This study aims to clarify the characteristics and temporal patterns of colonic barrier damage in mice following a single simulated high-intensity exercise in hot and humid environment, and further investigate the protective effects of baicalein (BAI) intervention on colonic injury, thereby providing experimental evidence for the development of medical protection time windows and pharmaceutical intervention protocols for personnel engaged in high-intensity exercise and operations in hot and humid environments.

①Thirty SPF-grade 8-week-old male C57BL/6J mice were randomly divided into 5 groups (n=6): control group, 30 min post-exercise group, 4 h post-exercise group, 12 h post-exercise group, and 24 h post-exercise group. The mice in the exercise groups were subjected on a treadmill in a heat chamber (temperature: 38℃, humidity: RH 75%) at 80% maximal exercise velocity (Vmax) (exercise 12 min, rest 8 min, 3 cycles; slope 10°). Subsequently, the colon tissues were harvested for morphological observation by HE staining in each group; ELISA was used to detect the serum levels of inflammatory factors (TNF-α, IL-6, IL-10, and IL-1β) to observe the inflammatory response of each group. Intestinal fatty acid binding protein (I-FABP) and lipopolysaccharide (LPS) were detected to observe intestinal permeability; Microassay was performed to determine the concentrations of reduced glutathione (GSH) and malondialdehyde (MDA), as well as the activity of catalase (CAT) and superoxide dismutase (SOD) in the colon tissues; RT-qPCR was applied to detect the expression levels of intestinal tight junction protein related genes Occludin, ZO-1, Claudin-1, inflammation and oxidative stress related genes Tlr4, TNF-α, IL-1β, IL-6, IL-10, heme oxygenase-1 (HO-1), and superoxide dismutase1 (SOD1), as well as key transcription factors HSF1, NF-κB, PARP-1, and NRF2 in heat stress regulation; Western blotting was utilized to detect the protein of colonic tight junction protein Occludin. ② For BAI intervention study, 24 SPF-grade 8-week-old male C57 BL/6J mice were randomly divided into 4 groups (n=6) control group, exercise group (high temperature and high humidity exercise, gavage with deionized water 200 μL), exercise+low- and high-dose intervention groups (BAI gavage 200 μL at 10 and 40 mg/kg per day). At 4 h after the end of exercise, colonic and blood samples were collected for serum levels of urea nitrogen (BUN) content, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) to evaluate the safety of BAI. Other detection sites, indicators, and methods were the same as aforementioned.

① The core body temperature was significantly elevated in the exercise group (P<0.05); the structural damage, along with inflammatory cell infiltration was observed in colonic mucosa; the expression of colon Occludin, ZO-1, and Claudin-1 was downregulated (P<0.05), and the protein level of Occludin was decreased (P<0.05), and the serum levels of I-FABP and LPS were increased (P<0.05); The serum levels of TNF-α, IL-1β, IL-6, and IL-10 were increased (P<0.05), and the expression of IL-1β and IL-10 in the colon was upregulated (P<0.05); The concentration of MDA was increased (P<0.05), while the concentration of GSH and the activities of CAT and SOD were decreased (P<0.05); The expression of HSF1, PARP-1, NF-κB, Tlr4, TNF-α, IL-6, and SOD1 in the colon was upregulated (P<0.05), while the expression of NRF2 and HO-1 was downregulated (P<0.05). These changes were present at 30 min post-exercise, most prominent at 4 h post-exercise, and markedly recovered by 24 h. ② After BAI intervention, the core body temperature was significantly decreased (P<0.05), and the serum levels of ALT and AST were decreased (P<0.05); colonic mucosal injury was alleviated; the expression of Occludin at mRNA and protein levels in the colonic tissues were increased (P<0.05); the expression of ZO-1 and Claudin-1 were upregulated (P<0.05), and serum levels of I-FABP and LPS were decreased (P<0.05); the serum level of IL-1β was decreased (P<0.05), and the expression of IL-1β in the colon was downregulated (P<0.05); The concentration of MDA was decreased (P<0.05), while the concentration of GSH and activities of CAT and SOD in the colon were increased (P<0.05); The expression of HSF1, PARP-1, NF-κB, Tlr4, TNF-α, and IL-6 in the colon was downregulated (P<0.05), while the expression of NRF2 and HO-1 was upregulated (P<0.05). These results indicated that BAI possesses anti-inflammatory and antioxidant effects, with better efficacy in the low-dose group.

Peak colonic barrier injury occurs at 4 h after simulated high-intensity exercise in a hot and humid environment, and prophylactic use of BAI (10 mg/kg per day) can significantly inhibit inflammation and oxidative stress and alleviate injury, providing evidence for time windows and intervention basis for colonic injury induced by heat stress.

To investigate the alterations in skeletal muscle function and mass in an experimental mouse model of high-intensity exercise in a hot and humid environment.

Twenty-four male C57BL/6J mice (7~8 weeks old, weighing 21.30±0.67 g) were randomly assigned to a control group (CON group), a normal temperature and humidity exercise group (NE group), and a high temperature and humidity exercise group (HE group), with 8 mice in each group. The HE group was subjected to a hightemperature simulation chamber, maintaining a temperature of 37~39 ℃ and humidity of 70%~80%, for a 60-minute exercise intervention at a 10° incline and 80% of maximum velocity (12 min of exercise followed by 8 min of rest, for 3 cycles). The CON group did not exercise, while the NE group exercised in the same manner in a normal temperature and humidity environment. The overall condition of the mice was evaluated by monitoring their body weight and analyzing their body composition. Their serum creatinine and urea levels were detected using an automated biochemical analyzer. After exercise, skeletal muscle function in the mice of each group was assessed by measuring their grip strength and exhaustion time. The skeletal muscle contractility and resistance to fatigue were evaluated using an in situ/in vivo/ex vivo muscle testing system. HE staining was employed to observe the morphological and structural changes in the skeletal muscles, and the average cross-sectional area and diameter of the muscle fibers were analyzed. Genes related to protein synthesis (Eif4ebp1, p70S6k) and breakdown (Foxo3, Fbxo32, Trim63) and heat stress-related genes (Hsf-1, Hspa1a, Hsp90aa) were quantified using RT-qPCR.

(1) Compared with the CON and NE groups, the HE group exhibited significant decreases in body weight (P < 0.01) and lean body mass (P < 0.05), an upward trend of creatinine level (P < 0.05), and increases in the urea content (P < 0.01). (2) The mice in the HE group had notably reduced grip strength (P < 0.001), diminished skeletal muscle contraction, and weakened resistance to fatigue (P < 0.05) than the CON and NE groups. (3) The HE group demonstrated a reduction in the average cross-sectional area of muscle fibers (P < 0.05) and a decrease in average fiber diameter (P < 0.05), with particular up-regulation of Fbxo32, Trim63 and Eif4ebp1 (P < 0.01) and downregulation of p70S6k (P < 0.05) in comparison to the NE and CON groups.(4) The expression levels of heat stress-related genes were higher in the HE group than the CON and NE groups (P < 0.05).

Highintensity exercise in a hot and humid environment can lead to a decline in skeletal muscle function and mass in mice, potentially due to the disturbance of skeletal muscle protein synthesis and degradation triggered by excessive heat stress.