Longan Aril Reverses H2O2 Cytotoxicity in PC12 Cells via RAS/MEK/ERK Signaling Pathway

Tianrong LEI, Yang CAO, Chang LI, Jianghua ZHANG, Honghe XIAO, Cheng SONG, Hongyan LI,4*

1. Pharmaceutical College, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China; 2. Dalian Huaxin Physicochemical Testing Centre Ltd., Dalian 116600, China; 3. College of Light Industry and Chemical Engineering, Dalian Polytechnic University, Dalian 116034, China; 4. Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China

Abstract [Objectives] To explore the neuroprotective effects and mechanism of Longan Aril (LA) effective parts on PC12 cells injured by H2O2. [Methods] The neuroprotective effects of LA were evaluated by the cell viability, SOD and MDA content, apoptosis assay and relative protein expression of Aβ and p-Tau. The neuroprotective mechanism of LA was studied by using metabolomics and network pharmacology, and the expressions of RAS/MEK/ERK signaling pathway-related proteins were detected by western blotting. [Results] LA could improve the cell survival rate and SOD content, and reduce apoptosis and expression of Aβ and p-tau. Inhibition of RAS/MEK/ERK signaling pathway is a possible mechanism of LA neuroprotective effects. [Conclusions] LA has a neuroprotective effects in vitro and be likely to inhibit the process of AD by inhibition of RAS/MEK/ERK signalling pathway.

Key words Longan Aril (LA), RAS/MEK/ERK pathway, Alzheimer’s disease (AD), Neuroprotective effects

1 Introduction

Alzheimer’s disease (AD), an age-related neurodegenerative disease, is clinically characterized by impairment of memory and cognitive function and impaired mobility[1]. Current clinical drugs can improve symptoms of patients with mild-to-moderate AD, but they are scarcely able to modulate the disease process[2].

Longan Aril (LA), the aril of longan (DimocarpuslonganLour.), has antioxidant, antitumor, strong immunemodulatory, and anti-inflammatory effects[3-5]. Its anti-AD effects have also been reported in recent years[6], but the mechanism is still unclear.

Extracellular signal-regulated kinase1/2 (ERK1/2) is a subfamily of mitogen-activated protein kinase (MAPK) signaling pathways, plays an important role in cell proliferation, survival, and apoptosis[7], and may be involved in synaptic plasticity and memory processes[8]. In the previous animal experiments, the anti-AD pathways of LA were explored preliminarily through the metabolomics and we found that LA could improve learning and memory abilities in AD model mice via RAS/MEK/ERK pathway[9]. In this study, we conducted further exploration of this pathwayinvitro. We compared neuroprotective effects of water, n-butanol and ethyl acetate extraction of LA by the cell testsinvitro, and studied the neuroprotective mechanism of water extraction of the major active part, in order to lay a foundation for further study on the anti-AD active components and mechanism of LA.

2 Materials and methods

2.1 Anti-AD components analysis of LALA was purchased from Anhui Yishengyuan TCM Decoction Technology Co., Ltd. (Easyyoung), Anhui, China, and endorsed by Professor Li Feng (College of Pharmacy, Liaoning University of TCM, Dalian, China). LA was decocted three times by deionized water at a volume ratio of 1∶10. The components analysis of LA were performed using a LC-Q-TOF-MS, and the data were analyzed using an R software platform[9]. A network pharmacology method was used to screening the possible components and targets of anti-AD of LA. The operation was as follows: ChEMBL and UniProt databases were used to screen the human targets of components; OMIM, GeneCards and CTD databases were used to screen AD related targets; venny tools were used to comparing component targets with AD targets, and the targets associated with LA anti-AD were identified.

2.2 Contents determination of major components in LAThe contents of monosaccharides, amino acids, nucleotides and flavones of LA were determined by HPLC (Agilent Technologies, USA) with C18column (4.6 mm×250 mm×5 μm, Shiseido, Japan). The samples pre-treatment of LA and the conditions of chromatographic separation were performed as previously described[9].

2.3 Cellular model and treatmentsAfter the contents determination to extract the crude LA, the concentrated sample, successively extracted with different polar solvents (ethyl acetate and n-butanol) until the upper layer became colorless[10]. All of the above-LA extracts were treated by vacuum condensation and dried at 60 ℃.

The PC12 cells were divided into normal control group (control), model group (H2O2), treatment group (water, n-butanol or ethyl acetate), positive control group (Hup A) and agonist group (ceramide C6). The PC12 cells in all groups except the control group were exposed to 200 μM H2O2for 12 h. As for the treatment groups, they were treated with water, n-butanol or ethyl acetate extracts of LA 0.5 or 1.0 mg/mL for 2 h before adding H2O2; the positive control group was treated with 2 μM Hup A (Cayman Chemical, USA) using the same method. In the agonist group, an ERK activator ceramide C6 10 μM (Avanti, USA) was added after treatment with the water extraction of LA. PC12 cells (purchased from Shanghai Cell Therapy Group, China), were cultured in DMEM high-glucose media (Gibco, USA) containing 10% fetal bovine serum (FBS) (every green, China) and 1% Penicillin-Streptomycin (Genview, USA) at 37 ℃ in a humidified 5% CO2atmosphere.

2.4 Cell viability assayThe cells were treated with MTT (Solaibio, China) and the optical densities (OD) were measured by a MR-96A microplate reader (Mindray, China) at 492 nm. The cell viability was expressed as a percentage of the control group[11].

2.5 SOD and MDA contentsThe SOD and MDA contents in culture supernatant were detected by SOD and MDA assay kits (Nanjing Jiancheng Bioengineering Institute, China) using methods described in the instructions, and all determinations were done in triplicate.

2.6 Apoptosis assayCell apoptosis was assessed with annexin V-FITC/PI apoptosis detection kit (Genview, USA) and performed on the NoVocyte451141112066 flow cytometer (ACEA Biosciences Inc., USA).

2.7 Western blot analysis

2.7.1Protein expression analyses of Aβ & p-Tau. Western blot analysis was performed as previously described[12-13]. The membranes were probed with primary antibodies anti-Aβ (1∶1 000) and p-Tau (1∶1 000) (Abclonal, USA). GAPDH (1∶1 000) was used as loading control. The proteins were detected by using horseradish peroxidase conjugated (HRP, 1∶5 000) anti-rabbit secondary antibodies (Genview, USA) and visualized with electrochemiluminescence (ECL) reagent (Millipore, USA). All images were analyzed with the Image J 1.38 software (NIH, USA).

2.7.2Analysis of protein expression related to RAS/MEK/ERK signaling pathway. The membranes were probed with primary antibodies anti-HRAS (1∶1 000), anti-MEK (1∶1 000), anti-p-MEK (1∶1 000), anti-ERK (1∶1 000), and anti-p-ERK (1∶1 000) (Abclonal, USA). GAPDH (1∶1 000) was used as loading control.

2.8 Statistical analysisData analysis was conducted by using the SPSS v.25.0 statistics software. Experiments needed to be repeated at least three times. Differences among multiple groups were compared by the one-way analysis of variance (ANOVA), and statistical significance of two groups was evaluated by the two-tailed Student’st-test.P<0.05 is considered statistically significant.

3 Results and analysis

3.1 Components and contents of LAThe LA decoction was freeze-dried and its extraction yield was calculated at (49.27±3.98)%. Based on the mass value in Excel sheet and metlin network database in LC-Q-TOF-MS experiment, the total of 178 compounds were acquired, and there were 67 compounds detected within Δppm 10. The 67 components were input into the ChEMBL database to screen 52 components with 254 human targets, and 285 AD-related human targets were selected by OMIM database,etc. After comparison and analysis of venny tools, 11 common targets were found (Fig.1A), that is, 52 possible anti-AD components were preliminarily determined to regulate 11 anti-AD targets (Fig.1B).

Note: A. The common targets of LA components and AD; B. Anti-AD component-target network of LA. The network consisted of 52 potential anti-AD components (gray circles) and 11 anti-AD targets (black squares), which were connected by lines.

According to the full spectrum identification and literatures[14], the contents of major components of LA, such as carbohydrates, amino acids, nucleotides, and flavonoids were detected by HPLC (Fig.2). The results showed that the level of carbohydrates accounted for 26.110% of LA (Fig.2C and 2D), including amylaceums, mannoses, arabinoses,etc. The content of amino acids accounted for 2.794% of LA (Fig.2E), including prolines, alanines, glutamates,etc., and meanwhile no cystine was detected. The level of nucleotides accounted for 0.051% (Fig.2F), including adenines and uridines, uracils, cytidines,etc., while thymus purines, cytosines and thymidines were not found. Flavonoids, including dihydromyricetins, taxifolins, aromadendrins, myricetins, eriodictyols, quercetins, naringenins, luteolins, kaempferols and apigenins, were not detected either.

Note: A. The total components distribution; B. The distribution of the detected components, three different components were detected, carbohydrates, amino acids and nucleotides; C. The carbohydrates distribution; D. The components distribution of other carbohydrates rather than amylaceums; E. The amino acids components distribution; F. The components distribution of nucleotides.

3.2 Yield extraction of LA by water, n-butanol & ethlyacetateThe results indicated that, yield of the water media was the highest (60.53±5.88)%; both the n-butanol and ethyl acetate media had lower yield than water media (24.33±2.02)% and (23.50±1.96)%), respectively.

3.3 LA improving the cell survivalMTT assay showed that the cells survival rates of H2O2group were observably lower than that of the control group. After being treated with Hup A, and water, n-butanol or ethyl acetate extracts of LA, the cell survival rates were significantly improved (P<0.001,P<0.01, Fig.3A-C). The average concentration of extracts media was 1.9 mg/mL, which was respectively equivalent to 3.3 (water extract), 7.7 (n-butanol extract) and 7.9 (ethyl acetate extract) mg/mL of LA crude drug. Correspondingly, the average cell survival rates, as shown in Fig.3D, showed that the promoting proliferation effects were in descending order, followed by ethyl acetate (82.22±18.96)%, n-butanol (69.18±20.30)% and water (56.45±6.71)% extracts media. In contrast, if it was calculated based on the dosage of LA crude drug, the effects of water extract on proliferation was the most significant.

3.4 LA improving the SOD contentFig.3(A-C) illustrated that by using 0.8 mg/mL concentration of either water or ethyl acetate as extract media, obvious cell protection observed, while using n-butanol no protection was observed. Thus, the concentration was set in the range of 0.5-1.0 mg/mL for further study. As shown in Fig.4(A-D), SOD content in cell culture supernatant decreased after H2O2injury (P<0.05), and increased markedly after being treated with water and n-butanol media of LA and Hup A (P<0.05 orP<0.01). The average SOD content was applicable to the highest value in water extraction group. There was no significant difference in MDA content among all groups (P>0.05, Fig.4E-H).

Note: A-C. Cell viability in each group. 0.8, 1.6 and 3.2 were the concentrations of the extracts, which were equivalent to the crude drug dosage of LA 1.4, 2.8 and 5.6 mg/mL (water extract); 3.3, 6.6 and 13.2 mg/mL (n-butanol extract); 3.4, 6.8 and 13.6 mg/mL (ethyl acetate extract). D. Average cell viability. The mean concentrations of the extracts, namely the average of 0.8, 1.6 and 3.2 mg/mL was 1.9 mg/mL, which was equivalent to the crude drug dosage of LA 3.3 (water), 7.7 (n-butanol) or 7.9 (ethyl acetate) mg/mL, respectively. Average cell survival was the cell survival at average concentration. ***P<0.01 compared with control group; ##P<0.01, ###P<0.001 compared with H2O2 group.

Note: A-C. SOD contents in each group; D. Average SOD contents; E-G. MDA content in each group; H. Average MDA contents. The extracts media concentrations were 0.5 and 1.0 mg/mL. The average SOD or MDA contents were the contents of SOD or MDA when the concentration of extract was 0.75 mg/mL (it was the average of 0.5 and 1.0 mg/mL). *P<0.05 compared with control group; #P<0.05, ##P<0.01 compared with H2O2 group; ΔP<0.05 compared with water extraction group.

3.5 LA inhibiting the apoptosisThe results showed that after H2O2injury, the numbers of living cells decreased markedly, while those of apoptosis and necrosis cells increased significantly (P<0.01 orP<0.001, Fig.5A-E). Water, n-butanol and ethyl acetate extracts media of LA significantly improved cell survival and inhibited cell apoptosis and necrosis (P<0.05,P<0.01 orP<0.001). Among them, except the inhibitory effects on cell necrosis, the improvement of cell survival and the inhibitory effects on cell apoptosis of water and ethyl acetate extracts were significantly higher than that in n-butanol (Fig.5B-D).

Note: A. Apoptosis images; B. Rate of living cells; C. Rate of early apoptotic cells; D. Apoptosis cell rate in middle and late stage; E. Cell necrosis rate. The concentrations of water, n-butanol and ethyl acetate extracts media were all at 1.0 mg/mL.**P<0.01, ***P<0.01 compared with control group; #P<0.05, ##P<0.01, ###P<0.001 compared with H2O2 group.

3.6 LA reducing relative protein expressions of Aβ & p-Tau

As shown in Fig.6(A-C), the results showed that the expression levels of Aβ and p-Tau proteins induced by H2O2(Fig.6B and Fig.6C) were markedly higher than that in control group (P<0.01), and in the treatment groups were significantly reduced compared with the model group (P<0.05 orP<0.01), among them the water extract had the best inhibition effects on Aβ protein (P<0.01, Fig.6B). As for p-Tau protein (Fig.6C), the ethyl acetate and water extract media groups were the best (P<0.05). Expression of Aβ and p-Tau were lower than model group after being treated with Hup A (P<0.05).

Note: A. Aβ & p-Tau protein levels images; B. The relative expression of Aβ protein; C. The relative expression of p-Tau protein. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with H2O2 group.

3.7 LA extracted by water inhibiting the expression of target proteins related to the RAS/MEK/ERK signaling pathwayThe expression of ERK1/2 pathway related proteins, such as HRAS, MEK, p-MEK, ERK and p-ERK (Fig.7A-H), were analyzed by western blot test. The results indicated that the relative expression levels of HRAS (Fig.7B), p-MEK (Fig.7D) and p-ERK (Fig.7G) protein, and expression ratios of p-MEK/MEK (Fig.7E) and p-ERK/ERK (Fig.7H) in the model group were markedly higher than those in the control group (P<0.01 orP<0.001). Moreover, LA extracted by water could significantly inhibit the expression levels of these proteins compared with the model group (P<0.05 orP<0.01). And we found that the expression of p-MEK and the ratio of p-MEK to MEK in the agonist group were significantly reduced after H2O2injury (P<0.01), while there was no significant difference with the treatment group (P>0.05). Besides, compared with the treatment group, the agonist could ameliorate markedly phosphorylation of ERK (P<0.01), and the expression of p-ERK was lower than the model group (P<0.05). Therefore, we considered that LA could still inhibit the ERK pathway in the AD model cells after adding the ceramide C6. Nevertheless, this effects was restrained by the agonist.

Note: A. Images of target protein levels; B. The relative expression of HRAS protein; C. The relative expression of MEK protein; D. The relative expression of p-MEK protein; E. The ratio of p-MEK/MEK; F. The relative expression of ERK protein; G. The relative expression of p-ERK protein; H. The ratio of p-ERK/ERK. **P<0.01, ***P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with H2O2 group; ΔP<0.05, ΔΔP<0.01 compared with water extraction group.

4 Discussion

PC12 cells are widely used in diseases of the nervous system because of the reversible responses to neuronal growth factors[15]. To examine the anti-AD effective parts of LA, the PC12 AD cell model was utilized to evaluate the neuroprotection of water, n-butanol and ethyl acetate extraction of LA. The MTT, contents of SOD and other experiments showed that the water and ethyl acetate extract of LA, had better inhibitory effects on AD (P<0.05,P<0.01 orP<0.001, Fig.3-5). Since the yield of the water extract was much higher than that of ethyl acetate or n-butanol extract, and water decoction was widely used in TCM, the water extract of LA was used as the effective part for subsequent studies, and its anti-AD mechanism via RAS/MEK/ERK signaling pathway was studied by a western blot. The ceramide C6 was a kind of ERK agonist, and the concentration of ceramide C6 was selected according to the literatures[16].

The 11 targets regulated by 52 components of LA were enriched by DAVID database to obtain MAPK, PI3K-Akt and NOD-like receptor signaling pathway,etc(results were not shown). And the metabolomic results of animal experiments in another article showed that the anti-AD effects of LA were related to the RAS/MEK/ERK signaling pathway[9]. Therefore, this signaling pathway was selected to further study the mechanism of LA anti-AD.

5 Conclusions

In this study, we found that LA has a neuroprotective effects anti-ADinvitro, and water extract of LA could be the effective part and be likely to inhibit the process of AD by the inhibition of RAS/MEK/ERK signaling pathway.