Neuroprotective Mechanism Of Procyanidins Mediated By Nrf2/ARE Signaling Pathway On PC12 Cells And Zebrafish Model

Neurodegenerative diseases are caused by chronic,progressive degeneration or loss of neurons in the brain.Although the mechanism underlying neurodegenerative diseases is not fully understood,there is considerable evidence that oxidative damage caused by excessive production of reactive oxygen radicals（ROS）,damaged antioxidant defense systems,or both play a key role in these diseases.Antioxidant supplementation may help prevent or alleviate neurodegenerative diseases.Procyanidins（PCs）are natural antioxidants.Their antioxidant capacity is stronger than that of vitamin C and vitamin E.The Nrf2/ARE pathway is an important antioxidant pathway.However,whether PCs can prevent or alleviate neurodegenerative diseases by regulating the Nrf2/ARE pathway remains unclear.In this study,PC12 and zebrafish model were used to explore the neuroprotective effect and mechanism of PCs.The main research contents and results are as follows:1.The H₂O₂-induced damage model was used to evaluate the neuroprotective effect of PCs on PC12 cells.Results showed that,compared with model group,PCs（2/4μg/m L）could significantly improve cell viability,reduce the intracellular ROS level,reduce the production of MDA,and increase the activity levels of GSH-Px,CAT,and SOD（p<0.05）.Studying the neuroprotective mechanism of PCs at the protein level.The experimental group was compared to model group.Results showed that PCs（4μg/m L）could activate Nrf2/ARE pathway,facilitate transfer of Nrf2 from the cytoplasm to the nucleus,allow Nrf2 to accumulate in the nucleus,increase the expression of phase II detoxification enzyme protein downstream of Nrf2/ARE pathway（p<0.05）.2.The zebrafish damage model induced by H₂O₂ was used to study whether PCs could activate Nrf2/ARE pathway as in the PC12 cell model induced by H₂O₂.The experimental group was compared to model group.Results showed that PCs（8/16μg/m L）could significantly reduce the production of ROS and MDA in zebrafish induced by H₂O₂,increase the activity levels of antioxidant enzymes such as GSH-Px,CAT,and SOD,and alleviate the motor behavior disorder（p<0.05）.PCs（16μg/m L）also upregulated the transcription level of Nrf2 gene and related genes downstream of Nrf2/ARE pathway:GCLC,GCLM,HO-1 and NQO1.3.A Parkinson’s disease（PD）model of PC12 cells was used to study the neuroprotective effect of PCs.The experimental group was compared to the model group.The results showed that PCs（2/4μg/m L）could significantly improve the viability of PC12 cells,reduce the production of ROS and MDA,and increase the activity levels of antioxidant enzymes such as GSH-Px,CAT,and SOD（p<0.05）.Studying the neuroprotective mechanism of PCs at the protein level.The experimental group was compared to the model group.Results showed that PCs（4μg/m L）could activate Nrf2/ARE pathway,facilitate transfer of Nrf2 from the cytoplasm to the nucleus,allow Nrf2 to accumulate in the nucleus,increase the expression of phase II detoxification enzyme protein downstream of Nrf2/ARE pathway（p<0.05）.Zebrafish PD model was used to study whether PCs can play the same neuroprotective role as PC12 cell Parkinson’s disease model.The experimental group was compared to the model group.Results showed that PCs（16μg/m L）could significantly reduce the production of ROS and MDA,increase the activity levels of antioxidant enzymes such as GSH-Px,CAT,and SOD,alleviate motor behavior disorder,and reduce the rate of apoptosis in dopaminergic neurons.PCs of 16μg/m L could also upregulate the transcription level of Nrf2/ARE signaling pathway（p<0.05）.4.To further study the relationship between the neuroprotective effect of PCs and the structural characteristics of PCs.The results showed that the neuroprotective effects of catechin（c）,epicatechin（EC）and epicatechin gallate（ECG）were no obviously different from the model group（p<0.05）,PCs dimer:procyanidin B1（B1）,procyanidin B2（B2）,procyanidin B3（B3）,procyanidin B4（B4）,procyanidin Procyanidin B1-3-O-gallate（B1-G）,Procyanidin B2-3-O-gallate（B2-G）and PCs trimer:procyanidins C1（C1）were significant difference with the model group（p<0.05）.The neuroprotective effect of PCs trimer C1 treatment group was better than that of PCs dimer（B1,B2,B3,B4,B1-G,B2-G）treatment group（p<0.05）.The neuroprotective effect of PCs is positively correlated with the degree of polymerization of PCs.In conclusion,PCs can improve the antioxidant capacity by activating Nrf2/ARE pathway and play a neuroprotective role by reducing oxidative damage.The degree of polymerization of PCs is an important factor for PCs to play a neuroprotective role.