Proline promotes prolyl 4-hydroxylase subunit alpha 1 via transforming growth factor beta 1 to increase the synthesis and deposition of collagen in grass carp (Ctenopharyngodon idellus) muscle fibroblasts: Insights from integrated bioinformatics and functional analysis.

The present study aimed to clarify the molecular characteristics of prolyl 4-hydroxylase subunit alpha 1 (p4ha1) and elucidate its regulatory role in collagen synthesis in grass carp (Ctenopharyngodon idellus) muscle. To this end, the full-length coding DNA sequence (CDS) of muscle p4ha1 was first cloned and subjected to bioinformatics analysis, with sequence characterization specifically performed via prediction of open reading frame (ORF) and corresponding amino acid sequence, analysis of physical and chemical property, identification of conserved domain, alignment of amino acid sequence, and construction of the phylogenetic tree. The results revealed that the p4ha1 CDS is 1623 bp, encoding 540 amino acids, with a molecular weight of approximately 61.78 kDa and a theoretical isoelectric point of 6.47. The sequence includes a signal peptide and a prolyl 4-hydroxylase alpha subunit homologues domain (P4Hc). Furthermore, grass carp prolyl 4-hydroxylase subunit alpha 1 (P4HA1) shares the highest similarity with P4HA1 from redfin culter (Chanodichthys erythropterus, 95.96%) and blunt snout bream (Megalobrama amblycephala, 95.77%). Additionally, grass carp P4HA1 clusters closely with P4HA1 from other fish species, while house mouse (Mus musculus) and human (Homo sapiens) as mammals were clustered in another branch. Subsequently, in order to explore the effects of p4ha1 and transforming growth factor beta 1 (tgfb1) on collagen synthesis and its nutritional regulation in the muscle of grass carp, siRNA-mediated knockdown of p4ha1 and tgfb1 was performed in grass carp muscle fibroblasts to evaluate their effects on collagen synthesis. And then, the total collagen content was measured using Sirius Red staining, and the expression levels of collagen genes were evaluated using real-time quantitative PCR (RT-qPCR). Subsequently, proline was added to the medium at different concentrations to investigate its effect on collagen synthesis. The results showed a significant decrease in both collagen content and gene expressions (collagen type I alpha 1 chain [col1a1], collagen type I alpha 2 chain [col1a2], collagen type Ⅴ alpha 1 chain [col5a1], and p4ha1) following p4ha1 or tgfb1 knockdown (P < 0.05). And an appropriate amount of proline (0.9 mmol/L) could increase the total collagen content in muscle fibroblasts while activating the transforming growth factor β (TGFβ)/SMAD family members (SMADs) signaling pathway and promoting collagen synthesis (P < 0.05). Furthermore, p4ha1 and tgfb1 knockdown resulted in a decrease in the aforementioned indexes, which were significantly up-regulated after replacement of the medium containing 0.9 mmol/L proline (P < 0.05). Therefore, it was concluded that proline up-regulated the p4ha1 expression via tgfb1, thus increasing the collagen content in grass carp muscle.