Bushen Huoxue decoction (补肾活血汤) improves the reproduction of endometriosis-associated infertility by regulating Homeobox A10 and αlpha(v)beta(3) integrin expression

doi:10.19852/j.cnki.jtcm.2024.06.004

Abstract

Abstract:

OBJECTIVE: To investigate the mechanism of Bushen Huoxue decoction (补肾活血汤, BSHXD) to treat endometriosis-induced infertility.

MEDHODS: The main compounds of BSHXD were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The effect of BSHXD on Homeobox A10 (HOXA10) and alpha(v)beta(3) (αvβ3) integrin expression of Ishikawa cells, mouse model, and endometriosis-associated infertility women was evaluated by using Western blot analysis, immunohistochemistry and Real-Time quantitative polymerase chain reaction (RT-qPCR). The efficacy of BSHXD on embryo attachment were examined by using the BeWo spheroid and mouse embryo attachment assay. HOXA10 concentration in uterine flushing fluid of endometriosis-associated infertility women treated with BSHXD was measured by Enzyme-Linked immunosorbent assay (ELISA).

RESULTS: BSHXD improved BeWo spheroid and mice blastocysts attachment to Ishikawa cells and increased embryo implantation rates in mice and pregnancy rates in women with endometriosis-associated infertility. BSHXD enhanced HOXA10 and αvβ3 integrin expression in Ishikawa cell, endometriosis mouse model, and endometriosis-associated infertility women, which potentially improved endometrial receptivity.

CONCLUSIONS: BSHXD could improve endometrial receptivity of endometriosis-associated infertility in a dose-dependent manner by regulating HOXA10 and αvβ3 integrin expression.

Key words: infertility, endometriosis, homeobox A10 proteins, integrin alphaVbeta3, Bushen Huoxue decoction

TANG Weiwei, LIU Kaili, FAN Xumei, ZHU Li, ZENG Zheng, SUN Jiali, SHI Jie, ZHANG Zhenzhen, GUI Tao, WAN Guiping. Bushen Huoxue decoction (补肾活血汤) improves the reproduction of endometriosis-associated infertility by regulating Homeobox A10 and αlpha(v)beta(3) integrin expression[J]. Journal of Traditional Chinese Medicine, 2024, 44(6): 1137-1145.

Figures/Tables 5

Table 1 Main components of Bushen Huoxue decotion identified by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS)

Num	Compound	RT (min)	Molecular weight (Da)	Formula	Ionization model	Class I	Class II
1	Cryptochlorogenic acid (4-O-Caffeoylquinic acid)	2.79	3.54E+02	C16H18O9	[M-H]-	Phenolic acids	Phenolic acids
2	N-Feruloylagmatine	2.91	3.06E+02	C15H22N4O3	[M+H]+	Alkaloids	Phenolamine
3	3,4-Dihydroxybenzoic acid (Protocatechuic acid)	2.58	1.54E+02	C7H6O4	[M-H]-	Phenolic acids	Phenolic acids
4	Protocatechualdehyde	3.12	1.38E+02	C7H6O3	[M-H]-	Others	Aldehyde compounds
5	Luteolin-4'-O-glucoside	4.06	4.48E+02	C21H20O11	[M+H]+	Flavonoids	Flavones
6	Brevifolin carboxylic acid	3.05	2.92E+02	C13H8O8	[M-H]-	Phenolic acids	Phenolic acids
7	Gallic acid	1.81	1.70E+02	C7H6O5	[M-H]-	Phenolic acids	Phenolic acids
8	Paeonilactone C	4.34	3.18E+02	C17H18O6	[M+H]+	Terpenoids	Monoterpenoids
9	Quercetin-5-O-β-D-glucoside	3.82	4.64E+02	C21H20O12	[M+H]+	Flavonoids	Flavonols
10	Quercetin-3-O-glucoside (Isoquercitrin)	3.82	4.64E+02	C21H20O12	[M+H]+	Flavonoids	Flavonols
11	Cistanoside A	3.48	8.00E+02	C36H48O20	[M-H]-	Phenolic acids	Phenolic acids
12	Luteolin-7-O-glucoside (Cynaroside)	4.09	4.48E+02	C21H20O11	[M+H]+	Flavonoids	Flavones
13	Kaempferol-4'-O-glucoside	4.05	4.48E+02	C21H20O11	[M+H]+	Flavonoids	Flavonols
14	Quercetin-3-O-galactoside (Hyperin)	3.82	4.64E+02	C21H20O12	[M+H]+	Flavonoids	Flavonols
15	Saikosaponin A	6.37	7.80E+02	C42H68O13	[M-H]-	Terpenoids	Triterpene Saponin
16	Isoferulic Acid	4.03	1.94E+02	C10H10O4	[M-H]-	Phenolic acids	Phenolic acids
17	Echinacoside	3.19	7.86E+02	C35H46O20	[M-H]-	Phenolic acids	Phenolic acids
18	Saikogenin Q	5.54	4.88E+02	C30H48O5	[M+H]+	Terpenoids	Triterpene
19	Calycosin-7-O-glucoside	3.56	4.46E+02	C22H22O10	[M+H]+	Flavonoids	Isoflavones
20	Catalpol	1.19	3.62E+02	C15H22O10	[M-H]-	Terpenoids	Monoterpenoids

Figure 1 BSHXD promotes embryo attachment in vitro and in vivo A: adhesion experiments with BeWo spheroids attached to Ishikawa cell monolayers. The attached spheroids were counted and the attachment rate was calculated as a percentage of the total number of BeWo spheroids (% adhesion). Data were presented as mean ± standard deviation (n = 4). B: endometriosis mouse models were treated with dydrogesterone and different dose BSHXD. After 28 d treatment, the mice were mated. The pregnant mice were euthanized on the 14th day of gestation and total number of embryos was calculated. Statistical analysis was carried out using ANOVA tests among these groups. Data were presented as mean ± standard deviation (n = 7) C: Mouse embryo (black arrow) attached to Ishikawa cells (× 100). D: Adhesion experiments of mouse embryos attached to the Ishikawa cell monolayer. The attachment score of each group was described as median with the interquartile range (n = 5). In Figures A and D: Control group: Ishikawa cell cultured in basal medium; Dydrogesterone group: Ishikawa cell cultured in presence of 10 nmol/L dydrogesterone; Low dose BSHXD group: Ishikawa cell cultured in presence of 5% BSHXD serum; Normal dose BSHXD group: Ishikawa cell cultured in presence of 10% BSHXD serum; High dose BSHXD group: Ishikawa cell cultured in presence of 20% BSHXD serum. In Figure B: Control group: sham surgery mice were treated with water; Model group: endometriosis mouse models were treated with water; Dydrogesterone group: endometriosis mouse models were treated with dydrogesterone (3.03 mg·kg-1·d-1); Low dose BSHXD group: endometriosis mouse models were treated with low dose BSHXD (6.218 g·kg-1·d-1); Normal dose BSHXD group: endometriosis mouse models were treated with normal dose BSHXD (12.437 g·kg-1·d-1); High dose BSHXD group: endometriosis mouse models were treated with high dose BSHXD (24.873 g·kg-1·d-1). BSHXD: Bushen Huoxue decotion; ANOVA: analysis of variance. Statistical analysis was carried out using Kruskal-Wallis with Dunn’s multiple comparison tests among these groups. Compared with the Control group, aP < 0.05; compared with the Model group, bP < 0.01.

Figure 2 BSHXD induces HOXA10 and αvβ3 integrin expression in Ishikawa cells A: Ishikawa cells were treated with Dydrogesterone and different dose BSHXD serum for 24 h and the level of HOXA10 and ITGB3 were detected using RT-qPCR; B: Expression of HOXA10 and αvβ3 integrin were detected by western blotting; C: HOXA10 expression of western blotting was quantitated by using Image J software; D: αvβ3 integrin expression of western blotting was quantitated. Control group: Ishikawa cell cultured in basal medium; Dydrogesterone group: Ishikawa cell cultured in presence of 10 nmol/L dydrogesterone; Low dose BSHXD group: Ishikawa cell cultured in presence of 5% BSHXD serum; Normal dose BSHXD group: Ishikawa cell cultured in presence of 10% BSHXD serum; High dose BSHXD group: Ishikawa cell cultured in presence of 20% BSHXD serum. BSHXD: Bushen Huoxue decotion; HOXA10: Homeobox A10; αvβ3: alpha(v)beta(3) integrin; ITGB3: integrin beta(3); RT-qPCR: real-time quantitative polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ANOVA: analysis of variance. ANOVA tests were used among these groups. Data were presented as mean ± standard deviation (n = 3). Compared with the Control group, aP < 0.05; compared with the Low dose group, bP < 0.05; compared with the Nomal dose dose group, cP < 0.05.

Figure 3 BSHXD increases HOXA10 and αvβ3 integrin expression in endometriosis mouse model A: endometriosis mouse models were treated with dydrogesterone and different dose BSHXD, after 28 d treatment, the mice were mated. The pregnant mice were euthanized on the 14th day of gestation and the endometrium was subjected to immunohistochemical staining using HOXA10 antibody (× 100). A1: Control group; A2: Model group; A3: Dydrogesterone group; A4: Low dose BSHXD group; A5: Normal dose BSHXD group; A6: High dose BSHXD group. B: The endometrium was subjected to immunohistochemical staining using αvβ3 integrin antibody (× 100). B1: Control group; B2: Model group; B3: Dydrogesterone group; B4: Low dose BSHXD group; B5: Normal dose BSHXD group; B6: High dose BSHXD group. C: the intensity of HOXA10 staining was quantitated by using Image J software. D: the intensity of αvβ3 integrin staining was quantitated. E: the expression level of Hoxa10 was determined using RT-qPCR. F: the expression level of Itgb3 was determined by RT-qPCR. Control group: sham surgery mice were treated with water; Model group: endometriosis mouse models were treated with water; Dydrogesterone group: endometriosis mouse models were treated with dydrogesterone (3.03 mg·kg-1·d-1); Low dose BSHXD group: endometriosis mouse models were treated with low dose BSHXD (6.218 g·kg-1·d-1); Normal dose BSHXD group: endometriosis mouse models were treated with normal dose BSHXD (12.437 g·kg-1·d-1); High dose BSHXD group: endometriosis mouse models were treated with high dose BSHXD (24.873 g·kg-1·d-1). BSHXD: Bushen Huoxue decotion; HOXA10: Homeobox A10; αvβ3: alpha(v)beta(3) integrin; Itgb3: Integrin beta(3); RT-qPCR: real-time quantitative polymerase chain reaction; D-HSCORE: digital histological score; mRNA: messenger ribonucleic acid; ANOVA: analysis of variance. Statistical analysis was carried out using ANOVA tests among these groups. Data were presented as mean ± standard deviation (n = 7). Compared with the model group, aP < 0.05.

Figure 4 BSHXD induces HOXA10 and αvβ3 integrin expression in endometriosis-associated infertility women A: The mid-secretory endometrial biopsy specimens were obtained from twenty endometriosis-associated infertility women before and after BSHXD treatment for three menstrual cycles and were subjected to immunohistochemical staining by using HOXA10 antibody (×100). A1: Control group; A2: BSHXD group. B: The intensity of HOXA10 staining was quantitated by using Image J software. Control group: patients not treated with BSHXD; BSHXD group: patients treated with BSHXD (82 g/d) for three menstrual cycles. BSHXD: Bushen Huoxue decotion; ELISA: enzyme-linked immunosorbent assay; D-HSCORE: digital histological score. Student’s t-tests were used for comparisons of two groups. Data were presented as mean ± standard deviation (n = 20). Compared with the control group, aP < 0.05.

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