Resveratrol Promotes Goat Myoblast Differentiation via PROX1-Mediated Inhibition of NOTCH Signaling

Resveratrol (RES), a natural polyphenolic compound, has been reported to regulate cellular differentiation; however, its role in skeletal muscle development in ruminants remains unclear.

This study aimed to elucidate the molecular mechanism by which RES regulates myoblast differentiation in goats, with a focus on the role of PROX1 and its downstream signaling pathways.

Primary myoblasts isolated from a 3-mo-old fetal Nubian goat were used as an in vitro model and treated with 20 μmol/L RES, with untreated cells serving as controls. RNA sequencing was performed to identify differentially expressed genes, and RT-qPCR, molecular docking, as well as gain- and loss-of-function assays were conducted to investigate the role of PROX1 in myogenic differentiation. For in vivo validation, 20 healthy castrated male Nubian goats (180 ± 3 d of age; n = 10/group) were fed either a basal diet (CON) or a basal diet supplemented with 150 mg/kg RES for 120 d. Longissimus dorsi muscle samples were collected at the end of the experiment for subsequent analyses. Myogenic markers and signaling pathways were analyzed, and relative gene expression was calculated using the 2^-ΔΔCt method.

RNA-seq identified 221 differentially expressed genes after RES treatment, among which PROX1 was significantly upregulated (Log2Fold Change = 1.7, ∼3.25-fold compared with control, P < 0.0001). Molecular docking analysis indicated a stable interaction between RES and PROX1. Both in vitro and in vivo experiments showed that RES significantly increased PROX1 expression by ∼182% and 105%, respectively (P < 0.001). Functional analyses demonstrated that PROX1 overexpression promoted myogenic differentiation, increasing the mRNA expression levels of MyoD, MyoG, and MyHC to 2.70-, 1.75-, and 1.80-fold of control levels, respectively (P < 0.001), whereas MyoD protein expression increased by ∼80% (P < 0.001) and myotube formation was significantly enhanced (P < 0.001). In contrast, PROX1 knockdown markedly inhibited myogenic differentiation, reducing the mRNA expression of MyoD, MyoG, and MyHC by 75%, 48%, and 51%, respectively (P < 0.05), and decreasing MyoD protein expression by ∼43% (P < 0.001). Mechanistically, PROX1 promoted myogenic differentiation through suppression of the NOTCH1/HES1 signaling pathway (P < 0.05). Moreover, RES partially rescued the differentiation impairment induced by PROX1 deficiency through activation of PROX1 (P < 0.05). In vivo validation yielded results consistent with those observed in vitro.

RES promotes myoblast differentiation in goats through PROX1 activation and modulation of NOTCH signaling. These findings identify a PROX1-associated regulatory mechanism in skeletal muscle development in goats.