Preventive Effects And Mechanism Of Lotus Seedpod Procyanidins On Acrylamide-induced Nerve Cell Injury

Lotus seedpod procyanidins?LSPCs?are flavonoids extracted from lotus plants.Their molecular structure contains electron-rich hydroxyl groups,which have strong antioxidant activity and preventive effect on neurotoxicity.However,there are a few studies on its preventive mechanism.Acrylamide?ACR?is a kind of food-borne toxicant that people will inevitably ingest in their daily life,which is mainly a Maillard reaction product.Studies have shown that ACR can induce neurotoxicity,through oxidative stress reaction.It has become one of the effective strategies to prevent the food-borne toxicity of ACR by screening the nutritional components from the natural products which can prevent the neurotoxicity of ACR.Based on this,in the present study,the inhibition or scavenging effect of LSPCs on ACR production was studied by chemical system and food system.And the astrocyte/hippocampal neuron co-culture system was selected to clarify the preventive effect and the mechanism of LSPCs on ACR-induced neurotoxicity,which may provide a theoretical basis for the development of LSPCs in preventing ACR-induced neurotoxicity.The main research contents and results are as follows:1.The inhibition or scavenging effects of LSPCs on ACR in different systemsThree different simulation systems were established.After adding different doses of LSPCs?0.05%,0.1%,0.25%,0.5%,1.0%?to the reaction system,the ACR was brominated to 2,3-Dibromopropionamide?2,3-DBPA?.The content of 2,3-DBPA was determined by gas chromatography to verify the inhibition or scavenging effect of LSPCs on ACR.In the simulated body temperature system,no significant effect of LSPCs addition on ACR content was found,indicating that LSPCs had no scavenging effect on generated ACR.In the simulated Maillard reaction system,the results showed that LSPCs could inhibit the formation of ACR of fructose/asparagine at different temperature.The maximum inhibition rates at different temperature were respectively 31.79%,34.00%,44.83%,42.42%and 39.61%.However,the inhibition rates were not linear with the addition of LSPCs,but increased with the increase of LSPCs dosage at 100¹20?.When the temperature reached 140?,the inhibition rate of high dose group began to decrease in varying degrees.In food system,the results showed that LSPCs blocked the formation of ACR,the maximum inhibition rate was 43.21%,but when the LSPCs content reached 1.0%,the inhibition rate decreased.2.Selection of Neurocyte Injury Model Induced by ACRAstrocyte model,hippocampal neuron cell model and astrocyte/hippocampal neuron co-cultured cell model were established.The cells were damaged by different concentrations of ACR?2,4,6,8,10?g/mL?.Three cell models were evaluated by immunofluorescence staining,morphological observation and MTT assay.The results showed that the cell purity of astrocyte model and hippocampal neuron could reach90%,and ACR could obviously damage the cell models.Among them,the co-cultured cell model is more stable than astrocyte model and hippocampal nerve cell model,and more close to the complex environment of human body,which is suitable for subsequent experiments.Therefore,the IC50 value of ACR was 6.78?g/mL,which was applied to co-cultured cells for 48 h.3.Preventive effect and mechanism of LSPCS on ACR-induced co-cultured cell injuryThe experiment was divided into control group,injury group and prevention group.The control group did not do any treatment.The injury group was treated with the damage conditions previously obtained.The prevention groups were pretreated with LSPCs?2.5,5,10?g/mL?for 4 hours in advance,then ACR was added to treat the co-cultured cells.?1?To verify the preventive effect of LSPCs on ACR-induced nerve cell injury,we observed the morphological changes,cell viability,intracellular[Ca²⁺]i,ROS content,mitochondrial membrane potential,glutathione?GSH?and glutathione peroxidase?GSH-Px?content of co-cultured cells.The results showed that after LSPCs pretreatment,the morphology of nerve cells was significantly improved and the cell viability was increased.Meanwhile,compared with the injured group,mitochondrial membrane potential increased significantly?P<0.01?,intracellular[Ca²⁺]i,ROS content decreased significantly?P<0.01?,GSH content and GSH-Px activity increased significantly?P<0.01?.In summary,LSPCs have a good inhibitory effect on the neurotoxicity of ACR.?2?Western blot was used to detect the changes of oxidative stress-related proteins?Nrf2,HO-1,cytoplasmic Nrf2?,inflammatory factor NF-?B,synaptic function-related proteins?SYN,Arc,?-III-Tubulin and PSD95?in co-cultured cells,to verify the toxic mechanism of LSPCs on ACR-induced nerve injury.The results showed that the expression of Nrf2,HO-1 and cytoplasmic Nrf2 were significantly increased under ACR exposure?P<0.01?,while the expression of NF-?B and cytoplasmic NF-?B were significantly increased?P<0.01?.Pretreatment with LSPCs,the expression of HO-1 was significantly decreased?P<0.01?,and nuclear metastasis of cytoplasmic Nrf2 and cytoplasmic NF-?B occurred.Exposure to ACR,the expression of synaptic function-related protein decreased,and the expression up-regulated pretreatment with LSPCs.The results showed that ACR exposure resulted in the accumulation of ROS,the depletion of glutathione and HO-1,the oxidative stress pathway was activatedand,synaptic function disorder.Pretreatment with LSPCs promoted the nuclear transfer of Nrf2 and NF-?B in nerve cells,and protected the synaptic function.