Distinctive and Synergistic Gut Microbiota Regulation by Combined Treatment of Hyperthermia and Fish Oil Intake in Old Female and Ovariectomized Mice

Objectives: Emerging evidence strongly links gut microbiota with host metabolism, highlighting the pivotal roles of diet and lifestyle modification. Hyperthermia (HT) is a therapeutic strategy that shows promise in mitigating obesity and inflammation associated with aging, especially when paired with fish oil supplementation. However, the influence of gut microbiota in mediating these metabolic advantages remains unclear. This study aims to explore the effects of HT, both alone and in combination with fish oil supplements, on the gut microbiota of aged and ovariectomized (OVX) mice. Methods: Female C57BL/6 mice, both aged ten months and OVX, were divided into groups (n=6 each) and subjected to a 6-12 week high-fat diet (HF, 45% calories from fat), with or without fish oil supplementation. For lifestyle modification, mice underwent daily 30-minute whole-body HT at 40 °C. Cecal content was collected for 16s rRNA sequencing. Qiime2 and R were used for sequencing data analysis and visualization. The Spearman-ranked correlation was performed to assess the relationship between the abundance of the bacterial genus and host metabolic factors. Results: HT alone significantly lowered core body temperature (∼0.5°C) across all diets, leading to a reduced Firmicutes to Bacteroidetes ratio and altered gut microbiota richness and phylum composition. Independently, fish oil supplementation enhanced the abundance of Rosburia, a genus known for short-chain fatty acid production. Both HT and fish oil intake correlated with a decrease in Aplistipes, implicated in obesity and inflammation pathways. Notably, combining HT with fish oil supplementation provided enhanced protection against diet-induced obesity, insulin resistance, and lipopolysaccharide-triggered acute infections (p< 0.05). Conclusions: HT and dietary intervention with fish oil uniquely affected gut microbiota diversity and composition, offering combined metabolic and immune system benefits against the challenges of a high-fat diet and infection in aged and OVX mice. This study presents preclinical evidence supporting the role of HT and fish oil in promoting a healthy gut microbiota, crucial for combating metabolic and immune vulnerabilities induced by aging and menopause.