Neuroprotective And Anti-alzheimer’s Disease Studies Of Ginseng Peptides

Alzheimer’s disease(AD)is a slowly progressive neurodegenerative disease.Numerous studies have shown that oxidative stress plays an indispensable role in the development of AD.Various free radicals and reactive oxygen species(ROS)are produced during normal metabolic activities of the organism.These small amounts of ROS can coordinate with the antioxidant enzyme system and non-enzymatic antioxidant factors to maintain redox homeostasis in the body.However,once the ROS level exceeds the antioxidant capacity of cells,oxidative stress will occur.Neurons are more susceptible to direct damage from oxidative stress due to their high oxygen consumption and relatively low levels of antioxidants.Oxidative stress can promote Aβaggregation and tau protein hyperphosphorylation,resulting in the formation of amyloid plaques and neurofibrillary tangles,which in turn lead to neuronal cell death and further oxidative stress.Therefore,the use of antioxidants to maintain redox homeostasis within neuronal cells is also an effective way to treat Alzheimer’s disease.Due to the side effects of synthetic antioxidants,the search for natural antioxidants has become a current research hotspot.Bioactive peptides have attracted extensive attention for their high biosafety,low immunogenicity,easy synthesis,and antioxidant function.Ginseng peptides are biofunctional peptides obtained from ginseng or ginseng proteolytic products,which have important roles in antioxidant,anti-inflammatory,and immunomodulation.Thus,it may be both an antioxidant and a therapeutic or adjunctive treatment to slow down the progression of the disease.According to the above research,this study prepared ginseng peptides mixture by enzymatic digestion,systematically investigated its neuroprotective effects and mechanisms,and then isolated ginsenoside monomers and explored the potential function of monomeric peptides on Alzheimer’s disease in cellular and worm models.The main components of this study are as follows:In this study,ginseng fibrous root enzymatic hydrolysate(GFREH)was firstly prepared by enzymatic digestion,and then the molecular weight distribution of peptides in GFREH was measured by high-performance liquid chromatography(HPLC),and the antioxidant effect of GFREH was investigated in vitro and in vivo using free radical scavenging assay and worms oxidative stress model,respectively.The results showed that about 70% of the polypeptides in GFREH had molecular weights less than 2000 Da.In vitro,antioxidant assays illustrated that GFREH had good scavenging ability against 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),2,2-Diphenyl-1-picrylhydrazyl(DPPH),superoxide anion,and hydroxyl radicals.In addition,compared with juglone-damaged worms,GFREH improved the survival rate of worms,reduced the ROS level,up-regulated the expression of antioxidant enzyme SOD,reduced the accumulation of lipid and lipofuscin,and prolonged the lifespan of worms,which indicates that GFREH has effectively antioxidant effects.We next established a cellular oxidative injury model using neuroblastoma cells(SH-SY5 Y cells)and explored the protective effects and mechanisms of GFREH in oxidatively injured cells.The results proved that pretreatment with GFREH significantly increased cell viability,inhibited Ca2+ influx,ameliorated mitochondrial functional damage,reduced ROS accumulation,and reduced cell apoptosis compared with L-Glu-induced SH-SY5 Y cells.In addition,GFREH up-regulated the expression of nuclear factor-associated factor 2(Nrf2)and heme oxygenase 1(HO1),enhanced the activities of superoxide dismutase(SOD)and catalase(CAT),and down-regulated the expression of Keap1,inhibited the release of lactate dehydrogenase(LDH)and the production of malondialdehyde(MDA)compared with L-Glu alone exposed SH-SY5 Y cells.These results suggested that GFREH can alleviate L-Glu-induced oxidative damage by activating the Nrf2/Keap1 signaling pathway.To explore the antioxidant and neuroprotective effects of the active ingredients,we further isolated and identified the peptides,and the sequence of the purified peptides was determined to be Ile-Thr-Gly-Tyr-Ala-Pro(ITGYAP),Leu-Thr-Gly-Tyr-Ala-Pro(LTGYAP),Ile-Thr-Gly-Tyr-Pro-Ala(ITGYPA),and Leu-Thr-Gly-Tyr-Pro-Ala(LTGYPA).The neuroprotective effects of ITGYAP,LTGYAP,ITGYPA,and LTGYPA were investigated in AD cell model and worm model.The results demonstrated that all four ginseng peptides improved intracellular Ca2+ homeostasis,increased mitochondrial membrane potential,reduced intracellular oxidative stress levels,and inhibited cell apoptosis compared with Aβ1-42 oligomers treated cells.In the Th T staining assay,the fluorescence intensity of the ginseng peptide treated group was significantly lower than that of the Aβ1-42 alone culture group,indicating that the four ginseng peptides could inhibit the aggregation of Aβ1-42 in vitro.In the AD worm model,ITGYAP,LTGYAP,ITGYPA,and LTGYPA significantly delayed paralysis,reduced ROS content,improved worm health,extended lifespan,and reduced the accumulation of β-amyloid.In addition,molecular docking simulations indicated that ginseng peptides could bind to Aβ1-42 through intermolecular hydrogen bonding and hydrophobic interaction.In summary,GFREH exerted antioxidant activities and neuroprotective effects by activating the Nrf2/Keap1 signaling pathway.The ginseng peptide monomers ITGYAP,LTGYAP,ITGYPA,and LTGYPA inhibited the aggregation of Aβ in vitro and in vivo and alleviated the symptoms of AD,which may provide a new idea for the development of anti-AD drugs.