SCN and peripheral tissues; and oxidative stress due to the loss of antioxidant protection provided by melatonin [37,38,39,40,41,42].

Importantly, unlike models involving complete pinealectomy, dark deprivation is functional, reversible, and ethically more acceptable as it does not require surgical intervention.

The relationship between desynchronosis and accelerated aging has been intensively investigated over the past decade. It has been shown that in elderly individuals, the amplitude of circadian rhythms is reduced, and nighttime melatonin levels are significantly lower than in young subjects. In experimental models, constant lighting accelerates the appearance of age-associated phenotypes: sarcopenia, osteoporosis, thymic involution, and cognitive deficits. However, data on the comprehensive impact of long-term dark deprivation specifically on the liver, with emphasis on markers of cellular senescence and the circadian status of hepatocytes, remain insufficient in the literature. Most studies are confined either to biochemical analysis or to isolated molecular pathways, failing to provide an integrative picture. Given that the central pathogenic link in dark deprivation is endogenous melatonin deficiency, exogenous hormone administration is regarded as a pathogenetically justified corrective strategy. Numerous studies have confirmed that melatonin at doses of 10–20 mg/L of drinking water can: restore circadian rhythm amplitude; exert antioxidant and anti-inflammatory effects; improve mitochondrial function; suppress activation of senescence pathways (p53/p21, p16INK4a/Rb) [43,44,45,46,47,48,49].

However, the question of whether a long-term (three-month) course of melatonin can completely prevent the formation of a pro-senescent hepatic phenotype in young animals under conditions of permanent light stress remains open.

We hypothesized that chronic dark deprivation induces a complex of structural and metabolic changes in the liver that meet the criteria of accelerated aging (hypertrophy, polyploidization, steatosis, mitochondrial dysfunction, elevated p16/p21, reduced regenerative potential), with melatonin deficiency serving as the key trigger, and that exogenous melatonin, by replenishing this deficiency, is capable of blocking the entire cascade of pathological reactions.

Despite active research into the relationship between desynchronosis and aging, data on the comprehensive impact of long-term dark deprivation specifically on the liver, with emphasis on markers of cellular senescence and the circadian status of hepatocytes, remain insufficient in the literature. Most studies are confined either to biochemical analysis or to isolated molecular pathways, failing to provide an integrative picture that spans from systemic hormone levels to ultrastructural organization. Furthermore, the question of whether a long-term (three-month) course of melatonin can completely prevent the formation of a pro-senescent hepatic phenotype in young animals under conditions of permanent light stress remains open. The novelty of the present study lies in its multi-level, integrative approach—combining biochemical, histological, ultrastructural, and molecular analyses—to demonstrate for the first time that chronic dark deprivation induces a complex of structural and metabolic changes in the liver that meet the criteria of accelerated aging, with melatonin deficiency serving as the key trigger. We hypothesized that exogenous melatonin, by replenishing this deficiency, is capable of blocking the entire cascade of pathological reactions. Therefore, the aim of the present study was a comprehensive evaluation of the effects of three-month dark deprivation and the corrective action of exogenous melatonin on the morphofunctional, ultrastructural, and molecular state of the liver in young adult rats.

The present study provides a comprehensive characterization of structural and functional changes in the rat liver under conditions of chronic light deprivation (constant illumination for three months) and demonstrates a pronounced protective effect of exogenous melatonin. The obtained data indicate that desynchronosis, induced by suppression of endogenous melatonin secretion, initiates a cascade of pathological processes at the systemic, tissue, cellular, and molecular levels, the totality of which meets the criteria for accelerated liver aging.

Three-month housing under constant lighting conditions led to a significant decrease in plasma melatonin levels, confirming the adequacy of the selected model for studying the consequences of light pollution. It is known that light inhibits the activity of AANAT (aralkylamine N-acetyltransferase) in pinealocytes, which is a key mechanism for suppressing the nocturnal melatonin peak [50]. The review by Srinivasa et al. [51] emphasizes that the liver functions as a peripheral clock closely connected to the suprachiasmatic nucleus (SCN), and disturbances in circadian rhythms are directly associated with the development of metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC). Importantly, melatonin interacts with nuclear receptors ROR-α (retinoic acid receptor-related orphan receptor-alpha), which are critical for maintaining circadian rhythms and possess anti-inflammatory and potentially anti-tumor properties [52]. Chronic melatonin suppression in our model thus creates conditions for multiple disturbances in hepatocellular homeostasis through dysregulation of both receptor-mediated (MT1/MT2, ROR-α) and non-receptor mechanisms.

Modern humans face a dual effect of the artificial light environment: on one hand, excessive nighttime illumination (ALAN) suppresses melatonin synthesis, and on the other hand, lack of daytime sunlight (especially its blue-violet spectrum) disrupts circadian system synchronization and leads to vitamin D deficiency. This combination creates a “perfect storm” for the development of so-called “diseases of civilization,” including metabolic syndrome and liver pathology. Our experimental data fully confirm this concept at the tissue and molecular levels [53].

The identified biochemical shifts in animals of the DD group (hypoglycemia, hypoproteinemia, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, increased AST, ALT, LDH, and ALP) indicate profound metabolic disorders and hepatocyte damage. These data are consistent with current understanding of the role of circadian dysregulation in the pathogenesis of MASLD [54,55,56].

Increased activity of AST, ALT, and LDH in the DD group is a marker of cytolysis and indicates damage to hepatocyte membranes, which at the ultrastructural level is confirmed by mitochondrial swelling and cristae destruction. Complete normalization of all biochemical parameters in the DD + Mel group underscores the key role of melatonin in maintaining hepatic metabolic homeostasis.

The increase in hepatocyte area by 42.5% with unchanged nuclear area and the decrease in N/C ratio by 30.4% in the DD group indicate cellular hypertrophy. Hepatocyte hypertrophy is often considered an adaptive response to metabolic stress; however, it can also be a precursor to pathological remodeling and fibrosis [57,58,59].

A twofold increase in the proportion of connective tissue confirms the development of fibrosis. The work of Kim and Cheon [60] sheds light on the molecular mechanisms of the antifibrotic action of melatonin. The authors showed that melatonin, through activation of the MT2 receptor, increases the expression of BMAL1 and antioxidant enzymes, suppressing TGF-β1-induced activation of hepatic stellate cells (HSCs). The use of an MT2 antagonist or siRNA against MT2 completely abolished these effects, proving that the MT2-BMAL1 signaling pathway is critical for the antifibrotic action of melatonin. In our model, fibrosis induced by light deprivation was completely prevented by melatonin, which is consistent with the described mechanism. Pramong et al. [61] also demonstrated that in aged rats, the expression of collagen type I in the liver increases, and melatonin treatment reduces this parameter.

Pronounced steatosis in the DD group and an increase in the proportion of hepatocytes with fatty degeneration confirm that light pollution is a risk factor for the development of MASLD. Melatonin, by restoring circadian regulation of lipid metabolism and possessing antioxidant properties, significantly reduced steatosis in the DD + Mel group (0.90 points). The meta-analysis by Singh [62] confirms these data, demonstrating clinically significant improvement in the lipid profile of patients with MASLD during melatonin administration. Nevertheless, the proportion of hepatocytes with fatty degeneration in our work remained slightly but significantly elevated relative to the control, which may indicate the need for longer therapy for complete elimination of lipid inclusions or the existence of a pool of cells with irreversible changes [63].

The decrease in the proportion of binuclear hepatocytes in the DD group may reflect disruption of the cytokinesis process or depletion of the pool of cells capable of forming binuclear forms, which are considered a reserve for polyploidization and regeneration [64]. Work using BTBR mice [65] clearly demonstrated that three populations of cells are present in the liver: mononuclear diploid, binuclear, and mononuclear tetraploid, and their ratio is an important indicator of the functional state of the organ. Interestingly, in the DD + Mel group, the proportion of binuclear cells was significantly higher not only compared to DD but also to the control. This may indicate activation of regenerative processes and an increase in the functional reserve of the liver under the influence of melatonin [66].

The trend toward an increase in mean ploidy in both experimental groups (to 3.22 and 3.20 n) is consistent with the concept of stress-induced polyploidization as an adaptive mechanism allowing hepatocytes to increase functional capacity without division [67]. The paradoxical increase in the Ki-67 proliferation index in the DD group against the background of pronounced activation of cellular senescence markers (p16, p21) may be explained by compensatory hyperplasia in response to chronic damage and cell death. However, such proliferation is likely “unbalanced” and does not lead to effective restoration of the population of functionally competent hepatocytes. Normalization of Ki-67 in the DD + Mel group indicates stabilization of cell renewal and elimination of chronic damage [68,69].

The most significant result of this work is the demonstration that chronic light deprivation induces a cellular senescence phenotype in the liver, as evidenced by a sharp increase in the expression of p16 and p21, as well as p53. p16 and p21 are universally recognized biomarkers of aging, and their expression is minimal in young tissues [70,71]. González-Gallego et al. [72], in a series of works, demonstrated that melatonin is capable of reducing apoptotic changes in the liver of aging rats through inhibition of the mitochondrial apoptosis pathway and preventing oxidative stress. Activation of these pathways in our study indicates the initiation of cellular senescence programs in a significant portion of hepatocytes.

Simultaneous profound suppression of BMAL1 and CLOCK in the DD group demonstrates the disintegration of the molecular circadian oscillator. A study in mice [73] showed that Clock mutation leads to arrhythmic expression of Per1 and Per2 in the liver and skeletal muscle, despite preserved rhythms in the SCN, emphasizing the tissue-specific requirements for clock components and the absolute dependence of peripheral oscillators on functional CLOCK. In our model, suppression of endogenous melatonin led to a similar effect—desynchronization of hepatic clocks with preserved (presumably) SCN function. The increase in PER2 in the DD group may be a consequence of disruption of feedback mechanisms in the circadian cycle.

The complete normalization of all molecular markers (p16, p21, p53, BMAL1, CLOCK, PER2, Ki-67) in the DD + Mel group indicates that exogenous melatonin not only compensates for the deficiency of the endogenous hormone but also restores the regulatory circuits controlling aging and circadian rhythmicity, likely through receptor-mediated mechanisms, including the MT2-BMAL1 axis [74,75,76] and modulation of ROR-α activity [77]. Indeed, it has been shown that melatonin, through interaction with membrane receptors, modulates the expression of clock genes, including BMAL1 and CLOCK, in liver cells, and that nuclear ROR-α receptors, also under the control of melatonin, are critically important for maintaining circadian rhythms and possess anti-inflammatory properties.

Moreover, fundamental studies demonstrate a direct link between clock genes (BMAL1, CLOCK, PER2) and the regulation of aging markers p16, p21, and p53, which explains the complete normalization of these parameters in our model upon restoration of circadian rhythmicity by exogenous melatonin [78,79,80,81].

Electron microscopy data provided direct visualization of the damage underlying the observed functional disturbances. Accumulation of lipofuscin—the “aging pigment”—is a marker of oxidative stress and reduced autophagy efficiency [82,83,84]. The study by Manikonda et al. [85] showed that with age (in 12- and 24-month-old rats), lipid peroxidation increases in the liver, the GSH/GSSG ratio and the activity of antioxidant enzymes decrease, and the daily rhythms of these parameters are disrupted. Melatonin administration partially restored the amplitude and acrophases of these rhythms. In our study, young (6-month-old) rats subjected to light deprivation exhibited ultrastructural changes similar to age-related ones, confirming the concept of “accelerated aging” under the influence of desynchronosis.

Mitochondrial damage (polymorphism, swelling, cristae destruction, tendency to decreased numerical density) confirms the key role of mitochondrial dysfunction in pathogenesis. Yang et al. [86], in a model of atrazine-induced toxic liver damage, showed that melatonin restores the level of Rab8a—a protein critical for regulating fatty acid utilization in mitochondria. Rab8a knockdown abolished the protective effect of melatonin, indicating a new molecular mechanism of mitoprotection. The ability of melatonin to accumulate in mitochondria at high concentrations and its direct antioxidant activity explain the preservation of organelle ultrastructure in the DD + Mel group [87].

Reduction of the Golgi complex (decreased number of cisternae and profile area) in the DD group correlates with hypoalbuminemia and impaired liver secretory function. Restoration of Golgi complex morphology under the influence of melatonin underscores its role in maintaining not only the energy but also the secretory apparatus of the cell [88,89,90].

The totality of the obtained data allows us to propose the following sequence of events: chronic light exposure, melatonin suppression, disruption of receptor signaling (MT2, ROR-α), disintegration of circadian oscillators in hepatocytes (decrease in BMAL1/CLOCK), metabolic dysregulation (dyslipidemia, impaired carbohydrate and protein metabolism), oxidative stress and mitochondrial dysfunction (decrease in Rab8a, antioxidant enzymes), activation of cellular senescence pathways (p53/p21, p16), hepatocyte hypertrophy and polyploidization, steatosis and fibrosis (through HSC activation), compensatory proliferation (increase in Ki-67) against the background of depleted regenerative reserve (decrease in proportion of binuclear cells).

Exogenous melatonin, by replenishing the hormone deficiency, acts at several levels:

The obtained results have direct relevance to human health. Given that more than 80% of the world’s population lives under conditions of light pollution [86], and the prevalence of MASLD reaches 38% in the population, the identification of melatonin as an effective agent for the prevention and correction of desynchronosis-associated liver damage opens new therapeutic horizons. Moreover, clinical studies are currently investigating the effect of melatonin on sleep and cognitive functions in patients with liver cirrhosis and hepatic encephalopathy. This underscores the growing interest in the therapeutic potential of melatonin in severe liver diseases and expands the range of possible clinical applications beyond metabolic disorders.

Thus, our study not only confirms the concept of light pollution as a factor in accelerated liver aging but also details the molecular, cellular, and ultrastructural mechanisms of this process, and demonstrates the powerful protective potential of melatonin, paving the way for clinical studies in at-risk groups. The obtained results have direct relevance to human health, paving the way for clinical studies in night shift workers, residents of megacities, and patients with MASLD.

Several limitations of this study should be acknowledged.

Conceptualization, D.A.A., A.I.A. and V.P.C.; methodology, D.A.A., M.A.K. and V.P.C.; formal analysis, investigation, A.I.A. and M.A.K.; writing—original draft preparation, D.A.A.; writing—review and editing, D.A.A. All authors have read and agreed to the published version of the manuscript.

This study was performed in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986). Ethical approval for the research was granted by the Bioethical Committee of the Avtsyn Research Institute of Human Morphology of FSBSI “Petrovsky National Research Centre of Surgery” (Protocol No. 5, 23 May 2025).

Not applicable.

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

The authors declare no conflicts of interest.

This research was performed as part of a state-funded assignment of “Avtsyn Research Institute of Human Morphology of Federal State Budgetary Scientific Institution ‘Petrovsky National Research Centre of Surgery’”, No. 124021600054-9.