Investigation of the active ingredients and mechanism of Shuangling extract in dextran sulfate sodium salt induced ulcerative colitis mice based on network pharmacology and experimental verification

doi:10.19852/j.cnki.jtcm.20240408.003

Abstract

Abstract:

OBJECTIVE: To explore the pharmacodynamic effects and potential mechanisms of Shuangling extract against ulcerative colitis (UC).

METHODS: The bioinformatics method was used to predict the active ingredients and action targets of Shuangling extract against UC in mice. And the biological experiments such as serum biochemical indexes and histopathological staining were used to verify the pharmacological effect and mechanism of Shuangling extract against UC in mice.

RESULTS: The Shuangling extract reduced the levels of seruminterleukin-1β (IL-1β), tumor necrosis factor-α (TNF-N), interleukin-6 (IL-6) and other inflammatory factors in UC mice and inhibited the inflammatory response. AKT Serine/threonine Kinase 1 and IL-6 may be the main targets of the anti-UC action of Shuangling extract, and the TNF signaling pathway, Forkhead box O signaling pathway and T-cell receptor signaling pathway may be the main signaling pathways.

CONCLUSION: The Shuangling extract could inhibit the inflammatory response induced by UC and regulate intestinal immune function through multiple targets and multiple channels, which provided a new option and theoretical basis for anti-UC.

Key words: colitis, ulcerative, pharmacodynamic study, network pharmacology, molecular docking simulation, Shuangling extract

CHU Mengzhen, WANG Yu, LIN Zhijian, Lyu Jintao, ZHANG Xiaomeng, ZHANG Bing. Investigation of the active ingredients and mechanism of Shuangling extract in dextran sulfate sodium salt induced ulcerative colitis mice based on network pharmacology and experimental verification[J]. Journal of Traditional Chinese Medicine, 2024, 44(3): 478-488.

Figures/Tables 6

Table 1 KEGG enrichment analysis of related pathways

ID	Pathway	Gene ratio	P value
hsa05200	Pathways in cancer	29/38	4.67E-28
hsa05161	Hepatitis B	22/38	1.37E-26
hsa05212	Pancreatic cancer	16/38	6.01E-22
hsa05219	Bladder cancer	14/38	4.64E-21
hsa05205	Proteoglycans in cancer	20/38	2.97E-20
hsa04917	Prolactin signaling pathway	15/38	2.02E-19
hsa05142	Chagas disease (American trypanosomiasis)	16/38	1.22E-18
hsa04668	TNF signaling pathway	16/38	1.91E-18
hsa04620	Toll-like receptor signaling pathway	15/38	7.87E-17
hsa05215	Prostate cancer	14/38	2.68E-16
hsa05213	Endometrial cancer	12/38	1.09E-15
hsa04915	Estrogen signaling pathway	14/38	1.33E-15
hsa04660	T cell receptor signaling pathway	14/38	1.52E-15
hsa05133	Pertussis	13/38	1.65E-15
hsa05160	Hepatitis C	15/38	2.19E-15
hsa04068	FoxO signaling pathway	15/38	2.42E-15
hsa05210	Colorectal cancer	12/38	8.93E-15
hsa05140	Leishmaniasis	12/38	4.38E-14
hsa05220	Chronic myeloid leukemia	12/38	5.15E-14
hsa05223	Non-small cell lung cancer	11/38	1.62E-13

Figure 1 Molecular docking research A: structural formula of the main active ingredient. A1: pachymic acid; A2: astilbin; A3: luteolin; A4: lactucopicrin. B: molecular docking diagram of active components of Shuangling extract and key targets. B1-B5: docking results of MK-2206 dihydrochloride, pachymic acid, astilbin, luteolinandlactucopicrin active ingredients with AKT1 target protein. B6-B10: docking results of Corylifol A, pachymic acid, astilbin, luteolinand lactucopicrin active ingredients with IL-6 target protein. IL-6: interleukin-6; AKT1: AKT serine/threonine kinase 1.

Table 2 Molecular docking results of active Ingredients of Shuangling extract and core targets

Compound	Estimated ΔG (kcal/mol)
Compound	MK-2206 dihydrochloride	Corylifol A	pachymic acid	astilbin	luteolin	lactucopicrin
AKT1	-12.02	-	-7.41	-9.59	-7.09	-9.87
IL-6	-	-6.65	-5.39	-4.2	-4.95	-5.45

Figure 2 Serum inflammatory cytokine levels and histopathological HE staining A: levels of serum TNF-α; B: levels of serumIL-1β; C: levels of serumIL-6; D: colon tissue pathology HE staining score; E: representative HE stained slices of Shuangling extract in anti-UC (× 400). E1: CON; E2: MOD; E3: MSL; E4: SL-H; E5: SL-L. CON group: normal operation (n = 6); MOD group: 3% DSS without treatment (n = 6); MSL group: 3% DSS rats treated with Mesalazine water solution at a daily dose of 400 mg/kg (n = 6); SL-H group: 3% DSS rats treated with Shuangling extract at a daily dose of 18.34 g/kg for 7 d (n = 6). SL-L group: 3% DSS rats treated with Shuangling extract at a daily dose of 9.17 g/kg for 7 d (n = 6). TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6; HE: hematoxylin-eosin. CON: control; MOD: model; MSL: mesalazine; SL-H: Shuangling extract high-dose; SL-L: Shuangling extract low-dose; DSS: dextran sulfate solution; UC: ulcerative colitis. The statistical significances were assessed using a one-way analysis of variance followed by Dunnett's test. All results were presented as mean ± standard deviation. aP < 0.01, compared with the CON group; bP < 0.01 and cP < 0.05, compared with the MOD group.

Figure 3 Expression of the CD3 and Foxp3 protein in the colon A: immunohistochemical staining of CD3 protein in mouse colon (× 400). A1: CON; A2: MOD; A3: MSL; A4: SL-H; A5: SL-L. B: immunohistochemical staining of FOXP3 protein in mouse colon (× 400). B1: CON; B2: MOD; B3: MSL; B4: SL-H; B5: SL-L. C: average optical density of CD3 protein in colon tissue of mice in each group; D: average optical density of FOXP3 protein in colon tissue of mice in each group. CON group: normal operation (n = 6); MOD group: 3% DSS without treatment (n = 6); MSL group: 3% DSS rats treated with Mesalazine water solution at a daily dose of 400 mg/kg (n = 6); SL-H group: 3% DSS rats treated with Shuangling extract at a daily dose of 18.34 g/kg for 7 d (n = 6). SL-L group: 3% DSS rats treated with Shuangling extract at a daily dose of 9.17 g/kg for 7 d (n = 6). CD3: cluster of differentiation 3; FOXP3: forkhead box p3; DSS: dextran sulfate solution; UC: ulcerative colitis; CON: control; MOD: model; MSL: mesalazine; SL-H: Shuangling extract high-dose; SL-L: Shuangling extract low-dose; DSS: dextran sulfate solution. The statistical significances were assessed using a one-way analysis of variance followed by Dunnett's test. All results were presented as mean ± standard deviation. aP < 0.05, compared with the CON group; bP < 0.05, compared with the MOD group.

Figure 4 Expression of the AKT and p-AKT protein in the colon A: immunohistochemical staining of AKT protein in mouse colon (× 400). A1: CON; A2: MOD; A3: MSL; A4: SL-H; A5: SL-L; B: immunohistochemical staining of p-AKT protein in mouse colon (× 400). B1: CON; B2: MOD; B3: MSL; B4: SL-H; B5: SL-L; C: average optical density of AKT protein in colon tissue of mice in each group; D: average optical density of p-AKT protein in colon tissue of mice in each group. CON group: normal operation (n = 6); MOD group: 3% DSS without treatment (n = 6); MSL group: 3% DSS rats treated with Mesalazine water solution at a daily dose of 400 mg/kg (n = 6); SL-H group: 3% DSS rats treated with Shuangling extract at a daily dose of 18.34 g/kg for 7 d (n = 6). SL-L group: 3% DSS rats treated with Shuangling extract at a daily dose of 9.17 g/kg for 7 d (n = 6). AKT: AKT serine/threonine kinase; p-AKT: phospho-Akt Serine/threonine kinase; UC: ulcerative colitis; CON: control; MOD: model; MSL: mesalazine; SL-H: Shuangling extract high-dose; SL-L: Shuangling extract low-dose; DSS: dextran sulfate solution. The statistical significances were assessed using a one-way analysis of variance followed by Dunnett's test. All results were presented as mean ± standard deviation. aP < 0.05, cP < 0.01, compared with the CON group; bP < 0.05, dP < 0.01, compared with the MOD group.

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