| Tartary buckwheat is a rare medicine food homology plant. Tartary buckwheat protein is one of the main functional components of tartary buckwheat. In recent years,studies have shown that tartary buckwheat protein has physiological activities such as antioxidant, hypolipidemic, blood pressure and so on. In this paper, the buckwheat hydrolysates were isolated and purified by ultrafiltration and gel chromatography for preparation of antioxidant peptides, and the model of oxidative stress and lipid metabolic disorder in HepG2 cells was established by hydrogen peroxide and oleic acid treatment respectively, so the effects of tartary buckwheat bioactive peptides on the regulation of oxidative stress and lipid metabolism in model cells were investigated,and its possible mechanism was studied by RT-PCR. The possible action pathway of tartary buckwheat peptide to regulate blood lipid was revealed from two aspects of antioxidation and lipid metabolism, and the aim was to provide theoretical basis for further development of tartary buckwheat protein. The experiment results were as follows:(1) Using in vitro antioxidant capacities (ferric reducing antioxidant power,hydroxyl radical scavenging capacity and ABTS radical scavenging capacity) as evaluation index. tartary buckwheat peptide was prepared by using gel chromatography and ultrafiltration separation technology. Then the molecular weight distribution of the peptide was determined by gel exclusion chromatography (GPC). The results showed that the ferric reducing ability of plasma, hydroxyl radical scavenging capacity and ABTS radical scavenging capacity were 37.8±4.2 mg TE (trolox equivalents)/g 77.3±4.1 mg TE/g and 67.3±5.8 mg TE/g respectively. The molecular weight of TBPH-II was mainly distributed at 1 KDa to 3 KDa, and the polypeptide with a weight average molecular weight of 1.29 KDa accounted for 61.4 % of TBPH-II.(2) The oxidative stress model was successfully established with 0.1 mM H2O2 for 24 hours to HepG2 cells. Then the cells were treated with different concentrations of tartary buckwheat bioactive peptide TBPH-II, and the redox state and antioxidant enzyme activity were detected. The mechanism was explored through RT-PCR. The results showed that after 2.0 mg/mL TBPH-II treatment, the activities of SOD and CAT in oxidative stress model cells were extremely significantly increased by 33.5 % and 60.9 % (P<0.01). The content of MDA and the ratio of GSSG/T-GSH was extremely significantly decreased 40.9 % and 47.4 % respectively (P<0.01). The mRNA expression of PPARa and HO-1 was significantly up-regulated (P<0.01). Our results demonstrated that TBPH-? could effectively improve the oxidative stress of HepG2 cells induced by H2O2,and it might improve the oxidative stress by activating the PPAR?/HO-1 signaling pathway.(3) HepG2 cells were treated with 0.75 mg/mL oleic acid 24h to establish a model of abnormal lipid metabolism in HepG2 cells. Then the cells were treated with different concentrations of tartary buckwheat bioactive peptide TBPH-? to detect the lipid metabolism. The mechanism was explored through RT-PCR. The results showed that after 2.0 mg/mL TBPH-? treatment, the content of total cholesterol and triglyceride in abnormal lipid metabolism model cells were decreased extremely significantly by 24.6 %and 34.4 % respectively (P<0.01),and Oil red O staining showed that lipid aggregation was improved. The mRNA expression of ABCA1 was significantly up-regulated(P<0.05),and the mRNA expression of HMGCR was significantly down-regulated(P<0.05). Our results demonstrated that TBPH-? could effectively improve the lipid metabolism abnormality induced by oleic acid in HepG2 cells. It might promote the cholesterol efflux of HepG2 cells by activating ABC A1 signaling pathway, and reduce cholesterol synthesis through the HMGCR signaling pathway in HepG2 cells.'In conclusion, tartary buckwheat peptide could effectively improve the oxidative stress and regulate the lipid metabolism in hepatocyte. |
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