Investigation of the Active Ingredients and Mechanism of Hudi Enteric-Coated Capsules in DSS-Induced Ulcerative Colitis Mice Based on Network Pharmacology and Experimental Verification

Abstract

Background: Hudi enteric-coated capsule (HDC) is a Chinese medicine prescribed to treat ulcerative colitis (UC). However, its anti-inflammatory active ingredients and mechanisms remain unknown. This study aimed to investigate the active components of HDC and explore its potential mechanisms against UC by integrating network pharmacology and experimental verification.

Methods: A DSS-induced colitis murine model was established to validate the efficacy of HDC by detecting disease activity index (DAI) and histopathological changes. Network pharmacological analysis was performed to identify the active compounds and core targets of HDC for the treatment of UC. The main compounds in HDC were identified by high-performance liquid chromatography. The relative expressions of HDC's core targets were also determined in vivo. Finally, molecular docking was applied to model the interaction between HDC and target proteins.

Results: In an in vivo experiment, HDC, especially the middle-dose HDC, effectively reduced clinical symptoms of UC, including weight loss, bloody stool, and colon shortening. Besides, the severity of colitis was considerably suppressed by HDC as evidenced by reduced DAI scores. A total of 118 active compounds and 69 candidate targets from HDC closely related to UC progression were identified via network pharmacology. Enrichment analysis revealed that the key targets of HDC correlated with the expressions of PTGS2, TNF-α, IL-6, and IL-1β. Meanwhile, these cytokines were enriched in various biological processes through the IL-17/JAK2/STAT3 signaling pathway. The middle-dose HDC contributed more to ameliorating DSS-induced colitis through this signaling pathway than other dosages. Nine components binding to JAK2, STAT3, IL-17 and IL-6 were identified by molecular docking, confirming again the inhibition effects of HDC on the IL-17/JAK2/STAT3 signaling pathway.

Conclusion: The HDC treatment, particularly the middle-dose, exerted an anti-UC effect in a multi-component, multi-target, and multi-mechanism manner, especially inhibiting the IL-17/JAK2/STAT3 signaling pathway to downregulate the secretion of proinflammatory cytokines.

Keywords: Hudi enteric-coated capsule; IL-17/JAK2/STAT3 pathway; network pharmacology; ulcerative colitis.

Graphical abstract

Figure 1

HDC attenuates the development of DSS-induced colitis. (A) The time course of DSS administration and different treatment in mice. C57BL/6 mice were provided with water or 2.5% DSS-containing water from day 1 to 7. At the same time, mice were orally administered with different dosages of HDC or 5-ASA. (B) Body weight changes in each group. (C) DAI scores in each group. (D) Images of colonic tissues in each group. (E) Colonic lengths in each group. HDC L, HDC M, and HDC H indicate 40, 80, and 160mg/kg dosage of HDC, respectively. Data are represented as mean ± SEM (n = 4–6). ^###P < 0.001, compared with the control group; *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the DSS group.

Figure 2

The H&E staining of colonic tissues (400×). (A) The control group. (B) The DSS group. (C) The low-dose HDC group. (D) The medium-dose HDC group. (E) The high-dose HDC group. (F) The 5-ASA group. n = 6.

Figure 3

Construction of candidate-target network for HDC against UC. (A) Distribution of HDC potential targets and UC targets. (B) Herb-compound-target network of HDC. Orange diamonds indicate targets. The colorful octagons show the major components of seven herbs: Bai Hua She She Cao (green), Bai Jiang Cao (pink), Bai Ji (blue), Di Yu (yellow), Zhu Sha Qi (deep red), Gan Cao (yellow-green), and HuZhang (light pink), respectively. Grey lines indicate the interrelationships between compounds and targets.

Figure 4

PPI analysis of candidate targets for HDC against UC. Network of 69 key targets based on central network evaluation. The size of nodes is proportional to the degree of centrality by the topology analysis.

Figure 5

Enrichment analysis of candidate targets for HDC against UC. (A) GO enrichment analysis for 69 key targets. (B) KEGG enrichment analysis for 69 key targets. (C) Ingredient-target-pathway network. Pink squares, ingredients; green rounds, protein targets; blue triangles, pathway. The color and size of the nodes reflect the degree value.

Figure 6

HPLC fingerprinting of the major compounds of HDC. (A) HPLC profiles of single standards. (B) HPLC profiles of mixed standards. (C) HPLC profiles of HDC aqueous extract. (D) Structures of the analytes and internal standards, and the sequence is consistent with (A).

Figure 7

The mRNA relative expressions of inflammatory cytokines in colonic tissues assessed by RT-qPCR. (A) IL-1β, (B) IL-6, (C) TNF-α, (D) PTGS2, and (E) IL-17. Data are represented as mean ± SEM (n = 3–6) of three parallel measurements. ^###P < 0.001, compared with the control group; *P < 0.05 and **P < 0.01, compared with the DSS group.

Figure 8

The protein relative expressions of p-JAK2, JAK2, p-STAT3, and STAT3 in colonic tissues assessed by Western Blot. (A) Representative Western blot images. Western blot quantification analysis of p-JAK2/JAK2 (B) and p-STAT3/STAT3 (C). Data are represented as mean ± SEM (n = 3) of three parallel measurements. ^##P < 0.01 and ^###P < 0.001, compared with the control group; *P < 0.05, **P < 0.01, ***P < 0.001 and ***P < 0.0001, compared with the DSS group.

Figure 9

Molecular docking of HDC compounds establishing interactions with target proteins. Binding modes of HDC compounds, including polydatin, gallic acid, quercetin, naringenin, luteolin, kaempferol, isorhamnetin, and formononetin, with (A) JAK2, (B) STAT3, (C) IL-17, and (D) IL-6. (E) Binding force scores for HDC compounds and target proteins.