Vitamin D3 Improves Hypothalamic–Pituitary–Adrenal Axis Function, Immunological Responses, and Gut Dysbiosis in Sleep Desynchrony

Shift work plays a crucial role in the modern working environment. It supports sustainability, customer service, public safety, and health care, and offers economic advantages. This type of work is essential in sectors such as health care, transportation, industry, commerce, and hospitality (Kalkanis et al. 2023). Currently, approximately 20% of the global workforce is engaged in shift work. Statistical analyses have revealed a 15% increase in the prevalence of shift work across several European countries. In the United States, the incidence of shift work has reached 20% among men and 11% among women (Soltanzadeh et al. 2024). Notably, shift work has several psychophysiological effects that are primarily related to circadian misalignments and sleep disturbances, and shift‐work sleep disorder is estimated to affect 10% to 38% of the workforce (Sateia 2014; Pallesen et al. 2021). The effects are particularly strong for individuals working rotating night shifts, for whom sleep is often prematurely interrupted because of the influence of circadian rhythms, leading to severe sleepiness and reduced performance capacity (Åkerstedt 1990).

Sleep is regulated by the two‐process model, in which the homeostatic process reflects the accumulation of sleep pressure during wakefulness and the circadian process follows a 24‐h rhythm aligned with the light–dark cycle. Shift workers often experience irregular sleep schedules that cause misalignments between the two processes, leading to chronic sleep deprivation (CSD), impaired sleep quality, insomnia, and excessive daytime sleepiness, all of which contribute to decreased alertness and poorer cognitive performance (Drake et al. 2004). Sleep desynchrony considerably affects the mental health of shift workers. Studies have reported that shift workers are twice as likely as day‐only workers to report depressive symptoms (Driesen et al. 2010). Furthermore, shift workers have a 1.5‐fold greater risk of experiencing severe depressive symptoms (Lee et al. 2016). Although some studies have identified an increased risk of anxiety and depression among shift workers (Lin et al. 2012; Bara and Arber 2009), others have indicated better psychological well‐being among shift workers compared with non‐shift workers (Bildt and Michélsen 2002; Nabe‐Nielsen et al. 2011). These conflicting findings may reflect variations in the contexts, occupational settings, and population characteristics of the aforementioned studies. In addition to mental health problems, shift workers are at a higher risk of experiencing other health conditions, such as metabolic syndrome (De Bacquer et al. 2009; Pietroiusti et al. 2010), cardiovascular diseases (particularly coronary heart disease) (Torquati et al. 2018), and gastrointestinal disorders (Drake et al. 2004; Knutsson and Bøggild 2010). Therefore, employing effective interventions to mitigate these health risks is crucial. Research has indicated that sufficient high‐quality sleep after night shifts can reduce fatigue and enhance recovery, particularly among nurses (Silva‐Costa et al. 2011; Konya et al. 2022). Thus, for shift workers, implementing targeted interventions may help mitigate the adverse health effects of sleep desynchrony and improve their quality of life and cognitive performance.

Vitamin D supplementation has been explored as a potential intervention for enhancing sleep quality. Majid et al. (2018) reported that 8 weeks of vitamin D supplementation reduced sleep latency and increased sleep duration in middle‐aged individuals. Furthermore, dietary vitamin D intake was demonstrated to significantly reduce the risk of anxiety and sleep disorders, although no significant association between dietary vitamin D intake and depression was identified (Arabshahi et al. 2024). Shift work was also reported to be linked to lower vitamin D levels, more depressive symptoms, and poorer sleep quality (Park et al. 2019). A study suggested that sleep deprivation can have lethal effects in model organisms, with these effects primarily resulting from the accumulation of reactive oxygen species (ROS), particularly in the gut (Vaccaro et al. 2020). Because of its ability to reduce anxiety, alleviate sleep disorders, and act as an antioxidant to counter ROS accumulation, the current study selected vitamin D for the experimental treatment following sleep desynchrony. Moreover, vitamin D has been shown to modulate gut barrier integrity and intestinal inflammation through cytokine regulation and microbiota interaction, highlighting its potential in gastrointestinal and systemic immune regulation (Vernia et al. 2022). While the individual roles of vitamin D3 in modulating immune responses, regulating stress pathways, and influencing gut microbiota have been reported, no study to date has comprehensively evaluated these systems in an integrated manner under conditions of chronic sleep desynchrony. Therefore, this study investigated the effects of continuous sleep desynchrony and subsequent interventions, including vitamin D3 treatment and sleep recovery, on the body weight, metabolic activity, stress response, immune function, and gut microbiota composition in mice.

CSD was induced for 28 days using a sleep fragmentation device (Lafayette Instrument Company, Lafayette, IN, USA), following a protocol adapted from Cabrera‐Aguilera et al. (2020) with minor modifications. The device comprised a nearly silent mechanical motor that moved a horizontal bar across the floor of a standard mouse cage. This bar gently swept back and forth just above the cage floor to disrupt sleep while ensuring minimal noise and stress for the animals. The automated system could be operated without the need for human interaction, further reducing potential stress. The bar was programmed to sweep once per minute during the mice's designated sleep period (8 a.m. to 6 p.m., 10 h in total), and this protocol was applied continuously for 28 days.

Dexamethasone, an exogenous steroid, suppresses the secretion of adrenocorticotropic hormone by activating negative feedback on the pituitary gland, which renders it a useful tool for evaluating the function of the hypothalamic–pituitary–adrenal (HPA) axis (Pariante and Lightman 2008). In the present experiment, corticosterone levels were measured following intraperitoneal injections of dexamethasone to assess HPA axis activity.

On Day 18, the mice received an intraperitoneal injection of saline, and plasma samples (100 µL) were collected from the cheek after 1 h, with these samples serving as the baseline (blank) group. The following day (Day 19), the same mice were injected intraperitoneally with dexamethasone, and after 1 h, plasma samples (100 µL) were collected again, with these samples representing the test group. Corticosterone suppression efficiency was calculated using the formula: 100 − ([corticosterone level] after dexamethasone injection/[corticosterone level] after saline injection) × 100.

At the conclusion of the experiment, the corticosterone levels of the plasma samples were quantified using the Corticosterone AssayMax enzyme‐linked immunosorbent assay (ELISA) Kit (Assaypro, Saint Charles, MO, USA).

RNA samples were extracted from various brain regions, including the prefrontal cortex (PFC), hippocampus, hypothalamus, bed nucleus of the stria terminalis, and cerebral cortex, and preserved in RNA Save (Biological Industries, Kibbutz Beit Haemek, Israel) before being stored at −80°C for subsequent analysis. Total RNA extraction was conducted using the EZ‐RNA II total RNA isolation kit (Biological Industries). After extraction, 1 µg of RNA in a 20‐µL reaction mixture was reverse transcribed into cDNA using the ToolsQuant II Fast RT Kit (TOOLS, Taipei, Taiwan). Quantitative PCR (qPCR) was subsequently performed using the TOOLS 2X SYBR qPCR Mix (TOOLS) and analyzed with a LightCycler 480 system (Roche, Basel, Switzerland). The resulting cDNA was used to measure the expression of key gene categories, including inflammatory genes (TNF‐α, IL‐6, and IFN‐γ) and circadian genes (Bmal‐1, Clock, Cry1, Cry2, and Per2). The relative mRNA expression levels of each target gene were quantified on the basis of threshold cycle values and normalized to the expression of glyceraldehyde‐3‐phosphate dehydrogenase.

All antibodies used in the present study were derived from naive CD4⁺ T cells isolated from mouse spleens in accordance with the protocol outlined by Yang et al. (2018). Spleen tissues from each experimental mouse were minced into small fragments and mechanically disrupted to release cells. A single‐cell suspension was prepared by filtering the disrupted tissue through 200‐mesh screens. The procedures for labeling, permeabilization, and fixation were performed in accordance with the relevant kit instructions. The antibodies used comprised MS FITC Rat anti‐mouse CD4 (Clone GK1.5, Biolegend, San Diego, CA, USA), MS PE anti‐mouse NK 1.1 (Clone PK136, Biolegend), MS PE anti‐mouse IFN‐γ (Clone XMG1.2, Biolegend), and MS PE anti‐mouse IL‐4 (Clone PC61, BD Biosciences, San Jose, CA, USA). Flow cytometric analysis was conducted using a Beckman Coulter FC500 cytometer (Beckman Coulter, Brea, CA, USA), with data analysis performed using the software CXP.

Protein expression levels in the proximal colon were analyzed using Western blotting, in accordance with the method described by Ho et al. (2019). Membranes were incubated with primary antibodies targeting ZO‐1, occludin, claudin, and β‐actin at 4°C for 12 h on a shaker. The primary antibodies used in the present study comprised anti‐ZO‐1 (Cat# 21773‐1‐AP, 1:1000, Proteintech), anti‐occludin (Cat# 27260, 1:1000, Proteintech), anti‐Claudin (Cat# AF0127, 1:1000, Affinity), and anti‐β‐actin (Cat# 4970S, 1:3000, Cell Signaling). Immunoreactive bands were detected using enhanced chemiluminescence, and protein expression levels were quantified through densitometry by using the software ImageJ (Wayne Rasband, Madison, WI, USA). The results were normalized to β‐actin levels and expressed as fold changes relative to the control group.

The amplification and library preparation of the 16S rRNA gene were performed in accordance with Illumina's recommended protocols (https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html↗). Specifically, the universal primers 341F (forward primer; 5'‐CCTACgggNggCWgCAg‐3') and 805R (reverse primer; 5'‐gACTACHCggg‐TATCTAATCC‐3'), which include Illumina overhang adapter sequences, were used to amplify the V3–V4 region of the bacterial 16S rRNA gene through PCR with a limited number of cycles. After amplification, the Nextera XT Index Kit (Illumina, San Diego, CA, USA) was employed to add Illumina sequencing adapters and dual‐index barcodes to the resulting amplicons. The quality and quantity of the constructed library were evaluated using a Qsep100 Analyzer (BiOptic, Taipei, Taiwan). For additional details on the methodology, including database use, validation, and data analysis, refer to Tung et al. (2021). Functional profiling was conducted with PICRUSt2 (v2.3.0_b), which predicts gene family and pathway abundances (e.g., KEGG Orthologs and MetaCyc pathways) from phylogenetic placement of ASVs. Differential abundance analyses of predicted functions were carried out using ALDEx2 (v1.18) in R (v 4.5.1), applying centered log‑ratio (clr) transformation and Monte Carlo sampling to account for compositionality. Group comparisons were assessed using Welch's t‐test and Wilcoxon rank‐sum test, with p values corrected by Benjamini–Hochberg false discovery rate (FDR) correction. Beta diversity was assessed using weighted UniFrac and generalized UniFrac (α = 0.5) distance metrics. Principal coordinate analysis (PCoA) was used for visualization. Group differences were statistically tested using PERMANOVA via the adonis function (vegan v 2.6‐4) in R (v 4.5.1).

The endotoxin marker LPS was detected using a ToxinSensor Chromogenic LAL Endotoxin Assay Kit (Cat# L00350, GenScript, Piscataway, NJ). ELISA procedures were performed in accordance with the manufacturer's instructions.

Fecal samples weighing 40 mg were collected and immediately stored in 400 µL of water containing 0.5% phosphoric acid. After collection, the samples were rapidly frozen at −80°C. Prior to analysis, the samples were thawed, homogenized, and centrifuged at 14,000 rpm for 15 min, after which the supernatant was carefully collected. An equal volume of ethyl acetate was subsequently added to the supernatant, followed by vigorous vortexing for 2 min. The mixture was centrifuged again at 14,000 rpm for 15 min, and the organic phase was isolated and stored at −80°C until further analysis. Gas chromatography was subsequently performed in accordance with the protocol established by Tung et al. (2021). Short chain fatty acids (SCFAs) were identified by comparing the recorded retention times with those of established standards, including those for acetic acid, propionic acid, and butanoic acid.

Statistical analysis was conducted using the software GraphPad Prism (Version 6, GraphPad Software, San Diego, CA, USA), with the data expressed as means ± standard errors of the mean (SEMs). Data were analyzed by performing the Mann–Whitney U test, a nonparametric statistical test. p values of <0.05 were regarded as indicative of significant differences.

The findings of the present study reveal that 28 days of sleep desynchrony did not significantly alter the body weight, food intake, or water consumption in the mice. The mice in all groups (NS, CSD, CSDR, and CSDVD) exhibited normal and steady increases in body weight over the course of the experiment, and no significant differences between initial and final body weight were identified among the groups. Similarly, no significant differences in food and water intake were detected, suggesting that prolonged sleep disruption did not substantially influence the fundamental metabolic processes in these animals. Despite experiencing extended periods of circadian misalignment, the mice appeared to maintain metabolic homeostasis. This stability may be attributable to the capacity of the mice to adaptively regulate energy expenditure in response to sustained stress, thereby ensuring the preservation of vital physiological functions; the mice may have employed intrinsic regulatory mechanisms to offset energy deficits induced by CSD.

The secretion of corticosterone, a key stress hormone, is regulated by the HPA axis, and its function can be evaluated using the dexamethasone suppression test. In the NS group, the administration of dexamethasone led to a significant decrease in corticosterone levels, indicating a normal HPA axis suppression response. However, in the CSD group, dexamethasone failed to effectively suppress corticosterone secretion, suggesting HPA axis dysfunction. This could have resulted from a weakened negative feedback mechanism, reflecting desensitization or tolerance of the HPA axis after chronic sleep desynchrony. Notably, in the mice treated with vitamin D3, dexamethasone significantly suppressed corticosterone secretion, indicating that vitamin D3 supplementation helped to ameliorate HPA axis dysfunction induced by sleep desynchrony. The beneficial effects of vitamin D3 may stem from its anti‐inflammatory and antioxidant properties (Silva and Lazaretti‐Castro 2022). Studies have reported that emotional disorders such as anxiety and depression are often associated with a reduction in antioxidant enzyme activity in the HPA axis (Rasmus and Kozłowska 2023). Therefore, as an antioxidant, vitamin D3 may enhance antioxidant enzyme activity, thereby restoring the HPA axis's sensitivity to stress. One study also suggests that vitamin D helps to regulate the stress axis (Rolf et al. 2018). In addition, vitamin D3 was revealed to suppress glucocorticoid‐induced transcription and cytotoxicity in hippocampal cell cultures (Eyles et al. 2005). Studies focused on the human central nervous system have reported that the highest expression of vitamin D receptors and activating enzymes occurred in the hypothalamus, particularly in the paraventricular nucleus (Eyles et al. 2005; Smolders et al. 2013). Smolders et al. (2013) indicated that the corticotrophin‐releasing hormone‐positive neurons in the paraventricular nucleus, which are part of the stress axis, also express vitamin D 24‐hydroxylase (Smolders et al. 2013), suggesting that these neurons are responsive to vitamin D. Our finding is consistent with those of previous studies on the role of vitamin D3 in maintaining stress homeostasis and protecting against stress‐related damage. Moreover, in the current study, after 28 days of sleep desynchrony, no significant differences in baseline corticosterone levels were identified among the NS, CSD, and CSDVD groups. This suggests that sleep desynchrony impairs HPA axis regulation but does not lead to significant changes in baseline corticosterone levels in the short term. These results indicate that vitamin D3 treatment can effectively improve HPA axis dysfunction caused by sleep desynchrony and restore normal stress response regulation.

Although vitamin D3 supplementation exerted beneficial effects on behavior, immune responses, and gut barrier integrity, its impact on central stress regulation—particularly hypothalamic cytokine expression and HPA axis normalization—remained limited. This partial restoration may reflect the complexity of neuroendocrine feedback circuits and the need for additional interventions to fully restore central sensitivity. To enhance HPA axis resilience, several adjunctive strategies have shown promise in modulating stress responsivity via complementary mechanisms. Adaptogens—natural compounds such as Rhodiola and Schisandra—have been reported to stabilize HPA axis function by attenuating excessive glucocorticoid release and normalizing stress‐induced neuroendocrine signals, including heat shock proteins and FOXO transcription factors (Tóth‐Mészáros et al. 2023). Melatonin, a well‐established chronobiotic, not only entrains circadian rhythms but also directly inhibits CRH and ACTH secretion, thereby dampening corticosterone elevation under acute and chronic stress (Konakchieva et al. 1997). Additionally, psychobiotics such as Lactiplantibacillus plantarum PS128 can modulate the gut–brain axis via vagal afferents and immune signaling, leading to reduced peripheral cortisol levels and improved stress resilience (Wu et al. 2021b). Omega‐3 polyunsaturated fatty acids (PUFAs), notably EPA and DHA, have been shown to attenuate neuroinflammation, enhance glucocorticoid receptor sensitivity, and normalize HPA axis function through hippocampal modulation (Zhou et al. 2022a). These interventions may synergize with vitamin D3 by targeting distinct levels of HPA regulation—omega‐3s reinforcing receptor‐level feedback sensitivity, psychobiotics modulating upstream stress perception, and adaptogens or melatonin stabilizing central output. Therefore, future studies should consider combinatorial approaches incorporating vitamin D3 with these agents to achieve more comprehensive restoration of neuroendocrine homeostasis under chronic circadian disruption.

In the present study, behavioral tests were used to evaluate the effects of sleep desynchrony on cognition, locomotor activity, and anxiety‐like behaviors in mice, with a focus on the effects of vitamin D3 treatment. In the present study's experiment, the NS and CSDVD mice constructed higher‐quality nests with more complete structures, suggesting improved overall health in these groups. By contrast, the CSD group exhibited a slight decline in nest‐building ability, suggesting that sleep desynchrony negatively affects motivation and physical condition. By contrast, the CSDVD group exhibited improvements in nest quality, suggesting that vitamin D3 supplementation can restore cognitive function and motivation. In addition, the OFT results indicated that the NS group exhibited greater locomotor activity, as evidenced by a wider range of movement within the test arena. By contrast, the CSD group exhibited a significant reduction in movement range, suggesting that sleep desynchrony impaired the mice's locomotor capacity and exploratory behavior. The CSDR and CSDVD groups exhibited partial recovery of activity levels. In particular, the CSDVD group exhibited a significant increase in distance traveled compared with the CSD group. This suggests that vitamin D3 plays a role in improving locomotor function and reducing anxiety‐like behaviors. In the EPM test, the CSD group exhibited a significant reduction in total distance traveled, which is consistent with the observed reduction in locomotor activity. By contrast, the CSDR and CSDVD groups did not exhibit significant differences in total distance traveled relative to the CSD group.

Although the EPM, OFT, and NBTs are commonly used to assess anxiety‐like behaviors in mice, they each reflect distinct behavioral domains. The OFT and NBTs are more sensitive to changes in general activity, motivation, and mild anxiety, whereas the EPM focuses on approach–avoidance conflict and risk assessment under high‐stress conditions. As noted by Figueiredo Cerqueira et al. (2023), the OFT captures exploratory behavior in novel environments, while the EPM reflects behavioral inhibition in threatening contexts. However, Shoji and Miyakawa (2021) reported that EPM results are more susceptible to environmental factors and prior testing experience, potentially limiting their consistency. Therefore, the absence of significant differences in the EPM in this study—despite anxiolytic‐like trends observed in the OFT and NBTs—may be due to the EPM's distinct sensitivity to risk‐related behaviors. This highlights the importance of using multiple behavioral assays to capture various aspects of anxiety.

A study reported that insufficient vitamin D intake is linked to various mental disorders, including anxiety (McCann and Ames 2008). Another reported that moderate‐dose vitamin D supplementation (≥800 IU daily) had beneficial effects in treating depression, particularly when serum vitamin D levels were lower than the normal range (Spedding 2014). Additionally, vitamin D supplementation over a period of 2 years was associated with improved sleep in 1500 patients experiencing neurological conditions (Gominak and Stumpf 2012). A double‐blind clinical trial demonstrated that vitamin D3 supplementation significantly improved anxiety, depression, and physical health in university students but had no significant effect on sleep quality (Karami‐Mohajeri et al. 2021). By contrast, another study did not identify any significant differences between the vitamin D3 and placebo groups in terms of depression incidence, recurrence, or changes in mood scores, suggesting that vitamin D3 supplementation did not help prevent depression (Okereke et al. 2020). In the present study, the mice that received vitamin D3 supplementation built higher‐quality nests and exhibited improved locomotor activity relative to the other groups, suggesting that vitamin D3 promoted cognitive recovery and motor function. Notably, the potential of vitamin D3 to improve anxiety and depression has been supported by other studies, such as those reporting that moderate doses of vitamin D supplementation helped to treat depression, particularly when vitamin D levels were lower than normal. However, several long‐term studies have indicated that vitamin D3 has limited preventive effects on depression and does not significantly improve sleep quality. Emerging evidence suggests that both higher dosages and longer durations of vitamin D intervention may be required to elicit more robust neurobehavioral and immunomodulatory outcomes. For instance, Gao et al. (2018) reported that high‐dose vitamin D supplementation led to significant improvements in depressive symptoms in individuals with major depressive disorder. A recent preclinical study showed that extending vitamin D3 treatment duration in rats significantly improved neuroinflammation and behavioral deficits induced by chronic stress (Arabshahi et al. 2024). Similarly, a clinical study in Iranian students found greater improvements in psychological well‐being after extended administration of higher‐dose vitamin D3 (Mansouri et al. 2024). Therefore, although the results of the present study suggest that vitamin D3 supplementation contributes to the partial recovery of locomotor function, its role in regulating anxiety may be limited in the short term. Future studies with varied doses and longer intervention duration should be conducted to enable more comprehensive assessment of such effects.

Sleep desynchrony led to an increase in neutrophils and a decrease in lymphocytes, indicating a shift in the immune system toward a more pro‐inflammatory state. Vitamin D3 treatment helped to counteract this by reducing neutrophils and increasing lymphocytes, thereby contributing to the re‐establishment of immune homeostasis. A study reported an inverse relationship between 25‐hydroxyvitamin D levels and inflammation, as indicated by a negative correlation between vitamin D levels and the neutrophil‐to‐lymphocyte ratio (Akbas et al. 2016). Collectively, these findings highlight the potential of vitamin D3 in modulating immune responses and reducing inflammation. Regarding immune cell modulation, CSD slightly increased the number of NK cells and altered the proportion of T helper cells, both of which play crucial roles in regulating immune responses, and sleep recovery reduced Th17 cells. Notably, although Th1 cells significantly decreased after sleep recovery, vitamin D3 treatment appeared to slightly elevate the levels of these cells, suggesting it plays a role in balancing Th1 and Th2 responses. However, it did not fully restore the Th1/Th2 ratio to normal levels. These findings suggest that sleep desynchrony significantly affected immune function, shifting immune cell populations toward a more inflammatory state. However, the effects of vitamin D3 on specific immune cells, such as Th1, Th2, and NK cells, appeared to be limited.

In this study, sleep desynchrony did not significantly alter the expression of inflammation‐related genes in the hypothalamus, as evidenced by the absence of significant differences in the expression levels of inflammatory factors, such as TNF‐α, IL‐6, and IFN‐γ, among the NS, CSD, CSDR, and CSDVD groups. This suggests that short‐term sleep desynchrony did not induce a pronounced systemic inflammatory response. However, the CSD group exhibited a significant increase in the expression of clock genes, indicating that sleep desynchrony affected the expression of circadian gene. This finding further highlights the close interaction between sleep and circadian rhythms. The upregulation of these genes may be related to an imbalance in the internal time regulation mechanism caused by sleep disruption. Notably, both sleep recovery and vitamin D3 treatment restored Bmal‐1 expression to near‐normal levels, suggesting that these interventions had a positive effect on the normal functioning of circadian genes. Therefore, although sleep desynchrony did not significantly affect the expression of inflammatory genes in the hypothalamus, it significantly affected the expression patterns of circadian genes.

This study analyzed the effect of sleep desynchrony on gut microbiota composition and the potential role of vitamin D3 in regulating gut microbiota imbalance. Although no significant differences in alpha diversity indices (e.g., observed species and Chao1, Shannon, and Simpson indices) were identified among the NS, CSD, CSDR, and CSDVD groups, beta diversity analysis (based on GUniFrac and weighted UniFrac PCoA plots) revealed significant differences in gut microbiota composition between the CSD and NS groups. This indicates that sleep desynchrony altered the structure of the gut microbiome. Notably, the gut microbiota composition in the CSDVD group, which underwent vitamin D3 treatment, shifted closer to that of the NS group, suggesting that vitamin D3 treatment helps mitigate dysbiosis induced by circadian rhythm disruption. By contrast, the microbiota composition in the CSDR group, which underwent sleep recovery alone, remained similar to that of the CSD group, indicating that sleep recovery alone had limited effects on restoring gut microbiota. At the phylum level, the CSD group exhibited a significant reduction in Bacteroidota and an increase in Firmicutes, indicating gut dysbiosis due to sleep desynchrony. Vitamin D3 treatment significantly increased Bacteroidota and decreased the Firmicutes percentage in gut microbiota. Also, this study observed a notable increase in the F/B ratio in the CSD group that reflected a severe microbiome imbalance. Vitamin D3 treatment significantly reduced this ratio, bringing it closer to the levels observed in the NS group. Ciubotaru et al. (2015) suggested that vitamin D3 supplementation alters the microbiota composition by reducing the abundance of the phylum Firmicutes. In contrast, Mandal et al. (2016) reported that vitamin D treatment can increase the Proteobacteria/Firmicutes ratio. However, both studies reported an increase in the population of Bacteroidetes.

At the family level, the increase in Lachnospiraceae and the decrease in Muribaculaceae in the CSD group indicated gut microbiota dysregulation, but vitamin D3 treatment effectively reversed these changes, confirming its role in re‐establishing gut microbial balance. One study (Ko et al. 2019) reported an increase in the relative abundance of Lachnospiraceae when the severity of sleep apnea–hypopnea syndrome increased. In another study, the relative abundance of Lachnospiraceae was, on average, negatively correlated with sleep efficiency and total sleep time (Smith et al. 2019). One study suggested that vitamin D is associated with specific genera within the Lachnospiraceae family, including Blautia, Roseburia, Dorea, and Coprococcus (Tangestani et al. 2021). Muribaculaceae, which belongs to the phylum Bacteroidetes, is a beneficial bacterium found within the intestinal microbiota of mice. Our previous study indicated that the relative abundance of Muribaculaceae was lower in mice subjected to acute sleep deprivation (Yang et al. 2023). Several studies have demonstrated that this family contributes to the extension of lifespan in mice (Lagkouvardos et al. 2019; Smith et al. 2021). Studies involving ruminants have reported that the relative abundance of Muribaculaceae correlates significantly with circadian oscillations in the rumen of dairy cows because of the influence of melatonin, and its higher abundance has been linked to potential benefits in treating subclinical mastitis in these animals (Ouyang et al. 2021; Wang et al. 2021). However, another study reported a reduction in Muribaculaceae in hemodialysis patients with sarcopenia (Zhou et al. 2022b). Yet another study reported that short‐term calcitriol treatment did not reduce intestinal bacterial overgrowth but induced significant changes in bacterial diversity and promoted the enrichment of Muribaculaceae, Bacteroidales, Allobaculum, Anaerovorax, and Ruminococcaceae (Lee et al. 2021).

Notably, the PICRUSt‐predicted functional alterations are consistent with recent studies showing that sleep deprivation disrupts gut microbial composition, reduces beneficial taxa, and promotes the expansion of opportunistic bacteria, which are associated with intestinal inflammation and impaired barrier function (Wang et al. 2022; Liu et al. 2025). Decreased gluconeogenesis and fermentation pathways may reflect impaired microbial SCFAs output, disrupting epithelial energy supply and immune balance (Tremaroli and Bäckhed 2012). Furthermore, recent evidence indicates that brain peptidoglycan (PG) levels are dynamically regulated by sleep and wake states as well as circadian rhythms (English and Krueger 2025). For instance, PG concentrations decrease in regulatory regions such as the hypothalamus and brainstem during acute sleep deprivation, but increase in cortical areas, suggesting region‐specific shifts in PG localization. Sleep deprivation also alters the expression of PG recognition proteins, such as Pglyrp1, implying heightened host immune surveillance under disrupted sleep conditions (Rehman et al. 2001). These changes may trigger neuroimmune cascades that sustain low‐grade inflammation. The observed enrichment in bacterial polysaccharide biosynthesis and Mixed acid fermentation pathways may contribute to increased microbial immunogenicity and enhanced inflammatory signaling, reflecting a greater antigenic potential. These bacterial structural components may activate host pattern recognition receptors and initiate mucosal immune responses, contributing to sustained low‐grade inflammation. In particular, metabolites derived from bacterial polysaccharide biosynthesis, such as LPS and capsular polysaccharides, have been shown to cross a compromised gut–brain barrier and stimulate microglial activation, contributing to neuroinflammation (Farmen et al. 2021). Similarly, end products of Mixed acid fermentation, including acetate, succinate, and propionate, can modulate glial cell activity and cytokine release in the central nervous system via G‐protein‐coupled receptors or histone deacetylase inhibition (Blad et al. 2012). Collectively, these findings suggest that the activation of pro‐inflammatory microbial pathways under conditions of sleep desynchrony and vitamin D3 supplementation may reflect a neuroimmune response linking microbial metabolism to host inflammatory tone and sleep regulation.

The observed enrichment in microbial pathways related to amino acid biosynthesis and glycolysis following vitamin D3 supplementation may underlie the behavioral improvements seen in the CSDVD group. Notably, the upregulation of L‐arginine biosynthesis III in the CSDVD group suggests enhanced microbial contribution to systemic arginine availability. L‐arginine is a semi‐essential amino acid critical not only for protein synthesis but also as a precursor for nitric oxide (NO), a mediator of neuronal communication, vascular tone, and immune regulation (Wu et al. 2021a). Adequate arginine–NO signaling is essential for maintaining neurovascular health and cognitive function, while its deficiency has been linked to oxidative stress, neuroinflammation, and impaired neuroplasticity. In murine models, dietary L‐arginine supplementation has demonstrated anti‐stress effects by reducing oxidative stress and improving emotional behavior (Pervin et al. 2021). Moreover, recent clinical evidence suggests that disrupted L‐arginine metabolism contributes to NO deficiency, impaired cerebral perfusion, and heightened neuroimmune burden in critically ill patients, emphasizing the relevance of restoring arginine homeostasis to support neurological recovery (Zinellu et al. 2024). Together, these findings imply that vitamin D3–induced microbial shifts promoting arginine biosynthesis may buffer stress‐induced neuroendocrine dysregulation and enhance behavioral resilience through NO‐mediated mechanisms, providing a plausible link to the partial recovery observed in the CSDVD group.

Additionally, the enrichment of glycolysis‐related pathways, such as Glycolysis I, in the CSDVD group may reflect enhanced microbial support for host energy metabolism under stress. Zhu et al. (2025) demonstrated that maternal vitamin D promotes glycolytic reprogramming of offspring CD4⁺ T cells, enhancing immune tolerance and reducing inflammation, with downstream effects on reducing autism spectrum disorder (ASD)‐like behaviors. This highlights the critical role of metabolic fitness in neuroimmune resilience. In our study, the observed increase in microbial Glycolysis I and II pathway abundance in the CSDVD group may reflect a similar mechanism: Vitamin D3–induced microbial shifts potentially enhance glycolytic capacity, thereby supporting central and peripheral immune recovery under chronic stress. Such metabolic reprogramming may improve energy availability and modulate inflammation, providing a plausible mechanistic link to the behavioral improvements observed in vitamin D3–treated sleep‐disrupted mice. Furthermore, the capacity of vitamin D3 supplementation to modulate microbial energy metabolism pathways may reinforce host mitochondrial function and ATP production, facilitating neuronal and immune recovery. Together, these data suggest a microbiota–immune–behavior axis whereby vitamin D3 shapes microbial metabolic output (e.g., glycolysis), which in turn supports host immune cells and neurobehavioral resilience under sleep–wake disruption.

In the current study, sleep desynchrony significantly reduced the expression of tight junction proteins ZO‐1, occludin, and claudin in the proximal colon, indicating impairment of intestinal barrier function. Vitamin D3 treatment significantly restored the expression levels of ZO‐1 and claudin, suggesting a protective role of vitamin D3 in maintaining intestinal barrier integrity. By contrast, sleep recovery alone did not have significant effects on the levels of these proteins. Studies have indicated that an increase in the abundance of Firmicutes could lead to gut barrier dysfunction. (Filippo et al. 2010; Schippa and Conte 2014). Therefore, researchers have hypothesized that vitamin D3 treatment can modulate the microbiota composition, resulting in higher and lower levels of Bacteroidetes and Firmicutes, respectively, which could help maintain the intestinal barrier integrity. In addition, in vitro and animal studies have indicated that vitamin D signaling enhances epithelial barrier integrity by regulating the expression of various junctional proteins, defensins, and mucins (Vernia et al. 2022). Finally, although no significant differences in LPS levels were identified among the NS, CSD, CSDR, and CSDVD groups in the current study, sleep desynchrony affected the production of SCFAs in all groups. The CSD group exhibited a significant reduction in propionic acid and butyric acid levels, and vitamin D3 treatment significantly restored these levels. One study reported that the abundance of Muribaculaceae was positively correlated with the concentration of propionic acid (Smith et al. 2019; Yang et al. 2023). In our study, Muribaculaceae abundance was significantly reduced in the CSD group but restored to normal levels following vitamin D3 administration, suggesting that vitamin D3 reversed sleep‐induced microbial loss and subsequently enhanced propionic and butanoic acid production.

Vitamin D3 may exert its protective effects through both direct and indirect mechanisms. Directly, activation of the vitamin D receptor (VDR) has been shown to upregulate tight junction proteins such as occludin and claudin, suppress NF‐κB signaling, and reduce intestinal inflammation, thereby enhancing epithelial barrier function independently of microbial changes (Cai et al. 2025). Indirectly, vitamin D3–induced shifts in microbiota composition may promote the expansion of beneficial SCFAs‐producing taxa, which are known to support gut barrier integrity and regulate systemic immune responses (Gao et al. 2023). Notably, SCFAs can also modulate the hypothalamic–pituitary–adrenal (HPA) axis by reducing CRH and cortisol levels, attenuating neuroinflammation, and further preserving epithelial integrity (Wang et al. 2023). In the present study, vitamin D3 supplementation partially restored SCFAs levels, with a trend toward increased propionic acid compared with the CSD group, supporting a potential role in both gut and neuroendocrine regulation. These proposed pathways merit further investigation using microbial depletion, fecal transplantation, or VDR knockout models to establish causal relationships.

The physiological effects of sleep desynchrony or sleep deprivation vary substantially with its duration. Short‐term deprivation typically induces reversible changes in stress responses and neuroplasticity, as shown by Murata et al. (2024) and Popescu et al. (2025), who reported that rebound sleep restored hippocampal function and normalized gene expression patterns. In contrast, CSD becomes a common workstyle among workers and students, leading to more severe and potentially irreversible impairments that often go unnoticed. Tang et al. (2024) and Zheng et al. (2025) found that prolonged sleep loss caused oxidative damage, visual deficits, and reproductive dysfunction, which were not readily reversed by short‐term recovery sleep. These findings suggest that while acute sleep loss may be recoverable, chronic deprivation can result in lasting structural and metabolic damage. Nonetheless, differences in experimental methods, sleep deprivation protocols, and recovery conditions complicate direct comparisons across studies, highlighting the need for standardized models to better characterize the impacts of sleep loss and recovery.

Despite the clear biological trends observed in behavior, immunological markers, and gut microbiota composition, this study is limited by its small sample size (n = 6 per group), which may reduce the statistical power to detect subtle differences, especially in highly variable datasets such as the microbiome. This limitation may have contributed to the lack of significant group differences observed in certain outcomes, such as plasma LPS levels and specific inflammatory markers. Another important limitation is the use of only male mice, which may not fully capture potential sex‐specific responses to vitamin D3, especially in mental health and HPA axis regulation. Prior studies suggest that vitamin D metabolism, stress responsivity, and behavioral outcomes can differ between sexes due to hormonal and receptor‐level variations (Bertone‐Johnson 2009). Although our sample size is consistent with standard exploratory animal studies, future research should include larger cohorts and both male and female animals to validate and extend these findings while enhancing translational relevance. Additionally, although acute and chronic sleep deprivation are often distinguished by duration, with acute deprivation typically referring to experimental protocols lasting less than 3 days and chronic deprivation involving sustained or repeated sleep restriction for 3 days or more, the boundary between short‐term and long‐term effects remains difficult to delineate (Alhola and Polo‐Kantola 2007). This ambiguity is partly attributable to methodological heterogeneity across studies, including variations in sleep disruption paradigms, recovery schedules, and behavioral outcome measures.

Nevertheless, this study provides an integrative perspective on the physiological consequences of chronic sleep desynchrony and highlights the potential of vitamin D3 supplementation as a partial corrective intervention. To our knowledge, this is the first animal study to simultaneously evaluate behavioral performance, metabolic status, stress responses, immune parameters, and gut microbial profiles in the context of chronic sleep desynchrony and vitamin D3 intervention. This multidimensional approach not only improves our understanding of the interplay between circadian health and systemic physiology but also supports the translational potential of vitamin D3 in mitigating the health impacts of shift work and related sleep disturbances.

The findings of the present study signify the adverse effects of chronic sleep desynchrony, which simulates the effects of long‐term shift work, on various physiological, behavioral, and immunological parameters, including its effects in disrupting gut microbiota composition and intestinal barrier integrity. Chronic sleep desynchrony led to imbalances in immune function and gut microbiota, and subsequent vitamin D3 treatment mitigated these effects. Vitamin D3 treatment effectively restored gut microbial balance, improved intestinal barrier function, and enhanced the levels of SCFAs such as propionic and butanoic acids. Additionally, vitamin D3 treatment helped to normalize HPA axis dysfunction and partially restore locomotor function impaired by sleep disruption. Overall, these findings highlight the therapeutic potential of vitamin D3 in restoring physiological homeostasis and protecting against the detrimental effects of sleep desynchrony.

Changwei W. Wu: conceptualization, project administration, resources, supervision, writing – review and editing. Yen‐Ju Huang: resources, supervision, writing – review and editing. Hsien‐Yu Fan: methodology, data curation, formal analysis. Jin‐Wei Xu: data curation, writing – review and editing. Jun‐Lan Zeng: investigation, methodology, validation, visualization. Yu‐Chen S. H. Yang: investigation, methodology, validation, visualization. Yu‐Tang Tung: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, writing – review and editing.

This work was financially supported by a research grant from the National Science and Technology Council (NSTC112‐2320‐B‐005‐009‐MY3), Ditmanson Medical Foundation Chia‐Yi Christian Hospital (R111‐40), and National Health Research Institutes (NHRI‐EX113‐11129EI).

All experimental procedures involving animals were thoroughly examined and approved by the IACUC of National Chung Hsing University, and the protocol guidelines 111‐004R were approved by the ethics committee of the IACUC. The current study was carried out in compliance with the ARRIVE guidelines.

The authors have nothing to report.

The authors declare no conflicts of interest.