The Effect of 13 Weeks Long-Distance Bicycle Riding on Inflammatory Response Indicators Related to Joint Cartilage and Muscle Damage

his study was to investigate the effects of 13 weeks of long-distance cycling on biomark- ers of joint cartilage, muscle damage and inflammation. All subjects in this study were seven participants of the “One Korea New-Eurasia Peace Cycle Expedition”, in which they rode cycles from Berlin, Germany to Seoul, Korea for thirteen weeks. The total course of the expedition was divided into three sub-courses: course 1 (from Berlin to Moscow), course 2 (from Moscow to Ulaan- baatar) and course 3 (from Ulaanbaatar to Seoul). All the selected participants rode 87.4 km/day (course 1), 70.4 km/day (course 2) and 57.6 km/day (course 3) on average, respectively. We collected their blood samples before the expedition in Seoul (S1), after course 1 in Moscow (M), after course 2 in Ulaanbaatar (U) and after the expedition in Seoul (S2), to analyze biomarkers of joint cartilage damage (Cartilage Oligomeric Matrix Protein; COMP), muscle damage (Creatine Phosphokinase; CPK, Lactate Dehydrogenase; LDH, Myoglobin), inflammation (Interleukin-6; IL-6, Interleukin-1 ; IL-1 , Tumor Necrosis Factor- ; TNF- , C-Reactive Protein; CRP) and stress hormone (Cortisol). According to this result,

Moscow (M), after course 2 in Ulaanbaatar (U) and after the expedition in Seoul (S2), to analyze biomarkers of joint cartilage damage (Cartilage Oligomeric Matrix Protein; COMP), muscle damage (Creatine Phosphokinase; CPK, Lactate Dehydrogenase; LDH, Myoglobin), inflammation (Interleukin-6; IL-6, Interleukin-1 ; IL-1 , Tumor Necrosis Factor- ; TNF- , C-Reactive Protein; CRP) and stress hormone (Cortisol). According to this result, COMP (S1; 188.37 46.68 ng/mL) showed a sig- nificant increase after the expedition course 1 (M; 246.69 51.69 ng/mL,p= 0.012) andcourse 2 (U; 237.09 62.57 ng/mL,p= 0.047), and recovered to the stable state after expeditioncourse 3 (S2; 218.46 34.78.p= 0.047). Biomarkers of muscle damage (CPK, LDH and Myoglobin) and inflammation (IL-6, IL-1 , TNF- and CRP) were not significantly changed in all courses, but CRP (S1; 1.07 0.76 ng/mL) showed a tendency to decrease after the expedition course 1 (M; 0.3 0.1 mg/mL ,p= 0.044). Lastly, the Cortisol level significantly increased in all courses (per p< 0.05), but the Cortisol level after expedition course 3 (S2; 21.00 3.65 mg/mL) was lower than that of after the expedition course 1 (M; 24.23 2.47 mg/mL,p= 0.028). In summary, it seems that repetitive and continuous 50–90 km/day of cycling can increase joint cartilage damage risk and stress hormone temporarily. However, this result suggests that the appropriate intensity of cycling for thirteen weeks does not increase physical damage, and rather enhances the human body to adapt to exercise training. Keywords:long-distance bicycle riding; in ammatory response; joint cartilage; muscle damage 1. Introduction Endurance training improves cardiopulmonary endurance and immune function, and is also effective in preventing various metabolic diseases, such as obesity, diabetes and cardiovascular disease. Given these bene ts, more people are participating in endurance exercise [1,2], but long-distance endurance training without proper preparation can increase the probability of sudden death, and can also cause acute liver damage, muscle damage, bone and joint damage, hematuria, dehydration and gastrointestinal disease [3,4]. Thus, the safety of participation in long-distance endurance competition and sports should be investigated. Int. J. Environ. Res. Public Health2022,19, 16314.

can increase the probability of sudden death, and can also cause acute liver damage, muscle damage, bone and joint damage, hematuria, dehydration and gastrointestinal disease [3,4]. Thus, the safety of participation in long-distance endurance competition and sports should be investigated. Int. J. Environ. Res. Public Health2022,19, 16314.

Int. J. Environ. Res. Public Health2022,19, 16314 2 of 12 Cartilage oligomeric matrix protein (COMP) is a non-collagenous extracellular matrix protein that maintains collagen structure in joint cartilage. It is secreted into the blood when joint cartilage is damaged, and is thus used as an index of joint cartilage damage [5,6]. In other words, the influence of long-distance endurance training on indicators of joint cartilage damage may differ depending on exercise method, intensity and duration. Moreover, research has been lacking on the influence of exercises in which the effects of bodyweight on joints are limited, such as cycling or swimming, on joint cartilage damage. Cârdova et al. (2015) collected blood samples from athletes before and after a 3-day cycling competition (123 km for day 1, 128 km for day 2 and 100 km for day 3) to measure serum CPK, myoglobin and LDH concentrations; the authors found that CPK did not change significantly after the competition on day 1 and before the competition on day 2, but significantly increased after the competition on day 2 until recoveryday 1 after the competition on day 3 [7]. Furthermore, Kim et al. (2009) showed that the concentration significantly increased up to 4 days after the completion of the 100–200 km ultramarathon [8]. In contrast, serum myoglobin concentration increased significantly after each competition, but returned to normal before the competition on the next day. Cytokines are regulatory proteins secreted by cells in response to various stimuli, which act as major regulatory factors in various aspects, such as immune response, muscle atrophy and development, insulin sensitivity and fat metabolism [9–11]. Interleukin 6 (IL-6) and 1 beta (IL-1 ), which are pro-inflammatory cytokines, and tumor necrosis factor alpha (TNF- ), which is an inflammatory cytokine, are major indicators of muscle damage and inflammatory response [12,13]. IL-6 is known to be involved in various responses associated with inflammation and damage, including its role in increasing cortisol and C-reactive protein (CRP), another inflammatory marker [14]. Chiu et al. (2015) reported that in the concentration of IL-6 and TNF- , CRP was significantly increased from immediately after exercise to

major indicators of muscle damage and inflammatory response [12,13]. IL-6 is known to be involved in various responses associated with inflammation and damage, including its role in increasing cortisol and C-reactive protein (CRP), another inflammatory marker [14]. Chiu et al. (2015) reported that in the concentration of IL-6 and TNF- , CRP was significantly increased from immediately after exercise to 24 h after exercise in 24 men who completed a 100 km ultramarathon [15]. Although one-time long-distance endurance exercise has been reported to increase the serum concentrations of IL-6, TNF- and CRP [12,16,17], changes in inflammatory markers with long-distance exercise still remain controversial. In summary, with increases in the participation of non-professional athletes in en- durance exercise, as well as increases in competitions and sports events, it is important to elucidate the in uence of long-distance endurance exercise on markers of tissue damage in the body, in order to set safe exercise plans. Nevertheless, the in uence of long-distance endurance exercise on joint cartilage damage, muscle damage and level of in ammation according to exercise type, distance and duration remains unclear, with a lack of relevant re- search. Therefore, this study aimed to investigate the in uence of 13 weeks of long-distance cycling on in ammatory markers associated with muscle and joint cartilage damage. 2. Materials and Methods 2.1. Study Participants The participants of this study were adults aged from in their 20s to their 50s who did not have any musculoskeletal disorder, but had more than 7 years of experience with cycling. Furthermore, they were healthy individuals who had not participated in an exercise program or diet for the 3 months, and no speci c disease was found in the medical examination. The participants were screened for their cycling abilities to complete the ultra- long-distance cycling event across Europe and Asia (One Korea New-Eurasia Peace Cycle Expedition, Figure). The participants consisted of 6 male and 1 female cyclist selected for the expedition (Table). The selected participants all received detailed explanation of the study purpose and procedures, and provided written consent. Ethical permission was granted from the Korea National Sport

abilities to complete the ultra- long-distance cycling event across Europe and Asia (One Korea New-Eurasia Peace Cycle Expedition, Figure). The participants consisted of 6 male and 1 female cyclist selected for the expedition (Table). The selected participants all received detailed explanation of the study purpose and procedures, and provided written consent. Ethical permission was granted from the Korea National Sport University's Ethics Committee (Korea National Sport University Industry-Academic Cooperation Foundation-985), and all procedures were in accordance with the Declaration of Helsinki for research on human subjects.

Int. J. Environ. Res. Public Health2022,19, 16314 3 of 12Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 3 of 12 Figure 1. The participant recruitment and selection process. Table 1. The characteristics of subjects No. Sex Age (Yr.) Height (cm) Fat (%) S1 (kg) M (kg) U (kg) S2 (kg) 1 Male 23.0 176.1 14.5 67.7 69.1 70.0 72.9 2 Male 51.6 174.0 22.1 79.7 81.0 83.5 81.0 3 Male 22.8 177.4 10.0 75.4 76.8 81.2 81.0 4 Male 45.1 174.0 17.7 71.8 73.8 74.3 73.9 5 Female 33.9 156.9 32.7 59.5 58.3 58.9 59.6 6 Male 29.7 180.5 14.4 73.4 72.2 75.8 79.0 7 Male 25.5 177.0 30.8 90.3 86.4 85.8 89.8 S1: Before start in Seoul, M: Moscow U: Ulaanbaatar S2: Return to Seoul. 2.2. Study Design and Participants 2.2.1. Cycling According to the program of the One Korea New-Eurasia Peace Cycle Expedition, the participants cycled 4869 km from the 15,000 km-long expedition. The total course of the expedition was divided into three sub-courses: course 1 (from Berlin to Moscow); course 2 (from Moscow to Ulaanbaatar); and course 3 (from Ulaanbaatar to Seoul). Alt- hough the original plan was to cycle for 5 days and have 1 day of rest, some changes were made depending on environmental conditions (weather, route conditions and cycling per- missions). Figure 2 (Google map) and Table 2 show the sub-courses and cycling content. Figure 1.The participant recruitment and selection process. Table 1.The characteristics of subjects. No. Sex Age (Yr.) Height (cm) Fat (%) S1 (kg) M (kg) U (kg) S2 (kg) 1 Male 23.0 176.1 14.5 67.7 69.1 70.0 72.9 2 Male 51.6 174.0 22.1 79.7 81.0 83.5 81.0 3 Male 22.8 177.4 10.0 75.4 76.8 81.2 81.0 4 Male 45.1 174.0 17.7 71.8 73.8 74.3 73.9 5 Female 33.9 156.9 32.7 59.5 58.3 58.9 59.6 6 Male 29.7 180.5 14.4 73.4 72.2 75.8 79.0 7 Male 25.5 177.0 30.8 90.3 86.4 85.8 89.8 S1: Before start in Seoul, M: Moscow U: Ulaanbaatar S2: Return to Seoul. 2.2. Study Design and Participants 2.2.1. Cycling According to the program of

Male 45.1 174.0 17.7 71.8 73.8 74.3 73.9 5 Female 33.9 156.9 32.7 59.5 58.3 58.9 59.6 6 Male 29.7 180.5 14.4 73.4 72.2 75.8 79.0 7 Male 25.5 177.0 30.8 90.3 86.4 85.8 89.8 S1: Before start in Seoul, M: Moscow U: Ulaanbaatar S2: Return to Seoul. 2.2. Study Design and Participants 2.2.1. Cycling According to the program of the One Korea New-Eurasia Peace Cycle Expedition, the participants cycled 4869 km from the 15,000 km-long expedition. The total course of the expedition was divided into three sub-courses: course 1 (from Berlin to Moscow);course 2 (from Moscow to Ulaanbaatar); and course 3 (from Ulaanbaatar to Seoul). Although the original plan was to cycle for 5 days and have 1 day of rest, some changes were made depending on environmental conditions (weather, route conditions and cycling permissions). FigureInt. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 3 of 12 Figure 1. The participant recruitment and selection process. Table 1. The characteristics of subjects No. Sex Age (Yr.) Height (cm) Fat (%) S1 (kg) M (kg) U (kg) S2 (kg) 1 Male 23.0 176.1 14.5 67.7 69.1 70.0 72.9 2 Male 51.6 174.0 22.1 79.7 81.0 83.5 81.0 3 Male 22.8 177.4 10.0 75.4 76.8 81.2 81.0 4 Male 45.1 174.0 17.7 71.8 73.8 74.3 73.9 5 Female 33.9 156.9 32.7 59.5 58.3 58.9 59.6 6 Male 29.7 180.5 14.4 73.4 72.2 75.8 79.0 7 Male 25.5 177.0 30.8 90.3 86.4 85.8 89.8 S1: Before start in Seoul, M: Moscow U: Ulaanbaatar S2: Return to Seoul. 2.2. Study Design and Participants 2.2.1. Cycling According to the program of the One Korea New-Eurasia Peace Cycle Expedition, the participants cycled 4869 km from the 15,000 km-long expedition. The total course of the expedition was divided into three sub-courses: course 1 (from Berlin to Moscow); course 2 (from Moscow to Ulaanbaatar); and course 3 (from Ulaanbaatar to Seoul). Alt- hough the original plan was to cycle for 5 days and have 1 day of rest, some changes were made depending on environmental conditions (weather, route conditions and cycling per- missions). Figure 2

the expedition was divided into three sub-courses: course 1 (from Berlin to Moscow); course 2 (from Moscow to Ulaanbaatar); and course 3 (from Ulaanbaatar to Seoul). Alt- hough the original plan was to cycle for 5 days and have 1 day of rest, some changes were made depending on environmental conditions (weather, route conditions and cycling per- missions). Figure 2 (Google map) and Table 2 show the sub-courses and cycling content. Figure 2. The course of One Korea New-Eurasia Peace Cycle Expedition. 1st section, from Berlin to Moscow (black square), 2nd section, from Moscow to Ulaanbaatar (red square) and 3rd section, From Ulaanbaatar to Seoul (blue square).

Int. J. Environ. Res. Public Health2022,19, 16314 4 of 12 Table 2.Cycling content. 1st Section (1) 2nd Section (2) 3rd Section (3) Cycling distance (km) 2097 1678 1094 Total days 29 34 30 Cycling days 24 24 19 Mean cycling (km) 87.4 70 57.6 The longest cycling (km) 132 147 117 The shortest cycling (km) 20 14 10 Mean temperature ( F) 16.5 1.7 1.6 Maximum temperature ( F) 22 15 18 Minimum temperature ( F) 1 7 7 (1) From Berlin to Moscow, (2) From Moscow to Ulaanbaatar, (3) From Ulaanbaatar to Seoul. 2.2.2. Time and Method for Sample Collection Blood samples were collected in the morning of the day after cycling after fasting at the following time points: before the expedition (in Seoul); during the expedition in Moscow (day 29) and Ulaanbaatar (day 63); and on the last day of expedition in Seoul (day 93). The collected samples were immediately placed into serum tubes and EDTA tubes and centrifuged (Beckman, Brea, CA, USA, 15 min/3000 rpm). The supernatant serum and plasma were separated and carried on dry ice ( 78.5 C), and stored at 80 C in a freezer until analysis. 2.2.3. Sample Analysis In order to assess the level of joint cartilage and muscle damage from long-distance cycling, the concentration of COMP, an index of joint cartilage damage, was measured using Human COMP Quantikine ELISA kits (R&D systems Minneapolis, Minneapolis, MN, USA) through an enzyme-linked immunosorbent assay (ELISA). The levels of CPK, LDH and myo- globin, which are markers of muscle damage, were analyzed using Modular Analytics (Roche, Mannheim, Germany) through UV assay. Inflammatory markers (IL-6, IL-1 andTNF- ) were analyzed using Human High Sensitivity Cytokine kits (R&D systems Minneapolis, Min- neapolis, MN, USA) through Luminex assay, and CRP was analyzed using Modular Analytics (Roche, Germany) through immunoturbidimetric assay. The concentration of cortisol, a stress hormone, was analyzed using a fully automated chemistry analyzer (Beckman, Brea, CA, USA). This study was conducted as a joint study. The results pertaining to serum cortisol have already been published through Gil's study [18]. However, because the results were obtained from

and CRP was analyzed using Modular Analytics (Roche, Germany) through immunoturbidimetric assay. The concentration of cortisol, a stress hormone, was analyzed using a fully automated chemistry analyzer (Beckman, Brea, CA, USA). This study was conducted as a joint study. The results pertaining to serum cortisol have already been published through Gil's study [18]. However, because the results were obtained from the same participants and because evidence for the influence of this study's exercise type on tissue damage response was needed, the results have been cited in this study as well. 2.2.4. Data Analysis Mean and standard deviation were calculated for the collected data using SPSS/PC+Version 20.0. One-way repeated measures ANOVA was used to assess the differences in serum markers according to cycling distance. When differences were present, they were analyzed through Duncan multiple test analysis, and the level of statistical significance was set asp< 0.05. 3. Results 3.1. Changes in Markers of Joint Cartilage Damage When serum COMP concentration, which is an indicator of joint cartilage damage, was analyzed (Figure), the concentration was 188.37 46.68 ng/mL before the expedition, 246.69 51.69 ng/mL after course 1 (Berlin–Moscow), 237.09 62.57 ng/mL aftercourse 2 (Moscow–Ulaanbaatar) and 218.46 34.78 ng/mL after course 3 (Ulaanbaatar–Seoul). Compared to pre-expedition value, the concentration increased after course 1 (p= 0.012) and course 2 (p= 0.047). Compared to course 1, the concentration decreased after course 3 (p= 0.036).

Int. J. Environ. Res. Public Health2022,19, 16314 5 of 12Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 5 of 12 3. Results 3.1. Changes in Markers of Joint Cartilage Damage When serum COMP concentration, which is an indicator of joint cartilage damage, was analyzed (Figure 3), the concentration was 188.37 ± 46.68 ng/mL before the expedi- tion, 246.69 ± 51.69 ng/mL after course 1 (Berlin–Moscow), 237.09 ± 62.57 ng/mL after course 2 (Moscow–Ulaanbaatar) and 218.46 ± 34.78 ng/mL after course 3 (Ulaanbaatar– Seoul). Compared to pre-expedition value, the concentration increased after course 1 (p = 0.012) and course 2 (p = 0.047). Compared to course 1, the concentration decreased after course 3 (p = 0.036). Figure 3. Serum COMP concentration in different locations. Values are mean ± S.D. Significance (p < 0.05) is denoted by a: relative to S1 and b: relative to M. S1: Before start in Seoul, M: Moscow U: Ulaanbaatar S2: Return to Seoul. 3.2. Changes in Markers of Muscle Damage 3.2.1. Changes in CPK When serum CPK concentration, a marker of muscle damage, was analyzed (Figure 4), the concentration was 156 ± 85.84 U/L before the expedition, 267.28 ± 197.66 U/L after course 1 (Berlin–Moscow), 227.71 ± 155.22 U/L after course 2 (Moscow–Ulaanbaatar) and 259.57 ± 227.94 U/L after course 3 (Ulaanbaatar–Seoul). There was no significant difference noted at subsequent measurements compared to the baseline (p > 0.05). Figure 4. Serum CPK concentration in different locations. Values are mean ± S.D. S1: Before start in Seoul, M: Moscow U: Ulaanbaatar S2: Return to Seoul. 3.2.2. Changes in Myoglobin When serum concentration of myoglobin, an indicator of muscle damage, was ana- lyzed (Figure 5), the concentration was 23.14 ± 8.49 U/L before the expedition, 26.05 ± 12.83 U/L after course 1 (Berlin–Moscow), 19.17 ± 6.56 U/L after course 2 (Moscow–Ulaanbaatar) and 26.35 ± 9.71 U/L after course 3 (Ulaanbaatar–Seoul). The concentration did not differ significantly from the baseline at each of the subsequent measurements (p > 0.05). Figure 3. Serum COMP concentration in different locations. Values are mean S.D. Significance (p< 0.05) is