The sea cucumber collagen contains a high content of hydrophobic amino acids, which play essential roles in various bioactivities. A total of 2647 unknown active peptide fragments (2–20 amino acids) were obtained via virtual enzymolysis from 16 known collagen sequences in Apostichopus japonicus . Then, the novel bifunctional hexapeptide (DCDPRL, 717.788 Da) with hypoglycemic and antioxidant activities was identified via molecular docking and pharmacokinetics. DCDPRL revealed strong radical scavenging capacity in vitro with IC 50 values for DPPH, ABTS, OH, and O 2 − of 206.12 μM, 186.06 μM, 1.52 mM, and 247.71 μM, respectively. In addition, DCDPRL (250 μM) effectively reduced blood glucose levels and oxidative damage in zebrafish larvae through glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic peptide receptor (GIPR) and Kelch-like ECH-associated protein 1 (Keap1) targets. Furthermore, the structure–activity relationship analysis revealed that sulfur-containing cysteine, hydrophobic amino acids (proline and leucine), and repeated acidic aspartic acid were essential for bifunctional activities.
Song Wang,Liu Song,Yaru Li,Zhenxin Zhao,Ting Xue,Guoxiang Luan,Huarong Guo
Holothurian cell culture remains unsuccessful due to the lacking of suitable medium and tissues. Here, a reliable in vitro cell culture system has been developed for Apostichopus japonicus by optimizing the basal and growth media as well as the sourced tissues. Blood vessel and longitudinal muscle tissues were first found to be the best two sourced tissues. Subsequently, the composition and content of free amino acids and carbohydrates in holothurian coelomic fluid were analyzed and the obtained data were used to design and obtain an optimal basal medium (SCBM-4). Next, an optimal growth medium (SCGM-24) was further developed by supplementing 1 % FBS, 25 % coelomic fluid and 20 % body wall enzymatic hydrolysate of sea cucumbers into SCBM-4. Primary blood vessel (SCBV) cells could be efficiently isolated by mechanical dissociation in tissue dissociation apparatus and survive healthily for 22 d in SCBM-4, and 58 d in SCGM-24 with a gradually decreased proliferation activity. Primary longitudinal muscle (SCLM) cells could only be efficiently isolated by explant culture and healthily survive for 52 d in SCBM-4 but with no proliferation activity, whereas their viabilities decreased sharply in SCGM-24. The epithelial-shaped SCBV cells could be successfully sub-cultured for 3 times using 4 °C PE solution, whereas the loosely attached round-shaped SCBV and SCLM cells could be passaged for 6 and 4 times by pipetting, respectively. Additionally, shrimp covert mortality nodavirus (CMNV) could cross-species infect and multiply in SCBV cells, and acute heat shock treatment could significantly upregulate the transcriptional expression of heat shock protein genes.
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