Immunologic, Anti-Inflammatory, and Anti-Muscle Damage Profile of Supplemented Vitamin D3 in Healthy Adults on Strenuous Endurance Exercise

tedly, strenuous endurance exercise can depress the immune system and induce in ammation and muscle damage. Therefore, this double-blinded, matched-pair study aimed to investigate the impact of vitamin D 3supplementation on immune response (leukocyte, neutrophil, lymphocyte, CD4 + , CD8 + , CD19 + , and CD56 + counts), in ammatory pro le (TNF- and IL-6), muscle damage (CK and LDH levels), as well as aerobic capacity after strenuous endurance exercise in 18 healthy men taking 5000 IU of vitamin D 3(n= 9) or placebo (n= 9) daily for 4 weeks. Total and differential blood leukocyte counts, levels of cytokines, and muscle damage biomarkers were determined before, immediately after, and 2, 4, and 24 h after exercise. The IL-6, CK, and LDH levels were signi cantly lower in vitamin D 3group at 2, 4, and 24 h post exercise (p< 0.05). Maximal and average heart rates during exercise were also signi cantly lower (p< 0.05). In the vitamin D 3 group, the CD4 + /CD8 + ratio after 4 weeks of supplementation was

24 h after exercise. The IL-6, CK, and LDH levels were signi cantly lower in vitamin D 3group at 2, 4, and 24 h post exercise (p< 0.05). Maximal and average heart rates during exercise were also signi cantly lower (p< 0.05). In the vitamin D 3 group, the CD4 + /CD8 + ratio after 4 weeks of supplementation was only signi cantly lower at post-0 than at baseline and signi cantly higher at post-2 than at baseline and post-0 (allp< 0.05). Taken together, 5000 IU of daily vitamin D 3supplementation for 4 weeks exhibited positive effects in terms of increased blood 25(OH)D levels, CD4 + /CD8 + ratio (immune response), and aerobic capacity while inhibiting in ammatory cytokines and CK and LDH (muscle damage) in people performing strenuous endurance exercise. Biology2023,12, 657.

Biology2023,12, 657 2 of 18 Keywords: strenuous endurance exercise; CD4 + /CD8 + ratio; in ammation; creatine kinase; heart rate; Interleukin-6; lactate dehydrogenase 1. Introduction An increasing body of evidence implies that despite various bene cial attributes, cer- tain types of exercise may impose signi cant physiological stresses. In particular, repeated bouts of strenuous endurance exercise have been associated with suppressed immune function, in ammation, and muscle damage [1,2]. The immune system prevents infection and is involved in the maintenance of tissue repair, metabolism, sleep, fatigue, and mental health systems [3]. Notably, studies have demonstrated muscle damage events initiated even within 24 h through in ammatory events. Speci cally, in the early hours of the recovery period, neutrophils dominate the in ammatory cell pro le, acting to clear cellular debris and propagating cytokine-secreted in ammatory response [2]. Further, mast cells also in ltrate muscle tissue, releasing histamine and chemo-attractants. Within4 to 24 h after muscle damage, proin ammatory macrophages in ltrate the muscle and release proin ammatory cytokines. These macrophages also remove damaged tissue through phagocytosis and stimulate the proliferation of myoblasts. After 24 h, proin ammatory macrophages are replaced by anti-in ammatory macrophages and CD8 + and T-regulatory lymphocytes. These cells secrete anti-in ammatory cytokines, recruit macrophages, and stimulate myoblast proliferation and expansion of the satellite cell pool. Studies have investigated the incidence of upper respiratory tract infection (URTI) and cellular immunity after high-intensity exercise [4,5]. Lymphocytes are cellular immunity components that are divided into helper T (CD4 + ), cytotoxic T (CD8 + ), B (CD19 + ), and natu- ral killer (NK; CD56 + ) cells. Notably, strenuous exercise could reduce the CD4 + /CD8 + ratio and therefore may represent a relevant marker of immunological change [6–9].A decrease in CD4 + , CD8 + , CD19 + , and CD56 + lymphocytes during the recovery period (1–2 h) post high-intensity exercise indicates the immunosuppressive effect, which is also known as an open-windowimpact [3,10,11]. In ammation is a primary immune response involved in the prevention of infection, damage, and noxious conditions for the maintenance of tissue repair

[6–9].A decrease in CD4 + , CD8 + , CD19 + , and CD56 + lymphocytes during the recovery period (1–2 h) post high-intensity exercise indicates the immunosuppressive effect, which is also known as an open-windowimpact [3,10,11]. In ammation is a primary immune response involved in the prevention of infection, damage, and noxious conditions for the maintenance of tissue repair and homeostasis [12,13]. Reportedly, in ammation-related markers such as tumor necrosis factor- (TNF- ) and interleukin (IL)-6 have been found to have considerably increased post strenuous exercise [14–16]. Remarkably, strenuous exercise generates free radicals, reactive oxygen (ROS), and nitrogen species in tissues and cells [17]. Further, high-intensity exercise could destroy tissues, damage cells, and inhibit muscle growth along with an increase in creatine kinase (CK) and lactate dehydrogenase (LDH) levels [18]. Vitamin D, an essential fat-soluble vitamin, occurs in two main forms: ergocalciferol (D2), obtained from vegetables or supplements, and cholecalciferol (D3) [19,20]. Vitamin D3synthesis is triggered in the skin after ultraviolet B exposure. Additionally, it could be acquired from oily sh, forti ed food, and oral supplements, and according to an Inter- national Olympic Committee statement in 2018, vitamin D3supplements among athletes could promote immune health and assist with training, recovery, muscle soreness, and injury management [21]. However, vitamin D de ciency is common among the general public as well as in athletes worldwide [22–26]. Vitamin D de ciency leads to mitochon- drial dysfunction, decreased adenosine triphosphate (ATP) production, increased ROS production, oxidative damage, muscle atrophy, and impaired muscle function [3,4]. Re- markably, activated (hydroxylated) vitamin D [1,25(OH)D] stimulates an abundance of vitamin D receptors (VDR) in satellite cells and central myonuclei during muscle regenera- tion. Moreover, vitamin D inhibits B-cell proliferation, differentiation, and immunoglobulin secretion [27]. In addition, T-cell proliferation is also inhibited, indicating a resultant shift from a Th1 to a Th2 phenotype [27,28]. Notably, the inhibition of Th17 cell development and facilitation of T regulatory cells increases the production of anti-in ammatory cy- tokines such as IL-10 while decreasing the production of in ammatory cytokines (IL-17 and

T-cell proliferation is also inhibited, indicating a resultant shift from a Th1 to a Th2 phenotype [27,28]. Notably, the inhibition of Th17 cell development and facilitation of T regulatory cells increases the production of anti-in ammatory cy- tokines such as IL-10 while decreasing the production of in ammatory cytokines (IL-17 and

Biology2023,12, 657 3 of 18 IL-21) [29–32]. Vitamin D also inhibits the monocyte production of in ammatory cytokines such as TNF- , IL-1, IL-6, IL-8, and IL-12 [33]. In one study, a 14-week vitamin D3supplementation (at 5000 IU/day) in athletes led to an increase in blood 25(OH)D levels and ameliorated immune function [34]. Sim- ilarly, vitamin-D-de cient taekwondo athletes on 4-week vitamin D3supplementation (at 5000 IU/day) demonstrated improved immune health through signi cantly elevated 25(OH)D levels and a decrease in URTI risk [35]. Notably, several studies have revealed mixed results on the effects of vitamin D supplementation on in ammatory cytokines and muscle damage [36–39]. Speci cally, some studies indicated a reduction in the in- ammatory response and muscle damage [36,38], whereas others reported the absence of signi cant differences [37,39]. Thus, the above literature suggests that vitamin D3may regulate in ammation, immunological responses, and muscle damage. However, the conclusion on the impacts of vitamin D on the maintenance and restoration of muscle damage/strength and in ammation remains obscure. Therefore, we hypothesize that the outcomes of our study will help in reaching a clear consensus on the impact of vitamin D3supplementation on immune response, in ammatory cytokine production, and muscle damage after strenuous endurance exercise. We determined the levels of CD4 + /CD8 + , CD19 + , CD56 + , leukocytes, neutrophils, and lymphocytes for assessing immune response. The in ammatory response was estimated through TNF- and IL-6, while muscle dam- age creatine kinase (CK) and lactate dehydrogenase (LDH) represented muscle damage. Additionally, we measured blood 25(OH)D, maximal heart rate (HRmax), average heart rate (AHR), and maximal oxygen consumption (VO2max) to assess the extent of vitamin D3's impact on aerobic capacity. We anticipate that the nding of this study will contribute to determining the role of vitamin D3supplementation to improve the quality of life and performance during strenuous endurance exercise. 2. Materials and Methods 2.1. Participants Using G*Power software (v.3.1.9.2) and a 2 2 mixed-design analysis of variance (ANOVA) with an effect size of 0.4, an alpha error of 0.05, and a power of 0.80, the total number of

will contribute to determining the role of vitamin D3supplementation to improve the quality of life and performance during strenuous endurance exercise. 2. Materials and Methods 2.1. Participants Using G*Power software (v.3.1.9.2) and a 2 2 mixed-design analysis of variance (ANOVA) with an effect size of 0.4, an alpha error of 0.05, and a power of 0.80, the total number of subjects was calculated to be 16; however, 18 subjects were nally recruited above the cut-off value. Individuals with diabetes or cardiovascular, liver, renal, or au- toimmune diseases; blood 25(OH)D levels > 30 ng/mL; or maximal oxygen consumption VO2max< 40 mL/kg/min were excluded. None of the included participants regularly consumed supplements during the experimental period. Participants were requested to maintain a regular lifestyle and avoid the consumption of alcohol and other nutritional supplements. All participants were asked to complete the study between March and May 2021 to minimize variability due to UV exposure. The participant characteristics are listed in Table. Table 1.Participant characteristics. Variable Vitamin D 3Group Placebo Group p-Value Age (years) 21.9 1.4 22.1 2.0 0.784 Body height (cm) 173.1 6.3 173.3 6.8 0.944 Body weight (kg) 64.2 9.1 69.1 8.2 0.248 Data are presented as the mean standard deviation (SD);n= 9 in each group. 2.2. Experimental Design and Procedure Figure performed a graded exercise test (GXT) until exhaustion on a cycle ergometer to deter- mine VO2max1 day before supplementation. A double-blinded, matched-pair design was adopted to divide all 18 participants into the vitamin D3(n= 9) and placebo (n= 9) groups based on their VO2maxlevel. The VO2maxwas measured post 4 weeks of supplementation. A strenuous endurance exercise test (SEET) was conducted 2 days after the GXT, and blood

Biology2023,12, 657 4 of 18 samples were collected before (baseline), immediately after (post-0), and 2 h (post-2), 4 h (post-4), and 24 h after (post-24) exercise. Immune response biomarkers (i.e., leukocyte, neutrophil, and lymphocytes (CD4 + , CD8 + , CD19 + , and CD56 + ) counts and in ammatory cytokines (TNF- and IL-6 levels)) were analyzed at pre, post-0, post-2, post-4, and post-24, while muscle damage biomarkers (i.e., CK and LDH levels) were analyzed at pre, post-0, post-4, and post-24. The time to exhaustion, maximal heart rate (HRmax), and average heart rate (AHR) were also recorded during the exercise test.Biology 2023, 12, 657 4 of 19 2.2. Experimental Design and Procedure Figure 1 illustrates the schematic representation of the present study. All participants performed a graded exercise test (GXT) until exhaustion on a cycle ergometer to deter- mine VO2max 1 day before supplementation. A double-blinded, matched-pair design was adopted to divide all 18 participants into the vitamin D3 (n = 9) and placebo (n = 9) groups based on their VO2max level. The VO2max was measured post 4 weeks of supplementation. A strenuous endurance exercise test (SEET) was conducted 2 days after the GXT, and blood samples were collected before (baseline), immediately after (post-0), and 2 h (post- 2), 4 h (post-4), and 24 h after (post-24) exercise. Immune response biomarkers (i.e., leuko- cyte, neutrophil, and lymphocytes (CD4 + , CD8 + , CD19 + , and CD56 + ) counts and inflamma- tory cytokines (TNF-α and IL-6 levels)) were analyzed at pre, post-0, post-2, post-4, and post-24, while muscle damage biomarkers (i.e., CK and LDH levels) were analyzed at pre, post-0, post-4, and post-24. The time to exhaustion, maximal heart rate (HRmax), and aver- age heart rate (AHR) were also recorded during the exercise test. Figure 1. Experimental scheme. GXT, graded exercise test; SEET, strenuous endurance exercise test. 2.3. Vitamin D3 Supplementation The supplementation protocol and dosage were applied according to the method ex- plained in another study [40]. During the experiment period, individuals in the supple- mentation group were asked to consume 5000 IU of vitamin

(AHR) were also recorded during the exercise test. Figure 1. Experimental scheme. GXT, graded exercise test; SEET, strenuous endurance exercise test. 2.3. Vitamin D3 Supplementation The supplementation protocol and dosage were applied according to the method ex- plained in another study [40]. During the experiment period, individuals in the supple- mentation group were asked to consume 5000 IU of vitamin D3 in the oil form (Liquid Shield Vitamin D3+E; Panion & BF Biotech, Taipei, Taiwan) after lunch for 4 weeks. The placebo group received medium-chain triglycerides (Panion & BF Biotech). The supple- ments provided to both groups had identical taste and color. 2.4. GXT Protocol The current GXT protocol, based on another study [41], was used to determine the intensity of the exercise test (65% VO2max) on a cycling ergometer (Monark LC6; Monark Exercise AB, Sweden). After a warm-up for 5 min at 50 W (velocity = 70 ± 5 rpm), the workload was increased by 25 W every 2 min. Strong verbal encouragement was provided to the participants to maintain 70 rpm speed. The test was terminated when the partici- pants were not able to maintain at least 60 rpm. Exchange volume, VO2, and VCO2 were measured using gas analysis (MetaMax 3B; Cortex, Leipzig, Germany). In addition, heart rate (S610; Polar, Kempele, Finland) was also measured simultaneously. Each partici- pant’s rating of perceived exertion (RPE) was also recorded during every stage on a stand- ard Borg scale [42]. Individual VO2max was achieved when at least two of the following criteria were met: (1) heart rate ≤ 15 beats/min of individual predicted HRmax, (2) RPE > 18, and (3) respiratory exchange ratio > 1.1 [43]. 2.5. Strenuous Endurance Exercise Test (SEET) SEET was conducted 2 days after the participants completed the GXT. The partici- pants rested for 30 min before the test and wore a Polar 610 to monitor their heart rates. Participants exercised at 65% VO2max for 2 h after a 5 min warm-up to a heart rate of 150 beats/min. Based on the participants’ heart rate and VO2max, the workload of the cycle er- gometer was adjusted to

the GXT. The partici- pants rested for 30 min before the test and wore a Polar 610 to monitor their heart rates. Participants exercised at 65% VO2max for 2 h after a 5 min warm-up to a heart rate of 150 beats/min. Based on the participants’ heart rate and VO2max, the workload of the cycle er- gometer was adjusted to maintain the demand intensity of effort. After a 2 h cycling exer- cise at 70 rpm, we verbally encouraged the participants to increase their exercise speed to Figure 1. Experimental scheme. GXT, graded exercise test; SEET, strenuous endurance exercise test. 2.3. Vitamin D3Supplementation The supplementation protocol and dosage were applied according to the method explained in another study [40]. During the experiment period, individuals in the supple- mentation group were asked to consume 5000 IU of vitamin D3in the oil form (Liquid Shield Vitamin D3+E; Panion & BF Biotech, Taipei, Taiwan) after lunch for 4 weeks. The placebo group received medium-chain triglycerides (Panion & BF Biotech). The supple- ments provided to both groups had identical taste and color. 2.4. GXT Protocol The current GXT protocol, based on another study [41], was used to determine the intensity of the exercise test (65% VO2max) on a cycling ergometer (Monark LC6; Monark Exercise AB, Sweden). After a warm-up for 5 min at 50 W (velocity = 70 5 rpm), the workload was increased by 25 W every 2 min. Strong verbal encouragement was provided to the participants to maintain 70 rpm speed. The test was terminated when the participants were not able to maintain at least 60 rpm. Exchange volume, VO2, and VCO2were measured using gas analysis (MetaMax 3B; Cortex, Leipzig, Germany). In addition, heart rate (S610; Polar, Kempele, Finland) was also measured simultaneously. Each participant's rating of perceived exertion (RPE) was also recorded during every stage on a standard Borg scale [42]. Individual VO2maxwas achieved when at least two of the following criteria were met: (1) heart rate 15 beats/min of individual predicted HRmax, (2) RPE > 18, and (3) respiratory exchange ratio > 1.1 [43]. 2.5. Strenuous Endurance Exercise Test (SEET)

simultaneously. Each participant's rating of perceived exertion (RPE) was also recorded during every stage on a standard Borg scale [42]. Individual VO2maxwas achieved when at least two of the following criteria were met: (1) heart rate 15 beats/min of individual predicted HRmax, (2) RPE > 18, and (3) respiratory exchange ratio > 1.1 [43]. 2.5. Strenuous Endurance Exercise Test (SEET) SEET was conducted 2 days after the participants completed the GXT. The partici- pants rested for 30 min before the test and wore a Polar 610 to monitor their heart rates. Participants exercised at 65% VO2maxfor 2 h after a 5 min warm-up to a heart rate of 150 beats/min. Based on the participants' heart rate and VO2max, the workload of the cycle ergometer was adjusted to maintain the demand intensity of effort. After a 2 h cycling exercise at 70 rpm, we verbally encouraged the participants to increase their exercise speed to 100 rpm until exhaustion at the present workload (de ned by the failure to maintain a speedof 90 rpm). Their oxygen consumption, AHR, and HRmaxwere recorded. During the test, the participants were provided with 300 mL of water every 15 min if needed. The criteria of volitional exhaustion were identical to the evaluation criteria for VO2max.

Effects of vitamin D3 on immune response and muscle damage in endurance exercise.