EXPLORING THE MECHANISMS OF KC14 PEPTIDE FROM COMMON CARP BIOINFORMATICS AND IN VIVO ZEBRAFISH MODEL

Abstract

Oxidative stress can damage cellular macromolecules, leading to neurodegeneration. Utilizing antioxidant therapeutic approaches presents a novel and effective strategy to mitigate both neurodegeneration and oxidative stress. This research explored the antioxidant and neuroprotective properties of the KC 14 peptide, derived from the liver-expressed antimicrobial peptide (LEAP 2A) of Cyprinus carpio L. The antioxidant potential of KC 14 was assessed through molecular docking studies. Concentrations of the peptide ranging ti"om 5 to 45 f!M were tested in vitro and in vivo using a zebra fish model. The study focused on evaluating the developmental and neuroprotective effects of KC14 peptide on zebrafis~ larv~e__:xp_osed to H202, followed by treatment with KC14. Results showed that KC 14 enhanced and restored antioxidant enzyme levels in comparison to untreated larvae subjected to oxidative stress conditions. Molecular docking was employed to determine the binding affinities and docking scores with antioxidant enzymes, revealing significant interactions. Additionally, KC 14 was found to reduce neuronal damage by lowering lipid peroxidation and nitric oxide production, while enhancing acetylcholinesterase activity. To further confirm the neuroprotective properties ofKC 14, intracellular ROS levels in zebrafish larvae were measured using DCFH-DA and DPPP staining techniques, which demonstrated reduced ROS levels and diminished lipid peroxidation. Overall, the study confirmed the potent antioxidant activity of the KC 14 peptide, � suggesting its potential as a promising therapeutic candidate for treating neurodegenerative diseases driven by oxidative stress.

Specification

THE FIELD OF INVENTION (Pharmaceuticals)
This invention aligns with recent advancements in the biotechnology and pharmaceutical sectors,
particularly in the use of antioxidants and the development of novel pep tides from natural sources. It
centers on reducing oxidative stress and enhancing antioxidant activity, with promising applications
in drug development, pharmaceuticals, and functional supplements.
BACKGROUND OF THE INVENTION .
Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS)
and the body's capacity to detoxifY these reactive intermediates, such as catalase and superoxide
dismutase. This imbalance is a major contributor to neurodegenerative processes. Antioxidants play
a vital role in neutralizing ROS and preventing oxidative damage. Fish-derived antimicrobial peptides
(AMPs) have shown potential as antioxidants due to their diverse biological activities. The common
carp (Cyprinus carpio L.), a significant dietary fish in China and other countries, offers a promising
source of such peptides. Research on AMPs suggests their potential as effective therapeutic agents
for treating various diseases. Among the AMPs identified in common carp are two hydrophobic
peptidcs derived from P-defensin genes, skin mucus, and eDNA sequences of hepcidin and LEAP
2A. The LEAP 2A eDNA encodes a sequence of92 amino acids, with an open reading frame (ORF)
of 279 bp within a 1325 bp length. mRNA expression analysis of LEAP 2A has shown gene
expression across various tissues, including the skin, liver, gills, head, and kidney. Exploring the
antioxidant properties of the KC 14 peptide could pave the way for new strategies in managing
oxidative stress and discovering novel pharmaceutical drugs.
SUMMARY OF THE INVENTION
The invention focuses on evaluating the antioxidant potential of a short-chain sequence of KC 14
peptide derived from LEAP 2A from Cyprinus carpio, using a zebrafish larvae model. In this study,
zebra fish larvae exposed to oxidative stress by hydrogen peroxide (H202) are treated with the KC 14
peptide. The effectiveness of the peptide in enhancing antioxidant activity is assessed by measuring
changes in key antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT),
glutathione (GSH), glutathione peroxidase (GPx), and glutathione S-transferase (GST), which are
essential components of the antioxidant defense system. The study aims to elucidate the active
mechanism of the KC 14 peptide concerning its therapeutic potential and neuroprotective properties .
This is achieved through a combination of computational approaches, antioxidant assays, and
behavioral analysis in in vivo zebrafish larval models. To further understand the response at the
transcriptional level, the expression of antioxidant genes such as SOD, CAT, GPx, and glutathione
reductase (GSR) is examined in zebrafish larvae using the RT -PCR technique. This research offers
potential for advancing antioxidant therapies and developing functional foods and pharmaceutical
products targeting oxidative stress-related disorders
Specifications
The LEAP 2A peptide was derived from the eDNA sequence found in the previously established
transcriptome library of Cyprinus carpio. With accession number CF662655, the discovered
sequence for LEAP 2A was added to the nucleotide sequence database maintained by the European
Molecular Biology Laboratory (EMBL). LEAP 2A typically comprises 41 amino acid residues,
featuring four cysteines situated at conserved positions within the anticipated mature peptides. With
the help of HeliQuest tool the amino acid sequence of different peptide with helical structure was
predicted.
-----�--""7PeptiaeRanker, atool specializing in predicting the bioactivity ofpeptides,\vas employed to assess.-�
and rank the sequences generated by HeliQuest. Based on the results of PeptideRanker the short�
cha�in KC 14 peptide (1 KRALLA WCLLFL VC14) sequence was selected and then the 3D structure of
the common carp-derived LEAP 2A peptide was generated using the Chimera tool.
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�The KC 14 peptide was manufactured in GenScript, USA, and confirmed the purity as >80% pure
using HPLC and an MS detector (supplied by the company). After dissolving the peptide in .0.1%
DMSO and a I mM concentration stock was made and kept at -20�C for future research purposes.
� In-silico molecular docking analysis supported the in-vitro cell free and in-vivo zebrafish enzyme
estimation results. KC 14 peptide's molecular docking results revealed strong binding energies and
amino acid interactions with antioxidant proteins.
� The KCI4 peptide derived from common carp was synthesized and its antioxidant potential was
evaluated in the in vivo zebrafish embryo and larvae model. The hydrogen peroxide (HzOz) was used
to induce oxidative stress in this study.
� The stress-induced zebrafish larvae were treated with KC 14 peptide derived from common carp in
a semi-static exposure manner up to 96 hours post fertilization (hpt).
� Assessment of antioxidant enzyme levels like superoxide dismutase (SOD) and catalase (CAT)
glutathione S-transferase (GST), glutathione (GSH), and glutathione peroxidase (GPx) enzymes in
zebrafish larvae following treatment with KC 14 peptide was carried out�5.
Introducing KC 14, a novel short-chain bioactive peptide derived from LEAP 2A in Cyprinus carpio
(common carp), which exhibits exceptional antioxidant and neuroprotective properties. KC 14
represents a significant advancement in antioxidant therapy, providing robust protection against
oxidative stress-induced damage. Its molecular structure, predicted using the HeliQuest tool and
validated through 3D modeling with Chimera, highlights its potential as a therapeutic agent. With its
helical conformation and bioactive nature, KC14 effectively modulates intracellular reactive oxygen
species (ROS) levels and enhances the activity of key antioxidant enzymes, including superoxide
dismutase (SOD), catalase (CAT), and glutathione-related enzymes such as GSH, GPx, and GST.
The ncuroprotective effects of KC 14 have been evaluated using a novel Ice-tray method, which
analyzes the locomotor response of oxidative stress-induced zebrafish larvae treated with the peptide.
The studies reveal that KC14 significantly reduces nitric oxide levels and lipid peroxidation. In
experiments using zebrafish embryo and larvae models, KC 14 demonstrates promising results by
providing protection against hydrogen peroxide (H202)-induced oxidative stress. The observed
upregulation of antioxidant gene expression further confirms its potential to enhance antioxidant
defenses, positioning KC 14 as a powerful candidate for developing new therapies targeting oxidative
stress-related conditionsvi
We Claim
I. Claim: A method for synthesizing the KC 14 peptide from Cyprinus carpio involves usmg
bioinformatics tools to identifY the peptide sequence and optimize its bioactivity for enhanced
antioxidant properties.
2. Claim: A formulation with KC 14 peptide for biomedical research, demonstrating its capability to
modulate antioxidant enzyme levels.
3. Claim: A method for assessing the effects of KCI4 peptide from common carp on superoxide
cliBinutaseJ:SQQ),_catalase (CAT), and g-lutathione family enzyme levels in a zebrafish larvae model -~~-~~--~-~--~--~----
exposed to oxidative stress.
4. Claim: A pharmaceutical composition with KC 14 peptide for ()xidative .stress-related disorders,
providing neuroprotection by reducing intracellular ROS, restoring acetylcholinesterase (AChE)
levels, and lowering nitric oxide (NO) and lipid peroxidation in zebrafish larvae.
5. Claim: A novel, sustainable, and eco-friendly KCI4 peptide serves as an effective antioxidant
�agent, offering environmental and health benefits.

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