Mechanism of Fructus Mume Pills Underlying Their Protective Effects in Rats with Acetic Acid-Inducedulcerative Colitis via the Regulation of Inflammatory Cytokines and the VEGF-PI3K/Akt-eNOS Signaling Pathway

Abstract

Background: Fructus mume pills (FMPs) have been clinically proven to be effective for treating ulcerative colitis (UC). However, the therapeutic and protective mechanisms have not been fully studied.

Aim: We aimed to explore the mechanism of FMPs in an acetic acid (AA)-induced ulcerative colitis rat model.

Methods: The targets, GO terms, and KEGG pathways for the FMPs and UC were screened and constructed using network pharmacology. A possible mechanism was verified in a 4% AA-induced colitis rat model. Colitis activity and state were evaluated using the disease activity index, and colon ulceration and intestinal mucosal damage were determined by histopathological observation through HE, AB-PAS, and Masson pathological staining. The concentrations of TNF-α, IL-6, IL-8, IL-10, MPO, MMP9, CXCR1, eNOS, and VEGF were measured to evaluate vascular permeability effects.

Results: The network pharmacology results showed 108 active compounds, and 139 FMP-related targets were identified. Twenty-nine targets were identified for FMPs against UC, which included MMP9, MMP3, ESR1, PTGS1, PPARA, MPO, and NOS2. A total of 1,536 GO terms and 41 pathways were associated with FMP treatment of UC. The pharmacological evaluation showed that FMPs attenuated inflammation in AA-induced colitis by reducing the serum concentrations of TNF-α, IL-6, IL-8, and IL-10 and the colonic concentrations of MPO, MMP9, and CXCR1. FMPs ameliorated hyperpermeability by reducing the colonic VEGF and eNOS concentrations. FMPs also significantly decreased the VEGFA, VEGFR2, Src, and eNOS protein expressions in colon tissue through the VEGF-PI3K/Akt-eNOS signaling pathway.

Conclusion: These results suggest that FMPs control UC inflammation by regulating inflammatory cytokine concentrations. FMPs alleviate AA-induced UC by regulating microvascular permeability through the VEGF-PI3K/Akt-eNOS signaling pathway.

Figure 1

UPLC profiles of standards and FMP aqueous extract. (a) Chromatograms of citric acid, phellodendron hydrochloride, coptisine hydrochloride, berberine hydrochloride, ferulic acid, lobetyolin, cinnamic acid, hydroxy-α-sanshool, 6-gingerol, ß-asarum ether, and a-asarum standards under 285 nm detection wavelength. S1: standard chromatograms, S2 : FMP chromatograms. (b) Chromatograms of benzoylaconine, benzoyl neoaconitine, and benzoyl hypoaconitine standards under 235 nm detection wavelength. S3: standard chromatograms, S4 : FMP chromatogram.

Figure 2

Experiment process design. Control, model, H-FMP, and L-FMP groups were set up on day 1, UC rat model was established with 4% acetic acid on day 8, and FMP administration lasted for seven days. Pharmacological evaluation of FMP was conducted using the disease activity index, colon histopathological changes, UC basic pathological mechanisms, and network pharmacology results for predicted targets and pathways.

Figure 3

Herb-compound-target network for FMP. The FMP herb-compound-target network contained 10 herbs, 108 compounds, and 139 targets. The herbs are depicted as pink squares. The purple rectangle shapes represent compounds. The yellow triangle shapes represent FMP targets. The lines represent the relationship between the compounds and target nodes.

Figure 4

FMP-UC target network. (a) Distribution of FMP and UC targets. (b) FMP-compound-target-UC network, purple triangles representing shared targets between FMP and UC, Earth yellow octagons representing FMP compounds, and grass green representing herbs. (c) FMP-UC PPI network.

Figure 5

GO and KEGG enrichment results. (a) Top 10 BP enrichment. (b) Top 10 MF enrichment. (c) Top 10 CC enrichment. (d) KEGG pathway enrichment. This graph represents the interaction between enrichment paths, and the arrows indicate the upstream and downstream signal relationships between the pathways. (e) KEGG pathway distribution diagram.

Figure 6

Dynamic changes in the characteristic manifestations of each group of rats. (a) Bodyweight changes. (b) Rectal blood changes. (c) Diarrhea changes. (d) DAI score. ^∗∗p < 0.001 compared with the control group, ^∗p < 0.05 compared with the control group, #p < 0.05 compared with the control group.

Figure 7

FMP relieved the histopathologic damage of AA-induced UC rats. (A1–E1) Macroscopic pathologic observations by the naked eye. (A2–E2) H&E pathological staining of colon tissue from each group of rats. Arrow symbols indicate the infiltration of neutrophils and mononuclear inflammatory cells; triangle symbols show the disappearance, distortion, and exfoliation of epithelial tissue; pentagram symbols represent crypt structure changes. (A3–E3) AB-PAS pathological staining of colon tissue from each group of rats; in AB-PAS staining, various glycoproteins of glycogen neutral mucins were purple-red. Acidic mucins, proteoglycans, and hyaluronic acid were blue. (A4–E4) Masson pathological staining colon tissue from each group of rats. Fibrous tissue was stained blue, and the cells, cytoplasm, erythrocytes, muscle tissue, eosinophilic granules, and connective tissue were stained red.

Figure 8

Effect of FMP on serum inflammation cytokines and predicted targets. ^∗p < 0.05, ^∗∗p < 0.001 compared with the control group and #p < 0.05, ##p < 0.001 compared with the model group.

Figure 9

The effects of two FMP dosages on VEGFA, VEGFR2, Src, PI3k, Akt, and eNOS in colon tissue in AA-induced UC rats. Data are expressed as the mean ± SEM (n = 3). ^∗p < 0.05, ^∗∗p < 0.001 compared with the control group, and #p < 0.05 compared with the model group. The thresholds of detection were as follows: VEGFA, 25 kDa; VEGFR2, 200 kDa; Src, 55 kDa; PI3K, 80 kDa; Akt, 60 kDa; eNOS, 130 kDa; GAPDH, 37 kDa; a-tubulin, 55 kDa; and ß-actin, 42 kDa.