Neuroprotective role of hyperforin on aluminum maltolate-induced oxidative damage and apoptosis in PC12 cells and SH-SY5Y cells

Accepted Manuscript Neuroprotective role of hyperforin on aluminum maltolate-induced oxidative damage and apoptosis in PC12 cells and SH-SY5Y cells Haoran Wang, Bing Shao, Hongyan Yu, Feibo Xu, Peiyan Wang, Kaiyuan Yu, Yanfei Han, Miao Song, Yanfei Li, Zheng Cao PII:

S0009-2797(17)31159-6

DOI:

https://doi.org/10.1016/j.cbi.2018.11.016

Reference:

CBI 8468

To appear in:

Chemico-Biological Interactions

Received Date: 25 October 2017 Revised Date:

17 October 2018

Accepted Date: 23 November 2018

Please cite this article as: H. Wang, B. Shao, H. Yu, F. Xu, P. Wang, K. Yu, Y. Han, M. Song, Y. Li, Z. Cao, Neuroprotective role of hyperforin on aluminum maltolate-induced oxidative damage and apoptosis in PC12 cells and SH-SY5Y cells, Chemico-Biological Interactions (2018), doi: https://doi.org/10.1016/ j.cbi.2018.11.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Neuroprotective Role of Hyperforin On Aluminum Maltolate-Induced

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Oxidative Damage and Apoptosis in PC12 Cells and SH-SY5Y Cells

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Haoran Wanga, Bing Shaoa, Hongyan Yua, Feibo Xua, Peiyan Wanga, Kaiyuan Yua,

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Yanfei Hana, Miao Songa, Yanfei Lia,*, Zheng Cao a,*

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Medicine, Northeast Agricultural University, Harbin 150030, China

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Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine. College of Veterinary

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Corresponding author at: College of Veterinary Medicine, Northeast Agricultural University, NO. 600 Changjiang Road, Harbin 150030, China. Tel.: +86 18846180665; E-mail address: [email protected] (Y.F. Li). *

Corresponding author at: College of Veterinary Medicine, Northeast Agricultural University, NO. 600 Changjiang Road, Harbin 150030, China. Tel.: +86 13895776045; E-mail address: [email protected] (Z.C).

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Abstract Many reports demonstrated that aluminum maltolate (Almal) has potential toxicity to human

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and animal. Our study has demonstrated that Almal can induce oxidative damage and apoptosis in

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PC12 cells and SH-SY5Y Cells, two in vitro models of neuronal cells. Hyperforin (HF) is a

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well-known antioxidant, anti-inflammatory, anti-amyloid and anti-depressant compound extracted

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from Hypericum perforatum extract. Here, we investigated the neuroprotective effect of HF

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against Almal-induced neurotoxicity in cultured PC12 cells and SH-SY5Y cells, mainly caused by

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oxidative stress. In the present study, HF significantly inhibited the formation of reactive oxygen

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species (ROS), decreased the level of lipid peroxide and enhanced the activities of superoxide

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dismutase (SOD) and glutathione peroxidase (GSH-Px) compared with Almal group in PC12 cells

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and SH-SY5Y cells. Additionally, HF suppressed the reduction of the mitochondrial membrane

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potential (MMP), cytochrome c (Cyt-c) release, activation of caspase-3, and the down-regulation

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of Bcl-2 expression and up-regulation of Bax expression induced by Almal in PC12 cells and

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SH-SY5Y cells. In summary, HF protects PC12 cells and SH-SY5Y cells from damage induced by

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Almal through reducing oxidative stress and preventing of mitochondrial-mediated apoptosis.

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Key words: Aluminum maltolate; Hyperforin; Neuroprotective; PC12 cells and SH-SY5Y cells;

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Oxidative damage; Mitochondrial-mediated apoptosis

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1. Introduction Aluminum (Al) is one of the most abundant elements in the earth crust and has been well

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known as a potent neurotoxicant [1]. It’s widely used increases human and animal exposure to Al,

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and the potential risk related to toxicity [2, 3] through natural processes (soil erosion, acid rain and

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volcanic eruptions), and man-made sources (food, medicine, industry, water purifier and packing

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containers) [4, 5]. Al exposure causes

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respiratory and immune system [6]. The brain is the primary target organ of Al accumulation [7].

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Al can cross the blood-brain barrier (BBB) and enter into the central nervous system (CNS)

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causing brain damage [8]. A large number of epidemiological investigations and animal studies

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have reported that excessive intake of Al can lead to decreasing learning function, and cognitive

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impairment [9-13]. Al-induced spatial memory disorders are closely associated to the Al content of

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brain and the forms of Al compounds [14]. Different Al-containing compounds exhibit different

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toxicity [15]. Among the Al salts, Al lactate, citrate and chloride are commonly used in in vivo and in

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vivo neurotoxicity study. These salts are hydrolyzed under normal pH

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which

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years, Aluminum Maltolate (Almal) has been significantly investigated for Al neurotoxicity.

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This Al complex is extensively used in vitro mechanistic studies as it does not form insoluble

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precipitates of Al-hydroxide at physiological pH and release a large amount of Al3+ [17].

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Numerous previous studies have shown that Almal is more toxic to animals and neuronal cells

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than Al lactate or Al chloride [18-20]. Maltolate is a common ingredient of the human diet and it

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can be formed during sucrose pyrolysis or thermal degradation of starch [21]. It is an approved

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food additive in the US and Australia, used as a flavor enhancer in coffee, chocolate milk and as a

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the nervous hematopoietic, skeletal,

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toxicities

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and form Al(OH)3 precipitate,

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may remarkably reduce the Al content of the brain and their bioavailability[16]. In recent

ACCEPTED MANUSCRIPT flavoring agent in breads and cakes [22]. Its high affinity to Al, there is a potentiality to form

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Almal in the gastrointestinal tract [23]. The gastrointestinal tract is main pathway for Al

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absorption[24], and is mainly distributed in the brain and followed by liver, spleen, kidney and

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bone [7]. Furthermore, maltolate can increase the solubility and subsequently enhances the

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bioavailability of Al, leading to a higher absorption of Al3+, and ultimately high accumulation of

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Al3+ in the brain due to its lipophilic characteristic [25]. Thus, Almal-induced neurotoxicity is

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relevant to human health. Although the exact mechanism of Al neurotoxicity remains unclear, it is

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now generally accepted that mainly involved in oxidative stress, apoptosis, plasticity destruction,

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amyloid beta-protein generation and inflammation [26-29]. Oxidative stress plays a core role in the

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induction of Al neurotoxicity [30]. Al-induced oxidative stress can further promote Aβ protein

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production [31], destruction of synaptic plasticity [32], activation of inflammation [33] and

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apoptosis [34]. Thus, treatment for oxidative stress may be effective in relieving Al neurotoxicity.

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Hyperforin (HF) is the main active company of HPE [35] and exerts significant biological

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properties including anti-cancer and anti-bacterial [36-38]. Our previous study found that

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Hypericum perforatum extract (HPE) can effectively alleviate pathological changes in aluminum

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chloride-induced Alzheimer's disease rat- through its antioxidant, anti-inflammatory and

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anti-amyloid effects [39]. In recent years, the neuroprotective potential of HF has attracted the

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interest of researchers. HF can easily cross the BBB and exerts its pharmacological effects due to

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its lipophilic properties [40]. Lee et al. reported that HF can attenuate microglia activation and

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inhibits p65-Ser276 NFκB phosphorylation in the rat piriform cortex following status epileptics

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[37]. Data from Huang et al. shown that HF attenuated Al-induced Aβ protein production via

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reducing the activity of BACE1 in PC12 cell [41]. HF can promote mitochondrial function and

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ACCEPTED MANUSCRIPT oligodendrocytes development[42]. It is also an effective therapeutic agent for mild and moderate

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depression [43, 44]. Although some studies have shown that HF has strong antioxidant properties,

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however, neuroprotective effects of HF are not largely elucidated especially on Al-induced

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neurotoxicity. A recent study revealed that HF has strong radical scavenging activity and can

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significantly relieve Fe2+-induced DNA damage in vitro [45]. Haag et al. reported that regular

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application of the hyperforin-rich cream can reduce radical formation in skin cell [46]. HF also

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inhibits the generation of reactive oxygen species (ROS) in human isolated polymorphonuclear

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leukocytes (PMNL) [47]. Data based on the reviewed in the above suggested that HF may have a

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strong neuroprotective effects against neurotoxicity, and we assume that HF can further relieve

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Al-induced neurotoxicity via suppressing oxidative stress.

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PC12 cell is derived from the rat adrenal pheochromocytoma, whose receptor and synthetic

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transmitter are similar to neurons, so it has been widely used as an in vitro neuronal model system

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for

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neuro-pathological including Alzheimer's disease and Parkinson's disease-relevant research

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[51-53]. PC12 is often used to study the mechanism of Al-neurotoxicity [54-57]. Our previous

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study demonstrated that HF can attenuate Almal-induced Aβ production and Tau phosphorylation

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via regulating Akt/GSK-3β signaling pathways PC12 cells [41]. This represents that the PC12 cell

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a suitable model for Al-induced-neurotoxicity study in in vitro. The SH-SY5Y, a

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catecholaminergic neuroblastoma human cell line, has been used to explores the mechanism of

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nerve cell injury in in vitro and the protective effect of drugs on neuronal cells because of its

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typical characteristics of neuroendocrine cells. It is also a commonly used cell line to study

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Al-induced-neurotoxicity [58-61]. Add SH-SY5Y cell line can further confirm the protective

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neuro-physiological

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ACCEPTED MANUSCRIPT mechanism of HF on Almal-induced neurotoxicity. Therefore, our study was designed to assess

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the neuroprotective effect of HF on Al-induced PC12 cells and SH-SY5Y cells toxicity and its

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potential mechanisms.

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2. Materials and Methods

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2.1. Almal preparation

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Almal was prepared from Aluminum chloride hexahydrate (AlCl3• 6H2O) (Sigma) and

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Maltolate (3-hydroxy-2-methyl-4-pyrone) (Sigma) as described previously by Bertholf et al. [62].

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Almal stock solution was prepared in double distilled water and sterilized using 0.22

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micron-pore-size filters.

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2.2 Cell culture and treatment

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PC12 cells and SH-SY5Y cells were supplied by the Cell Bank of the Chinese Academy of

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Sciences (Shanghai, China). PC12 cells and SH-SY5Y cells were cultured in DMEM medium

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(HyClone, Logan, USA) supplemented with 10 % fetal bovine serum (HyClone, Logan, USA),

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and 100 U/ml antibiotic mixture of penicillin/streptomycin, at 37 °C in a humidified atmosphere

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with 5 % CO2. After 24 h of cell attachment in microplates (Corning, USA) or in cell culture

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bottles (Corning, USA), then cells were randomly divided into control group, HF group,

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Almal+HF group and Almal group, then the cells were cultured in the medium containing 0 µM

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HF + 0 µM Almal (control group), 1 µM HF + 0 µM Almal (HF group), 1 µM HF + 0 µM Almal

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(HF+Almal group) and 0 µM HF + 0 µM Almal (Almal group) for 12h, respectively. Then, the

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culture medium was refreshed and the cells were cultured in the medium containing 0 µM HF + 0

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µM Almal (control group), 0 µM HF + 0 µM Almal (HF group), 0 µM HF + 500 µM Almal

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(HF+Almal group), 0 µM HF + 500 µM Almal (Almal group) for another 48 h, respectively.

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Afterwards, the PC12 cells and SH-SY5Y cells were collected for determination. There were ten

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replicates for each treatment.

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2.3. Cell viability assay The protective effect of HF on Almal-induced reduction of PC12 cell and SH-SY5Y cells

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viability was detected by CCK-8 assay according to the manufacturer’s instructions. Briefly, 1 ×

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106 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in 96-well plates and incubated for 24 h.

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After different treatment, 10 µl CCK-8 solution was added to each well, and incubated at 37 °C in

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5 % CO2 for 30 min. Then the optical density of each sample was measured at 450 nm using an

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auto-microplate reader (Infinite M200, Tecan, Austria). Cell viability was expressed as a

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percentage of control.

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2.4. Assessment of LDH Activity.

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The LDH leakage was determined by using commercially available kit (Jiancheng

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Bioengineering, Nanjing, China). Briefly, 1 × 106 PC12 cells and 8 × 105 SH-SY5Y cells were

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seeded in 6-well plates and incubated for 24 h. After different treatment, the supernatant was

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collected and centrifuged at 3200 g for 5 min and the OD value of LDH in the medium was

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determined by the colorimetric method according to LDH assay kit. LDH activity was expressed

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as a percentage of control.

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2.5. Assessment of cell morphology

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Briefly, PC12 cells were seeded at a density of 1 × 106 cells/ml in 35 mm cell culture dishes

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(Thermo Fisher Scientific, USA) and incubated for 24 h. After different treatment, PC12 cells

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were fixed for scanning electron microscopy (SEM) preparation; all samples were fixed in the

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same way. Then, PC12 cells were dehydrated through a graded ethanol series, prior to desiccation,

ACCEPTED MANUSCRIPT using a critical-point drying. Fully desiccated samples were mounted on a specimen stub using a

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carbon tab. After critical-point drying, using CO2 and sputter-coating with gold according to

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standard procedures, specimens were operating at 5 kV accelerating voltage. Then, the

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morphology of cells was monitored under a scanning electron microscopy. (BDS200-pH Inverted

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Microscope, Chongqing Optec Instrument Co., Ltd, China).

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2.6. Flow cytometric apoptosis assay

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Flow cytometry analysis was performed to identify and quantify the apoptotic cells by using

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Annexin V-FITC/PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China)

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according to the manufacturer’s instruction. Briefly, 1 × 106 PC12 cells and 8 × 105 SH-SY5Y

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cells were seeded in in 6-well plate at 37 °C and incubated for 24 h. After different treatment, cells

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were harvested, washed in ice cold PBS two times, re-suspended in binding buffer and incubated

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with Annexin V-FITC and PI in dark at room temperature in for 40 min. Then, the cells were

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analyzed using a flow cytometer (FACS-caliber, Becton Dickinson, San Jose, CA, USA) and all

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cells samples were analyzed by Mod Fit software.

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2.7. Measurement of ROS and MDA

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The levels of ROS were examined using the oxidation sensitive fluoroprobe 2′,

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7′-dichlorofluorescein diacetate kit (DCFH-DA, JianCheng Institute of Biotechnology, Nanjing,

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China) according to the manufacturer’s instructions. DCFH-DA is a non-fluorescent compound

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that is freely taken up by cells and hydrolyzed by esterase to 2′, 7′-dichlorofluorescein (DCFH).

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Then, it is oxidized to fluorescent 2′, 7′-dichlorofluorescein (DCF) in presence of peroxides,

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thereby indicating the level of intracellular ROS. In briefly, 1 × 106 PC12 cells and 8 × 105

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SH-SY5Y cells were seeded in in 6-well plate at 37 °C and incubated for 24 h. After different

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ACCEPTED MANUSCRIPT treatment, cells were washed in PBS three times and incubated with 10 µM DCFH-DA for 30 min

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at 37 °C in dark room. The fluorescence was determined using a fluorescence microplate reader

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(JASCO, FP-6500, Japan) at 488nm excitation and 525 nm emission wavelength. The

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fluorescence intensity of DCF represented the quantity of intracellular ROS. The intracellular

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ROS level was expressed as a ratio of the control group. All data are from three separate

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experiments.

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The MDA content were determined using commercially available assay kit (Jian Cheng

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Institute of Biotechnology, Nanjing, China). Briefly, 1 × 106 PC12 cells and 8 × 105 SH-SY5Y

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cells were seeded in in 6-well plate at 37 °C and incubated for 24 h. After different treatment,

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PC12 cells were washed for two times with PBS， and then scraped from the plate into cold PBS.

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The cells in PBS were homogenized by using an ultrasonic cell disruptor (Ningbo Scientz

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Biotechnology Co. Ltd.). The homogenate was centrifuged at 8000 g for 15 min at 4 °C, and then

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the supernatant was collected for MDA level analysis. Protein content was measured using BCA

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protein assay kit (BSA, Beijing Institute of Biotechnology, Nanjing, China). The MDA results

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were expressed as nmol/mg protein. All data were from three separate experiments.

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2.8. Measurement of SOD and GSH-Px activities

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The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in cell

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were measured by using commercially available assay kit (Jian Cheng Institute of Biotechnology,

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Nanjing, China). Briefly, 1 × 106 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in in 6-well

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plate at 37 °C and incubated for 24 h. After different treatment, PC12 cells were washed twice with

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PBS then scraped from the plate into cold PBS. The cells in PBS were homogenized by using an

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ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co. Ltd.). The homogenate was then

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centrifuged at 8000 g for 15 min at 4 °C, and the supernatant was collected for SOD and GSH-Px

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activities.

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Biotechnology, Nanjing, China). The results were expressed as U/mg protein. All data are from

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three separate experiments.

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2.9. Measurement of mitochondrial membrane potential (MMP)

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Protein content was measured using BCA protein assay kit (BSA, Beijing Institute of

MMP was detected according to the instructions of JC-1 mitochondrial membrane potential

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assay kit (Jian Cheng Institute of Biotechnology, Nanjing, China). In briefly, 1 × 106 PC12 cells

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and 8 × 105 SH-SY5Y cells were seeded in in 6-well plate at 37 °C and incubated for 24 h. After

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different treatment, cells were washed and suspended in ice-cold PBS. Then the cell was added

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with JC-1 working solution (5 µg/ml) and serum-free medium at 37 °C in the dark and washed in

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PBS three times. The cellular fluorescence intensity of JC-1 in PC12 cells was measured using a

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flow cytometer (FACS-caliber, Becton Dickinson, San Jose, CA, USA). The cellular fluorescence

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intensity of JC-1 in SH-SY5Y cells was measured under a fluorescent microplate reader. The

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treatment cellular MMP was expressed as a percentage of control cells.

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2.10. Measurement of mRNA

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In briefly, 1 × 106 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in in 6-well plate at

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37 °C and incubated for 24 h. After different treatment, cells were harvested and washed in

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ice-cold PBS three times. The total RNA was extracted using Trizol Reagent (Invitrogen, USA),

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and cDNA synthesis was performed using a reverse transcription kit (TransScript First-Strand

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cDNA Synthesis SuperMix, TransGen Blotech, Beijing, China). The reactions were conducted

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with 20 µL reaction volumes (10 µL of DNA Master Mix, 9 µL of H2O, 0.3 µL of each 10 µM

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primer, and 0.4 µL cDNA). All primer sequences of PC12 cells were used as previously described

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ACCEPTED MANUSCRIPT [63]. All primer sequences of SH-SY5Y cells were used

as previously described [64, 65]. PCR

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was performed in an ABI PRISM 7500 Real-Time PCR System (Applied Bio systems, CA) using

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SYBR Green PCR Core Reagents (Roche). Amplification of GADPH and β-actin mRNA (as an

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endogenous control) were used to standardize the amount of sample added to the reactions. Each

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sample was examined in triplicate, and a mean value was calculated. Data were analyzed

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according to the 2-∆∆Ct method.

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2.11. Measurement of caspase-3 activity

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Caspases-3 is regarded as the most of the death signals converge to induce cell apoptosis and

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was determined with the caspase-3 assay kit (Jian Cheng Institute of Biotechnology, Nanjing,

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China). Briefly, 1 × 106 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in in 6-well plate at

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37 °C and incubated for 24 h. After different treatment, cells were harvested and caspase-3 activity

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were determined according to the manufacturer instructions. Caspase-3 activity was measured

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using a microplate reader at 405 nm. Caspase-3 activity was expressed as a percentage of control.

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All data were from three separate experiments.

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2.12. Western blot analysis

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Cyt-C release was tested by Western blot. Whole-cell protein extracts were obtained using

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super active RIPA lysis buffer containing 1 % PMSF (Beyotime Institute of Biotechnology,

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Jiangsu, China). Total proteins were obtained by centrifugation and cytoplasmic proteins were

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extracted using cytoplasmic protein extraction kit. Protein quantification was determined by BCA

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assay (Beyotime institute of biotechnology, Jiangsu, China). The protein was separated by 8%

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sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes.

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The membranes were blocked with 5% fat-free milk in TBST buffer at room temperature for 3h,

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ACCEPTED MANUSCRIPT incubated overnight at 4 °C with primary antibodies against cytochrome c (Santa Cruz

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Biotechnology, Santa Cruz, CA, USA;) in 5% non-fat milk in TBST. Then washings with TBST,

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15 min each time, the membranes were incubated with appropriate secondary antibodies

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conjugated to horseradish peroxidase (1: 3000) for 2 h at 37 °C. After washing again with TBST,

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the object protein was detected using the enhanced ECL reagent (Beyotime institute of

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biotechnology, Jiangsu, China). And GADPH and β-actin were used as the internal control.

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Quantitative analysis was carried out using Gel-Pro analyzer 4 image analysis system.

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2.13. Statistical Analysis

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Data are expressed as means ± SD of at least three separate experiments. Statistical

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significance was determined using one-way ANOVA followed by LSD post hoc tests. Difference

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was considered significant at p < 0.05.

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3. Results

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3.1. Effects of HF on survival in Almal-treated PC12 cells and SH-SY5Y cells

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As shown in Fig. 1A, when PC12 cells were treated with Almal at concentrations ranging

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from 100 to 800 µM for 24 and 48 h, cell viability declined in a time- and dose-dependent manner.

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In addition, we use SPSS22.0 software to calculate that IC50 value of Almal was 504 µM when

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PC12 cells were treated for 48 h. When SH-SY5Y cells were treated with Almal at concentrations

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ranging from 5 to 2000 µM for 24 and 48 h, cell viability declined in a time- and dose-dependent

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manner (as shown in Fig. 1a). In addition, we use SPSS22.0 software to calculate that IC50 value

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of Almal was 518 µM when SH-SY5Y cells were treated for 48 h.

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To determine the appropriate dose to protect Almal-induced damage, PC12 cells and

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SH-SY5Y cells were pretreated with HF at concentrations ranging from 0.25 to 20 µM for 12 or

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24 h and then exposed to Almal for 48 h, respectively. After different treatment, we found that

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pretreatment with 0.25 to 2.5 µM HF significantly attenuated the Almal-induced cell viability

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decrease (as shown in Fig. 1B and Fig. 1b). As shown in Fig. 1C, PC12 cells and SH-SY5Y cells proliferation rates were all higher in the

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HF group compared with the control group (P<0.01). The percentage survival rate for cells treated

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with Almal alone for 48 h was 60.37 ± 5.5% and 59.02 ± 3.8% respectively compared with the

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control group (P<0.01). However, treatment with 1 µM HF the viability of PC12 cells was

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significantly increased to 81.9 ± 5.4% and 86.1 ± 3.4% respectively compared with the Almal

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group (P<0.01). And as shown in Fig. 1c, the LDH activity of PC12 cells and SH-SY5Y cells

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were all slightly lower than the control but did not have statistically significant. The LDH activity

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for cells treated with Almal alone for 48 h was increased to 250.1 ± 4.5% and 322.6 ± 5.1%

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respectively compared with the control group (P<0.01). However, treatment with 1 µM HF the

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LDH levels of PC12 cells and SH-SY5Y cells was significantly decreased to 180.5 ± 3.1% and

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203.2 ± 3.1% respectively compared with the Almal group (P<0.01). These obvious differences

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indicated that HF can promote cell proliferation and effectively reduce Almal-induced cell

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viability decreased and damage.

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PC12 cells damage induced by Almal was accompanied by the cellular morphological

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changes, and the protective effects of HF were shown through scanning electron microscope

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observation. Results in Fig. 2 showed that the PC12 cells were star or pyramid-shaped and grew

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and spread perfectly in the control group. There was no obvious morphological change between

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control and HF group, this indicated that HF had no toxic effect on PC12 cells. Treatment with

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500 Almal showed that cells became round up, cell body shrinkage, and loss in cell membrane

ACCEPTED MANUSCRIPT integrity. Bleb formation on the cell surface, one of the characteristics of so-called apoptotic

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bodies can be observed in the HF+Al or Al group (Apoptotic bodies are indicated by arrows).

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However, pretreatment with 1µM HF can effectively alleviate the production of apoptotic bodies.

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These obvious differences indicated that HF can effectively alleviate Almal-induced cell damage.

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3.3. Effects of HF on Apoptosis in Almal-treated PC12 cells and SH-SY5Y cells

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Cells apoptotic rate was evaluated by flow cytometry analysis using Annexin V-FITC/PI

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staining. As shown in Fig. 3, the rate of normal cells was 95.9% and 95.2% in the control group.

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The rate of apoptosis in the HF group was slightly lower than the control but did not have

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statistically significant. The apoptosis rate significantly increased to 13.7% and 13.8 %

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respectively compared with the control group (P<0.01) after treatment with 500µM Almal for 48 h.

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However, when pretreated with 1 µM HF, the apoptosis rate was significantly decreased to 8.4%

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and 7.9 % compared with the Almal group (P<0.01). These obvious differences indicated that HF

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can protect PC12 cells and SH-SY5Y cells from apoptosis induced by Almal.

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3.4. Effects of HF on the change of ROS and MDA in Almal-treated PC12 cells and SH-SY5Y cells

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To determine whether HF exerts its protection by inhibiting ROS production, we assayed the

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generation of ROS and MDA. As shown in Fig. 4A and B, in PC12 cells the level of MDA in the

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HF group was slightly lower than the control but did not have statistically significant, and in

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SH-SY5Y cells the level of MDA in the HF group was significantly lower than the control

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(P<0.05). The level of ROS was significantly reduced compared with the control group (P<0.01),

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this indicated that HF has a strong free radical scavenging ability as previously described [45].

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And when PC12 cells exposure to 500µM Almal for 48 h, the generation of ROS and the content

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of MDA were markedly increased compared with the control group (P<0.01). However,

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ACCEPTED MANUSCRIPT pretreatment with HF markedly reduced ROS and MDA generation compared with the Almal

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treated group (P<0.01). These results suggested that HF is effective to alleviate oxidative damage

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in PC12 cells and SH-SY5Y cells induced by Almal.

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3.5. Effects of HF on SOD and GSH-Px activities in Almal-treated in PC12 cells and SH-SY5Y

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cells

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Cells can against oxidative stress and damage by their own defense mechanisms antioxidant

298

system, including antioxidant enzymes including SOD and GSH-Px etc. As shown in Fig. 5A and

299

B, the activity of SOD of PC12 cells and SH-SY5Y cells in the HF group was significantly higher

300

than the control (P<0.01). The activity of GSH-Px was significantly increased compared with the

301

control group (P<0.01). When treated with 500 µM Almal for 48 h, the SOD activities were

302

significantly decreased by 45.91% and 35.5% respectively compared with the control group

303

(P<0.01) and the GSH-Px activities were significantly decreased by 63.8% and 49.2%

304

respectively compared with the control group (P<0.01). However, pretreatment with 1 µM HF for

305

12h, the GSH-Px activities in cells were significantly restored to 88.9% and 80.5% respectively

306

compared with the Almal group (P<0.01), the SOD activities in cells were significantly

307

respectively restored to 75.5% and 60.2% compared with the Almal group (P<0.01). These results

308

suggested that HF is effective to alleviate oxidative damage in PC12 cells and SH-SY5Y cells

309

induced by Almal mainly via enhancing the activities of SOD and GSH-Px.

310

3.6. Effects of HF on Mitochondrial Membrane Potential in Almal-treated in PC12 cells and

311

SH-SY5Y cells

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Mitochondria are central to both normal cell function and the regulation of cell apoptosis.

313

Apoptosis is often accompanied by mitochondrial dysfunction, which is reflected by MMP. Flow

ACCEPTED MANUSCRIPT cytometry analysis results as indicated in Fig. 6 A and B, the MMP of PC12 cells in the HF group

315

was slightly higher than the control but did not have statistically significant (P>0.05). When

316

treated with 500 µM Almal for 48 h, the MMP of PC12 cells was significantly reduced compared

317

with the control group (P<0.01). However, pretreatment with HF for 12h markedly reduced MMP

318

when compared with the Almal treated group (P<0.05). These results suggested that HF is

319

effective to alleviate mitochondrial dysfunction in PC12 cells induced by Almal.

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As shown in Fig. 6 C, the MMP of SH-SY5Y cells in the HF group was slightly lower than

321

the control but did not have statistically significant (P>0.05). When treated with 500 µM Almal

322

for 48 h, the MMP of SH-SY5Y cells was significantly reduced compared with the control group

323

(P<0.01). However, pretreatment with HF for 12h markedly reduced MMP of SH-SY5Y cells

324

when compared with the Almal treated group (P<0.01). These results suggested that HF is

325

effective to alleviate mitochondrial dysfunction in SH-SY5Y cells induced by Almal.

326

3.7. Effects of HF on the mRNA expression of Bax, Bcl-2 and caspase-3 in Almal-treated in PC12

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cells and SH-SY5Y cells

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In PC12 group, treatment with HF for 12h alone, Bcl-2 mRNA expression was significantly

329

increased compared with the control group (P<0.01) and Bax mRNA expressions was

330

significantly decreased compared with the control group (P<0.05). In SH-SY5Y group, treatment

331

with HF for 12h alone, Bcl-2 mRNA expression was significantly increased compared with the

332

control group (P<0.05) and Bax mRNA expressions was slightly lower than the control but did

333

not have statistically significant. And Almal can significantly increase the mRNA levels of Bax

334

and reduced the Bcl-2mRNA levels in PC12 cells and SH-SY5Y cells, respectively. However,

335

when the cells pretreated with HF for 12h, the Bcl-2 mRNA expression was significantly

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ACCEPTED MANUSCRIPT increased and the Bax mRNA expressions was significantly decreased compared with the Almal

337

group (P<0.01). These results suggested that HF relieves Almal-induced apoptosis by altering the

338

expression of the Bcl-2 family. Significant changes in caspase-3 mRNA expression were observed

339

in HF/ HF+Almal group also indicated that HF alleviates Almal-induced apoptosis.

340

3.8. Effects of HF on the activity of caspase-3 in Almal-treated PC12 cells and SH-SY5Y cells

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The activity of caspase-3 in the HF group was slightly lower than the control but did not have

342

statistically significant. When PC12 cells and SH-SY5Y cells exposed to 500 µM Almal for 48,

343

the activity of caspase-3 was significantly decreased compared with the control group (P<0.01).

344

However, pretreatment with HF, PC12 cells and SH-SY5Y cells showed a significant decrease in

345

caspase-3 activity compared with the Almal-treated group (P<0.01). These results suggest that HF

346

relieves Almal-induced apoptosis by inhibiting caspase cascade activation.

347

3.9. Effects of HF on the protein expression of Cytochrome c (Cyt-c) in Almal-treated in PC12

348

cells and SH-SY5Y cells

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As shown in Fig. 9, the level of Cyt-c was significantly increased compared with the control

350

group (P<0.01). When PC12 cells or SH-SY5Y cells treated with Almal, the protein expression of

351

Cyt-c was significantly increased compared with control group (P<0.01). However, pretreatment

352

with HF significantly decreased Cyt-c protein expression caused by in Almal-treated PC12 cells or

353

SH-SY5Y cells.

354

4. Discussion

355

4.1 Determination of Almal and HF treatment time and dose in PC12 Cells and SH-SY5Y cells

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At present, the dose of Al is usually between 250-750 µM in Al-neurotoxicity in vitro studies

357

[66-68]. In order to determine the appropriate Al dose, PC12 cells were treated with 100-800 µM

ACCEPTED MANUSCRIPT Almal for 24 h or 48 h. Results showed that the viability of PC12 cells induced by Almal

359

decreased in a dose- and time-dependent manner. The IC50 of Almal to PC12 cells was 504 µM at

360

48 h. In summary, we combined the doses and time of Almal in other studies and the IC50 results

361

of our pre-experiments, and we finally chose 500 µM Almal treatment PC12 cells for 48 h for

362

follow-up experiments. Similarly, we designed a pre-experiment to determine the appropriate

363

aluminum dose for SH-SY5Y cells. After SH-SY5Y cells were treated with 5-2000 µM Almal for

364

24 h or 48 h, CCK-8 cell viability assay kit results showed that Almal-induced SH-SY5Y cell

365

viability decreased in a dose- and time-dependent manner. We also calculated that the IC50 of

366

Almal to SH-SY5Y cells was 518 µM at 48 h. In summary, we combined the IC50 results of our

367

pre-experiments, and we finally chose 500 µM Almal treatment SH-SY5Y cells for 48 h for

368

follow-up experiments. Dinamarca et al. found that after treatment of primary hippocampal

369

neurons with a gradient dose of HF for 24 h, HF at a concentration of 0.01-1.0 µM had no

370

significant effect on cell viability, but cell viability was significantly reduced at concentrations

371

greater than 10 µM [69]. In addition, Christian et al. It was found that pretreatment with 0.1 to 3

372

µM HF for 20 min significantly inhibited the production of excess ROS in polymorphonuclear

373

leukocytes in a dose-dependent manner[70]. Thus, the neuroprotective effect of HF is closely

374

related to its concentration and treatment time. In order to choose the optimal dose of HF against

375

Al-neurotoxicity, PC12 cells or SH-SY5Y cells were treated with 0.25 to 20 µM HF for 12 h and

376

24 h respectively and then exposed to 500 µM Almal for 48 h. CCK-8 kit assay cell viability

377

results show that 0.25 to 2.5 µM HF can significantly reduce Al-malt-induced PC12 cell viability

378

decreased. We combined the doses and time of HF in other studies and the results of our

379

pre-experiments, and we finally chose 1 µM HF pretreatment PC12 or SH-SY5Y cells for 12 h for

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ACCEPTED MANUSCRIPT 380

follow-up experiments.

381

4.2 Effects of HF on damage in Almal-exposed PC12 cells and SH-SY5Y cells The effect of HF on the proliferation of PC12 cells was measured by the CCK-8 assay.

383

Results showed that when cells were treated with 1 µM HF, to some extent, could promote the

384

proliferation of PC12 cells. When the PC12 cells exposed to 500 µM Almal for 48 h significantly

385

decreased the cell viability and increased the apoptosis. However, HF pretreatment in PC12 cells

386

for 12h remarkably increased the cell viability. The results implied that HF observably protected

387

PC12 cells against Almal-induced cytotoxicity. Scanning electron microscope was adopted to

388

observe cellular morphology. The results showed that Almal could cause damage to PC12 cells,

389

leading to shrinkage of cell bodies, disruption of dendritic networks and increase of intercellular

390

space and apoptotic bodies. However, pretreatment of cells with HF significantly attenuated

391

Almal-induced morphological damage and decreased the number of apoptotic bodies. When the

392

cell membrane was damaged, the intracellular LDH was released into the culture medium, and the

393

content of LDH was an important index to detect the cell death. In this study, we found that the

394

level of LDH was significantly increased in the Almal group, while pretreatment with HF for 12 h

395

the LDH level in the supernatant were all significantly reduced in PC12 cells. These results all

396

showed that HF could prevent PC12 cells from damage induced by Almal. Further, we chose

397

another cell line, SH-SY5Y, to verify whether HF protects against Almal-induced cell damage by

398

measuring cell viability and release of LDH. The results showed that HF also significantly

399

alleviated the increased LDH levels and decreased cell viability in Al-malt exposed SH-SY5Y

400

cells. In comparison with PC12, LDH results suggested that SH-SY5Y cells appeared to be more

401

sensitive to Almal and HF. In conclusion, we preliminarily concluded that HF can effectively

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ACCEPTED MANUSCRIPT 402

relieve cell damage induced by Almal exposure in PC12 cells and SH-SY5Y cells.

403

4.3 Effects of HF on oxidative damage in Almal-exposed PC12 cells and SH-SY5Y cells Some in vitro studies have demonstrated that Almal can induce ROS generation and

405

oxidative damage [20, 61]. In order to elucidate whether HF could prevent Almal-induced ROS

406

generation and the oxidative damage, we first investigated the levels of ROS via measuring a

407

conversion of DCFH2-DA to dichlorofluorescein (DCF). The fluorescent signal of DCF liberated

408

in the cells was enhanced after Almal treatment in PC12 cells. Pretreatment with HF markedly

409

reverse this increase in fluorescent intensity in PC12 cells, which indicated the reduction of ROS

410

generation. In addition, exposure of HF alone can also significantly reduce the production of ROS

411

in PC12 cells. These results suggest that the neuroprotective effects of HF may result from

412

inhibition of intracellular ROS production. Oxidative cellular damage caused by ROS is normally

413

accompanied by an increase in lipid peroxide, which reduces membrane fluidity. A breakdown

414

product of lipid peroxides, MDA, was most commonly used as a parameter to evaluate the extent

415

of lipid peroxide [71]. In the present experiment, the level of MDA was significantly enhanced

416

after Almal treatment. Pretreatment with HF markedly reversed this increase. In addition,

417

treatment with HF alone can also significantly reduce the level of MDA. These results suggested

418

that HF effectively attenuates Almal-induced oxidative damage in PC12 cells. To further

419

determine the protective effect of HF on Almal-induced oxidative damage, we also measured the

420

levels of ROS and MDA in different treatment groups in SH-SY5Y cells. The results showed that

421

HF can effectively relieve the ROS excess production and MDA levels caused by in

422

Almal-exposed SH-SY5Y cells. Similarly, treatment with HF alone can also reduce MDA levels.

423

These results indicated that HF effectively attenuates Almal-induced oxidative damage. However,

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ACCEPTED MANUSCRIPT 424

its antioxidant mechanism remains unclear To explore its antioxidant mechanism, we also examined the activity changes in antioxidant

426

enzyme including SOD and GSH-Px. SOD is generally regarded as the first line of defense against

427

tissue and cellular damage caused by ROS [72]. It catalyzes the dismutation of O2•− to H2O2. The

428

activity of SOD indirectly indicates the level of intracellular free radicals. GSH-Px is the second

429

line of defense by transforming H2O2 into water and molecular oxygen [73]. Reduced antioxidant

430

enzyme activity can lead to oxidative stress. Our result indicated that Almal treatment greatly

431

decreased the activities of SOD and GSH-Px in PC12 cells and SH-SY5Y cells as same as the

432

previous study [74, 75], while pretreatment of PC12 cells with HF can inhibit the decrease of the

433

activities of these enzymes. These results suggested that HF protects PC12 cells and SH-SY5Y

434

cells against oxidative damage induced by Almal via enhancing the activities of SOD and

435

GSH-Px.

436

4.4 Effects of HF on apoptosis in Almal-exposed PC12 cells and SH-SY5Y cells

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Almal-induced oxidative damage can further lead to cell apoptosis via the mitochondrial

438

pathway [74]. Briefly, Almal-induced ROS can enter mitochondria to destroy key substrates in the

439

mitochondrial electron transport chain, leading to the increased ROS generation and MMP loss

440

[76, 77]. In turn, excessive ROS further destroys mitochondria and forms a vicious cycle that

441

finally leads to apoptosis. In our experiments, flow cytometry results showed that cultured with

442

500 µM Almal for 48 h significantly cause PC12 cells apoptosis. Scanning electron microscopy

443

showed a significant increase in apoptotic bodies in the Almal group. And we found that HF

444

significantly decreased the apoptosis rate and the numbers of apoptotic bodies compared with

445

Almal group. These results proved that HF effectively reduced apoptosis in Almal-treated PC12

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ACCEPTED MANUSCRIPT 446

cells. In SH-SY5Y cells, HF can also significantly reduce the increase in apoptosis rates induced

447

by Almal and these results are similar to those of PC12 cells. However, HF can attenuate

448

Almal-induced apoptosis whether via the mitochondrial pathway remain unclear. To explore the underlying mechanism of anti-apoptotic effects of HF, we examined the

450

changes in MMP. The change of mitochondrial membrane potential is recognized as an early

451

manifestation of mitochondrial pathway apoptosis [78]. In our study, we demonstrated that Almal

452

induced the MMP loss of PC12 cells and SH-SY5Y cells. In contrast, pretreatment with HF

453

significantly prevented the changes, which preliminary proved that HF can alleviate

454

mitochondrial-mediated apoptosis in Almal-treated PC12 cells and SH-SY5Y cells.

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The loss of MMP further causes releases apoptosis substances like Cyt-c from mitochondria

456

into the cytosol, results in the activation of the caspase cascade, which would finally lead to

457

apoptosis [79]. In this study, treatment with HF alone significantly reduced the Cyt-c protein

458

expression in PC12 cells. When the cells exposed to Almal, Cyt-c protein expression was

459

significantly inhibited in PC12 cells. In contrast, pretreatment with HF significantly prevented the

460

changes. Similarly, the same trend was found by examining the expression of Cyt-c protein in

461

SH-SY5Y cells which further proved that HF can further alleviate mitochondrial-mediated

462

apoptosis via inhibiting the release of Cyt-c.

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Caspases-3 is regarded as the most of the death signals converge to induce cell apoptosis [80].

464

Caspase-3 can be activated by Cyt-c. Pretreatment with HF significantly reduced the activity and

465

mRNA expression of caspase-3 compared with the Almal group. These significant differences

466

proved that HF can alleviate mitochondrial-mediated apoptosis in Almal-treated PC12 cells and

467

SH-SY5Y cells via preventing activation of caspase-3.

ACCEPTED MANUSCRIPT In our experiment, we also examined changes in the expressions of apoptotic-related genes

469

when the cell pretreatment with HF to explore the potential role of anti-apoptotic in the

470

Almal-treated PC12 cells and SH-SY5Y cells. The Bcl-2 family of proteins, as a regulator of

471

apoptosis, is involved in positive and negative regulation of the mitochondrial apoptotic pathway.

472

Bcl-2 and bax proteins were anti-apoptotic proteins and pro-apoptotic proteins, respectively [81].

473

Bcl-2 and bax in a dynamic balance can maintain mitochondrial integrity and thereby reducing the

474

release of Cyt-c and inhibiting apoptosis [81]. In this study, pretreatment with HF significantly

475

inhibited the down-regulation of bcl-2 expression and up-regulation of bax expression induced by

476

Almal. Treatment with HF alone also can up-regulated Bcl-2 expression and bax expression.

477

These results indicated that HF can also relieve mitochondria-induced apoptosis in Almal-treated

478

PC12 cells and SH-SY5Y cells by regulating the expression of Bcl-2 and bax. Compared with the

479

SH-SY5Y cell line, the expression levels of bax and bcl-2 mRNA indicated that HF exerts a

480

relatively greater anti-apoptotic effect in PC12 cells. In summary, HF can effectively alleviate

481

mitochondrial-mediated apoptosis induced by Almal. However, since ROS inhibitors were not

482

added in this experiment, it is not clear whether HF exerts its anti-apoptotic effect via its

483

antioxidant potential. Therefore, further validation is needed in the future.

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In summary, our experiments show that HF exerts neuroprotection against oxidative damage

485

and mitochondrial-mediated apoptosis induced by Almal in PC12 cells. In addition, the increase in

486

SH-SY5Y cell line experiments further confirmed HF can effectively relieve Almal

487

exposure-induced oxidative damage and mitochondria-mediated apoptosis. HF will be redefined

488

as a neuroprotective agent that combines anti-oxidant property.

489

ACCEPTED MANUSCRIPT 490 491 492

Acknowledgements The study was supported by a Grant from the National Natural Science Foundation Project (31372496).

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Fig.1. (A) Effects of Amal alone on the viability of PC12 cells. PC12 cells were treated with

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the indicated concentrations (100, 200, 300, 400, 500, 600, 700 and 800 µM) of Almal for 24 or

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48h. (a) SH-SY5Y cells were treated with the indicated concentrations (5, 10, 50, 100, 250, 500, 1000 and 2000 µM) of Almal for 24 or 48h. (B) and (b) HF alleviated Almal-induced cell cytotoxicity. Cells were treated with 0, 0.25, 0.5, 1, 1.25, 2.5, 5, 10 and 20 µM HF for 12 or 24h and then exposure to 500 µM Almal for 48h. + indicates that the cells are treated with Al-malt, indicates that the cells are not treated with Al-malt. (C) HF alleviated Almal-induced cell cytotoxicity. Cells were treated with 1 µM HF for 12 h and then exposed to 500 µM Almal for 48h. (c) HF alleviated Almal-induced cell LDH release. Data are expressed as mean ± S.D. (n = 3).

*

ACCEPTED MANUSCRIPT indicates extremely significant difference from the corresponding Al-malt (p<0.05), extremely significant difference from the corresponding Al-malt (p<0.01); significant difference from the corresponding control (p<0.05),

##

#

**

indicates

indicates extremely

indicates extremely significant

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difference from the corresponding control (p<0.01).

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Fig.2. Effects of HF on morphologic changes in Almal-treated PC12 cells. The cells were observed by

scanning electron microscopy. Apoptotic bodies are indicated by arrows. Control: control, HF: Hyperforin, HF+Al:

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Hyperforin+Aluminum maltolate, Almal: Aluminum maltolate

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Fig.3. Effect of HF on Almal-induced apoptosis in PC12 cells and SH-SY5Y cells. Data are shown as mean ±

(p<0.05),

**

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S.D. from three separate experiments. * indicates extremely significant difference from the corresponding Al-malt

indicates extremely significant difference from the corresponding Al-malt (p<0.01);

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extremely significant difference from the corresponding control (p<0.05),

difference

from

the

corresponding

control

(p<0.01).

Hyperforin+Aluminum maltolate, Almal: Aluminum maltolate

Control:

##

#

indicates

indicates extremely significant

control,

HF:

Hyperforin,

HF+Al:

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Fig.4. (A) Effect of HF on ROS level in Almal-treated PC12 cells and SH-SY5Y cells. (B) Effect of HF on

experiments.

*

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MDA level in Almal-treated PC12 cells and SH-SY5Y cells. Data are shown as mean ± S.D. from three separate

indicates extremely significant difference from the corresponding Al-malt (p<0.05), #

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extremely significant difference from the corresponding Al-malt (p<0.01);

difference from the corresponding control (p<0.05),

##

**

indicates

indicates extremely significant

indicates extremely significant difference from the

corresponding control (p<0.01). Control: control, HF: Hyperforin, HF+Al: Hyperforin+Aluminum maltolate,

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Fig.5 (A) Effects of HF on SOD activity in Almal-treated PC12 cells and SH-SY5Y cells. (B) Effects of HF

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on GSH-Px activity in Almal-treated PC12 cells and SH-SY5Y cells. There results were expressed as U/mg

protein. Data are shown as mean ± S.D. from three separate experiments.

corresponding Al-malt (p<0.01);

(p<0.05),

##

#

**

indicates extremely significant

indicates extremely significant difference from the

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difference from the corresponding Al-malt (p<0.05),

*

indicates extremely significant difference from the corresponding control

indicates extremely significant difference from the corresponding control (p<0.01). Control: control,

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Fig.6. Effect of HF against Almal-induced the loss of MMP of PC12 cells by flow cytometry. (C) Effect of HF against Almal-induced the loss of MMP of SH-SY5Y cells by JC-1 staining. * indicates extremely significant

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difference from the corresponding Al-malt (p<0.05),

corresponding Al-malt (p<0.01);

##

**

indicates extremely significant difference from the

indicates extremely significant difference from the corresponding control

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maltolate

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(p<0.01). Control: control, HF: Hyperforin, HF+Al: Hyperforin+Aluminum maltolate, Almal: Aluminum

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Fig.7. Effect of HF on mitochondrial apoptosis related genes expression. (A) Bcl-2 mRNA expression; (B)

Bax mRNA expression; (C) Caspase mRNA expression. Data are shown as mean ± S.D. from three separate

experiments.

*

indicates extremely significant difference from the corresponding Al-malt (p<0.05),

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extremely significant difference from the corresponding Al-malt (p<0.01);

difference from the corresponding control (p<0.05),

##

#

**

indicates

indicates extremely significant

indicates extremely significant difference from the

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corresponding control (p<0.01). Control: control, HF: Hyperforin, HF+Al: Hyperforin+Aluminum maltolate,

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Fig.8. Effect of HF on the activation of caspase-3 in Almal-treated PC12 cells. Data are shown as mean ± S.D.

(p<0.05),

**

*

indicates extremely significant difference from the corresponding Al-malt

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from three separate experiments.

indicates extremely significant difference from the corresponding Al-malt (p<0.01);

##

indicates

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extremely significant difference from the corresponding control (p<0.01). Control: control, HF: Hyperforin,

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Fig.9. Effects of HF on Cyt-c in Almal-treated PC12 cells and SH-SY5Y cells. Data are shown as mean ± S.D.

from three separate experiments.

(p<0.05),

**

*

indicates extremely significant difference from the corresponding Al-malt

indicates extremely significant difference from the corresponding Al-malt (p<0.01);

##

indicates

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extremely significant difference from the corresponding control (p<0.01). Control: control, HF: Hyperforin,

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HF+Al: Hyperforin+Aluminum maltolate, Almal: Aluminum maltolate.

ACCEPTED MANUSCRIPT ·IC50 value of Almal is 504 µM when PC12 cells were treated for 48h. ·IC50 value of Almal is 518 µM when SH-SY5Y cells were treated for 48h. ·HF can attenuate oxidative damage and apoptosis induced by Almal in PC12 cells.

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·HF can attenuate oxidative damage and apoptosis induced by Almal in SH-SY5Y cells.