Procyanidins Extracted from Lotus Seedpod Ameliorate Amyloid-β-Induced Toxicity in Rat Pheochromocytoma Cells

Alzheimer's disease (AD) is a progressive neurodegenerative disease, which is characterized by extracellular senile plaque deposits, intracellular neurofibrillary tangles, and neuronal apoptosis. Amyloid-β (Aβ) plays a critical role in AD that may cause oxidative stress and downregulation of CREB/BDNF signaling. Anti-Aβ effect has been discussed as a potential therapeutic strategy for AD. This study aimed to identify the amelioration of procyanidins extracted from lotus seedpod (LSPC) on Aβ-induced damage with associated pathways for AD treatment. Rat pheochromocytoma (PC12) cells incubated with Aβ 25–35 serve as an Aβ damage model to evaluate the effect of LSPC in vitro. Our findings illustrated that LSPC maintained the cellular morphology from deformation and reduced apoptosis rates of cells induced by Aβ 25–35. The mechanisms of LSPC to protect cells from Aβ-induced damage were based on its regulation of oxidation index and activation of CREB/BDNF signaling, including brain-derived neurotrophic factor (BDNF) and phosphorylation of cAMP-responsive element-binding (CREB), protein kinase B (also known as AKT), and the extracellular signal-regulated kinase (ERK). Of note, by high-performance liquid chromatography-tandem mass spectroscopy (LC-MS/MS), several metabolites were detected to accumulate in vivo, part of which could take primary responsibility for the amelioration of Aβ-induced damage on PC12 cells. Taken together, our research elucidated the effect of LSPC on neuroprotection through anti-Aβ, indicating it as a potential pretreatment for Alzheimer's disease.


Introduction
Alzheimer's disease (AD), a progressive neurodegenerative disease, is characterized by extracellular senile plaque deposits, intracellular neurofibrillary tangles, and neuronal apoptosis. Progressive loss of memory and other cognitive functions are typical symptoms in AD [1]. According to the amyloid hypothesis, amyloid-β-(Aβ-) related toxicity and imbalance are cardinal reasons that contribute to synaptic dysfunction and subsequent neurodegeneration in AD [2,3]. Aβ has been, therefore, suggested as a potential therapeutic target for AD treatment [4].

Hoechst
Staining. Cells were seeded on 6-well plates. After intervention as three groups, each group was washed with PBS twice before 800 μL staining buffer was added and subsequently stained with Hoechst staining solution (5 μL) for 30 min in the dark. Cells were imaged on a fluorescence microscope (Olympus Corporation, Japan). Hoechst staining was executed according to the instructions (Beyotime, China).

Quantitative
Real-Time PCR (qRT-PCR). Total RNA was isolated from cells via RNAiso Plus (TaKaRa, China), and cRNA was extracted using the PrimeScript™ RT reagent Kit (TaKaRa, China), all of which were based on the instructions. qRT-PCR was carried out using the SYBR® Premix Ex Taq™ (TaKaRa, China) with an ABI 7900HT real-time thermocycler (Applied Biosystems, CA), as previously described [29]. The correlated expressions of genes were calculated by 2 -△△CT methods. Primers of specific genes, including BDNF (forward: 5 ′ -AGCAGGCTCTGGAATGATGT-3 ′ ; reverse: 5 ′ -GGATTTGAGTGTGGTTCTCCA-3 ′ ) and      and fasted for 12 h but had access to deionized water. For the control group, physiological saline was given by oral gavage daily; for LSPC group, LSPC (a brownish red power) was dissolved in physiological saline (20 mg/mL) and administered to rats at a dose of 200 mg/kg body weight by oral gavage daily for two weeks. Body weights were measured every two days. Rats were sacrificed two hours later after a final dose. Tissues (brain, cardiac, liver, kidney, spleen, and pancreas), intestine content, and plasma were harvested and stored at −80°C until analysis.
2.11. LC-MS/MS. For the extraction of LSPC and its metabolites, tissues (60 mg) were homogenized with 300 μL mixture (50 μL 1% (w/v) aqueous ascorbic solution and 250 μL 0.1% formic acid). Ethyl gallate was an internal standard. Each sample was hydrolyzed with a β-glucuronidase/sulfatase type H1 (1500 U/mL) from H. pomatia (Sigma, USA) for two hours at 37°C. Then, methanol (200 μL) was added to each sample followed by vibration (30 s) and centrifugation (12000 rpm, 10 min, 4°C), and the supernatant was collected. The extraction was repeated once. The combined supernatants were evaporated to dryness under vacuum at 35°C. The residue was reconstituted in 50 μL of solvent (methanol/water, 1 : 1, v/v) for LC-MS/MS analysis. The analysis was performed on a high-performance liquid chromatography-tandem mass spectroscopy (LC-MS/ MS, AB Sciex QTrap 4500, Applied Biosystems, Foster City, CA, USA). This method was in accordance with the reported studies [30][31][32]. Briefly, 5 μL samples were injected for LC-MS/MS, and the analytes were separated by BETASIL Phenyl Column (2.1 mm × 150 mm, 3 μm; Thermo Scientific, USA) at 35°C. The mobile phases composed (a) water with 0.2% acetic acid and (b) methanol with 0.2% acetic acid. Ionization was carried out by electrospray in the negative mode. The calibration curves of respective standards were utilized to quantify compounds. Transition ions, retention times, and mass-spectrometry parameters for all compounds were shown in Table S1; chemical structures of all compounds were exhibited in Figure S1.
2.12. Statistical Analysis. The data are presented as mean values ± standard error of the mean (SEM) and analyzed by ANOVA with Student-Newman-Keuls (SNK) or student t-test on SPSS software version 19.0. The level of significance was set for P value < 0.05. [25][26][27][28][29][30][31][32][33][34][35] and LSPC. Figure 1(a) demonstrated that 20 μM Aβ 25-35 had a significant effect on the survival rate of PC12 cells after 24 h intervention that was consistent with the previous report [33]. Thus, we chose the dosage of 20 μM Aβ 25-35 with the intervention period of 24 h on PC12 cells for further study. In order to testify a dosedependent manner of LSPC, we added 1, 2.5, 5, 10, 20, and 40 μg/mL LSPC into PC12 cells before Aβ 25-35 intervention, respectively. As shown in Figure 1   All data are mean ± SEM. * P < 0 05 for groups vs control group; # P < 0 05 for groups vs Aβ groups. (d) mRNA expression of intracellular BDNF in each group by qRT-PCR analysis. Cells were cultured as three groups as above, including control, Aβ, and LSPC. All data are mean ± SEM. * P < 0 05 for groups vs control groups; # P < 0 05 for groups vs Aβ groups. All the results above are the representative of the three independent experiments.  the damage from Aβ 25-35 remarkably. In flow cytometry analysis (Figures 2(h) and 2(i)), we further validated that the apoptosis rates of PC12 cells with 20 μM Aβ 25-35 , including early apoptosis rates (AE), later apoptosis rates (LA), and total apoptosis rates (TA), were higher than that in the control (P < 0.05), and addition of 10 μg/mL LSPC significantly lessened apoptosis rates augmented by Aβ 25-35 (P < 0 05).  (Figures 4(a) and 4(b)) and augmented BDNF expression (Figures 4(a) and 4(c)), indicating that LSPC could mitigate Aβ 25-35 -induced diminishment of CREB phosphorylation and BDNF expression. qRT-PCR analysis of BDNF mRNA (Figure 4(d)) demonstrated that Aβ 25-35 significantly attenuated BDNF mRNA expression compared with control group (P < 0 05) while LSPC counteracted the effect of Aβ 25-35 on BDNF mRNA expression.
To further identify CREB/BDNF signaling in neuroprotection of LSPC, we applied LY294002, an inhibitor of the PI3K/AKT pathway, and PD98059, an inhibitor of the ERK pathway. Cells were cultured as five groups: PC12 cells, PC12 cells with 10 μM LY294002, PC12 cells with 10 μg/mL
There was no significant difference in body weight between the control group and LSPC group after LSPC treatment ( Figure S2). Catechin and epicatechin were distinguished by LC-MS/MS according to distinctive retention time and transition ions.

Discussion
Recently, there has been an increasing interest in the discovery of potential flavonoids for preventing dementia or AD; nevertheless, the complexity and diversity of flavonoids restrict the understanding of their value on AD treatment. This study comprehensively verified its anti-Aβ neurotoxicity in vitro that could alleviate AD-related symptoms.
In AD, Aβ may contribute to oxidative stress in the brain [1,34] while the antioxidant activity is an outstanding feature  of flavonoids. PC12 cells with Aβ 25-35 , as an AD-like model, were performed to testify the abilities about anti-Aβ neurotoxicity of LSPC [35,36]. LSPC has no toxicity in vitro and in vivo that coincided with previous studies [27,37]. LSPC has exhibited its antioxidation effect in vitro that was consistent with Xu et al. [27]. Interestingly, a higher concentration of LSPC (20 mg/L) seemed to be less efficient in the decrease of MDA and LDH, and a dose-response could be seen regarding the SOD activity. This result could be partly due to the difference in antioxidant activity associated with doses of procyanidins, cell type, and time of exposure [38]. The inconsistency of different antioxidant enzymes activities has been reported by Puiggròs et al. [39]. Antioxidant reactions of flavonoids, as illustrated by many studies, may benefit the treatment and precaution of cancer [40], cardiovascular diseases [41,42], type 2 diabetes [41,42], and neurodegenerative diseases [43]. Since periphery anti-Aβ has been proposed as potential approaches to ameliorate impairment of Aβ [44,45] in the central nervous system that the liver and kidney have been tightly related to it [44,46], it is a high possibility that antioxidant effect of LSPC could contribute to alleviate Aβ toxicity in this pathway. Not only oxidative stress is attributed to accumulation and neurotoxicity of Aβ in AD but also downregulation of CREB/BDNF signaling [5,[12][13][14]. Several studies have shed the light on anti-Aβ effect of flavonoids [47][48][49]. Lin et al. have reported that Aβ could induce the death of cells [50], and in AD, it is a major damage resulted from Aβ aggregation [51]. According to Hoechst staining and flow cytometry in the present study, LSPC kept cellular morphology from deformation and suppressed the apoptosis of cells induced by Aβ. In addition, Aβ can reduce the expression of BDNF in AD [52], and CREB can mediate Aβ-induced BDNF downregulation [53] that are in accordance with our results. CREB/BDNF signaling was downregulated by Aβ but upregulated by LSPC. Through targeting phosphorylation of CREB, AKT, and ERK, the upstream of CREB/BDNF signaling can affect BDNF transcription [6,7]. Activations of both AKT and ERK were restrained by Aβ [8,9] but increased with treatment of LSPC in our study. CREB/BDNF signaling plays a vital role in neuron survival, and BDNF-based synaptic repair is proposed as a therapeutic strategy for AD [54]. LSPC could hence ameliorate Aβ-induced damage in AD through CREB/BDNF signaling. Notably, an interaction between CREB/BDNF signaling and oxidative stress has been confirmed [18,19]. Valvassori et al. have reported that increased BDNF in the brain can modulate oxidative stress [55]. Taken together, LSPC has both antioxidative effects and the ability to regulate CREB/BDNF signaling as a potential AD pretreatment. Several researches focusing on lotus also support that compounds from lotus may show neuroprotection [20]. Note: control and LSPC represent different intervention groups, respectively. PDB, ECG, EGC, HVA, 3,4-DHPA, p-HPPA, PCC, 3-HPAA, and 3-HBA stand for procyanidin dimer B, epicatechin gallate, epigallocatechin, homovanillic acid, 3,4-dihydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, protocatechuic acid, 3-hydroxyphenylacetic acid, and 3-hydroxybenzoic acid, respectively. Values represent the concentrations of metabolites in different rat tissues, and they were all presented as the means ± SEM (n = 7); ND = not detected; * , * * , * * * indicates significant differences between two groups with or without LSPC (p < 0 05, p < 0 01, and p < 0 001), respectively.  )propionic acid, protocatechuic acid, 3-hydroxyphenylacetic acid, and 3-hydroxybenzoic acid, respectively. Values represent the concentrations of metabolites in different rat tissues, and they were all presented as the means ± SEM (n = 7); ND = not detected; * , * * , * * * indicates significant differences between two groups with or without LSPC (p < 0 05, p < 0 01, and p < 0 001), respectively.
By LC-MS/MS, we found several detectable compositions accumulated in vivo and quantities of them were varied in rat tissues and plasma after consecutive LSPC administration. As reported, Aβ can aggravate in both central and periphery tissues and the relationship between AD and the peripheral system is indivisible [56,57]. AD has been called as "type 3 diabetes", concerning its association with insulin resistance [58]; it also has been related to the gut-brain axis [59]. The distribution of LSPC was only measured in rat urine before so it was profound to confirm the distribution of it in vivo. In the LSPC group, epicatechin and quercetin, resulted from quercetin-3-O-glucuronide in LSPC [25], were found to accumulate in the brain. Wang et al. [16] have reported 3'-Omethyl-epicatechin-5-O-β-glucuronide, the major metabolites of epicatechin in the brain, may promote long-term potentiation (LTP) through CREB signaling. Quercetin-3-Oglucuronide has been reported to cross the blood-brain barrier and accumulate in the brain [60,61]; deconjugation of it may contribute to the appearance of quercetin in tissues [61]. Quercetin-3-O-glucuronide has also been identified to inhibit Aβ aggregation [60] and reduce oxidative stress [61,62]. The increment of BDNF protein and AKT phosphorylation in the rat by quercetin-3-O-glucuronide has been observed by Baral et al. [63]. Serra et al. [64] have discussed the distribution of procyanidins from hazelnut extract after treatment once, reporting only p-HPPA is significantly increased in the brain. Conversely, our results showed that LSPC could lead to the accumulation of quercetin, epicatechin, gallic acid, vanillic acid, m-coumaric acid, protocatechuic, 3-HPAA, and pyrocatechol. This inconsistency could be ascribed to the difference between LSPC and hazelnut extract and intervention time.
Other compounds in the brain detected to increase in LSPC group, including gallic acid [65], vanillic acid [66], and protocatechuic acid [67], have been discussed to anti-Aβ neurotoxicity through multifarious pathways. Gallic acid could inhibit Aβ neurotoxicity through suppressing neuroinflammation [65]; vanillic acid is found to attenuate oxidative stress induced by Aβ [66]; protocatechuic acid may also minimize inflammatory response [67]. But evidences about these materials are insufficient. Further studies are required to discern and compare the effects of different compounds after LSPC treatment as an integral or as separated components.

Conclusion
Our research firstly affirmed anti-Aβ effectiveness of LSPC that indicated it as a promising pretreatment for AD and expounded LSPC distribution in vivo. Through cell experiments, our study not only proved anti-Aβ effects of LSPC through evaluation of cell viability and cellular morphology but also identified the antioxidant effect of LSPC and BDNF/CREB signaling in its anti-Aβ mechanisms ( Figure 8). We also applied LC-MS/MS in the detection of LSPC in vivo that contributed to explain its effect. Future studies still need to enrich our scientific recognition of LSPC and then establish the novel therapeutic strategies for AD.

Data Availability
The data is available on the website of Figshare and the access is https://figshare.com/s/fb5f71daf2ef08cdff42.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.