Differentiating Damp‐Heat and Cold‐Damp Diarrhea in Rat Models via Gut Microbiota Dysbiosis and Short‐Chain Fatty Acid Profiling

On the basis of gut microbiota and short-chain fatty acids (SCFAs), this study aims to identify diagnostic biomarkers for damp-heat diarrhea and cold-damp diarrhea. Rat models of damp-heat diarrhea and cold-damp diarrhea were established. Changes in body weight, body temperature, food intake, water consumption, and the diarrhea index were recorded. ELISA was employed to detect levels of IL-6, IL-1β, TNF-α, and IL-10. Histological evaluations were conducted using H&E staining and AB-PAS staining techniques. Transmission electron microscopy was utilized to observe ultrastructural changes in the colonic epithelium, while Western blot analysis was performed to assess the expression of Occludin, Claudin1, Claudin5, GPR41, GPR43, GPR109A, and NLRP3 in colon tissues. GC-MS analysis was carried out to determine the content of SCFAs in the cecal contents of rats; additionally, 16S rRNA sequencing was performed to analyze the composition of gut microbiota in these animals. Differential analysis methods were applied to evaluate similarities and differences in SCFAs profiles and gut microbiota between damp-heat diarrhea and cold-damp conditions. The body weight and food intake of rats with induced damp-heat diarrhea or cold-damp diarrhea significantly decreased over time as their diarrheal symptoms progressively worsened. However, following treatment with appropriate prescriptions tailored for each condition resulted in an improvement in diarrheal symptoms among the affected rats. In accordance with the "prescription-based syndrome differentiation" theory, the rat experimental animal models of damp-heat diarrhea and cold-dampness diarrhea were successfully established. The models exhibited characteristic diarrheal symptoms alongside increased levels of inflammatory factors indicative of severe histopathological damage; there was also a notable reduction in tight junction protein expression observed across all models studied. Furthermore, the Firmicutes/Bacteroidota ratio showed a significant decrease. Interestingly, differences between damp-heat diarrhea and cold-damp diarrhea manifested as follows: Both modeling groups showed an increase in the relative abundance of Lachnoclostridium and Marvinbryantia. In the damp-heat diarrhea group, the levels of Lachnoclostridium and Marvinbryantia were relatively low; however, these levels gradually increased after successful treatment. In contrast, in the cold-damp diarrhea group, the trends of Lachnoclostridium and Marvinbryantia were opposite. Mucosal color has the potential for clinical diagnosis of damp-heat diarrhea and cold-damp diarrhea. Moreover, Lachnoclostridium and Marvinbryantia are potential biomarkers for distinguishing between damp-heat diarrhea and cold-damp diarrhea. However, the diagnostic basis and accuracy of Lachnoclostridium and Marvinbryantia biomarkers still need to be further validated.