| Lead can contaminate food through the enrichment of plants and animals,residues in packaging or production equipment during food processing,food additives or ingredients,etc.Lead contamination is currently a food safety problem that seriously threatens the health of our residents.Traditional methods to improve lead toxicity can cause loss of essential trace elements,tissue damage and other adverse effects to the body,therefore,the development of new safe and effective methods to improve lead toxicity is the focus of current research in the field of food safety in China.It was found that based on the antagonistic effect of selenium and lead,selenium-enriched proteins and peptides have significant ameliorative effect on lead-induced oxidative damage in the body.As a natural selenium-enriched food,the selenium of selenium-enriched Pleurotus eryngii is mostly combined with peptides,but the relevant structural characteristics and the mechanism of action to ameliorate lead toxicity have not been systematically resolved.In the present study,we selected the active P.eryngii seleniumcontaining peptides fraction by constructing a model of lead-induced hepatocyte oxidative damage and investigated the structural characteristics of selenium-containing peptides and the mechanism of amelioration of lead toxicity,with the following main findings:1.The selenium-containing proteins were extracted from the selenium-enriched P.eryngii by alkaline extraction and acid precipitation,and the selenium-containing peptides were prepared by enzymatic digestion using trypsin.The fractions were selected and structurally characterized by lead-induced NCTC1469 hepatocytes model,ultrafiltration combined with multistage chromatography and mass spectrometry.The results showed that the active selenium-containing peptides of P.eryngii had an ameliorative effect on the oxidative damage induced by lead exposure in NCTC1469 cells.The selenium-containing peptides(SPH-Ⅰ-2)were structurally characterized by LC-MS/MS,and 12 selenium-containing peptides were identified using PEAKS Studio8.5 software.2.The mechanism of amelioration of lead toxicity by SPH-Ⅰ-2 was investigated by constructing a model of oxidative damage induced by lead exposure in NCTC1469 cells.The results showed that SPH-Ⅰ-2 could alleviate the lead-induced apoptosis and reduce the lead-induced increase in AST and ALT levels in hepatocytes by inhibiting the activation of caspase-3 and caspase-9 enzymes,which regulate apoptosis.SPH-Ⅰ-2could activate Nrf2 pathway-related genes and enhance the expression level of related proteins to improve the lead-induced oxidative damage in hepatocytes.Compared with the lead-treated group,the expression levels of Nrf2 and its downstream HO-1,GCLC,GCLM and NQO1 related genes and proteins were significantly increased in the SPH-Ⅰ-2-treated group.In the SPH-Ⅰ-2 treated group,ROS levels,NO and LDH release and MDA levels were significantly reduced,and intracellular GSH levels and SOD and CAT antioxidant enzyme activities were significantly increased(P < 0.05).3.The absorption and transport of SPH-Ⅰ-2 in the intestinal epithelium and the effect of SPH-Ⅰ-2 on the amelioration of lead toxicity in hepatocytes after absorption and transport were studied by constructing Caco-2 monolayer cell model and Caco-2/NCTC1469 cell co-culture system.The results showed that SPH-Ⅰ-2 was well absorbed in the intestinal epithelial cells,and SPH-Ⅰ-2 was transported across the membrane through the endocytosis of cell membrane.In the Caco-2/NCTC1469 cell co-culture system,SPH-Ⅰ-2 still had strong antioxidant capacity and protected hepatocytes after the uptake and transport by the Caco-2 monolayer cell membrane,which could inhibit the lead-induced oxidative stress in hepatocytes by activating the intracellular antioxidant system to alleviate the effect of lead toxicity. |
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