Molecular Characterization of Melatonin Receptors in Perccottus glenii and Its Potential Roles in Melatonin-Mediated Antioxidant and Anti-Inflammatory Responses During Post-Freezing Recovery.

In this study, we report for the first time the cloning and identification of three melatonin receptors (PgMtnr1a, PgMtnr1b, and PgMtnr1c) from the freeze-resistant fish Perccottus glenii, all of which encode typical GPCR proteins. These receptors are expressed widely in high-metabolism tissues such as the liver. During the freezing and recovery process at -2 °C, the expression of liver receptors exhibited dynamic changes: PgMtnr1a and PgMtnr1c were significantly upregulated during the middle of resuscitation, while PgMTNR1A reached its peak expression in the later stages, suggesting its involvement in stress repair. In vitro experiments confirmed that melatonin significantly enhances the activity of liver antioxidant enzymes in a receptor-dependent manner, an effect that can be inhibited by the antagonist luzindole. Anti-inflammatory analyses indicate that melatonin primarily inhibits inflammation-related genes through Mtnr1a and Mtnr1b. Furthermore, the overexpression of PgMTNR1A can synergistically activate ERK in the presence of melatonin, inhibit the JNK/p38 MAPK pathway, and downregulate the expression of NF-κB and COX2. Pathway inhibition experiments further validated that the MAPK/NF-κB axis, including ERK, JNK, and p38 pathways, mediates the anti-inflammatory effects of melatonin. This study systematically elucidates the molecular mechanisms by which the melatonin receptors of P. glenii play crucial antioxidant and anti-inflammatory roles during the later stages of freeze-thaw resuscitation, particularly by regulating the MAPK/NF-κB signaling axis through MTNR1A, thereby providing a novel basis for understanding the adaptation of vertebrates to extreme environments.