[Study on the effect of high-intensity interval exercise on ulcerative colitis in mice].

内容摘要

Objective: This study aims to systematically explore the protective effect and its underlying mechanism of high-intensity interval exercise (HIE) on the mouse model of ulcerative colitis (UC) induced by dextran sulfate sodium (DSS) through regulating the structure and function of gut microbiota. Methods: A simple randomized controlled allocation method was adopted. From July to August 2024, 24 male C57BL/6 mice were divided into the control group (Con), ulcerative colitis group (DSS), high-intensity interval exercise group (HIE), and high-intensity interval exercise-ulcerative colitis group (HIE-DSS) in the experimental animal room of the Second Department of Infectious Disease Prevention and Control in the Disease Prevention and Control Center of the Eastern Theater Command, with six mice in each group. During the experimental stage, the mice in the HIE and HIE-DSS groups first received 5-week high-intensity interval exercise training. Then, the mice in the DSS and HIE-DSS groups were given free access to a 3% DSS solution for 7 days to induce the UC model. The diarrhea status, body weight changes of the mice were observed, and the colon length was measured. The histopathology and morphology of the colon tissues were scored. H&E staining was used to observe the pathological changes in the colon tissues. An enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression levels of interleukin (IL) IL-10, IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) in the peripheral serum of the mice in each group. Real-time fluorescent quantitative PCR (qRT-PCR) was used to monitor the changes in the levels of IL-1 mRNA, IL-6 mRNA, IL-10 mRNA, and TNF-α mRNA in the mouse colon tissues. Kits were used to detect the changes in malondialdehyde (MDA), myeloperoxidase (MPO), catalase (CAT), glutathione (GSH), total superoxide dismutase (T-SOD), and reactive oxygen species (ROS) in the mouse colon tissues. 16S rRNA sequencing and gas chromatography-mass spectrometry (GC-MS) techniques were used to detect the changes in the gut microbiota and fecal metabolites. Immunohistochemical techniques and bacterial translocation experiments were used to evaluate the changes in the gut mucosal proteins Occludin and Muc2 and the gut barrier function. One-way analysis of variance (One-way ANOVA) was used for statistical analysis. Newman-Keuls multiple comparison test was used to evaluate the differences between groups. Results: The weight changes (g) of the Con group, DSS group, HIE group, and HIE-DSS group were (28.29±0.71), (23.71±0.67), (27.61±1.20), and (25.63±0.78) respectively; the colon lengths (cm) were (7.67±0.41), (4.87±0.18), (7.08±0.22), and (6.53±0.14) respectively; the morphological scores of colon tissues were (1.26±0.62), (2.01±0.41), (1.25±0.70), and (1.57±0.28) respectively; the pathological scores were (0.66±0.31), (1.33±0.42), (0.81±0.19), and (1.03±0.32) respectively. The weight change, colon length, colon morphological score, and colon tissue pathological score of the DSS group were significantly lower than those of the HIE-DSS group, with statistically significant differences (t=3.195, P=0.036), (t=4.478, P=0.008 6), (t=3.745, P=0.007 1), and (t=4.106, P=0.006 9).The contents of IL-1β (pg/ml) in the serum of the Con group, DSS group, HIE group, and HIE-DSS group were 19.10±0.89, 26.26±1.86, 221.65±1.43, and 22.46±1.54 respectively; the expression levels of IL-6 (pg/ml) were 30.82±7.15, 71.90±14.42, 28.70±6.62, and 37.40±4.58 respectively; the expression levels of TNF-α (pg/ml) were 22.78±6.11, 37.86±7.12, 24.68±4.07, and 27.75±6.32 respectively; the expression levels of IL-10 (pg/ml) were 59.13±9.02, 43.38±6.72, 53.17±5.27, and 51.92±4.18 respectively. Compared with the Con group, the expression levels of IL-1β, IL-6, and TNF-α in the serum of the DSS group were significantly increased, while the expression level of IL-10 was significantly decreased, with statistically significant differences (t=6.345, P=0.044), (t=5.115, P=0.038), (t=8.931, P=0.021), (t=6.612, P=0.039). The expression levels of IL-1β, IL-6, and TNF-α in the HIE-DSS group were significantly lower than those in the DSS group, while the expression level of IL-10 was significantly higher, with statistically significant differences (t=7.749, P=0.000 8), (t=5.327, P=0.007 4), (t=10.140, P=0.000 6), (t=7.718, P=0.000 5).The relative expression levels of ROS (U/mol) in the colon tissues of the Con group, DSS group, HIE group, and HIE-DSS group were 111.83±10.90, 185.16±25.56, 118.83±16.43, and 135.83±11.81 respectively; the expression levels of MPO (U/mgport) were 0.71±0.85, 2.76±0.62, 0.90±0.43, and 1.56±1.33 respectively; the expression levels of CAT (U/mgport) were 0.44±0.31, 0.08±0.21, 0.37±0.25, and 0.30±0.22 respectively; the expression levels of MDA (nmol/mgport) were 0.38±0.12, 0.92±0.22, 0.46±0.18, and 0.57±0.19 respectively; the expression levels of GSH (mg/port) were 17.80±6.12, 6.11±2.82, 14.71±4.18, and 12.28±5.011 respectively; the expression levels of T-SOD (U/mgport) were 3.69±2.12, 1.25±0.61, 3.23±0.55, and 2.73±0.47 respectively. Compared with the HIE-DSS group, the expression levels of ROS, MPO, and MDA in the colon of the DSS group were significantly increased, while the expression levels of CAT, GSH, and T-SOD were significantly decreased, with statistically significant differences (t=3.915, P=0.005 4), (t=4.543, P=0.000 4), (t=8.018, P=0.000 7), (t=12.510, P=0.000 6), (t=6.703, P=0.000 5), (t=5.784, P=0.000 8).The expression levels (IOD) of tight-junction protein Muc2 in the colon tissues of the Con group, DSS group, HIE group, and HIE-DSS group were 3 019.51±23.26, 2 080.25±11.18, 2 690.32±17.24, and 2 473.50±16.04 respectively; the expression levels (IOD) of Occludin were 2 881.04±12.07, 2 198.30±10.05, 2 720.30±14.02, and 2 680.30±11.23 respectively. The expression levels of Muc2 and Occludin in the DSS group were significantly lower than those in the HIE-DSS and Con groups, with statistically significant differences (t=8.743, P=0.000 7), (t=9.634, P=0.000 9). Conclusion: As an emerging exercise mode, high-intensity interval training may participate in alleviating the pathogenesis of ulcerative colitis by regulating gut microbiota and their metabolites. 目的： 旨在探讨高强度间歇运动（HIE）通过调节肠道菌群结构与功能，对葡聚糖硫酸钠（DSS）诱导的小鼠溃疡性结肠炎（UC）模型的保护效应及其潜在机制。 方法： 采取简单随机对照分配方式，于2024年7—8月在东部战区疾病预防控制中心传染病防控二科实验动物房将24只雄性C57BL/6小鼠分为对照组（Con）、溃疡性结肠炎组（DSS）、高强度间歇运动组（HIE）和高强度间歇运动-溃疡性结肠炎组（HIE-DSS），每组各6只。在实验阶段，HIE组与HIE-DSS组小鼠首先接受为期5周的高强度间歇运动训练，之后对DSS组和HIE-DSS组小鼠采用3%DSS溶液自由饮用7 d，以诱导UC模型。观察小鼠腹泻情况、体重变化并测量结肠长度。对结肠组织病理学和形态学进行评分；采用苏木精-伊红（H&E）染色观察结肠组织病理变化；采用酶联免疫吸附试验（ELISA）分析各组小鼠外周血清中白介素（IL）的IL-10、IL-1β、IL-6和肿瘤坏死因子-α（TNF-α）的表达水平；采用实时荧光定量PCR（qRT-PCR）监测小鼠结肠组织中IL-1 mRNA、IL-6 mRNA、IL-10 mRNA和TNF-α mRNA水平的变化；采用试剂盒检测小鼠结肠组织丙二醛（MDA）、髓过氧化物酶（MPO）、过氧化氢酶（CAT）、谷胱甘肽（GSH）、超氧化物歧化酶（T-SOD）和活性氧（ROS）的变化；采用16S rRNA测序和气相色谱-质谱联用（GC-MS）技术检测肠道菌群和粪便代谢物的变化。采用免疫组化技术和细菌移位实验评估肠道黏膜蛋白Occludin和Muc2及肠道屏障功能变化。采用单因素方差分析（One-way ANOVA）进行统计学分析。组间差异的评估使用Newman-Keuls多重比较检验。 结果： Con组、DSS组、HIE组、HIE-DSS组体重（g）变化分别为（28.29±0.71）、（23.71±0.67）、（27.61±1.20）、（25.63±0.78）；结肠长度（cm）分别为（7.67±0.41）、（4.87±0.18）、（7.08±0.22）、（6.53±0.14）；结肠组织形态学评分（score）分别为（1.26±0.62）、（2.01±0.41）、（1.25±0.70）、（1.57±0.28）；病理评分（score）分别为（0.66±0.31）、（1.33±0.42）、（0.81±0.19）、（1.03±0.32）。DSS组体重变化、结肠长度、结肠形态学评分、结肠组织病理评分均较HIE-DSS组显著降低，差异有统计学意义（t=3.195，P=0.036）、（t=4.478、P=0.0086）、（t=3.745、P=0.0071）、（t=4.106，P=0.0069）。Con组、DSS组、HIE组、HIE-DSS组血清中IL-1β含量（pg/ml）分别为19.10±0.89、26.26±1.86、221.65±1.43、22.46±1.54，IL-6表达量（pg/ml）分别为30.82±7.15、71.90±14.42、28.70±6.62、37.40±4.58，TNF-α表达量（pg/ml）分别为22.78±6.11、37.86±7.12、24.68±4.07、27.75±6.32，IL-10表达量（pg/ml）分别为59.13±9.02、43.38±.6.72、53.17±.5.27、51.92±4.18。与 Con组相比，DSS组血清中IL-1β、IL-6、TNF-α表达显著升高，IL-10表达显著降低，差异有统计学意义（t=6.345，P=0.044）、（t=5.115，P=0.038）、（t=8.931，P=0.021）、（t=6.612，P=0.039）；HIE-DSS组IL-1β、IL-6、TNF-α表达较DSS组显著下降，IL-10表达显著上升，差异有统计学意义（t=7.749，P=0.000 8）、（t=5.327，P=0.007 4）、（t=10.140，P=0.000 6、（t=7.718，P=0.000 5）。Con组、DSS组、HIE组、HIE-DSS组的结肠组织中ROS相对表达量（U/mol）分别为111.83±10.90、185.16±25.56、118.83±16.43、135.83±11.81，MPO表达量（U/mgport）分别为0.71±0.85、2.76±0.62、0.90±0.43、1.56±1.33，CAT表达量（U/mgport）分别为0.44±0.31、0.08±0.21、0.37±0.25、0.30±0.22，MDA表达量（nmol/mgport）分别0.38±0.12、0.92±0.22、0.46±0.18、0.57±0.19，GSH表达量（mg/port）分别为17.80±6.12、6.11±2.82、14.71±4.18、12.28±5.011，T-SOD表达量（U/mgport）分别为3.69±2.12、1.25±0.61、3.23±0.55、2.73±0.47。与HIE-DSS组相比，DSS组结肠中ROS、MPO、MDA表达显著升高，CAT、GSH、T-SOD表达显著降低，差异有统计学意义（t=3.915，P=0.005 4）、（t=4.543，P=0.000 4）、（t=8.018，P=0.000 7）、（t=12.510，P=0.000 6）、（t=6.703，P=0.000 5）、（t=5.784，P=0.000 8）。Con组、DSS组、HIE组、HIE-DSS组的结肠组织紧密连接蛋白Muc2蛋白表达量（IOD）分别为3 019.51±23.26、2 080.25±11.18、2 690.32±17.24、2 473.50±16.04。Occludin蛋白表达量（IOD）分别为2 881.04±12.07、2 198.30±10.05、2 720.30±14.02、2 680.30±11.23，DSS组中Muc2、Occludin蛋白表达量较HIE-DSS和Con组显著降低，差异有统计学意义（t=8.743，P=0.000 7）、（t=9.634，P=0.000 9）。 结论： 高强度间歇运动作为一种新兴的运动方式，可能通过调控肠道菌群及其代谢产物，参与减轻溃疡性结肠炎的发病机制。.