Abstract:

This study aimed to investigate the effects of the aldo-keto reductase family 1 member b1 (AKR1B1) gene on the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Bioinformatics tools were used to predict and analyze the structure and physicochemical properties of the AKR1B1-encoded protein. RNA was extracted from 8 tissues, including the duodenum, longissimus dorsi, and subcutaneous fat of 3-day-old piglets, and quantitative real-time PCR (qRT-PCR) was performed to determine AKR1B1 expression levels in each tissue. PSCs were isolated from leg muscles and longissimus dorsi and cultured in vitro, with samples collected at specific time points during proliferation and differentiation. Small interfering RNA (siRNA) was used to suppress AKR1B1 expression, and qRT-PCR, immunofluorescence, and Western blot analyses were conducted to evaluate AKR1B1′s role in PSCs proliferation and differentiation. The AKR1B1 protein consisted of 316 amino acids, was predominantly expressed in the cytoplasm, and was predicted to be a stable, highly hydrophilic, acidic protein. qRT-PCR results showed that AKR1B1 expression was highest in the longissimus dorsi and duodenum. During PSCs proliferation, AKR1B1 expression gradually increased, peaking on day 5 of differentiation before declining. Knockdown of AKR1B1 during proliferation significantly reduced the mRNA and protein levels of proliferation markers Ki67 and cyclin D1 (ccnd1) (P < 0.05) and decreased the proportion of EdU⁺ cells (P < 0.05). During differentiation, AKR1B1 knockdown significantly upregulated the mRNA and protein levels of differentiation markers myogenin (MyOG) and myosin heavy chain (MyH3) (P < 0.05). Immunofluorescence results showed a highly significant increase in the proportion of MyOG⁺ cells (P < 0.01) and an elevated differentiation index (P < 0.05). AKR1B1 knockdown significantly reduced the phosphorylation of AMPK at Ser182 (P < 0.05) while significantly increasing the phosphorylation of TSC2, RAPTOR, and mTOR at Ser2448 (P < 0.05). This led to a significant increase in the expression of S6K1 and S6 proteins (P < 0.05). In summary, AKR1B1 knockdown inhibited PSCs proliferation and positively regulated differentiation. AKR1B1 was found to mediate the AMPK/mTOR/S6 signaling pathway to regulate PSCs differentiation. This study provides insights into the specific effects of AKR1B1 on porcine skeletal muscle growth and development, offering a theoretical basis and scientific clues for healthy livestock management.

Key words: AKR1B1, porcine skeletal muscle satellite cells, skeletal muscle cell proliferation and differentiation, AMPK/mTOR/S6 pathway