• HIGHLIGHTS
- Combined treadmill exercise and vitamin D improved cognition, reduced neuroinflammation, preserved blood-brain barrier integrity, and enhanced mitochondrial efficiency in nonpathological mice.
- This combined strategy shows potential for improving brain health even in the absence of disease.
INTRODUCTION
The combined influence of lifestyle modifications and nutritional interventions has been associated with enhanced neuroprotection and improved brain health [1]. Among such approaches, the synergistic effects of physical exercise and vitamin D supplementation are particularly promising. Exercise may upregulate vitamin D receptor expression, thereby increasing tissue sensitivity to vitamin D [2]. The concurrent administration of exercise and vitamin D yields diverse physiological benefits, notably in the regulation of reactive oxygen species (ROS), modulation of inflammatory cytokines, and immune response control [3, 4]. Although ROS generated by exercise are often considered harmful, they also serve as signaling molecules that facilitate cellular adaptation when balanced by antioxidant systems, including those modulated by vitamin D [5, 6]. Moreover, both exercise and vitamin D supplementation can enhance mitochondrial efficiency, further aiding in the regulation of ROS production and promoting overall cellular health [5, 6]. Both interventions also increase the expression of anti-inflammatory cytokines, such as interleukin-10 (IL-10), which mitigate the detrimental effects of chronic inflammation and help modulate immune cell activation [4].
Recent studies have highlighted the effects of combined exercise and vitamin D supplementation on brain health, particularly in mitigating oxidative stress and regulating inflammation [4]. These effects play a crucial role in maintaining the integrity of the blood-brain barrier (BBB), an essential component for preserving cognitive and behavioral functions, which is further supported by both exercise and vitamin D supplementation [7, 8]. While substantial evidence from pathological models demonstrates these benefits, the impact of combined exercise and vitamin D treatment on brain function under normal (nonpathological) conditions remains unclear. Investigating these baseline effects may help clarify whether combined interventions simply restore impaired functions or actively enhance brain function even in the absence of disease.
Therefore, this study aimed to examine the effects of combined exercise and vitamin D supplementation in nonpathological mice, with a focus on modulating anti-inflammatory cytokine expression, maintaining BBB structural integrity, regulating oxidative stress, enhancing mitochondrial efficiency, and ultimately, evaluating cognitive function.
MATERIALS AND METHODS
Animals
Forty male C57BL/6 mice (4 months old) were housed under controlled conditions and randomly assigned to 4 groups: control (Con), exercise (EX), vitamin D (VD), and exercise + vitamin D (EXVD) (n = 10/group).
Fig. 1 presents the overall experimental design. Mice in the exercise groups performed treadmill running on a motorized rodent treadmill (Columbus Instruments, USA) 5 times per week for 20 weeks. Each exercise session lasted 30 minutes and included a 5-minute warm-up and cool-down at 8 m/min, followed by 20 minutes at 11 m/min, with monthly speed increases up to 15 m/min to prevent adaptation. This intensity range corresponds to approximately 70%–80% of maximal oxygen consumption, and this protocol has previously been shown to exert beneficial effects on brain function, including reduced neuroinflammation and improved BBB integrity [9, 10]. Vitamin D groups received intraperitoneal injections of ultra-high purity 1,25(OH)₂D₃ (Cayman Chemical Company, USA; Cat. 12079), dissolved in glycol (Sigma-Aldrich, Germany), at a dose of 5-μg/kg body weight, twice per week for 20 weeks [11]. Mice were then anesthetized and euthanized for blood and brain collection.
Western Blotting
Hippocampal proteins were extracted and quantified using the Bradford assay (Bio-Rad, Hercules, USA), then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad), blocked, and incubated overnight with primary antibodies (Table 1). The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-rabbit immunoglobulin G (IgG) (H+L)-HRP (1:1,000; Bio-Rad; Cat. 1706515), anti-mouse IgG (H+L)-HRP (1:1,000; Bio-Rad; Cat. 1706515), and anti-mouse IgG (H+L)-HRP (1:1,000; Bio-Rad; Cat. 1706516). The blots were developed using a chemiluminescent HRP substrate kit (Millipore, USA). Band intensities were quantified using ImageJ software (ver. 1.42; National Institutes of Health) and normalized to β-actin.
Immunofluorescence
Brain sections were blocked with BlockAid (Invitrogen, USA; Cat. B10710) and incubated overnight at 4°C with the following primary antibodies: anti-interleukin (IL)-10 (Thermo Fisher Scientific, USA; Cat. PA5-85660), anti-GLAST (Miltenyi Biotec, Germany; Cat. 130-118-483), anti-C3 (Novus Biologicals, USA; Cat. NBP1-32080), and anti-S100A10 (Thermo Fisher Scientific; Cat. PA5-95505). After phosphate-buffered saline washing, sections were incubated for 1 hour with secondary antibodies: anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific; Cat. A11034) and anti-rabbit IgG Alexa Fluor 594 (Thermo Fisher Scientific; Cat. A11001). Nuclear staining was performed using DAPI (Santa Cruz Biotechnology, USA; Cat. 62248). Slides were mounted with DAKO Fluorescence Mounting Medium and imaged using a Zeiss LSM 880 confocal microscope (Carl-Zeiss-Promenade 10, Germany).
High-Resolution Respirometry (Oroboros O2k)
Mitochondrial function in hippocampal tissues was evaluated using the Oroboros Oxygraph O2k high-resolution respirometer (Oroboros Instruments, Austria). Isolated hippocampal tissues were incubated in 2 mL of mitochondrial respiration medium (MiR05) adjusted to pH 7.0. Oxygen concentration in each chamber was stabilized, and mitochondrial respiration was assessed by sequential substrate addition using the Oroboros Oxygraph O2k. Respiration was initiated with 5mM pyruvate and 0.5mM malate to stimulate complex I-linked activity, followed by the addition of 2mM adenosine diphosphate (ADP) to evaluate oxidative phosphorylation capacity. Finally, 9mM succinate was added to assess complex II-linked respiration. Respiratory parameters, including basal respiration, adenosine triphosphate (ATP)-linked respiration, and maximal respiratory capacity, were recorded and normalized to tissue weight.
Flow Cytometry Analysis
Hippocampal tissues were digested in 10 mL of trypsin at 37°C for 15 minutes and dissociated into single cells. After stopping the reaction with complete Rosewell Park Memorial Institute medium, the suspension was filtered, centrifuged, and layered on a Percoll gradient. Astrocytes were isolated from the 70%/37% interface, washed, and labeled with GLAST antibodies. Labeled cells were analyzed on a FACSCanto II flow cytometer (BD Biosciences, USA), and data were processed using FlowJo version 7.6.5 (BD Biosciences).
Enzyme-Linked Immunosorbent Assay
Plasma cytokine concentrations were quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (Thermo Fisher Scientific). The following mouse-specific ELISA kits were used: tumor necrosis factor (TNF)-α (Cat. BMS607-3), IL-6 (Cat. KMC0061), IL-12 (Cat. BMS616), IL-10 (Cat. BMS614), IL-4 (Cat. BMS613), and transforming growth factor (TGF)-β1 (Cat. BMS608-4). Blood samples were collected via cardiac puncture prior to perfusion and centrifuged at 3,000 rpm for 15 minutes at 4°C to isolate plasma. Supernatants were stored at -80°C. Samples were thawed, diluted, and analyzed at 450 nm using a microplate reader, with cytokine concentrations calculated from standard curves.
Morris Water Maze Test
Spatial learning and memory were assessed using a modified Morris water maze (MWM). Mice were tested in a 120-cm diameter circular pool with water maintained at 22°C–23°C. An escape platform was fixed in the southeast quadrant, and visual cues were placed in all quadrants. Swimming behavior was recorded and analyzed using EthoVision XT software (Noldus Information Technology Inc., USA). Mice underwent 3 daily trials for 4 days, with 30-minute intervals between trials. Each trial lasted up to 60 seconds. Mice that failed to find the platform were guided to it and allowed to remain for 10 seconds. On day 5, a probe trial was conducted without the platform.
Statistical Analysis
Data are expressed as means ± standard error of the mean. Two-way analysis of variance (ANOVA) with the Greenhouse-Geisser correction (if sphericity was violated) was used to assess group ×time effects on escape latency in the MWM test. All other statistical analyses involving 1-way ANOVA followed by the least significant difference post hoc test to evaluate group differences. Significance was set at P=0.05. Analyses were conducted using IBM SPSS Statistics ver. 23.0 (IBM Co., USA).
RESULTS
Combining Treadmill Exercise With Vitamin D Injections Improved Cognitive Function in Nonpathological Mice
Fig. 2A illustrates spatial learning and memory performance as assessed by the MWM test. Two-way repeated-measures ANOVA revealed no significant interaction between group and time for escape latency, F(6.28, 75.38) =0.913, P =0.445, with the Greenhouse-Geisser correction applied (ε =0.698). However, the EXVD group demonstrated significantly greater learning acquisition compared to the Con (P =0.005), EX (P = 0.006), and VD (P = 0.007) groups.
Additionally, as shown in Fig. 2B, EXVD mice exhibited a significantly higher number of platform crossings than Con mice (P =0.02). There were no significant differences among groups in the time spent in the target quadrant.
Treadmill Exercise and Vitamin D Injections Influenced Levels of Anti-Inflammatory Cytokines in the Blood in Nonpathological Mice
As shown in Fig. 3, no significant differences were observed in proinflammatory cytokine levels among the groups. However, for anti-inflammatory cytokines, VD and EXVD mice showed significantly higher IL-10 levels than both Con mice (P<0.001 for both) and EX mice (P =0.001 and P <0.001, respectively). For TGF-β, EX and EXVD mice exhibited significantly lower levels than Con mice (P = 0.035 and P = 0.026, respectively). IL-4 levels did not differ significantly between any of the groups. These findings indicate that 20 weeks of treadmill running and vitamin D injections independently modulate plasma levels of anti-inflammatory cytokines, without affecting proinflammatory cytokines.
Treadmill Exercise and Vitamin D Injections Modulated the Hippocampal Levels of Inflammatory Cytokines in Nonpathological Mice
Fig. 4A presents representative protein expression levels of pro- and anti-inflammatory cytokines in the hippocampus. EX mice displayed significantly higher TNF-α levels compared to Con (P <0.001), VD (P <0.001), and EXVD mice (P =0.009). IL-6 levels were significantly lower in both VD and EXVD mice compared to Con (P < 0.001 for both) and EX mice (P = 0.001 for both). TGF-β and IL-10 levels did not show significant differences among the groups.
As shown in Fig. 4B, immunofluorescence analysis revealed that EX and VD mice had significantly smaller IL-10-positive areas than Con mice in the CA1 region (P = 0.042 and P = 0.030, respectively). In the CA3 region, IL-10-positive areas were significantly reduced in EX, VD, and EXVD mice compared to Con mice (P <0.001, P <0.001, and P =0.003, respectively). These findings suggest that in nonpathological mice, treadmill exercise alone may promote neuroinflammation, whereas vitamin D exerts anti-inflammatory effects in the hippocampus.
Treadmill Exercise and Vitamin D Injections Increased Inactive Astrocytes and Upregulated the Expression of Tight Junction Proteins in Nonpathological Mice
Fig. 5A shows flow cytometry plots depicting the relative number and percentage of astrocytes (GLAST+ cells) in the hippocampus. Con mice had significantly fewer astrocytes than VD (P=0.04) and EXVD mice (P=0.001), while EX mice also had fewer astrocytes than EXVD mice (P=0.009). For percentages, VD and EXVD mice exhibited significantly higher proportions of GLAST+ cells than Con (P=0.006 and P=0.005, respectively) and EX mice (P = 0.005 and P = 0.001, respectively).
Fig. 5B presents immunofluorescence images of the CA1 and CA3 hippocampal regions. In the CA1 region, Con mice showed a significantly larger C3-positive area than EXVD mice (P = 0.049). In the CA3 region, VD and EXVD mice displayed significantly reduced C3 staining areas compared to both Con (P =0.001 for both) and EX mice (P =0.028 and P =0.011, respectively). For S100A10 staining, EXVD mice showed significantly lower expression in both CA1 and CA3 regions than Con (P<0.001 for both), EX (P=0.004 and P=0.049), and VD mice (P =0.007 and P =0.028). Both EX (P =0.001 and P = 0.002) and VD mice (P=0.003 and P=0.004) also showed significantly lower S100A10 levels than Con mice in both regions.
Regarding hippocampal tight junction proteins, as shown in Fig. 5C, VD and EXVD mice exhibited significantly higher zonula occludens-1 (ZO-1) expression than Con (P = 0.005 and P =0.004, respectively). Occludin expression was significantly increased only in VD mice relative to Con (P=0.005), EX (P=0.001), and EXVD mice (P=0.002). For claudin, both VD and EXVD mice had significantly higher expression than Con and EX mice (P < 0.001 for all). These results suggest that vitamin D plays a dominant role in enhancing astrocyte support, reducing inflammation, and improving BBB integrity, while exercise contributes additional selective benefits, especially in combination with vitamin D.
Vitamin D Injections Decreased Antioxidant Levels, and Combining Treadmill Exercise With Vitamin D Injections Improved Mitochondrial Energy Production Efficiency
Fig. 6A displays representative expression levels for mitochondrial oxidative stress and antioxidant proteins. No significant differences were observed among groups for 4HNE, NRF2, or KEAP1. However, VD mice had significantly lower levels of the antioxidant enzymes SOD2, catalase, and GPX1 than Con (P < 0.001 for all) and EX mice (P=0.003, P<0.001, and P<0.001, respectively). Similarly, EXVD mice exhibited significantly lower expression of these antioxidants than Con (P =0.002, P <0.001, P < 0.001) and EX mice (P = 0.040, P < 0.001, P < 0.001).
Fig. 6B shows O2 consumption levels during mitochondrial energy production after substrate addition. Following pyruvate/malate (PM) and ADP addition, VD and EXVD mice demonstrated significantly lower O₂ consumption than Con mice (P = 0.020 and P=0.038 for PM; P=0.016 and P=0.031 for ADP, respectively). After succinate addition, only EXVD mice showed significantly lower O₂ consumption than Con mice (P =0.041). These findings indicate that vitamin D supplementation, with or without exercise, enhances antioxidant defense in the hippocampus without increasing oxidative stress, and also reduces mitochondrial oxygen consumption.
DISCUSSION
The current study investigated the combined effects of treadmill exercise and vitamin D injection on brain function in wild-type mice. Our findings demonstrate that the combined intervention led to significant improvements in cognitive function, evidenced by reduced escape latency and increased platform crossings in the MWM. The combined treatment elevated plasma levels of anti-inflammatory cytokines IL-10 and TGF-β, and reduced hippocampal proinflammatory cytokines TNF-α and IL-6, indicating anti-inflammatory effects. In the hippocampus, the intervention increased the proportion of GLAST+ astrocytes while decreasing both proinflammatory C3+ astrocytes and anti-inflammatory S100A10+ astrocytes, along with upregulation of tight junction proteins ZO-1 and claudin. Furthermore, mitochondrial oxygen consumption was reduced in EXVD mice, suggesting improved mitochondrial energy efficiency.
We found that combining exercise and vitamin D improved spatial learning and memory compared to either intervention alone, as evidenced by the MWM test. This result is consistent with prior studies demonstrating synergistic effects of exercise and vitamin D on cognitive function. For instance, Medhat et al. [4] reported that while vitamin D or exercise alone improved cognitive deficits, their combination led to greater improvements in cognitive function and neuropathology in an Alzheimer rat model. Similarly, Lipowski et al. [12] found that Nordic walking combined with vitamin D supplementation improved attention, executive function, and mental well-being in older women. These observations suggest a synergistic interaction, likely mediated by modulation of inflammation, support of BBB integrity, and improved mitochondrial efficiency, as also indicated by our results.
Peripheral inflammatory profiling revealed that proinflammatory cytokine levels remained stable, whereas vitamin D significantly increased IL-10 and exercise reduced circulating TGF-β. This indicates a shift toward an anti-inflammatory state without triggering compensatory proinflammatory responses, consistent with previous reports [13-15]. The suppression of TGF-β by exercise could represent a regulatory adaptation, especially given TGF-β’s dual roles and its association with adipose inflammation [16-18]. Alessi et al. [17] observed elevated TGF-β1 levels in both visceral and subcutaneous adipose tissues of severely obese individuals, with a strong positive correlation to body mass index. Widiastuti et al. [18] found that recreational cyclists had lower circulating TGF-β1 levels than sedentary individuals.
Exercise increased hippocampal TNF-α, while vitamin D reduced IL-6, suggesting that vitamin D may buffer potential neuroinflammatory responses induced by exercise. The increase in TNF-α after treadmill exercise may not necessarily reflect pathological inflammation but could indicate an acute, adaptive immune response consistent with the concept of exercise-induced hormesis, where transient proinflammatory signals yield long-term neuroprotective adaptations [19]. Ding et al. [20] reported that exercise-induced TNF-α may protect the brain by reducing inflammatory damage and preventing excessive inflammatory responses by limiting inflammatory mediator expression. These findings underscore the importance of distinguishing between transient, beneficial immune activation and chronic neuroinflammation. Furthermore, they highlight the necessity of evaluating both systemic and central immune responses, since peripheral cytokine levels may not accurately reflect neuroinflammation within the brain, potentially due to BBB integrity and glial cell activation [21].
Astrocyte analysis further supports a neuroprotective role for the combined treatment. Increased GLAST+ and reduced C3+ astrocyte populations in the VD and EXVD groups indicate a shift toward a more homeostatic state. Although exercise alone did not significantly alter A1 astrocyte markers, vitamin D appeared to suppress astrocyte activation when combined with exercise, reducing both A1 and A2 astrocyte phenotypes. This is consistent with previous findings showing that vitamin D attenuates reactive astrocytosis and inflammatory signaling in the CNS [22, 23]. Belaya et al. [22] reported that long-term voluntary exercise increased GFAP reactivity and the number of GFAP-positive astrocytes in the hippocampi of 5xFAD mice. Muraro et al. [23] demonstrated that combined vitamin D and aerobic exercise restored neuronal and glial structure in the hypothalamic nuclei of monosodium glutamate-treated rats.
The upregulated expression of tight junction proteins in the VD and EXVD groups indicates strengthened BBB integrity. Vitamin D’s role in maintaining tight junction structure, potentially via vitamin D receptor signaling and suppression of nuclear factor-kappa B, has been demonstrated in injury and disease models [8, 24]. In contrast, exercise alone did not affect BBB markers under normal conditions, consistent with previous findings that exercise-related BBB modulation is more pronounced in pathological states [25, 26].
Despite no changes in oxidative stress markers, antioxidant enzyme levels were reduced in vitamin D-treated groups, possibly reflecting decreased ROS production or improved redox balance due to enhanced mitochondrial efficiency. Mitochondrial assays showed reduced oxygen consumption in response to substrate stimulation in VD and EXVD groups, suggesting improved bioenergetic function without increased oxidative stress. Enhanced mitochondrial efficiency enables sufficient energy output [27, 28]. Mitochondrial oxygen consumption is closely linked to ATP synthesis, which is a process that generates ROS, but is also modulated by uncoupling proteins (UCPs), which reduce ROS production by facilitating proton leakage across the inner mitochondrial membrane [29]. UCPs thereby play a protective role by dissipating excess protons and preventing mitochondrial oxidative damage [30].
A discrepancy between Western blot and immunohistochemical findings for IL-10 expression highlights the limitations of each method. While total IL-10 levels were unchanged, region-specific reductions in IL-10-positive areas in the CA1 and CA3 subfields were detected via immunohistochemistry. These findings, together with reductions in A2 astrocytes in these regions, suggest spatially localized immune modulation that may be masked in homogenized samples [31, 32].
This study has several limitations. First, we focused exclusively on protein-level changes, without examining transcriptomic alterations [33]. Second, although mitochondrial changes were observed, further validation using more sensitive bioenergetic assays and markers such as PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is necessary [34, 35]. Finally, the mechanisms underlying the combined effects of exercise and vitamin D on neuroinflammation and mitochondrial function remain unclear and warrant further investigation.
In conclusion, exercise training and vitamin D supplementation exert complementary and synergistic effects on brain health in nonpathological mice. This intervention enhances cognitive function, supports BBB integrity, modulates systemic and central inflammation, and improves mitochondrial efficiency without increasing oxidative stress. These results underscore the clinical relevance of adequate exercise and vitamin D supplementation in promoting neuroprotection; however, additional research is needed to determine their applicability in humans.
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