Cuiru Keli improves bromocriptine-induced postpartum hypogalactia in rats by regulating the WTAP-Wnt5a-β-catenin signaling axis

doi:10.12092/j.issn.1009-2501.2026.09.006

Abstract

Abstract:

AIM: To study the effect and mechanism of Cuiru Keli (CRKL) on bromocriptine-induced postpartum hypogalactia in rats. METHODS: ELISA was used to detect the level of prolactin (PRL) in mammary tissue (MT) of postpartum hypogalactia. Cultivate rat mammary epithelial cells (RMECs) in vitro. The effect of CRKL on RMEC proliferation was detected by CCK8 method to determine the drug-containing serum. RT-qPCR and immunofluorescence detection of lactose, milk fat, and milk protein related gene expression. Network pharmacology predicts the signaling pathway of CRKL treatment for postpartum hypogalactia. Molecular docking and molecular dynamics verification of the binding between key components of CRKL and WTAP. RIP was used to study the interaction between WTAP and Wnt5a. RT-qPCR and Western blot were used to investigate the regulatory mechanism of CRKL on the Wnt/β-catenin signaling pathway. RESULTS: CRKL significantly increased the hourly milk production of female rats (P<0.05 or P<0.01). CRKL increases PRL levels in MT of model rats (P<0.01). Serum containing CRKL promotes the expression of PRLR, FASN, CSN2, and GLUT1 (all P<0.05 or P<0.01). Network pharmacology predicts a high correlation between the Wnt/β-catenin signaling pathway and CRKL treatment for postpartum hypogalactia. And the serum containing CRKL promotes the expression of key genes CCND1, c-Myc, and β-catenin in the Wnt signaling pathway (all P<0.05 or P<0.01). Molecular docking and molecular dynamics indicate that the key component of CRKL has strong binding ability with WTAP. Overexpression of WTAP in RMECs interferes with the function of CRKL (P<0.01). CONCLUSION: CRKL improves bromocriptine-induced postpartum hypogalactia in rats through the WTAP-Wnt5a-β-catenin signaling axis.

Key words: Cuiru Keli, postpartum hypogalactia, WTAP, Wnt5a, prolactin

CLC Number:

R965.2

Hui LI, Qiuyun XUE, Meiling YUAN, Qiying JIN, Jiaqing CHEN, Aixin XIA, Chenhao XU, Chenggui MIAO. Cuiru Keli improves bromocriptine-induced postpartum hypogalactia in rats by regulating the WTAP-Wnt5a-β-catenin signaling axis[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(9): 1200-1213.

Figures/Tables 11

Table 1 RT-qPCR primer sequences

Gene name (rat)	Primer sequence
PRLR	Forward primer	GAGGCGGATGAGAACAAGCAGATC
PRLR	Reward primer	GGAGGGTGGGTTGGAAATGAACTTC
FASN	Forward primer	GTGTGGTAGGCTTGGTGAACTGTC
FASN	Reward primer	GTGAGATGTGCTGCTGAGGTTGG
CSN2	Forward primer	GGTCTTCATCCTTGCCTGCCTTG
CSN2	Reward primer	CCTGTCCCATGAGTTTCACCTTCTG
GLUT1	Forward primer	CATCCACCACACTCACCACAC
GLUT1	Reward primer	GCCTGCCAAAGCGATTAACAAAGAG
CCND1	Forward primer	AGCAGCGACTCTGAAGAAGAACAAG
CCND1	Reward primer	GGATGACCCTGACTCGGACCTC
c-Myc	Forward primer	AAGAAGGCTCCACCATCGTTTGAAG
c-Myc	Reward primer	AGTCTGCGGTTGCTTGAGGTTG
β-actin	Forward primer	CACTATCGGCAATGAGCGGTTCC
β-actin	Reward primer	CAGCACTGTGTTGGCATAGAGGTC

Fig.1 CRKL promotes the secretion of milk in postpartum hypogalactia rats ($ {\overline x} \pm {s}$) Hourly milk production (A). Weight gain of offspring (B). PRL levels in MT was detected by ELISA (C). CCK8 cell proliferation activity (D). The mRNA expression of PRLR in RMECs was detected by RT-qPCR (E) (A-B, n=8; C-E, n=3). cP<0.01, compared with normal; eP<0.05, fP<0.01, compared with model. iP<0.01, compared with 1.0%.

Fig.2 CRKL promotes gene expression of milk lactose, milk fat synthesis, and milk protein in RMECs ($ {\overline x} \pm {s}$) The mRNA expression of FASN, CSN2, and GLUT1 in RMECs were detected by RT-qPCR (A, B, C). The protein expression of FASN, CSN2, and GLUT1 in RMECs were detected by immunofluorescence (D, E, F) (scale bar=50 µm) (n=3). cP<0.01, compared with normal; eP<0.05, fP<0.01, compared with model.

Fig.3 CRKL target predicted by network pharmacology Screening of CRKL components (A). Obtained 464 OGEs from the Venn diagram tool (B). Core target geneswere obtained by PPI network analysis (C, D). GO function analysis (E). KEGG enrichment analysis (F). Compound-Target-Pathway network (G).

Fig.4 CRKL promotes the expression of Wnt/β-catenin signaling pathway in RMECs ($ {\overline x} \pm {s}$) The mRNA expression of CCND1 and c-Myc in RMECs were detected by RT-qPCR (A, B). The protein expression of CCND1, c-Myc, and β-catenin in RMECs were detected by WB (C, D, E) (n=3). cP<0.01, compared with normal; eP<0.05, fP<0.01, compared with model.

Table 2 WTAP molecular docking

Mol ID	Molecule name	Herb name	Binding capacity
MOL008393	7-(beta-Xylosyl)cephalomannine_qt	C. pilosula root	?8.7
MOL013345	picraquassioside C	H. Fulva root	?8.7
MOL002157	wallichilide	L. Striatum root	?8.4
MOL013187	Cubebin	B. chinense root	?7.7
MOL004653	(+)-Anomalin	B. chinense root	?7.3
MOL000020	12-senecioyl-2E,8E,10E-atractylentriol	H. Fulva root	?7.3
MOL000371	3,9-di-O-methylnissolin	A. membranaceus root	?6.9
MOL000398	isoflavanone	A. membranaceus root	?6.8
MOL000438	(3R)-3-(2-hydroxy-3,4-dimethoxyphenyl)chroman-7-ol	A. membranaceus root	?6.6
MOL013343	hemerocallone	C. Pilosula root	?6.5

Fig.5 Molecular docking and molecular dynamics Molecular docking of WTAP with Picraquassioside C, 7- (beta Xylosyl) cephalomannine, Wallichilide, and Wallichilide (A, B, C, D). Stability analysis of WTAP molecular dynamics complex with four components (Picraquassiosis C, 7- (beta Xylosyl) Cephalomanine, Wallichilide, and Wallichilide) (E, F, G, H, I, J). Gibbs free energy analysis of WTAP molecular dynamics complexes with four components (Picraquassiosis C, 7- (beta Xylosyl) cephalomanine, Wallichilide, and Wallichilide) (K, L, M, N).

Table 3 Calculate the average binding free energy of four complexes using MM/PBSA method

Complex systems	ΔVDWAALS	ΔE_elec	ΔE_GB	ΔE_surf	ΔG_gas	ΔG_solvation	ΔTotal
WTAP_7-(beta-Xylosyl) cephalomannine	?37.14	?6.03	25.58	?4.70	?43.17	20.88	?22.29
WTAP_Cubebin	?28.94	?9.06	24.46	?4.02	?38.00	20.44	?17.56
WTAP_Picraquassioside C	?34.01	?30.03	36.97	?5.69	?64.03	31.28	?32.75
WTAP_Wallichilide	?36.00	1.29	11.80	?4.29	?34.71	7.51	?27.21

Fig.6 CRKL inhibits WTAP expression ($ {\overline x} \pm {s}$) The mRNA expression of WTAP in MT and RMECs were detected by RT-qPCR (A, B, C, D) (n=3). cP<0.01, compared with normal; fP<0.01, compared with model.

Fig.7 WTAP inhibits the expression of Wnt5a/β-catenin signaling pathway ($ {\overline x} \pm {s}$) The mRNA expression of Wnt5a in MT and RMECs were detected by RT-qPCR (A, B). RIP experiment (C). The mRNA expression of CCND1 and c-Myc in RMECs were detected by RT-qPCR (D, E). The protein expression of β-catenin in RMECs was detected by WB (F). The mRNA expression of FASN, CSN2, and GLUT1 in RMECs were detected by RT-qPCR (G, H, I) (n=3). cP<0.01, compared with normal; fP<0.01, compared with WTAP vector; iP<0.01, compared with anti-IgG group; lP<0.01, compared with anti-WTAP group.

Fig.8 Cuiru Keli improves bromocriptine-induced postpartum hypogalactia in rats via WTAP-Wnt5a-β-catenin pathway ($ {\overline x} \pm {s}$) The mRNA expression of CCND1 and c-Myc in RMECs were detected by RT-qPCR (A, B). The protein expression of β-catenin in RMECs was detected by WB (C). The mRNA expression of FASN, CSN2, and GLUT1 in RMECs were detected by RT-qPCR (D, E, F) (n=3). cP<0.01, compared with normal; fP<0.01, compared with CRKL.

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