Live-combined Bifidobacterium, Lactobacillus, and Enterococcus improves heat tolerance and attenuates systemic inflammatory injury in heat-stroke rats via modulation of gut microbiota and intestinal barrier

<bold>Objective</bold>To investigate the ameliorative effects of live-combined <italic>Bifidobacterium, Lactobacillus</italic>, and <italic>Enterococcus</italic> (LCBLE) on heat tolerance and systemic inflammatory injury in rats with heat stroke, and to explore its underlying mechanisms.<bold>Methods</bold>Rats were randomly divided into blank control, LCBLE control, model control, and LCBLE treatment groups. The LCBLE-treated groups received daily intragastric administration of LCBLE for 7 consecutive days, while the control groups received equal volumes of normal saline. On day 8, heat stroke was induced in the model control and LCBLE treatment groups. Core body temperature, body weight, and survival rate were monitored. Serum inflammatory cytokines were measured by enzyme-linked immunosorbent assay, and intestinal barrier function was assessed by blood biomarkers. Tight junction proteins zonula occludens-1 (ZO-1) and occludin were localized by immunofluorescence, intestinal ultrastructure was observed by transmission electron microscopy, and pathological morphology was evaluated by hematoxylin-eosin staining. Gut microbial composition was analyzed by 16S rRNA sequencing.<bold>Results</bold>LCBLE pretreatment significantly reduced peak core body temperature [(42.03±0.43) ℃ <italic>vs.</italic> (43.21±0.33) ℃, <italic>P</italic>&lt;0.01], attenuated post-model weight loss [(307.8±11.3) g <italic>vs.</italic> (286.7±12.6) g, <italic>P</italic>&lt;0.05 ], and improved 24 h survival rate (77.8% <italic>vs.</italic> 55.6%, <italic>P</italic>&lt;0.05) compared with the model control group. Serum levels of tumor necrosis factor-α, IL-6, and IL-1β were significantly lower in the LCBLE treatment group (all <italic>P</italic>&lt;0.01), along with reduced pathological scores in all intestinal segments (all <italic>P</italic>&lt;0.01). LCBLE also restored the expression and distribution of ZO-1 and occludin, preserved intestinal epithelial ultrastructure, and markedly decreased serum D-lactate, intestinal fatty acid-binding protein, and endotoxin levels (all <italic>P</italic>&lt;0.01). 16S rRNA sequencing revealed that LCBLE significantly increased the relative abundances of <italic>Bifidobacterium</italic> and <italic>Lactobacillus</italic> in the gut.<bold>Conclusion</bold>Pretreatment with LCBLE improves heat tolerance and alleviates systemic inflammation in heat-stroke rats, likely through regulating gut microbiota and maintaining intestinal barrier integrity.