Exploring the regulatory mechanisms of new Jinzhi on gut microbiota in heat-constipation type constipation rats via integrated 16S rRNA sequencing and metabolomics
GUO Zhi-yue,XIE Yi-xuan,LIU Shuang
Abstract:
Objective To elucidate the therapeutic mechanisms of new Jinzhi (fecal microbiota transplantation, FMT) in heat-constipation type constipation rats by integrating 16S rRNA sequencing and untargeted metabolomics to analyze gut microbiota and metabolic alterations and the enriched pathways of gut metabolites. Methods Thirty-two female Wistar rats were acclimatized for one week and then randomly allocated into four equal groups: Model group, FMT group, Lactulose group, and Blank control group. Rats in the FMT group were first rendered pseudo-germ-free using an antibiotic cocktail protocol (a mixture of bacitracin, neomycin, and streptomycin). Subsequently, the FMT group, Lactulose group, and Model group received a combination of heat-inducing Chinese herbs (Aconitum carmichaelii, Zingiber officinale, Evodia rutaecarpa, Cinnamomum cassia, Piper nigrum; crude drug concentration 20 g/kg) and loperamide hydrochloride (2 mg/kg) via gavage for two consecutive weeks to establish the heat syndrome constipation model. Following model establishment, interventions were administered for two weeks: the Lactulose group (positive control) received lactulose (10 mL/kg), and the FMT group received fecal microbiota suspension (10 mL/kg). Fecal water content and intestinal charcoal propulsion rate were calculated and compared among all four groups. Fecal samples from the Model group, FMT group, and Blank control group were subjected to 16S rRNA sequencing to characterize the gut microbiota and untargeted metabolomics to analyze enriched metabolic pathways. Results Compared to the blank group, the model group exhibited reduced fecal water content, decreased carbon propulsion rate, diminished Bacteroidota abundance, and abnormal proliferation of Proteobacteria (a conditional pathogen). FMT intervention significantly improved fecal water content and propulsion rate, restored microbial evenness (Shannon index) and Firmicutes/Bacteroidota ratio to near-baseline levels. LEfSe analysis identified Prevotellaceae and Rikenellaceae as signature taxa in the FMT group. Metabolomic profiling revealed significant enrichment of core metabolic pathways, including lipid metabolism, energy metabolism, and amino acid metabolism. Conclusion New Jinzhi alleviates heat-constipation by remodeling gut microbiota diversity, balancing microbial structure, activating metabolic functions, and reconstructing intestinal microecological homeostasis.
