The Study Of Antioxidant Mechanism Of Chickpea Bioactive Peptide

Large amounts of research have demonstrated that oxidative stress is intimately relatedwith a variety of chronic and acute diseases, such as cardiovascular disease, cancer,neurological disorders, diabetes, ischemic reperfusion and aging. The antioxidant peptidesparticipate in the scavenging of free radicals and thus prevent or reduce the damage ofreactive oxygen species. In this study, the antioxidant capacity of chickpea active peptide andthe regulation of Nrf2-Keap1signaling pathway was demonstrated by H2O2-induced Caco-2and HT-29cell lines. The main conclusions have been drawn as below:Firstly, the scavenging ability of chickpea active peptide on DPPH·and O2—·increasedin a concentration-dependent manner. The scavenging rate of DPPH·and O—2·at10mg/mLwere60.0%and79.2%, respectively.Secondly, Caco-2cells and HT-29cells were treated with different concentration ofchickpea active peptide, and then exposured in H2O2. Four indicators were determined: MDA,catalase (CAT) activity, glutathione reductase (GSH-Rx) activity and glutathione peroxidase(GSH-Px) activity. Compared to positive control (POS), the activities of three antioxidantenzymes elevated by40.2%、54.0%and101.5%in Caco-2cell line when treated with activepeptide at0.5mg/mL, respectively. In HT-29cells, they increased by33.2%、31.0%and102.0%. The results showed the protective effect of chickpea active peptide on H2O2-inducedcell lines.Thirdly, the mRNA expression level of Nrf2, NQO1, HO-1and γ-GCS were investigatedby PT-PCR to evaluate the antioxidant activity of this bioactive peptide on Nrf2andNrf2-mediated transcription in H2O2-induced Caco-2and HT-29cells. In Caco-2cells, themRNA expression level of Nrf2, HO-1, γ-GCS and NQO1had increased by3.1、3.0、3.4and3.5-fold compared to positive control, respectively. Nrf2, HO-1, γ-GCS and NQO1mRNAexpression in HT-29cells treated with0.5mg/mL peptide increased by2.9,3.0,3.6and3.2-fold. All of these results revealed that peptide treatment up-regulated the mRNA levels offour genes and supported the idea that peptide treatment can protect cells from H2O2-mediatedoxidative stress by activating antioxidant enzyme biosynthesis.Finally, modulation of the Nrf2-Keap1signaling pathway was demonstrated throughWestern Bolt. The protein expression levels of downstream antioxidant enzymes: HO-1,γ-GCS, NQO1and GAPDH were measured. Experimental results showed that the proteinexpression of antioxidant enzymes in Caco-2cells and HT-29cells increased with theincreasing concentration of chickpea peptide. Through the changes in protein expression andmRNA expression, we found that chickpea bioactive peptide can activate Nrf2-Keap1signaling pathway, which nake it easy for Nrf2to dissociate from Keap1and then might promote the transcription and expression of downstream genes. All the results might provethat chickpea bioactive peptide has significant protective effect on Caco-2cells and HT-29cells, and has a good prospect in the application of oxidative stress related chronic diseases.