In a recent study published in the journal Dr Scientific report, Researchers investigated the effect of inulin, a prebiotic, on gut microbial assembly and, consequently, skeletal muscle catabolism using murine models. Inulin is a naturally occurring carbohydrate found in more than 36,000 plant species, such as agave, asparagus, bananas, chicory, garlic, Jerusalem artichokes, onions, and wheat.
Laboratory rats suffered third-degree burns over 30% of their total body surface area (TBSA) and were divided into sham, case, and control groups. Experimental methods include immunofluorescence (IF) staining, immunohistochemistry (IH), and TUNEL staining for burn and atrophy visualization, and Western blotting, qRT-PCR, and metagenomic sequencing for gut microbiome characterization and assessment.
The results of the study revealed that burn injuries result in persistent and severe muscle wasting, weight loss, and protein breakdown. These observations can be traced back to the gut microbiota, especially changes in the general ratio between abundances. Firmicutes And Bacteridates (F/B ratio). Inulin has been found to support and increase Firmicutes– and reduce Parabacteroid distasonis accumulation, stabilizes the gut microbiome and reverses burn-induced weight loss and skeletal muscle atrophy.
Study: Dietary supplementation with inulin improves burn-induced skeletal muscle atrophy by regulating gut microbiota disorders. Image credit: Dorota Mileage/Shutterstock
Can the gut microbiome treat burns?
Burns, especially severe third-degree, are known to cause significant and persistent skeletal muscle degeneration and, more recently, have been reported to have profound and long-term effects on gut microbial assemblages. Skeletal muscle wasting is one of the central factors in burn pathophysiology that contributes both during recovery and to the severity of symptoms after recovery. This wasting is exacerbated by stress, systemic inflammatory responses, and decreased levels of physical activity, thereby delaying burn healing.
The gut microbiome, also called ‘gut flora’ and ‘gut microbiota’, refers to the entire assemblage of bacteria, fungi, archaea, and viruses that inhabit the digestive tract of animals. The gut microbiome plays an important role in health, with changes in community composition having particularly significant effects on the neuronal, digestive and cardiovascular systems. Recent studies have uncovered the role of burns in altering the diversity and abundance of gut microbiota, resulting in a significant reduction in probiotic genera and a corresponding increase in opportunistic pathogens.
Previous animal model experiments have illustrated that burn may contribute to microbial community dysbiosis and intestinal barrier dysfunction. Unfortunately, no study has yet explored the link between microbial dysbiosis and burn-induced muscle atrophy, leaving the mechanistic basis of this interaction unknown.
Inulin is a class of naturally occurring sugars (polysaccharides) belonging to fructans (fruit sugars). Previous studies have reported modulatory effects of these compounds on regulation Bacteridates And Bifidobacteria Microbiome populations. Separate work has elucidated the potential anti-inflammatory and muscle-damaging properties of inulin, highlighting it as a potentially safe and natural intervention to improve the quality of life (QoL) of burn victims.
About the study
The present study aimed to investigate the effects of inulin supplementation on gut microbiota dysbiosis and muscle atrophy in burned rats. Male Sprague-Dawley rats (mean age = seven weeks; mean weight = 195 g). Rats were divided into four experimental groups, namely 1. sham burn (S), 2. burn (B), 3. intervention (I), and 4. validation (V). All groups (except S) underwent anesthesia, followed by third-degree scald wounds of 30% total body surface area (TBSA).
Four days after the experiment, mice were euthanized to allow histochemical evaluation. These assessments included immunofluorescence (IF) staining to identify myofiber borders in the tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius (GAS) muscles. Immunohistochemistry (IH) investigation was then conducted to estimate the relative expression levels of ZO-1, occludin and claudin proteins from the excised tissue samples.
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining assay was used to detect DNA breaks when DNA fragmentation occurs during late stages of apoptosis. The characteristics of apoptotic cells were recorded and analyzed using high-resolution photography and ImageJ, respectively.
Protein and metagenomic sequencing analyzes were employed to characterize real-time gut microbial composition and metabolite secretion and monitor any changes during the study period. These include quantitative real-time polymerase chain reaction (qRT‒PCR), western blot, and enzyme-linked immunosorbent assay (ELISA).
Examination of daily rat weight, food intake, muscle weight, and fat weight showed that groups B and V lost significant body weight for the first six days after the burn procedure, after which the weight gradually increased. Weight loss was highest in group B, representing 7.28% of baseline weight. In contrast, inulin was shown to have a protective effect against weight loss, with group I losing only 2.94% of weight over the same period. After sham (no) burn, the S group presented a slower upward trend in weight gain.
Taxonomic analysis of gut microbiota in different treatment groups. (A) sparse curve analysis in four groups. (b) alpha diversity of four cohorts at the species level, measured in terms of the Chao1 index. (c) Beta diversity of four cohorts at the species level. (d) bar plot of taxonomic analysis among four groups at the taxonomic level of phylum. (e) bar plot of taxonomic analysis among four groups at the taxonomic level of species. The horizontal axis is the group, and the vertical axis is the relative abundance of specific species. (f, gLEfSe analysis LDA value data at each taxonomic level within different groups. The ordinate shows species with significant differences in LDA values greater than the current value (2.0) in different groups, i.e. biomarkers with significant differences. The length of the bar in the chart represents the effect of significantly different species. (h) Relative abundance of P. distasonis species in different groups. P values were determined by a two-tailed Wilcoxon rank-sum test, and data are presented as means ± SEM; *p<0.05. S Sham, B Burn, I Intervention, V Validation, LEfSe Linear Discriminant Analysis Effect Size, LDA Linear Regression Analysis, p. Distasonis parabacteroides_distasonis, SEM standard error of the mean.
Food intake assessments reported increasing consumption trends across groups B, I and V. Inulin’s benefits were more pronounced with the highest food consumption observed in the I cohort, suggesting that carbohydrates may indirectly accelerate recovery. Gene- and protein-level experiments revealed approximately threefold higher expression of muscle atrophy-related F-box (MAfbx) and muscle atrophy-related F-box (MuRF1) in the burn groups compared to the sham cohort. However, significantly lower MuRF1 and MAFbx levels were found in group I than their other burn cohorts, suggesting that inulin downregulates protein expression in the ubiquitin-proteasome signaling pathway.
Cytokine and intestinal tight junction (TJ) expression levels were further altered by inulin supplementation. Intestinal TJ, occludin and ZO-1 expression levels were found to be significantly lower in severe burns, but these values were significantly dampened and comparable to S H-scores in group I. Rarefaction analysis highlighted the phenomenon that inulin increases the alpha diversity of the gut microbial community and helps those with dysbiosis to return to baseline.
“Compared to the burn group, inulin increased the relative abundance of Firmicutes and Actinobacteria (30% and 3.3%, respectively) and decreased the abundance of Proteobacteria (9.7%) in the intervention group. We observed significant differences in the relative abundance of species across treatment groups. These results varied indicating differences in the gut microbial communities of mice in treatment groups.”
Functional analysis of the gut microbial community compared to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database presented that inulin was able to alter the functional repertoire of gut microbiota, resulting in higher expression of metabolic pathways involved in amino acid biosynthesis. Inulin further promoted skeletal muscle protein synthesis through modified phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling.
The present study investigates the association between the gut microbial community and skeletal muscle atrophy after severe burns. The results of the study indicate that burn causes a strong disruption in gut microbial assemblages, thereby altering systemic-level protein expression. Inulin, a plant-derived polysaccharide, has been shown to reverse these adverse effects, promote burn healing, and reduce skeletal muscle atrophy.
“These insights represent a critical first step and highlight the need to evaluate whether interventions targeting the gut microbiota could be a strategy for the treatment of burn skeletal muscle atrophy. Given the limited efficacy of current therapies and the significant impact of skeletal muscle atrophy on prognosis. Quality of life in burn patients, Innovative strategies to reverse skeletal muscle atrophy are urgently needed.”
- Sing, sing, etc. “Dietary supplementation with inulin improves burn-induced skeletal muscle atrophy by regulating gut microbiota disorders.” Scientific Reports, Vol. 14, no. 1, 2024, pp. 1-14, DOI: 10.1038/s41598-024-52066-8 https://www.nature.com/articles/s41598-024-52066-8