Heat stroke (HS) models in rats are associated with severe intestinal injury, which is often considered as the key event at the onset of HS. Probiotics can regulate the gut microbiota by inhibiting the colonization of harmful bacteria and promoting the proliferation of beneficial bacteria. Here, we investigated the preventive effects of a probiotic Bacillus licheniformis strain (BL, CMCC 63516) on HS rats as well as its effects on intestinal barrier function and gut microbiota. All rats were randomly divided into four groups: control (Con) + PBS (pre-administration with 1 ml PBS twice a day for 7 days, without HS induction), Con + BL group (pre-administration with 1 ml 1 × 10⁸ CFU/ml BL twice a day for 7 days, without HS induction), HS + PBS (PBS, with HS induction), and HS + BL (BL, with HS induction). Before the study, the BL strain was identified by genomic DNA analysis. Experimental HS was induced by placing rats in a hot and humid chamber for 60 min until meeting the diagnostic criterion of HS onset. Body weight, core body temperature, survival rate, biochemical markers, inflammatory cytokines, and histopathology were investigated to evaluate the preventive effects of BL on HS. D-Lactate, I-FABP, endotoxin, and tight-junction proteins were investigated, and the fluorescein isothiocyanate-dextran (FD-4) test administered, to assess the degree of intestinal injury and integrity. Gut microbiota of rats in each group were analyzed by 16S rRNA sequencing. The results showed that pre-administration with BL significantly attenuated hyperthermia, reduced HS-induced death, alleviated multiple-organ injury, and decreased the levels of serum inflammatory cytokines. Furthermore, BL sustained the intestinal barrier integrity of HS rats by alleviating intestinal injury and improving tight junctions. We also found that BL significantly increased the ratios of two probiotic bacteria, Lactobacillus and Lactococcus. In addition, Romboutsia, a candidate biomarker for HS diagnosis, was unexpectedly detected. In summary, BL pre-administration for 7 days has preventative effects on HS that may be mediated by sustaining intestinal barrier function and modulating gut microbiota.

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