To develop a composite nutritional preparation that can effectively improve exercise performance under calorie restriction (CR) condition.

A total of 24 male C57BL/6J mice (weighing 23~26 g, 8 weeks old) were randomly divided into control group (CON), CR, CR+basal nutrient group (CRN1), and CR+compound nutrient group (CRN2). All groups underwent moderate-intensity running training 5 d per week, for totally 3 weeks. The grip strength of the forelimbs were measured weekly, and in 3 weeks after training, exhaustion and post-exhaustion distance tests were conducted to evaluate exercise performance. Blood biochemical indicators, levels of skeletal muscle and liver redox biomarkers, and histopathological conditions were measured and observed.

After 21 d of intervention, the CR group and CRN1 group had the post-exhaustion running distance prolonged by 278% and 289%, respectively, reduced blood glucose level, and decreased muscle mass, subcutaneous fat and epididymal fat mass when compared with the CON group (P<0. 05). Compared with the CON group, the CRN1 and CRN2 groups demonstrated significantly higher gastrocnemius glycogen content. The CRN2 group obtained even longer post-exhaustion distance (increased by 52% and 36% respectively, compared with the CON group and CRN1 group, P<0. 05), enhanced grip strength of the forelimbs (raised by 9%, 17% and 15%, respectively than the CON, CR and CRN1 groups, P<0. 05), elevated brown fat mass (compared to the CON group and CRN1 group, P<0. 05), increased blood glucose level (compared to the CRN1 group, P<0. 05), decreased blood low-density lipoprotein cholesterol level (compared to the CON and CR groups, P<0. 05), and increased glutathione peroxidase content in the gastrocnemius muscle (compared to the CON group, P<0. 05).

Supplementing with compound nutritional supplements in mice under CR can promote exercise performance, including improving fatigue recovery after exhaustive exercise and enhancing forelimb grip strength.

To explore the efficacy of a compound nutrition preparation on wound healing after acute trauma and investigate the underlying mechanism primarily.

After skin wound model was successfully constructed on totally 60 male SD rats surgically, they were randomly divided into control group (CON group), whey protein group (WP group) and low-and high-dose nutritional formula groups (LDF and HDF groups), with 15 animals in per group. From the next day after surgery, the rats in each group were given corresponding nutritional interventions for 2 weeks. During the intervention, the wound conditions were observed and recorded, and the wound area was measured. The samples were collected on the 3rd, 7th and 14th days after surgery, respectively. Serum albumin (ALB), prealbumin (PA), total protein (TP), inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10), immunoglobulins (IgA, IgG and IgM) and hydroxyproline (Hyp) were detected by the corresponding reagent kits. The histopathologic changes of wound were observed with HE staining. Masson staining was used to observe the collagen fiber deposition in wound tissue. The angiogenesis of wound tissue was evaluated by immunohistochemical staining.

Compared with the CON group, the wound healing speed, collagen synthesis and angiogenesis speed were significantly accelerated in the WP group, LDF group and HDF group (P<0.05), and the effects were the most obvious in the HDF group. On the 3rd day after surgery, in the WP, LDF and HDF groups, the serum ALB, PA and TP levels were significantly increased (P<0.05), serum TNF-α and IL-1β levels were obviously decreased (P<0.05), and serum IL-10 level was notably increased (except WP group) when compared with the CON group. The serum IgG and IgM levels were significantly increased in the LDF group and HDF group (P<0.05) and significant increment of IgA was only observed in the HDF group (P<0.05). On the 7th day after modeling, the levels of ALB, PA and TP were significantly increased (P<0.05), the levels of TNF-α, IL-1β and IL-6 were obviously decreased (P<0.05), and the levels of IL-10 were remarkably increased (P<0.05) in the HDF group than the CON group; The LDF group and HDF group had significantly elevated serum IgG and IgM levels (P<0.05), but only the former group had statistically increased serum IgA level (P<0.05). On the 14th day after modeling, the HDF group had significantly increased levels of ALB and TP (P<0.05), decreased levels of IL-1β (P<0.05), and raised levels of IgG and IgM when compared with the CON group (P<0.05).

Our compound nutrition preparation promotes wound healing in rat model of acute trauma, which might be related to its improving the nutritional status, promoting collagen synthesis, and thus alleviating inflammatory response and enhancing immune function.

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.

To explore the role of indole-3-propionic acid (IPA) in the pathogenesis of metabolic associated fatty liver disease (MAFLD) induced by high-fat diet (HFD) in order to reveal the role and related mechanism of adipose tissue metabolism in the process.

A mouse model of MAFLD was induced by HFD. Male C57BL/6J mice (6~7 weeks old) were randomly divided into control group (CON), HFD group, and HFD+IPA intervention group (HFD+IPA). The CON group was fed with control diet, and the HFD group and HFD+IPA group were fed with 60% of high-fat diet. The experiment period was 12 weeks, and IPA was administered at 20 mg/(kg·d) for 6 weeks starting from the 7th week. The body weight and food intake of each group were monitored weekly. After the intervention, the body composition of mice was detected by animal body composition analyzer. After the mice were euthanized, the morphological and structural changes in the liver and adipose tissues were observed by HE staining, the indicators relevant to lipid metabolism in the serum, liver and adipose tissues were detected by automatic blood biochemical analyzer and biochemical kits, and the mRNA expression changes of lipid metabolism and inflammation related genes were detected by qRT-PCR.

Compared with the CON group, the HFD group had significantly increased body weight and body fat percentage, obvious lipid deposition in the liver, obviously elevated serum alanine aminotransferase, aspartate aminotransferase, liver triglyceride and total cholesterol levels (P<0.05), and raised mRNA levels of liver fatty acid transporter CD36 (P<0.05), while IPA intervention significantly reversed the above changes (P<0.05). IPA intervention significantly inhibited the HFD-induced enlargement of visceral and brown fat cells, reduced the content of visceral adipose tissue (VAT) and serum level of free fatty acids (P<0.05), and increased the mRNA expression levels of VAT lipolysis (HSL, CGI58), browning genes (Cidea, ND5, UCP1, Prdm16) (P<0.05), as well as those of brown adipose tissue (BAT) lipolysis (HSL, ATGL) and fatty acid beta oxidation (Cpt1a, PPARα) genes (P<0.05). Meanwhile, the mRNA levels of TNF-α, IL-1β, CXCL1 and CCL2 in VAT and BAT were decreased after IPA intervention (P<0.05).

IPA can improve the occurrence of MAFLD induced by HFD, and its mechanism may be closely associated with its regulation of BAT and VAT morphology, and the mRNA expression of metabolic function and inflammation related genes.

To investigate the effects of a 45% high-fat diet (HFD) with isocaloric intake on energy metabolism and endurance exercise capacity in SD rats.

Twenty-four male SD rats were randomly divided into normal chow diet group (CON), HFD group, normal chow diet+exercise training group (CONT), and HFD+exercise training group (HFDT). The CON and CONT groups received normal chow diet, while the HFD and HFDT groups received a 45% high-fat diet with isocaloric intake. The HFDT and CONT groups underwent an endurance training of moderate-intensity running for 6 weeks. Body weight, fat mass, and lean mass were measured weekly. Energy expenditure and basal metabolic rate during rest and exercise states were measured using Pheno Master/Calo Treadmill system. Blood glucose, lipids, and creatine kinase levels were detected after the exhaustion test.

In 6 weeks after intervention, the endurance exercise capacity was significantly enhanced in the HFDT group than the CONT group (P<0.05). There were no obvious differences in body weight and body composition among the groups under isoenergetic feeding conditions. At rest, no statistical differences were observed in total energy expenditure and basal metabolic rate among the groups. However, prior to the 4th week, the CON group primarily metabolized carbohydrates while the HFD group primarily metabolized fats. But the carbohydrate metabolism was decreased and then increased, and the substrate metabolism rates eventually reached similar levels between the 2 groups on the 5th to 6th week. The HFDT group primarily metabolized fats while the CONT group primarily metabolized carbohydrates, with significant differences persisting after 6 weeks of training (P<0.05). HFD led to elevated levels of serum cholesterol, triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), but, endurance training resulted in decreased lipid levels in the HFDT group, accompanied by an increase in β-hydroxybutyrate (βHB) level (P<0.05). Isoenergetic diets had no significant differences in their effects on liver and kidney function or muscle damage indicators.

An isoenergetic HFD can improve fat utilization ability and extend endurance exercise time in rats without altering body composition or affecting liver and kidney function.

To develop a nutritional formula on enhancing the endurance of heavy load exercise, and evaluate its efficacy comprehensively.

Sixty C57BL/6J male mice were randomly divided into control group (CON group) and low-, medium- and high-dose nutritional formula groups (LDF, MDF and HDF groups), with 15 mice in each group. Each group received intervention with nutritional formula at corresponding dose for 2 weeks, and underwent adaptive training and heavy load exercise in the 1st and 2nd weeks, respectively. Exhaustion exercise time, skeletal muscle antioxidant indicators (SOD, MDA, PC and GSH), fatigue related indicators (serum URA, LDH and LA), muscle glycogen, and serum exercise injury related indicators (ALT, AST, CK and CK-MB) were measured and detected in the mice, and comprehensive evaluation was conducted according to relevant evaluation standards.

The LDF group, MDF group and HDF group had significantly prolonged running exhaustion time than the CON group (P<0.05), with the HDF group showing the greatest improvement (P<0.05). Compared with the CON group, the activities of SOD and GSH in the skeletal muscles were significantly increased (P<0.05), while the levels of MDA and PC in skeletal muscles were obviously decreased in the 3 doses of nutritional formula groups (P<0.05). PAS staining of the skeletal muscles displayed that the glycogen content was significantly increased in the MDF group and the HDF group than the CON group (P<0.05), and the highest increase was observed in the HDF group (P<0.05). Biochemical test revealed that the levels of LDH, LA, ALT, AST, CK, and CK-MB were remarkably lower in the 3 doses of nutritional formula groups than the CON group (P<0.05).

The nutritional formula can significantly improve the endurance and skeletal muscle antioxidant capacity in mice under heavy load exercise, and has anti-fatigue and -injury protection effects. This nutritional formula can be used to support physical fitness during heavy load endurance exercise.

To construct a model of Fat-1/APPPS1 transgenic mice and a cellular model of microglia and explore the improvement effect and underlying mechanism of n-3 polyunsaturated fatty acids (n-3 PUFAs) on the learning and memory abilities of APPPS1 mice by regulating microglial activation and polarization.

After the male mice with heterozygous Fat-1 genotype were mated with the female ones with heterozygous APPPS1 genotype, genetic identification was used to screen the male offspring with Fat-1/APPPS1 genotype. Then after the male wild-type (WT) mice and those with Fat-1, Fat-1/APPPS1, and APPPS1 genotypes were bred until 9 months old, their learning and memory abilities were evaluated with Morris water maze (MWM) test. In addition, gas chromatography-mass spectrometry (GC-MS) was performed to detect the concentration of PUFAs in the brain, and immunohistochemistry (IHC) was applied to detect the deposition of β-amyloid protein (Aβ) in the hippocampal regions. Moreover, immunofluorescence assay, qRT-PCR, and enzyme-linked immunosorbent assays (ELISA) were conducted to measure inflammation, and transcription and expression of Iba-1 (indicating the microglial activation) and CD86 and CD206 (indicating microglial polarization) in central nervous system (CNS). After pretreated with DHA+EPA (25 μmol/mL ∶25 μmol/mL), microglial model of inflammatory injury was established in immortalized mouse BV2 cells induced by LPS (1 μg/mL). Afterwards, immunofluorescence assay, qRT-PCR and Western blotting were used to detect inflammation and microglial activation and polarization.

Compared with the APPPS1 mice, endogenous n-3 PUFAs effectively improved the learning and memory disorders in Fat-1/APPPS1 ones (P<0.05), remarkably alleviated Aβ deposition in the hippocampal regions (P<0.05), evidently reduced CNS inflammation and microglial activation (P<0.05) and transformed the activated microglia from M1 to M2 (P<0.05). Furthermore, BV2 cells with DHA+EPA pretreatment obviously resisted LPS-induced cellular inflammation and induced activated ones from M1 to M2 (P<0.05).

n-3 PUFAs inhibit the microglial activation, regulate the microglial polarization from M1 to M2, reduce CNS inflammation, and thus alleviate learning and memory disorders in APPPS1 mice.

To identify the effect of acute stress induced by composite factors on intestinal barrier dysfunction and explore the relevant mechanism.

Twenty male C57BL/6 mice (7-8 weeks old, body mass 20-22 g) were randomly divided into control group (CON group, n=10) and model group (MOD group, n=10). To establish an acute stress model of complex factors, the mice of the MOD group were given rest of 4 h, sleep deprivation of 15 h, and then followed by 3 h of noise (120 dB)/flash (2 000±500 lx) and 2 h of forced swimming (water temperature 26±1 ℃, water depth 45 cm) within 24 h. While the mice in the CON group were placed in a noiseless environment with constant temperature and humidity and normal circadian rhythm but been given no any treatment. At the end of forced swimming, the mice from the MOD group were anesthetized for blood samples and then euthanized. Serum markers of intestinal injury were detected by immunoenzyme-linked adsorption assay kit, morphological changes in colonic tissues were observed by HE staining, protein and mRNA levels of colonic tissue barrier function related molecules and inflammatory factors were detected by immunofluorescence, immunohistochemistry and RT-qPCR, and intestinal flora changes were analyzed by 16S rRNA technique.

Compared with the CON group, the serum markers of intestinal damage, lipopolysaccharide (LPS), intestinal fatty acid binding protein (I-FABP) and Zonulin were significantly increased in the MOD group (P < 0.05). HE staining showed that intestinal tissue damage was obvious in the MOD group. The results of immunofluorescence staining and RT-qPCR showed that the levels of mechanical barrier related molecules Zona occludens 1 (ZO-1) and Occludin were obviously decreased (P < 0.05). Immunohistochemistry and PAS staining indicated the number of chemical barriers associated goblet cells, and expression of mucin2 (Muc-2), antimicrobial peptide regenerating islet derived-3γ (Reg3γ) and regenerating islet derived-3β (Reg3β) were notably reduced (P < 0.05). Intestinal flora β diversity significantly changed, with harmful bacteria Bacteroidetes and Desulfovibrio significantly increased, while beneficial bacteria Rumenococcus family, norank_f_Erysipelotrichaceae, Gemella genus and Erysipelotrichaceae genus declined (P < 0.05). The levels of TNF-α, IL-6 and ROS were significantly enhanced (P < 0.05).

Acute stress of complex factors significantly induces intestinal injury and barrier dysfunction.