Network-based pharmacology and experimental validation to explore the mechanism of action of the Jiawei Pentongling formula (加味盆痛灵方) for the treatment of endometriosis-related pain

doi:10.19852/j.cnki.jtcm.20240806.004

Abstract

Abstract:

OBJECTIVE: To investigate the effects and mechanisms of the Jiawei Pentongling formula (加味盆痛灵方, JWPTL) in treating endometriosis-related pain using network pharmacology study and experimental validation.

METHODS: Active ingredients and relevant targets of JWPTL, as well as genes for endometriosis-related pain, were collected from public databases. Prediction of core targets and pathways of JWPTL against pain associated with endometriosis by protein-protein interaction (PPI) network work, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. The Sprague-Dawley rat endo-metriosis model was constructed using the autologous endosomal transplantation method, and the successfully modeled rats were randomly divided into the model group and the JWPTL group, with 8 rats in each group. Another 8 rats were set up in the sham group. Rats in the JWPTL group used the rectal delivery method with the addition of JWPTL (7.77 g·kg^-1·d^-1) once a day for 28 d. Rats in the model and sham-operated groups were given equal amounts of saline using the same administration method. The 50% paw withdrawal threshold (PWT) of the rats was measured at different time points. After the intervention, the expression of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) proteins and mRNA in endometriotic tissues was detected by immune-ohistochemistry and reverse transcription-polymerase chain reaction.

RESULTS: From GeneCards, Online Mendelian Inheritance in Man and other databases, a total of 964 endometriosis (EMs) -related pain targets were screened, 142 active ingredients of JWPTL, 605 targets, and 221 potential targets were obtained by intersection of Venn diagram; 44 core targets were identified by constructing PPI network. KEGG enrichment analysis showed that JWPTL mainly involves the PI3K-Akt signaling pathway, estrogen signaling pathway, hypoxia inducible factor-1 signaling pathway, tumour necrotizing factor signaling pathway, and other signaling pathways in the treatment of EMs-related pain. Animal experiments showed that JWPTL could up-regulate the mechanical pain threshold and reduce the expression of PI3K and Akt proteins and mRNA in ectopic endometrial tissues of model rats.

CONCLUSIONS: The present study preliminarily analyzed the pharmacological mechanism of the formula, and molecular docking and animal experiments showed the feasibility of this study, suggesting that the formula may inhibit the release of inflammatory factors and reverse the pain associated with EMs by downregulating the PI3K/Akt signaling pathway.

Key words: Endometriosis-related pain, phosphatidylinositol 3-kinase, proto-oncogene proteins c-akt, Signal Transduction, Jiawei Pentongling formula, network pharmacology

YU Siyun, ZHANG Shiwen, XIA Yu, LIU Xiaoqing, LIU Yajie, FU Jinrong. Network-based pharmacology and experimental validation to explore the mechanism of action of the Jiawei Pentongling formula (加味盆痛灵方) for the treatment of endometriosis-related pain[J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 991-999.

Figures/Tables 4

Table 1 Top ten major active ingredients of JWPTL for the treatment of pain associated with EMs

Molecular ID	Ingredient	Degree	Source	Caco-2	DL
MOL000098	quercetin	94	Chuanlianzi (Fructus Toosendan) Huzhang (Radix Polygoni Cuspidati) Yanhusuo (Rhizoma Corydalis Yanhusuo) Zaojiaoci (Spina Gleditsiae)	0.05	0.28
MOL000008	apigenin	45	Huzhang (Radix Polygoni Cuspidati)	0.43	0.21
MOL000006	luteolin	44	Huzhang (Radix Polygoni Cuspidati)	0.19	0.25
MOL002047	melianone	41	Chuanlianzi (Fructus Toosendan)	0.46	0.81
MOL003396	echinocystic acid	40	Zaojiaoci (Spina Gleditsiae)	0.09	0.74
MOL000422	kaempferol	39	Xixin (Herba Asari Mandshurici) Zaojiaoci (Spina Gleditsiae)	0.26	0.24
MOL000940	bisdemethoxycurcumin	31	Ezhu (Rhizoma Curcumae Phaeocaulis)	0.49	0.26
MOL013179	fisetin	31	Zaojiaoci (Spina Gleditsiae)	0.21	0.24
MOL006358	Stigmast-4-ene-3,6-dione	29	Zaojiaoci (Spina Gleditsiae)	0.96	0.79
MOL001996	Betulonic acid	28	Zaojiaoci (Spina Gleditsiae)	0.65	0.78

Figure 1 PWT in 3 groups of rats at different time points A: PWT of each group of rats before the operation and on days 1, 3, and 5 after the operation. B: comparison of PWT in each group of rats before surgery. C: comparison of PWT in each group of rats before and after modeling. D: PWT of each group of rats on days 1, 3, 5, 7, 14, 21, 28 after administration. E: comparison of PWT in each group of rats before and after administration. Sham group: treated with saline enemas; Model group: treated with saline enemas; JWPTL group: treated with JWPTL (7.77 g·kg -1·d -1). JWPTL: Jiawei Pentongling formula; PWT: paw withdrawal threshold. The t-test was applied for comparisons between two groups, data are presented as mean ± standard error of the mean (n = 8). aP <0.001, compared with baseline. bP <0.001, compared with the Model group.

Figure 2 Comparison of expression of PI3k and Akt protein in endometrium A-F: dyeing method of all pictures are the Immunohistochemical SP method, PBS was used as negative control (× 100). A: the expression of PI3K in normal endometrial tissue of Sham group; B: the expression of PI3K in ectopic endometrial tissue of Model group; C:the expression of PI3K in ectopic endometrial tissue of JWPTL group; D: the expression of Akt in normal endometrial tissue of Sham group; E: the expression of Akt in ectopic endometrial tissue of Model group; F: the expression of Akt of ectopic endometrial tissue of JWPTL group; G: relative expression of PI3K protein; H: relative expression of Akt protein. Sham group: treated with saline enemas; Model group: treated with saline enemas; JWPTL group: treated with JWPTL (7.77 g·kg -1·d -1). JWPTL: Jiawei Pentongling formula; PI3K: phosphatidylinositol 3-kinase; Akt: protein kinase B. The t-test was applied for comparisons between two groups, data are presented as mean ± standard error of the mean (n = 5). aP <0.05, compared with the Sham group; bP <0.05, compared with the Model group.

Table 2 Changes of PI3k and Akt mRNA in Endometrial tissue

Group	n	PI3K	Akt
Sham	5	0.99±0.16	1.04±0.13
Model	5	2.14±0.30^a	2.02±0.17^a
JWPTL	5	1.10±0.17^b	1.50±0.13^b

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