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. 2022 Apr 5:13:844685.
doi: 10.3389/fphar.2022.844685. eCollection 2022.

Network Pharmacology-Based Strategy to Identify the Pharmacological Mechanisms of Pulsatilla Decoction against Crohn's Disease

Jinguo Liu  1 , Lu Zhang  1 , Zhaojun Wang  1 , Shanshan Chen  1 , Shuyan Feng  1 , Yujin He  2 , Shuo Zhang  3
Affiliations

Network Pharmacology-Based Strategy to Identify the Pharmacological Mechanisms of Pulsatilla Decoction against Crohn's Disease

Jinguo Liu et al. Front Pharmacol. .

Abstract

Purpose: To explore pharmacological mechanisms of Pulsatilla decoction (PD) against Crohn's disease (CD) via network pharmacology analysis followed by experimental validation. Methods: Public databases were searched to identify bioactive compounds and related targets of PD as well as related genes in patients with CD. Analyses using the drug-compound-target-disease network, the protein-protein interaction (PPI) network, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to predict the core targets and pathways of PD against CD. Colon tissue resected from patients with CD and tissue samples from a mouse model of CD fibrosis treated with PD were assessed to verify the major targets of PD in CD predicted by network pharmacologic analysis. Results: A search of the targets of bioactive compounds in PD and targets in CD identified 134 intersection targets. The target HSP90AA1, which was common to the drug-compound-target-disease and PPI networks, was used to simulate molecular docking with the corresponding bioactive compound. GO and KEGG enrichment analyses showed that multiple targets in the antifibrotic pathway were enriched and could be experimentally validated in CD patients and in a mouse model of CD fibrosis. Assays of colon tissues from CD patients showed that intestinal fibrosis was greater in stenoses than in nonstenoses, with upregulation of p-AKT, AKT, p-mTOR, mTOR, p-ERK1/2, ERK1/2, p-PKC, and PKC targets. Treatment of CD fibrosis mice with PD reduced the degree of fibrosis, with downregulation of the p-AKT, AKT, p-mTOR, mTOR, p-ERK1/2, ERK1/2, and PKC targets. Conclusion: Network pharmacology analysis was able to predict bioactive compounds in PD and their potential targets in CD. Several of these targets were validated experimentally, providing insight into the pharmacological mechanisms underlying the biological activities of PD in patients with CD.

Keywords: Crohn’s disease; Pulsatilla decoction; fibrosis; network pharmacology; pharmacological mechanisms.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Network pharmacology framework of Pulsatilla decoction for treatment of Crohn’s disease.
FIGURE 2
FIGURE 2
Network construction, PPI network core screening, and molecular docking simulation. (A) Venn diagram of the five databases surveyed. (B) Drug–compound–target–disease network. Red: Pulsatilla chinensis; blue: Phellodendron chinense; green: Coptis chinensis; yellow: Cortex fraxini. (C) PPI network. (D) Screening of the PPI network core. (E) 2D structure of aureusidin. (F) 3D conformer of aureusidin. (G) 3D conformer of HSP90AA1 and molecular docking. The black arrow indicates the position of molecular docking.
FIGURE 3
FIGURE 3
GO and KEGG enrichment analyses. (A) Barplot of GO annotation. (B) Bubble diagram of GO annotation. (C) Barplot of KEGG enrichment analysis. (D) Bubble diagram of KEGG enrichment analysis. (E) PI3K-AKT signaling pathway. (F) mTOR signaling pathway.
FIGURE 4
FIGURE 4
Clinical data of patients with CD. (A) Abdominal enhanced CT. Arrows: ileocecal high signal focus (coronal and cross-sectional imaging). (B) Colonoscopy: ileocecal stenoses. (C) Sirius red staining. Red represents collagen fibers and yellow indicates muscle fibers as determined by light microscopy. S, stenoses; NS, non-stenoses. Scale bar: 250 μm, magnification: ×200. Arrows: disordered arrangement of intestinal glands and deformation and reduction of goblet cells. (D) Sirius red staining of collagen deposition. (E) Masson’s staining. Blue indicates collagen fibers and red represents muscle fibers as determined by light microscopy. Scale bar: 100 μm, magnification: ×200. Arrows: disordered arrangement of intestinal glands and deformation and reduction of goblet cells. (F) Masson’s staining of collagen deposition. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
FIGURE 5
FIGURE 5
Expression of AKT/mTOR and PKCs/ERK1/2 in stenoses and nonstenoses of CD patients. (A) Western blotting showing the relative protein intensities of p-AKT, AKT, p-mTOR, mTOR, p-ERK1/2, ERK1/2, p-PKCs, PKCs, vimentin, and α-SMA. (B–D) Real-time quantitative qPCR showing the relative levels of (B) AKT and mTOR mRNAs, (C) PKCs and ERK1/2 mRNAs, and (D) vimentin and α-SMA mRNAs. (E,F) Immunohistochemistry of (E) PKCs and (F) ERK1/2. Scale bars: 50 μm, magnification: ×400. S, stenoses; NS, non-stenoses. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
FIGURE 6
FIGURE 6
Assessment of the mouse colonic fibrosis model. (A) Schematic overview of the model. OA: oral administration; Pre: TNBS presensitization; EA: TNBS enteral administration. (B) Mouse weight change over time. (C) Mouse colon length over time. Blue arrows: local expansion and deformation of the intestinal cavity; black arrows: thickening of the intestinal walls and intestinal stenoses. (D) Sirius red staining of collagen fibers. PD: administration of Pulsatilla decoction. Scale bar: 500 μm, magnification: ×40. (E) Sirius red staining showing whole-layer collagen deposition. (F) Masson’s staining. Blue indicates collagen fibers. Scale bar: 500 μm, magnification: ×40. (G) Masson staining showing whole-layer collagen deposition. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
FIGURE 7
FIGURE 7
Expression of AKT/mTOR and PKCs/ERK1/2 in the mouse model of colonic fibrosis. (A) Western blotting showing the relative protein intensities of p-AKT, AKT, p-mTOR, mTOR, p-ERK1/2, ERK1/2, PKCs, vimentin, and α-SMA. (B–D) Real-time quantitative qPCR, showing the relative levels of (B) AKT and mTOR mRNAs, (C) PKCs and ERK1/2 mRNAs, and (D) vimentin and α-SMA mRNAs. (E,F) Immunofluorescence of (E) PKCs and (F) ERK1/2. Scale bars: 750 μm, magnification: ×50. PD: Pulsatilla decoction. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
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