Efficacy of active ingredients in Qingdai (Indigo Naturalis) on ulcerative colitis: a network pharmacology-based evaluation

doi:10.19852/j.cnki.jtcm.2023.01.011

Abstract

Abstract:

OBJECTIVE: To elucidate the protective effect of Qingdai (Indigo Naturalis, QD) on ulcerative colitis (UC) by means of in silico and in vivo approaches.
METHODS: A systems pharmacology analysis was per-formed to predict the active components of QD whereas the putative biological targets of QD against UC were obtained through target fishing, network cons-truction and enrichment analyses. Meanwhile, we examined the ameliorative effect of QD in a mouse model of dextran sulfate sodium (DSS)-induced colitis. During the 10-day experiment, the control and diseased mice were given with oral gavages of QD (1.3 g raw herbs·kg^-1·d^-1) or 5‐aminosalicylic acid (5-ASA, 100 mg·kg^-1·d^-1) every day. The underlying pharma-cological mechanisms of QD in UC were determined using polymerase chain reaction tests, histological staining, enzyme-linked immunoassays, and Western blotting analysis.
RESULTS: Searching from various network pharmacology databases, 29 compounds were identified in QD. According to the screening criteria suggested by TCMSP (i.e. OB ≥ 30% and DL ≥ 0.18), nine of them were considered the active ingredients that contribute to the ameliorative effects of QD on different mouse models of colitis. Most importantly, the protective effect of QD on DSS-induced colitis was significantly associated with modulations of the expression levels of glycogen synthase kinase 3-β (Gsk3-β) and forkhead box p3 (Foxp3), which are widely considered as important regulators of excessive inflammatory responses.
CONCLUSIONS: The results of this study provide solid scientific evidence for the use of QD or its core active components in the clinical management of UC.

Key words: Qingdai (Indigo Naturalis), colitis, ulcerative, glycogen synthase kinase 3 beta, signaling transduction, forkhead transcription factors, network pharmacology

LI Yue, WEN Shuting, ZHAO Runyuan, FAN Dongmei, ZHAO Dike, LIU Fengbin, MI Hong. Efficacy of active ingredients in Qingdai (Indigo Naturalis) on ulcerative colitis: a network pharmacology-based evaluation[J]. Journal of Traditional Chinese Medicine, 2023, 43(1): 124-133.

Figures/Tables 12

Table 1 Active compounds identified in QD

MOL_ID	molecule_name	OB (%)	DL
MOL000358	beta-sitosterol	36.91	0.75
MOL001781	Indigo	38.20	0.26
MOL001810	6-(3-oxoindolin-2-ylidene)indolo[2,1-b]quinazolin-12-one	45.28	0.89
MOL002309	indirubin	48.59	0.26
MOL002322	isovitexin	31.29	0.72
MOL011100	bisindigotin	41.66	0.39
MOL011105	indican	34.90	0.23
MOL011332	10h-indolo,[3,2-b],quinoline	54.57	0.22
MOL011335	Isoindigo	94.30	0.26

Figure 1 Results of network pharmacology-based analyses A: after searching from a variety of databases, the Venn diagram summarizes the targets in UC and QD; B: by using the Cytoscape 3.7.1 visualization software, the PPI network of QD was generated with 56 nodes and 272 edges; C: the PPI network of UC analysis was generated with 1423 nodes and 13335 edges; D: the network diagram of core targets of QD treatment against UC, which include 17 nodes and 77 edges; E: 17 targets identified according to the degrees of the nodes presented in the previous figure; F: the ultimate TCM-active ingredients-targets-disease network diagram was generated upon the above analyses. BCL2L1: apoptosis regulator Bcl-2; CA2: carbonic anhydrase II; ESR1: estrogen receptor; F2: thrombin; GSK3B: glycogen synthase kinase-3 beta; JUN: transcription factor AP-1; MAPK14: mitogen-activated protein kinase 14; NOS2: nitric oxide synthase, inducible; NR3C1: glucocorticoid receptor; PDE3A: CGMP-inhibited 3',5'-cyclic phosphodiesterase A; PON1: serum paraoxonase/arylesterase 1; PPARG: peroxisome proliferator activated receptor gamma; PTGS2: prostaglandin G/H synthase 2; ESR1: estrogen receptor; KDR: vascular endothelial growth factor receptor 2; MAPK14: mitogen-activated protein kinase 14; AHR: aryl hydro-carbon receptor; CA2: carbonic anhydrase II; IFNG: interferon gamma; NOS3: nitric-oxide synthase, endothelial; TNF: tumor necrosis factor.

Figure 2 Results of enrichment analyses A: a total of 1028 GO entries were identified from 19 potential targets upon the GO enrichment analysis; FDR < 0.05; B: a total of 78 signal pathways were obtained upon the KEGG enrichment analysis, R language was used to plot the KEGG histogram. GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; FDR: false discovery rate; VEGF: vascular endothlial growth factor; PD-L1: programmed cell death-Ligand 1; TNF: tumor necrosis factor; NAD (P) H: dehydrogenase, quinone 2; FMN: flavinmononucleotide.

Figure 3 Ameliorative effect of QD on experimental colitis A: change of body weight of each group was calculated as the percent difference between the original body weight at day 0 and the weight on any particular day during the experimental period. Values represent the averages of 8 mice; B: DAI scores of different experimental groups of mice; C: lengths of colons were measured at the time of sacrifice. Control group treating saline treatment control for 10 d. DSS group consuming 2.5% DSS water for 7 d followed by an additional consumption period of normal drinking water for 3 d. (5-ASA+DSS) group fed with 2.5% DSS, and respectively given with 5-ASA (o.g.; 100 mg·kg-1·d-1) from day 0 till the end of experiment for a total of 10 d. (QD+DSS) group fed with 2.5% DSS, and respectively given with QD (o.g.; 1.3 g raw herbs·kg-1·d-1) from day 0 till the end of experiment for a total of 10 d. QD: Qingdai (Indigo Naturalis); DAI: disease activity index; DSS: dextran sodium sulfate; 5-ASA: 5-aminosalicylic acid. aP < 0.005, DSS group compared with Control groups; bP < 0.05, (5-ASA+DSS) group compared with DSS groups.

Figure 4 HE images revealing mucosal integrity in tissues in 10 d A, H: mucosal integrity in tissues (× 100 and × 200) of control group treating saline treatment control for 10 d. B, E: mucosal integrity in tissues (×100 and ×200) of DSS group consuming 2.5% DSS water for 7 d followed by an additional consumption period of normal drinking water for 3 d. C, G: mucosal integrity in tissues (× 100 and × 200) of (5-ASA+DSS) group fed with 2.5% DSS, and respectively given with 5-ASA o.g.; 100 mg·kg-1·d-1) from day 0 till the end of experiment for a total of 10 d. D, H: mucosal integrity in tissues (× 100 and × 200) of (QD + DSS) group fed with 2.5% DSS, and respectively given with QD (o.g.; 1.3 g raw herbs·kg-1·d-1) from day 0 till the end of experiment for a total of 10 d. I: Histological scores were given based on the severity of colonic damages, lymphocyte infiltration and crypt disruption (n = 8/group). HE: hematoxylin-eosin; QD: Qingdai (Indigo Naturalis); DSS: dextran sodium sulfate; 5-ASA: 5-aminosalicylic acid. aP < 0.005 DSS group compared with Control groups; bP < 0.05, (5-ASA + DSS) group compared with DSS groups; cP < 0.05, (QD + DSS) group compared with DSS groups.

Table 2 Expression levels of FOXP3, GSK-3β and p-GSK-3β in colonic by means of Western blotting ($\bar{x}\pm s$)

Group	n	FOXP3	GSK-3β
Control	8	0.10±0.04	0.05±0.14	0.10±0.04
DSS	8	0.55±0.95^a	0.38±0.08	0.55±0.95^a
5-ASA+DSSS	8	1.46±0.18^b	0.15±0.61^b	0.27±0.08^b
QD+DSS	8	1.37±0.18^c	0.13±0.50^c	0.31±0.08^c

Table 3 qRT-PCR results showed the regulation of mRNA levels of GSK-3β, TNF-α and FOXP3 in the colonic tissues of experimental mice (%,$\bar{x}\pm s$)

Group	n	GSK-3β	TNF-α
Control	8	1.00±0.07	1.03±0.13	1.04±0.13
DSS	8	2.15±0.22^a	4.29±0.60^a	0.43±0.06^a
5-ASA+DSS	8	1.46±0.18^b	2.65±0.73^b	0.82±0.06^b
QD+DSS	8	1.37±0.18^c	2.20±0.51^c	0.70±0.16^c

Table 4 Colonic levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-17A were evaluated using ELISAs (pg/mL mg per tissue, $\bar{x}\pm s$)

Group	n	IL-1β	TNF-α
Control	8	4.15±0.84	14.30±1.2	2.63±0.78
DSS	8	19.11±1.75^a	97.25±9.68^a	8.59±2.41^a
5-ASA+DSS	8	10.93±0.99^b	37.83±8.41^b	3.83±0.98^b
QD+DSS	8	11.20±0.55^c	47.24±6.05^c	3.24±1.32^c

Figure S1 Experimental design outlined

Figure S2 Expression levels of GSK-3β, p-GSK-3β and FOXP3 in colonic by means of Western blotting

Table S1 Scoring of DAI of each group of animals

Scoring	Change of body weight	Consistency of stool	Fecal occult blood
0	<1 %	Normal	Negative
1	1-5%
2	6-10%	Loose	Moderate
3	11-15%
4	>15%	Intensively loose	Severe

Table S2 The list of primer sequences used in the qRT-PCR experiment

Gene name	Primer	Sequence
β-actin	Forward	5′-GGGAAATCGTGCGTGAC-3′
β-actin	Reverse	5′-AGGCTGGAAAAGAGCCT-3′
GSK-3β	Forward	5′-CCCACCATCACCATTAAGA-3′
GSK-3β	Reverse	5′-AATCCACCTTGCTTTCCA-3′
Foxp3	Forward	5′-ACCATTGGTTTACTCGCATGT-3′
Foxp3	Reverse	5′-TCCACTCGCACAAAGCACTT-3′
TNF-α	Forward	5′-CTGAACTTCGGGGTGATCGG-3′
	Reverse	5′-GGCTTGTCACTCGAATTTTGAGA-3′

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