SHLP6: A Novel NLRP3 and Cav1 Modulating Agent in Cu-Induced Oxidative Stress and Neurodegeneration

Thamarai Kannan H Suganiya Umapathy Ieshita Pan ^*

• Saveetha School of Engineering, Saveetha University, Chennai, India

Exposure to copper sulfate exposure induces oxidative stress by triggering the overproduction of reactive oxygen species, leading to inflammatory responses, activation of neuroinflammatory mediators, and exacerbation of tissue damage and cellular dysfunction. Small humanin-like peptide-6, a mitochondrial-derived peptide, known for its anti-aging and anticancer properties,has not been explored for its protective role against copper sulphate in vivo. This study aims to investigate the antioxidant, anti-inflammatory and neuroprotective potential of small humanin-like peptide-6in mitigating copper sulfate toxicity in zebrafish larvae. Zebrafish larvae were exposed to copper sulfate and treated with varying concentrations of small humanin-like peptide-6 (10–50 µg/ml). Treatment with small humanin-like peptide-6 at a concentration of 40 µg/ml significantly reduced malformations, improved heart rate by 178 bpm, and increased survival rates by 85% in zebrafish larvae exposed to copper sulfate. Small humanin-like peptide-6demonstrated the highest inhibition of 58.7% and 74.3% inin-vitro reactive oxygen speciesscavenging assays. It also enhanced the antioxidant enzymes, such assuperoxide dismutase (68.3 U/mg), catalase (82.40 U/mg), and reduced glutathione (79.3 U/mg), while reducing lipid peroxidationand nitric oxide levels to 3.86 and 3.41 U/mg respectively. Furthermore, treatment with small humanin-like peptide-6 improved acetylcholine esterase levels (78.3 U/mg), prevented locomotor activity with a distance travelled of 43.53 m and modulated gene expression related to inflammation and neuroinflammation. Small humanin-like peptide-6upregulated TNF-α (2.16-fold), NLRP3 (1.78-fold), and Cox-2 (0.705-fold) gene expression whilealso upregulatingIL-10 by 1.84-fold, indicating regulationof neuroinflammation. Antioxidant genes SOD (1.3-fold), CAT (1.7-fold), GST (1.5-fold) and GSH (1.8-fold) were significantly upregulated by small humanin-like peptide-6, suggesting a role in boosting cellular antioxidant defenses. Therefore, small humanin-like peptide-6 shows promise as a potent neuroprotective and antioxidant agent against copper sulfate-induced oxidative stress, ultimately mitigating reactive oxygen species, production, inflammation and neurodegeneration.