[Estradiol

objectivesEstradiol is the principal form of estrogen, and its role in tissue injury and repair varies according to the regenerative capacity of the tissue. However, its mechanism of action in skeletal muscle, which has a strong regenerative capacity, remains unclear. This study aims to investigate the effects of estradiol signaling on myofiber regeneration and repair in a mouse model of cardiotoxin (CTX)-induced acute skeletal muscle inflammation.

methodsIn vivo experiments: A total of 80 wild-type male and 205 wild-type female C57BL/6 mice were included, of which 75 female mice underwent ovariectomy (OVX). Following the induction of acute skeletal muscle inflammation with CTX, the mice received intramuscular injections of β-estradiol, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), or other agents. The mice were divided into a control group, a β-estradiol group, a PHTPP group, a OVX group, and an OVX+β-estradiol group. Serum estradiol and creatine kinase (CK) levels were measured using enzyme-linked immunosorbent assays (ELISA). Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was used to determine the messenger RNA (mRNA) expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-13 in injured muscle from female mice. The expression of estrogen receptor (ER)α, ERβ, G protein-coupled estrogen receptor (GPR) 30, in injured muscle was assessed using real-time RT-PCR and Western blotting (WB). Flow cytometry was used to determine macrophage phenotypes and the proportions of M2 macrophages expressing insulin-like growth factor-1 (IGF-1) or hepatocyte growth factor (HGF) in injured muscle from female mice. Immunofluorescence staining was performed to evaluate monocyte/macrophage infiltration and the expression of phosphorylated IGF-1 receptor (p-IGF-1R) and embryonic myosin heavy chain (eMHC). In vitro experiments: C2C12 mouse myoblasts were differentiated into mature myotubes in medium containing 2% horse serum (HS) for 72 hours and then treated with interferon gamma (IFN-γ), β-estradiol, PHTPP, or combinations of these agents for 24 hours. Both monoculture and coculture systems were established. In the monoculture system, the main groups were control (C2C12+HS), IFN-γ, IFN-γ+β-estradiol, IFN-γ+PHTPP, and IFN-γ+β- estradiol+PHTPP. WB was used to assess the expression of mesenchymal-epithelial transition factor (c-Met), p-IGF-1R, paired box protein 7 (Pax7), and other proteins in C2C12 cells. In the coculture system, after 24 hours of treatment with IFN-γ, β-estradiol, PHTPP, or their combinations, C2C12 cells were cocultured with mouse peritoneal macrophages for 4 hours. The main groups were control (C2C12+HS+macrophages), IFN-γ, IFN-γ+β-estradiol, IFN-γ+PHTPP, and IFN-γ+β- estradiol+PHTPP. Flow cytometry was used to determine macrophage phenotypes and the proportions of M2 macrophages expressing HGF or IGF-1. WB was used to assess c-Met, p-IGF-1R, and Pax7 protein expression in C2C12 cells in the coculture system.

resultsVivo experiments showed that serum estradiol levels and ERβ expression in injured muscle were significantly higher in female mice than in male mice (both

conclusionsEstradiol-ERβ signaling axis promotes the infiltration of M2 macrophages into injured skeletal muscle and enhances their expression of IGF-1 in female mice, thereby alleviating inflammation and promoting myofiber regeneration and repair.