Relationship between Neuroprotective Effects and Structure of Procyanidins

This study evaluated the relationship between the neuroprotective effects of procyanidins and their structural characteristics. In vitro, a rat pheochromocytoma cell line (PC12) was exposed to the grape seed-derived procyanidin monomers: catechin (C), epicatechin (EC), and epicatechin gallate (ECG); the procyanidin dimers: procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), and procyanidin B2-3-O-gallate (B2-G); and the procyanidin trimers: procyanidin C1 (C1) and N-acetyl-l-cysteine (NAC) for 24 h. Cells were then incubated with 200 μM H2O2 for 24 h. In vivo, zebrafish larvae (AB strain) 3 days post-fertilization were incubated with NAC or procyanidins (C, EC, ECG, B1, B2, B3, B4, B1-G, B2-G, C1) in 300 µM H2O2 for 4 days. Different grape seed procyanidins increased the survival of PC12 cells challenged with H2O2, improved the movement behavior disorder of zebrafish caused by H2O2, inhibited the increase of ROS and MDA and the decrease of GSH-Px, CAT, and SOD activities, and up-regulated the Nrf2/ARE pathway. The neuroprotective effects of the procyanidin trimer C1 treatment group were greater than the other treatment groups. These results suggest that the neuroprotective effect of procyanidins is positively correlated with their degree of polymerization.


Introduction
Neurodegenerative disease refers to disorders caused by chronic, progressive degeneration or deletion of neurons in the human brain [1], mainly characterized by protein accumulation and synaptic loss or dysfunction of neurons in the central nervous system [2]. Because neurons in the central nervous system do not regenerate, damage to these cells cannot be reversed. Consequently, once neurodegenerative diseases occur, the effects are permanent [3]. Therefore, the identification of effective methods for the prevention of neurodegenerative diseases is essential. Although the underlying mechanisms of neurodegenerative disease are not fully understood, there is evidence that oxidative damage caused by excessive production of reactive oxygen species (ROS), free radicals, and damage to the antioxidant defense system play a key role. Therefore, antioxidant supplementation may prevent or alleviate the onset of neurodegenerative disease [4][5][6]. Natural bioactive compounds are generally safe and cause minimal to no side effects. Therefore, identification of antioxidant compounds from natural resources that may aid in the prevention of neurodegenerative diseases is an important research focus.
Procyanidins are natural antioxidants with an antioxidant capacity stronger than that of vitamins C and E [7][8][9]. Previous research from our team has shown that procyanidins exhibit neuroprotective effects [10]. Interestingly, individual procyanidins have distinct effects, which may be due to differences in their structure such as the number of hydroxyl groups, the positioning of the hydroxyl groups, the degree of polymerization, and the type of flavane-3-alcohol unit bond [11]. The bioavailability of procyanidins is also affected by AAGCCTCTGTCCTTTGCTCC TGCTGTGGTAATGCCGTAGG Nrf2: nuclear factor-erythroid 2-related factor 2; HO-1: heme oxygenase 1; NQO1: quinone oxidoreductase 1.

Statistical Analysis
The experimental results were statistically analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple range test; results are presented as means ± standard deviation (SD). A p < 0.05 was considered statistically significant. SPSS (version 20.0) was used for statistical analysis.

Statistical Analysis
The experimental results were statistically analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple range test; results are presented as means ± standard deviation (SD). A p < 0.05 was considered statistically significant. SPSS (version 20.0) was used for statistical analysis.

Effects of Procyanidins with Different Structures on H 2 O 2 -Induced Damage in PC12 Cells
The effects of grape seed procyanidins with different structures on the survival of PC12 cells induced by H 2 O 2 were studied. Compared with the model group, 2. Values with different letters above each bar represent significant differences (p < 0.05, one-way ANOVA). NAC, N-acetyl-L-cysteine.

Effects of Procyanidins with Different Structures on Oxidative Stress in PC12 Cells Treated with H2O2
ROS and MDA content in cells is an index that directly reflects the level of oxidative stress [29][30][31]. GSH-Px, CAT, and SOD are important components of the antioxidant system, which can antagonize and block free radicals [32,33]. The higher the activity of GSH-Px, CAT, and SOD, the stronger the ability to scavenge oxygen free radicals [34]. Therefore, the antioxidant capacity of cells can be reflected by detecting the activities of GSH-Px, CAT, and SOD. Compared with the model group, treatment with different procyanidins inhibited H2O2-induced ROS and MDA levels and increased the activities of GSH-Px, CAT, and SOD in PC12 cells. The treatment effect of the procyanidin trimer C1 was greater than that of the procyanidin dimers (B1, B2, B3, B4, B1-G, and B2-G) ( Figure 3, p < 0.05). The effect in the procyanidin monomers (C, EC, and ECG) treatment group was less than in the procyanidin dimers (B1, B2, B3, B4, B1-G, and B2-G) and procyanidin trimer C1 treatment groups ( Figure 3).

Effects of Procyanidins with Different Structures on Oxidative Stress in PC12 Cells Treated with H 2 O 2
ROS and MDA content in cells is an index that directly reflects the level of oxidative stress [29][30][31]. GSH-Px, CAT, and SOD are important components of the antioxidant system, which can antagonize and block free radicals [32,33]. The higher the activity of GSH-Px, CAT, and SOD, the stronger the ability to scavenge oxygen free radicals [34]. Therefore, the antioxidant capacity of cells can be reflected by detecting the activities of GSH-Px, CAT, and SOD. Compared with the model group, treatment with different procyanidins inhibited H 2 O 2 -induced ROS and MDA levels and increased the activities of GSH-Px, CAT, and SOD in PC12 cells. The treatment effect of the procyanidin trimer C1 was greater than that of the procyanidin dimers (B1, B2, B3, B4, B1-G, and B2-G) ( Figure 3, p < 0.05). The effect in the procyanidin monomers (C, EC, and ECG) treatment group was less than in the procyanidin dimers (B1, B2, B3, B4, B1-G, and B2-G) and procyanidin trimer C1 treatment groups ( Figure 3).

Effects of Procyanidins with Different Structures on the Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2)/Antioxidant Response Element (ARE) Pathway in PC12 Cells Treated with H2O2
The different structures of the procyanidin treatments may up-regulate the expression of the Nrf2 protein, leading to transfer from the cytoplasm to the nucleus and accumulation in the nucleus, thereby up-regulating the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) in H2O2 injured PC12 cells. Our results showed significant differences between the procyanidin dimers (B1, B2, B3, B4, B1-G, and B2-G) and procyanidin trimer C1 treatment groups compared to the model group ( Figure 4, p < 0.05).

Effects of Procyanidins with Different Structures on the Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2)/Antioxidant Response Element (ARE) Pathway in PC12 Cells Treated with H 2 O 2
The different structures of the procyanidin treatments may up-regulate the expression of the Nrf2 protein, leading to transfer from the cytoplasm to the nucleus and accumulation in the nucleus, thereby up-regulating the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) in H 2 O 2 injured PC12 cells. Our results showed significant differences between the procyanidin dimers (B1, B2, B3, B4, B1-G, and B2-G) and procyanidin trimer C1 treatment groups compared to the model group ( Figure 4, p < 0.05).

Verification of the Effect of Nrf2 in the Protection of Cells Treated with Procyanidins
We further evaluated the role of Nrf2/ARE pathway activation in procyanidin-mediated neuroprotection. Results showed that the expression of Nrf2 in PC12 cells transfected with Nrf2 siRNA decreased significantly, blocking the protective effect of procyanidins in PC12 cells challenged with H 2 O 2 ( Figure 5). These results suggest that procyanidins (B1, B2, B3, B4, B1-G, B2-G, and C1) may protect neurons from oxidative stress by activating the Nrf2/ARE pathway.

Effects of Procyanidins with Different Structures on Exercise Capacity in Zebrafish Treated with H 2 O 2
Neurodegenerative diseases often exhibit symptoms of motor behavior disorders [35][36][37]. Therefore, this experiment studied the effects of different structures of procyanidins on exercise capacity of zebrafish treated with H 2 O 2 . Overall, 25 µM of procyanidins (C, EC, ECG, B1, B2, B3, B4, B1-G, B2-G, C1) did not decrease the exercise capacity of zebrafish (Figure 6a,b) and had a protective effect on zebrafish damaged by H 2 O 2 (Figure 6 c,d). The protective effect of the procyanidin trimer C1 was better than that of the procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G) (Figure 6c,d, p < 0.05). There was no significant difference between the procyanidin monomer (C, EC, ECG) treatment groups and the model group (Figure 6c

Verification of the Effect of Nrf2 in the Protection of Cells Treated with Procyanidins
We further evaluated the role of Nrf2/ARE pathway activation in procyanidin-mediated neuroprotection. Results showed that the expression of Nrf2 in PC12 cells transfected with Nrf2 siRNA decreased significantly, blocking the protective effect of procyanidins in PC12 cells challenged with H2O2 ( Figure 5). These results suggest that procyanidins (B1, B2, B3, B4, B1-G, B2-G, and C1) may protect neurons from oxidative stress by activating the Nrf2/ARE pathway.   nidins on exercise capacity of zebrafish treated with H2O2. Overall, 25 μM of procyanidins (C, EC, ECG, B1, B2, B3, B4, B1-G, B2-G, C1) did not decrease the exercise capacity of zebrafish (Figure 6a,b) and had a protective effect on zebrafish damaged by H2O2 ( Figure  6 c,d). The protective effect of the procyanidin trimer C1 was better than that of the procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G) (Figure 6c,d, p < 0.05). There was no significant difference between the procyanidin monomer (C, EC, ECG) treatment groups and the model group (Figure 6c,d, p > 0.05).

Effects of Procyanidins with Different Structures on Oxidative Stress in Zebrafish Treated with H2O2
The contents of ROS and MDA in different procyanidin treatment groups were lower than the model group, and the activities of antioxidant enzymes (GSH-Px, CAT, and SOD)

Effects of Procyanidins with Different Structures on Oxidative Stress in Zebrafish Treated with H 2 O 2
The contents of ROS and MDA in different procyanidin treatment groups were lower than the model group, and the activities of antioxidant enzymes (GSH-Px, CAT, and SOD) were higher than the model group. There was no significant difference between the procyanidin monomer (C, EC, ECG) treatment groups and the model group (Figure 7, p > 0.05); however, there was a significant difference between the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) treatment groups and the procyanidin trimer C1 treatment group and the model group (Figure 7, p < 0.05). The effect of the procyanidin trimer C1 treatment group on reducing the content of ROS and MDA and increasing the activity of antioxidant enzymes (GSH-Px, CAT, and SOD) was greater than that of the other treatment groups (Figure 7). B3, B4, B1-G, B2-G) treatment groups and the procyanidin trimer C1 treatment gr the model group (Figure 7, p < 0.05). The effect of the procyanidin trimer C1 tr group on reducing the content of ROS and MDA and increasing the activity of ant enzymes (GSH-Px, CAT, and SOD) was greater than that of the other treatmen (Figure 7).

Effects of Procyanidins with Different Structures on the Nrf2/ARE Pathway in Zebrafish Treated with H2O2
Different procyanidin treatments can up-regulate the expression of Nrf2 and the related genes NQO1 and HO-1, which are downstream of the Nrf2/ARE pathway in H2O2induced zebrafish. There was no significant difference between the procyanidin monomer (C, EC, ECG) treatment groups and the model group (Figure 8, p > 0.05); however, there was a significant difference between the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) treatment groups and the procyanidin trimer C1 treatment group and the model group

Effects of Procyanidins with Different Structures on the Nrf2/ARE Pathway in Zebrafish Treated with H 2 O 2
Different procyanidin treatments can up-regulate the expression of Nrf2 and the related genes NQO1 and HO-1, which are downstream of the Nrf2/ARE pathway in H 2 O 2induced zebrafish. There was no significant difference between the procyanidin monomer (C, EC, ECG) treatment groups and the model group (Figure 8, p > 0.05); however, there was a significant difference between the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) treatment groups and the procyanidin trimer C1 treatment group and the model group ( Figure 8, p < 0.05). Up-regulation of the Nrf2, NQO1, and HO-1 genes in the procyanidin trimer C1 treatment group was greater than in the other treatment groups.

Discussion
Excessive production of ROS and oxidative damage caused by a damaged antio dant defense system plays a key role in neurodegenerative diseases. Antioxidant supp mentation may help to prevent or alleviate neurodegenerative diseases [4][5][6]. Procyani is a natural antioxidant with a stronger antioxidant capacity than vitamins C and E [7 The bioavailability of procyanidins is affected by their structure, and different plants c tain procyanidins with distinct structural types. Due to the large number of procyanid complex separation conditions, and difficulty with purification, previous studies h mainly focused on mixtures of procyanidins or individual procyanidins, and less on structure-activity relationship of a variety of procyanidins [15,16]. Previous studies h found that procyanidins can play a neuroprotective role by regulating the Nrf2/ARE pa way, but the relationship between the neuroprotective effect of procyanidins and structural characteristics of procyanidins is not clear [10]. Grape seed is one of the m abundant sources of procyanidins in nature [13]. Most procyanidin products are fr grape seed [14]. H2O2 can penetrate the cell membrane and cause oxidative stress. T method can thus be used to establish an oxidative stress model. Therefore, 10 proc

Discussion
Excessive production of ROS and oxidative damage caused by a damaged antioxidant defense system plays a key role in neurodegenerative diseases. Antioxidant supplementation may help to prevent or alleviate neurodegenerative diseases [4][5][6]. Procyanidin is a natural antioxidant with a stronger antioxidant capacity than vitamins C and E [7][8][9]. The bioavailability of procyanidins is affected by their structure, and different plants contain procyanidins with distinct structural types. Due to the large number of procyanidins, complex separation conditions, and difficulty with purification, previous studies have mainly focused on mixtures of procyanidins or individual procyanidins, and less on the structure-activity relationship of a variety of procyanidins [15,16]. Previous studies have found that procyanidins can play a neuroprotective role by regulating the Nrf2/ARE pathway, but the relationship between the neuroprotective effect of procyanidins and the structural characteristics of procyanidins is not clear [10]. Grape seed is one of the most abundant sources of procyanidins in nature [13]. Most procyanidin products are from grape seed [14]. H 2 O 2 can penetrate the cell membrane and cause oxidative stress. This method can thus be used to establish an oxidative stress model. Therefore, 10 procyanidins from grape seeds were selected for study of the relationship between the neuroprotective effects of procyanidins and their structural characteristics using H 2 O 2 -damaged PC12 cells and zebrafish modeling.
This study evaluated the effects of 10 grape seed-derived procyanidins on the survival of PC12 cells and the motor capacity of zebrafish challenged with H 2 O 2 . The results showed that treatment with 5 µM procyanidins resulted in a protective effect on the survival of PC12 cells, and treatment with 25 µM procyanidins resulted in a protective effect on the motor capacity of zebrafish challenged with H 2 O 2 . There was no significant difference between procyanidin monomers (C, EC, ECG) and procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G). Additionally, there was no significant difference between the procyanidin monomer (C, EC, ECG) treatment groups and the model group (p > 0.05). However, there was a significant difference between the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) and the procyanidin trimer C1 treatment groups and the model group (p < 0.05). The protective effect of procyanidin trimer C1 treatment on the survival rate of PC12 cells and the motor capacity of zebrafish challenged with H 2 O 2 was greater than that of the other treatment groups. This shows that the protective effect of procyanidins on the viability of PC12 cells damaged by H 2 O 2 , and the exercise ability of zebrafish damaged by H 2 O 2 , were positively correlated with the degree of procyanidin polymerization.
ROS is an indicator that directly reflects oxidative stress levels [29,30]. MDA is the end product of the peroxidation reaction of free radicals acting on membrane lipids under the catalysis of metal ions, which reflects the degree of oxidative damage [31]. We evaluated the effects of different procyanidins from grape seeds on the content of ROS and MDA in PC12 cells and zebrafish challenged with H 2 O 2 . Compared with the model group, procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G) and the procyanidin trimer C1 significantly inhibited H 2 O 2 -induced increases in ROS and MDA in PC12 cells and zebrafish (p < 0.05). GSH-Px, CAT, and SOD are important components of the antioxidant system, which can antagonize and block free radicals [32,33]. The higher the activity of GSH-Px, CAT, and SOD, the stronger the ability to scavenge oxygen free radicals [34]. Therefore, the detection of GSH-Px, CAT, and SOD activity can reflect antioxidant capacity. Results showed that compared with the model group, procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G) and the procyanidin trimer C1 increased the activity of GSH-Px, CAT, and SOD in PC12 cells and zebrafish treated with H 2 O 2 (p < 0.05). Procyanidin monomers (C, EC, ECG) also exhibited a reversal effect on the decrease of GSH-Px, CAT, and SOD activities in PC12 cells and zebrafish treated with H 2 O 2 ; however, there was no significant difference compared to the model group (p > 0.05). This shows that the ability of the procyanidin trimer to reduce ROS and MDA contents and increase the activity of GSH-Px, CAT, and SOD was better than that of the procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G) and procyanidin monomers (C, EC, ECG).
The Nrf2/ARE pathway is an important antioxidant pathway [38]. Under normal conditions, Nrf2 binds to the inhibitory protein Keap1 in the cytoplasm [39]. When the cysteine residue at the terminus of Keap1 is damaged by oxidation, a redox reaction occurs and the conformation of Keap1 changes, causing it to separate from Nrf2. Once Nrf2 is activated, it moves from the cytoplasm to the nucleus, inducing the transcription of ARE-dependent antioxidant genes and enhancing antioxidant capacity [40][41][42][43][44][45]. Our results showed that after treatment with different procyanidins from grape seeds, expression of the Nrf2 gene was up-regulated in zebrafish. In PC12 cells, the expression of total Nrf2 protein increased, Nrf2 protein in the cytoplasm gradually decreased, and Nrf2 protein in the nucleus gradually increased. There were significant differences between the model group and the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) and procyanidin trimer C1 groups. The presence of Nrf2 in the nucleus promotes its binding to ARE, catalyzing transcription and inducing the expression of the phase II detoxification enzyme genes NQO1 and HO-1. NQO1 is an antioxidant enzyme that uses NADH or NADPH as a reduction cofactor to participate in the two-electron reduction of endogenous quinones [46]. Chaperone HO-1 cooperates with cytochrome p450 to catalyze the degradation of heme to biliverdin, which is then converted to bilirubin. Both biliverdin and bilirubin exhibit antioxidant and immunomodulatory properties [47,48]. Our results showed that grape seed-derived procyanidin dimers (B1, B2, B3, B4, B1-G, B2-G) and procyanidin trimer C1 treatment in PC12 cells and zebrafish reversed the down-regulation of HO-1 and NQO1 expression induced by H 2 O 2 . Nrf2 siRNA treatment was used to further evaluate the role of Nrf2/ARE pathway activation in the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) and procyanidin trimer C1 treatment groups. Results showed that the expression of Nrf2 in PC12 cells transfected with Nrf2 siRNA decreased significantly, and the protective effect of the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) and procyanidin trimer C1 treatments in PC12 cells treated with H 2 O 2 was blocked. This shows that procyanidins activated the Nrf2/ARE pathway, causing transfer of Nrf2 from the cytoplasm to the nucleus where it accumulated, and increased expression of the phase II detoxification enzymes HO-1 and NQO1, which are downstream of the Nrf2/ARE pathway. The regulatory effect of procyanidins on the Nrf2/ARE pathway was positively correlated with the degree of procyanidin polymerization.

Conclusions
Procyanidins from grape seeds protected PC12 cells from H 2 O 2 induced decreases in cell viability, improved the movement behavior disorder of zebrafish caused by H 2 O 2 , reversed H 2 O 2 -induced increases in ROS and MDA and decreases in antioxidant enzyme (GSH-Px, CAT and SOD) activity, and regulated the Nrf2/ARE pathway. The neuroprotective effect in the procyanidin trimer C1 treatment group was greater than in the procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) treatment groups, and the neuroprotective effect of the procyanidin monomer (C, EC, ECG) treatment group was less than that of the procyanidin trimer C1 and procyanidin dimer (B1, B2, B3, B4, B1-G, B2-G) treatment groups. Procyanidins play a neuroprotective role through activation of the Nrf2/ARE pathway and its downstream detoxification enzymes (HO-1, NQO1) and antioxidant enzymes (GSH-Px, CAT, and SOD). The degree of procyanidin polymerization is an important factor affecting the neuroprotective effect of procyanidins.