Mechanism Study Of Wheat Germ Peptide Alleviating Oxidative Stress In PC12 Cells Exposed To Lead

Lead(Pb)is a heavy metal that is difficult to degrade and is widely present in industry,food,and the environment.Lead can enter the human body through diet,skin contact,and biological enrichment,accumulating in organs such as the liver,kidneys,and blood,causing damage to the body,especially the developing central nervous system,which can lead to adverse effects such as learning and memory deficits,changes in brain function structure,and decreased intelligence.It is reported that lead-induced damage is closely related to oxidative stress.Wheat germ is a byproduct of wheat milling that is rich in nutrients such as vitamin E,polyphenols,and carotenoids,making it the most nutritious part of wheat.It is reported that wheat germ peptides(WGP)obtained by enzymatic hydrolysis from wheat germ have high biological activity,and some studies have shown that wheat germ peptides have physiological functions such as antioxidant,anticancer,antibacterial,and blood pressure-lowering effects,but whether it can alleviate lead exposure-induced neurological damage has not been studied.Since the Phaeochromocytoma cell line 12(PC12)cells have typical neuronal characteristics and have been widely used as an in vitro model to study neuronal damage,this study establishes a lead exposure oxidative stress damage model using PC12 cells,and tests whether wheat germ peptides can alleviate oxidative stress damage caused by lead exposure to explore its specific mechanism of action.The main content and results of this study are as follows:Establishment of lead-induced oxidative stress damage model for subsequent studies.Firstly,this study found that lead at a concentration of 10μM caused a decrease in cell viability level(P<0.05)and an increase in reactive oxygen species(ROS)level in PC12 cells,as detected by cell viability and ROS assays.As the concentration of lead increased,lead exposure further reduced the level of cell viability(P<0.05)and increased the level of ROS(P<0.05).In order to determine whether lead interfered with the redox balance in PC12 cells,this study detected oxidative stress-related indicators in lead-exposed PC12 cells.The study found that lead exposure decreased the activity of catalase(CAT)and superoxide dismutase(SOD)(P<0.05).At the same time,due to the decreased activity of glutathione reductase(GR)and glutathione peroxidase(GPx)(P<0.05),the ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG)was also reduced(P<0.05),resulting in a significant increase in malondialdehyde(MDA)production(P<0.05)and ultimately leading to oxidative stress in PC12 cells.The effects of wheat germ peptide on oxidative stress-related indicators in lead-exposed PC12 cells were studied.Firstly,some biologically active WGP sequences were collected by literature search and synthesized by solid-phase synthesis by a company.The results of bioinformatics analysis showed that the WGP used in this study had no toxicity and good water solubility,and most of them had high activity scores.Cell viability tests showed that WGP had no toxicity to PC12 cells at concentrations ranging from 0-200μM,therefore the maximum concentration of200μM was chosen for subsequent tests.Cell viability tests with pre-incubation of WGP for 4 hours before lead exposure showed that WGP3,WGP8,and WGP9 increased the decrease in cell viability caused by lead exposure(P<0.05),thus these three peptides were selected for subsequent experiments.Detection of oxidative stress-related indicators in normal PC12 cells and lead-exposed PC12 cells found that treatment with WGP3,WGP8,and WGP9 alone did not disrupt the oxidative-reductive balance in normal PC12 cells.In lead-exposed PC12 cells,WGP3,WGP8,and WGP9 increased the activity of CAT and SOD(P<0.05).As the activity of GR and GPx in the glutathione redox system was elevated(P<0.05),the level of GSH/GSSG was also increased(P<0.05),which in turn reduced the levels of ROS and MDA(P<0.05),ultimately alleviating the oxidative stress caused by lead exposure in PC12 cells.The effect of wheat germ peptide on the Nrf2/Keap1/TXNIP signaling pathway in PC12 cells exposed to lead is investigated.Firstly,protein imprinting was used to detect whether wheat germ peptide regulates the expression of Nuclear factor erythroid 2-related factor 2(Nrf2),Kelch-like ECH-associated protein 1(Keap1),and Thioredoxin-interacting protein(TXNIP)in PC12 cells exposed to lead.The results showed that lead exposure downregulated Nrf2 protein expression and upregulated Keap1 and TXNIP protein expression(P<0.05).However,treatment with WGP3,WGP8,and WGP9 upregulated Nrf2 protein expression and downregulated Keap1 and TXNIP protein expression(P<0.05).To further investigate whether Nrf2 protein appeared to undergo translocation in this process,this study used immunofluorescence detection of Nrf2 protein localization.The results showed that the fluorescence intensity of Nrf2 protein in the nuclei of cells exposed to lead was higher than that in the control group,but without statistical difference(P>0.05).However,the fluorescence intensity of Nrf2 protein in the nuclei of cells treated with WGP3,WGP8,and WGP9 was higher than that in the control group and has statistical difference(P<0.05).Meanwhile,the results of the fluorescence intensity of total Nrf2 protein in cells were consistent with the results of protein imprinting.The specific mechanism by which wheat germ peptides WGP3,WGP8,and WGP9 alleviate oxidative stress in PC12 cells exposed to lead was studied.Lead exposure upregulates the TXNIP protein in the nucleus and transports it to the mitochondria where it binds with TRX,subsequently inhibiting the antioxidant capacity of Thioredoxin(TRX).Lead exposure also upregulates Keap1 protein,which binds a large amount of Nrf2 protein in the cytoplasm,preventing downstream SOD and CAT antioxidant activation.Additionally,GR and GPx in the glutathione oxidation-reduction system are also inhibited,preventing GSH from being reduced to GSSG in a timely manner.Hence,a large amount of ROS in the cell cannot be decomposed,leading to the production of lipid peroxides and ultimately oxidative stress in PC12 cells.WGP3,WGP8,and WGP9 downregulate TXNIP expression and inhibit its binding with TRX,allowing TRX to perform its antioxidant function.Simultaneously,they downregulate Keap1 protein and shift a large amount of Nrf2 protein into the nucleus,thus activating downstream SOD and CAT’s antioxidant capacity.In addition,the activity of GR and GPx is enhanced,maintaining the normal redox balance of the glutathione oxidation-reduction system.Therefore,a large quantity of ROS is broken down through the activation of antioxidant systems,reducing MDA production,and ultimately mitigating oxidative stress damage caused by lead exposure in cells.