Guizhi Fuling Capsule inhibits uterine fibroids growth by modulating Med12-mediated Wnt/ll-Catenin signaling pathway

Ethnopharmacological relevance: Guizhi Fuling Capsule (GFC) is a famous traditional Chinese medicine (TCM) formula recorded in Synopsis of the Golden Chamber, which has achieved obvious effects in the treatment of uterine fibroids (UFs).Aim of study: Mediator complex subunit 12 (Med12) mutations were closely related to UFs in 85% of fibroid cases. The Wnt/ll-Catenin signaling pathway plays an important role in the occurrence and development of UFs. This study aims to explore the pharmacological mechanism of GFC against UFs in which the Med12-mediated Wnt/ll-Catenin pathway is involved.Materials and methods: Med12 was silenced in uterine fibroid cells (UFCs) using a lentivirus-based Med12 gene specific RNA interference (RNAi) strategy. Cell proliferation was performed by CCK-8 assay, cell apoptosis and cell cycle were measured by flow cytometry. The rat model of UFs was established by injecting estradiol benzoate and progesterone. Forty-eight rats were divided into six groups, the low-dose GFC (L-GFC) group, the medium dose GFC (M-GFC) group and the high-dose GFC (H-GFC) group were intragastrically treated with GFC solution at 0.25 g/kg, 0.50 g/kg and 1.00 g/kg per day for 8 weeks, the positive control (PC) group was administrated with mifepristone (2.70 mg/kg/day), the normal control (NC) group and the model control (MC) group were given equal volume of normal saline once a day for 8 weeks. The histopathological changes of uterine tissues were evaluated by H&E staining. The expression of Med12 in uterine tissues were detected by immunohistochemistry. The protein and mRNA levels of associated genes were evaluated by western bolt and real time-PCR, respectively. Related indicators involved in Wnt/ll-Catenin pathway, such as Wnt1, ll-Catenin, Cyclin D1, TCF1/ TCF7 and C-myc, were compared among different groups. Results: The Wnt/ll-Catenin signaling pathway was inhibited after Med12 gene was knocked out in UFCs. GFCcontaining serum could induce cell apoptosis, make the cell cycle stagnated in G0/G1 phase to inhibiting the proliferation and reduce the expression of Wnt1, ll-Catenin, Cyclin D1, TCF1/TCF7, and C-myc in control-shRNA cells, while had no significant effect on Med12-shRNA cells. Compared with the MC group, the weight, endometrial thickness, and pathological structure of the uterus in the GFC treated groups were significantly improved. The expression of Med12, Wnt1, ll-Catenin, Cyclin D1, TCF1/TCF7, and C-myc that related to Wnt/ll-Catenin pathway in the GFC treated groups were decreased with the increase of dosage administration.Conclusions: GFC inhibited UFs growth, which was directly associated with Med12 modulated Wnt/ll-Catenin signaling pathway. This study provided new perspective to understand the therapeutic mechanism of UFs.