Protein hydrolysates from Pleurotus geesteranus obtained by simulated gastrointestinal digestion exhibit neuroprotective effects in H ₂ O ₂ ‐injured PC12 cells

Abstract

Neurodegenerative diseases are considered to be among the diseases most threatening to human beings. Increasing evidence shows that antioxidant hydrolysates/ peptides with neuroprotective effects may relieve neurodegenerative diseases.However, related research in mushrooms, one of the richest sources of antioxidant hydrolysates/peptides, is in its infancy. Therefore, the in vitro neuroprotective effects of protein hydrolysates from Pleurotus geesteranus were researched in this study.Proteins were extracted from P. geesteranus and then hydrolyzed by simulated gastrointestinal digestion. The neuroprotective effects of the protein hydrolysates were evaluated by H 2 O 2 -injured PC12 cells. The hydrolysates showed a superior antioxidative ability and had a higher abundance of hydrophobic amino acids (e.g., leucine, alanine, and phenylalanine). Neither cytotoxicity nor the increase of ROS in PC12 cells was observed under treatment with the hydrolysates. However, pre-treatment with the hydrolysates in PC12 cells, which were then injured by H 2 O 2 , markedly attenuated ROS generation and enhanced the activities and mRNA expression of the endogenous antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD)), leading to a 26.68% increase in cell viability. The hydrolysates exhibited strong neuroprotective activity in H 2 O 2 -injured PC12 cells, possibly by reducing ROS generation and enhancing the activity of the antioxidant enzymatic system. Practical applicationsAntioxidant hydrolysates with neuroprotection were obtained from Pleurotus geesteranus proteins by simulating gastrointestinal digestion, which exhibited an excellent pre-protective effect in oxidatively damaged PC12 cells. Further study showed that hydrolysates pre-protection may exert antioxidant activities not only as an exogenous antioxidant to scavenge ROS but also as a gene regulator to modulate the endogenous antioxidant enzymes gene expression. These results indicated that the potential of antioxidant peptides, derived from P. geesteranus through gastrointestinal digestion, could serve as a source of bioactive molecules in the prevention, relief or even treatment of neurodegenerative disorders.

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“…Proteins and hydrolysates were prepared as previously described. 18 Se-CM powders (15 g/mL) were mixed with 5% ice-cold acetic acid solution and then stirred for 3 h at 25 °C. After that, the mixture was centrifuged at 4 °C, 8500 × g for 15 min to get the supernatant.…”

“…Hydrolysates of the proteins were prepared by simulated gastrointestinal (GI) digestion in vitro. 18 Protein solution (10 mg/mL) was hydrolyzed by pepsin (pH 2.0, 37 °C, 2 h) and pancreatin (pH 7.0, 37 °C, 2 h) sequentially. The protease/ substrate ratio was 4% (w/w) according to a previous study.…”

“…The protease/ substrate ratio was 4% (w/w) according to a previous study. 18 Finally, the hydrolysis was terminated at 100 °C for 10 min and then centrifuged at 4 °C, 8000 × g for 15 min. Afterward, the supernatant was collected and stored at −80 °C before use.…”

“…All fractions were concentrated using a rotary evaporator (EYELA CCA-1111, Japan) for bioactive activity assessment in an H 2 O 2 -injured PC12 cell model. The cell model was established as a previous study 18 with some modifications. The optimum concentration of the sample used in the cell model was 100 μg/mL (Figure S1A).…”

“…The SDS-PAGE, tricine-SDS-PAGE, TCA-soluble peptides, and determination of amino acid composition were performed following a previous study. 18 Mice and Administration. The experiment was approved by the Management and Ethics Committee of Experimental Animals of the Institute of Microbiology, Guangdong Academy of Sciences (Permit No.…”