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other·dry eye, ophthalmology, nutrition, antioxidants, clinical trial·PMC10454390

Isolation and Identification of a Novel Anti-Dry Eye Peptide from Tilapia Skin Peptides Based on In Silico, In Vitro, and In Vivo Approaches

International Journal of Molecular Sciences · 10 authors, 3 centres

AI SUMMARY

FIDELITY 88%

POPULATIONHuman corneal epithelial cells (HCECs) in vitro; C57BL/6 male mice (aged 6–7 weeks) with benzalkonium chloride (BAC)-induced dry eye disease in vivo

INTERVENTIONNGGPSGPR (NGG) peptide isolated from tilapia skin peptides (TSP); applied at 2.5, 5, and 10 μg/mL in vitro; 0.01% NGG eye drops in vivo

COMPARISONIn vitro: NaCl (100 mM) alone (M group); in vivo: DED group (BAC + saline), DED + SH (sodium hyaluronate eye drops) as positive control

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AGENT 01 · STRUCTURED SUMMARY

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TL;DR

This study identified a novel anti-dry eye peptide (NGGPSGPR, NGG) from tilapia skin peptides using in silico, in vitro, and in vivo methods. NGG restored cell viability and inhibited COX-2 expression in hyperosmolar-stressed human corneal epithelial cells, and in a mouse dry eye model it increased tear production, reduced tear ferning, prevented corneal thinning, and reduced goblet cell loss and apoptosis. These findings suggest NGG could be developed as a functional food ingredient or therapeutic agent for dry eye disease.

Full summary

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**Background:** Dry eye disease (DED) is a common ocular surface disorder driven by inflammation, with tear hyperosmolarity as a key stressor. Current anti-inflammatory treatments (e.g., corticosteroids, cyclosporine) have side effects. Tilapia skin peptides (TSP) have shown anti-DED activity in previous work, but the specific active peptides were unknown. This study aimed to isolate and identify the anti-DED peptide(s) from TSP using in silico, in vitro, and in vivo approaches.

**Methods:** TSP was fractionated by Sephadex G-25 chromatography into five fractions (TSP-I to TSP-V). The anti-DED activity of each fraction was tested in a hyperosmolar (100 mM NaCl) in vitro model using human corneal epithelial cells (HCECs) via MTT assay. The most active fraction (TSP-II) was analyzed by LC-MS/MS, yielding 131 peptides. In silico screening used ToxinPred (toxicity), PeptideRanker (bioactivity score ≥0.6), AIPpred (anti-inflammatory peptide prediction), and AllerTOP v.2.0 (allergenicity). Peptides with <11 amino acids, non-toxic, non-allergenic, and predicted anti-inflammatory were selected. Molecular docking against COX-2 (PDB: 5kir) was performed using HPEPDOCK. The top candidate, NGGPSGPR (NGG), was synthesized and tested in vitro (MTT, Western blot for COX-2) and in vivo in a BAC-induced DED mouse model (0.3% BAC, 14 days). Outcomes included tear production (phenol red thread), tear ferning score, corneal epithelial thickness (H&E), conjunctival goblet cell count (PAS), and corneal epithelial apoptosis (TUNEL).

**Key Results:** TSP-II (500 μg/mL) significantly restored HCEC viability under NaCl stress (p<0.05 vs. M group). LC-MS/MS identified 131 peptides in TSP-II (MW 634–1927 Da, 96.44% <1500 Da). In silico screening narrowed to three candidates: DFCPPGFNTK (DFC), EAPDPLRN (EAP), and NGGPSGPR (NGG). Molecular docking showed NGG had the highest HPEPDOCK score (−139.07) and bound within the COX-2 active pocket via four conventional hydrogen bonds and additional hydrophobic/electrostatic interactions. In vitro, NGG (2.5–10 μg/mL) significantly restored HCEC viability (p<0.01 vs. M group) and suppressed COX-2 protein expression (p<0.01 vs. M group). In vivo, NGG (0.01% eye drops) significantly increased tear production (p<0.05 vs. DED group), reduced tear ferning score (p<0.05), prevented corneal epithelial thinning (p<0.01), reduced conjunctival goblet cell loss (p<0.05), and decreased corneal epithelial apoptosis (p<0.05). NGG was comparable to sodium hyaluronate (SH) in most outcomes.

**Clinical Implications:** NGG is a novel, food-derived anti-DED peptide that acts at least partly by inhibiting COX-2-mediated inflammation. It shows promise as a safe, natural alternative or adjunct to current DED therapies. Further studies in other DED models and elucidation of its structure-activity relationship are warranted.

PICO

PPOPULATION

Human corneal epithelial cells (HCECs) in vitro; C57BL/6 male mice (aged 6–7 weeks) with benzalkonium chloride (BAC)-induced dry eye disease in vivo

IINTERVENTION

NGGPSGPR (NGG) peptide isolated from tilapia skin peptides (TSP); applied at 2.5, 5, and 10 μg/mL in vitro; 0.01% NGG eye drops in vivo

OOUTCOME

In vitro: cell viability (MTT assay), COX-2 protein expression (Western blot); in vivo: tear production (phenol red thread test), tear ferning score, corneal epithelial thickness (H&E), conjunctival goblet cell count (PAS), corneal epithelial apoptosis (TUNEL)

STUDY TYPE

other

SPECIALTY

dry eye

SUMMARISED BY

AI pipeline

FIDELITY CHECK

88% · B

ORIGINAL PAPER

PMC10454390

Isolation and Identification of a Novel Anti-Dry Eye Peptide from Tilapia Skin Peptides Based on In Silico, In Vitro, and In Vivo Approaches

International Journal of Molecular Sciences

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ABSTRACT

Tilapia skin is a great source of collagen. Here, we aimed to isolate and identify the peptides responsible for combating dry eye disease (DED) in tilapia skin peptides (TSP). In vitro cell DED model was used to screen anti-DED peptides from TSP via Sephadex G-25 chromatography, LC/MS/MS, and in silico methods. The anti-DED activity of the screened peptide was further verified in the mice DED model. TSP was divided into five fractions (TSP-I, TSP-II, TSP-III, TSP-IV, and TSP-V), and TSP-II exerted an effective effect for anti-DED. A total of 131 peptides were identified using LC/MS/MS in TSP-II, and NGGPSGPR (NGG) was screened as a potential anti-DED fragment in TSP-II via in silico methods. In vitro, NGG restored cell viability and inhibited the expression level of Cyclooxygenase-2 (COX-2) protein in Human corneal epithelial cells (HCECs) induced by NaCl. In vivo, NGG increased tear production, decreased tear ferning score, prevented corneal epithelial thinning, alleviated conjunctival goblet cell loss, and inhibited the apoptosis of corneal epithelial cells in DED mice. Overall, NGG, as an anti-DED peptide, was successfully identified from TSP, and it may be devoted to functional food ingredients or medicine for DED.

CITATION EXPORT

Jian Zeng, Cuixian Lin, Shilin Zhang et al.. Isolation and Identification of a Novel Anti-Dry Eye Peptide from Tilapia Skin Peptides Based on In Silico, In Vitro, and In Vivo Approaches. International Journal of Molecular Sciences. (2023). https://doi.org/10.3390/ijms241612772

AUTHORS

10 · 3 CENTRES

Jian Zeng, Cuixian Lin, Shilin Zhang, Haowen Yin, Kaishu Deng, Zhiyou Yang, Yongping Zhang, You Liu, Chuanyin Hu, Yun-Tao Zhao

1College of Food Science and Technology, Modern Biochemistry Experimental Center, Guangdong Ocean University, Guangdong Province Engineering Laboratory for Marine Biological Products, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Zhanjiang 524088, China
2College of Food Science and Engineering, Ocean University of China, Yu-Shan Road, Qingdao 266003, China
3Department of Biology, Guangdong Medical University, Zhanjiang 524023, China

EDITOR / REVIEWER

Bent Honoré