Role of timp2 in Ctenopharyngodon idellus muscle collagen deposition and the regulatory mechanism by Cu2+

Collagen serves as one of determinants of fish muscle quality. <i>Timp2</i> is a key enzyme in collagen metabolism. Its role in grass carp (<i>Ctenopharyngodon idellus</i>) and related nutritional regulation mechanism are unknown. Given the important role of copper as a micronutrient in collagen biosynthesis, it was hypothesized that Cu²⁺ up-regulates <i>timp2</i> expression through the TGFβ/Smads signaling pathway, thus modulating grass carp collagen content. In the present study, this hypothesis validation was performed through parallel experiments using the TGF-β/Smads inhibitor SB431542 and <i>timp2</i>-specific siRNA (si<i>timp2</i>) in cultured fibroblasts. Results showed that both si<i>timp2</i> and SB431542 treatment decreased the collagen content (to 90.2 % ± 0.52 % and 61.8 % ± 1.59 % of control, respectively), down-regulated <i>timp2</i> expression (by 53.8 % ± 1.30 % and 11.3 % ± 1.91 % relative to control) and up-regulated <i>mmp2</i> expression (by 33.1 % ± 9.70 % and 10.9 % ± 2.51 % relative to control). Additionally, SB431542 inhibited the TGFβ/Smads signaling pathway (<i>P</i> < 0.05). Coversely, Cu²⁺ supplementation significantly increased the collagen content, up-regulated <i>timp2</i> expression and enhanced TGFβ/Smads signaling pathway activity, while simultaneously suppressing <i>mmp2</i> expression (<i>P</i> < 0.05). Futhermore, after si<i>timp2</i> or SB431542 treatment, 10 μM Cu²⁺ reversed the changes in the relevant indicators induced by both si<i>timp2</i> and SB431542 treatment (<i>P</i> < 0.05). These results confirmed the hypothesis that Cu²⁺ enhanced collagen deposition through TGFβ/Smads-mediated up-regulation of <i>timp2</i>. The present study provided novel mechanistic insights into copper-dependent collagen regulation and suggested that dietary copper supplementation could be a practical nutritional strategy to improve muscle texture and product quality in farmed grass carp.