Molecular Assessment of Traditional Mongolian Medicine Tongola-5 in Lactulose Induced Diarrhea: An in vivo Study

Abstract

Propose: To elucidate the mechanisms underlying the therapeutic effects of Tonglaga-5 (TLG5) in the treatment of chronic diarrhea.

Methods: The chemical compositions of TLG5 in both in vitro and systemic circulation were analyzed using UPLC-Q/TOF MS^E. Network pharmacology was applied to predict the therapeutic effects of TLG5 on chronic diarrhea. Molecular docking was utilized to assess the binding affinity between key bioactive compounds and their corresponding targets. A total of 24 male Sprague-Dawley (SD) rats were randomly assigned to three groups: control, model, and TLG5 treatment. Corresponding administration included a standard diet, a high-lactose diet of varying concentrations, and TLG5 by gavage during modeling. Transcriptomic analysis was employed to assess gene expression changes in intestinal tissues. Alterations in the intestinal microbiota were evaluated using 16S rRNA sequencing. qPCR and immunohistochemistry technologies were adopted to analyze the expression of genes and proteins associated with intestinal barrier integrity. ELISA was performed to quantify levels of bile acids and short-chain fatty acids (SCFAs).

Results: UPLC-Q/TOF MS^E identified 118 compounds in vitro and 52 in the systemic circulation. Network pharmacological analysis revealed that the in vitro and in vivo components of TLG5 were associated with five core targets and five key compounds, respectively. RNA sequencing analysis showed that TLG5 treatment inhibited the inflammatory pathway. 16S rRNA sequencing demonstrated that TLG5 administration led to an upregulation of beneficial microbial populations and a concomitant downregulation of pathogenic bacteria. The TLG5 treatment group presented a reduction in necrotic areas and inflammatory cell infiltration, as well as preservation of the mucosal structure, with a marked decline in inflammatory lesions. Moreover, TLG5 treatment resulted in a significant increase in the levels of SCFAs and bile acids in the cecum.

Conclusion: TLG5 exerts therapeutic effects on chronic diarrhea through multiple mechanisms, including modulation of intestinal microbiota diversity, enhancement of intestinal barrier function, and attenuation of inflammatory responses.

Keywords: Mongolian medicine Tonglaga-5; chronic diarrhea; intestinal barrier; intestinal microbiota.

Graphical abstract

Figure 1

In Vitro Chemical Constituents of TLG5. (A) Total Ion Chromatogram (TIC) in positive ion mode. (B) TIC in negative ion mode (Z-FF indicates TLG5).

Figure 2

Network Pharmacology of In Vitro TLG5 Constituents in Chronic Diarrhea Treatment. (A) Venn diagram of in vitro TLG5 chemical constituent targets and chronic diarrhea disease targets. (B) Venn diagram of in vitro TLG5 chemical constituent targets and intestinal microbiota imbalance targets. (C) Venn diagram of in vitro TLG5 chemical constituent targets and intestinal barrier function targets. (D) PPI network of in vitro TLG5 chemical constituents targeting chronic diarrhea treatment. (E) PPI network of in vitro TLG5 chemical constituents targeting intestinal microbiota imbalance treatment. (F) PPI network of in vitro TLG5 chemical constituents targeting intestinal barrier treatment.

Figure 3

Analysis of In Vitro TLG5 Effective Chemical Constituent-Key Target-Related Disease Networks. (A) Analysis of the in vitro TLG5 effective chemical constituent-key target-chronic diarrhea network. (B) Analysis of the in vitro TLG5 effective chemical constituent-key target-intestinal microbiota imbalance network. (C) Analysis of the in vitro TLG5 effective chemical constituent-key target-intestinal barrier function network.

Figure 4

Functional Enrichment and Metabolic Pathway Analysis of In Vitro TLG5 Constituents in Chronic Diarrhea Treatment (and Related Symptoms). (A) GO analysis of potential targets of in vitro TLG5 chemical constituents for chronic diarrhea treatment (TOP 10). (B) GO analysis of potential targets of in vitro TLG5 chemical constituents for chronic diarrhea and related symptom treatment (TOP 10). (C) KEGG analysis of potential targets of in vitro TLG5 chemical constituents for chronic diarrhea treatment (TOP 30). (D) KEGG analysis of potential targets of in vitro TLG5 chemical constituents for chronic diarrhea and related symptom treatment (TOP 30).

Figure 5

Molecular Docking Results of In Vitro Active Compounds with Key Targets. (A) Molecular docking between STAT3 and Daucosterol. (B) Molecular docking between TNF and Isoquercitrin. (C) Molecular docking between IL-6 and Isoquercitrin. (D) Molecular docking between PIK3R1 and Isoquercitrin. (E) Molecular docking between PIK3CA and Daucosterol.

Figure 6

Transcriptomic Analysis of Colon Tissues in Rats with Chronic Diarrhea Treated with TLG5. (A) Volcano plot of DEGs between the control group and the model group. (B) Volcano plot of DEGs between the model group and the TLG5 treatment group. (C) Comparison of the relative fold changes among C, M and TLG5 using RNA-seq (* p < 0.05; **p < 0.01, ***p < 0.001, n=6). (D) Comparison of the relative fold changes among C, M and TLG5 using quantitative real time PCR (* p < 0.05; **p < 0.01, n=6). (E) KEGG enrichment analysis of downregulated DEGs in the model group compared to the control group. (F) KEGG enrichment analysis of downregulated DEGs in the TLG5 treatment group compared to the model group.

Figure 7

TLG5 improves microbial diversity and genus-specific abundance in rats with chronic diarrhea by remodeling intestinal flora structure. (A) Sparse curve analysis. (B) Shannon index comparison, (C) control group, M: model group, TLG5: TLG5 intervention group. (C) Wayne diagram analysis. (D) PCoA-based beta diversity. (E) Changes in genus-level abundance. (F) Heat map analysis: heat maps of relative abundance of species at the species level. (G) Histogram of LDA scores based on LEfSe analyses (LDA > 4 was considered to be a differential characteristic taxonomic unit); data are shown as means ± SDs (n = 6), differences between experimental groups were analyzed using a one-way ANOVA, and subsequent analyses were performed using Tukey’s multiple comparison test.

Figure 8

TLG5 Alleviates Chronic Diarrhea Symptoms, Enhances Intestinal Barrier Function, and Modulates Microbial Metabolites. (A) HE (×100) and Masson (×100) staining of rat colon tissues. (B) qPCR analysis of Claudin mRNA expression, with data presented as mean ± SD (* p < 0.05; **p < 0.01, n=6). (C) qPCR analysis of Occludin mRNA expression, with data presented as mean ± SD (* p < 0.05; **p < 0.01, n=6). (D) Immunohistochemical analysis of tight junction proteins (ZO-1, Claudin, and Occludin) in rat colon tissues (×100, n=6). (E) ELISA analysis of bile acid levels, with data presented as mean ± SD (* p < 0.05; ***p < 0.001, n=6). (F) ELISA analysis of SCFA levels, with data presented as mean ± SD (* p < 0.05; ***p < 0.001, n=6).

Figure 9

Network Pharmacology Analysis of In Vivo TLG5 Chemical Components. (A) Venn diagram of in vivo TLG5 chemical component targets and chronic diarrhea targets. (B) Venn diagram of in vivo TLG5 chemical component targets and inflammation targets. (C) PPI network of in vivo TLG5 chemical components targeting chronic diarrhea. (D) PPI network of in vivo TLG5 chemical components targeting inflammation.

Figure 10

Analysis of In Vivo TLG5 Active Component - Key Target - Associated Disease Networks. (A) Analysis of the in vivo TLG5 active component - key target - chronic diarrhea network. (B) Analysis of the in vivo TLG5 active component - key target - inflammation network.

Figure 11

Functional Enrichment Analysis of Intersection Targets. (A) GO analysis of potential targets for chronic diarrhea treatment associated with in vivo TLG5 chemical components (TOP10). (B) GO analysis of potential targets for chronic diarrhea and inflammation treatment associated with in vivo TLG5 chemical components (TOP10). (C) KEGG analysis of potential targets for chronic diarrhea treatment associated with in vivo TLG5 chemical components (TOP30). (D) KEGG analysis of potential targets for chronic diarrhea and inflammation treatment associated with in vivo TLG5 chemical components (TOP30).

Figure 12

Molecular Docking Results of In Vivo Active Components with Core Targets. (A) Molecular docking of HSP90AA1 and alpha-Onocerin. (B) Molecular docking of AKT1 and sitogluside. (C) Molecular docking of STAT3 and alpha-onocerin. (D) Molecular docking of MAPK1 and sitogluside. (E) Molecular docking of ESR1 and sitogluside.

Figure 13

Transcriptomic Analysis of Colon Tissues in Rats with Chronic Diarrhea Treated with TLG5. (A) KEGG enrichment analysis of upregulated DEGs in the control and model groups (TOP 20). (B) KEGG enrichment analysis of downregulated DEGs in the model and TLG5 treatment groups (TOP 20).

Figure 14

Inhibitory Effects of TLG5 on Inflammatory Cytokines Expression. (A)The level of inflammatory cytokine IL-1β. (B) The level of inflammatory cytokine IL-6. (C) The level of inflammatory cytokine TNF-α. (D) Expression of IL-1β mRNA in intestinal tissue. (E) Expression of IL-6 mRNA in intestinal tissue. (F) Expression of TNF-αmRNA in intestinal tissue.