1,8-cineol attenuated Aβ 25-35 -induced neuron injury through inhibiting IL-6, IL-8 release and NF-κB expression

attenuated Aβ25-35-induced neuron injury through inhibiting IL-6, IL-8 release and NF-κB expression. ABSTRACT: Objective: To explore the protective effect of 1,8-cineol against Amyloid beta 25-35 ( Aβ 25-35 )-induced cell injury in primary rat cortical neurons. Methods: Primary rat cortical neurons were cultured in vitro , treated with different concentrations of Aβ 25-35 (2.5, 5, 10 20, 40 μM) and 1,8-cineol (1, 3, 10 μM). Cell viability of neuronal cells were detected by MTT assay and cell death was detected by lactate dehydrogenase release (LDH). The production of IL-6 and IL-8 in the supernatant was measured by ELISA assay kits. NF-κB protein expression was detected by Western blotting. Results: In primary cultured neurons, Aβ 25-35 concentration dependently reduced cell viability and increased LDH release. 1,8-cineol with concentrations of 3 and 10 μM protected neuronal cells against Aβ 25-35 induced cell injury for 24 h. 3 and 10 μM of 1,8-cineol also significantly decreased the levels of IL-6 and IL-8 cytokine production in the supernatant. Increased NF-κB expression was also significantly reduced by 1,8-cineol treatment evaluated by Western blotting. Conclusions: Our results revealed a protective effect of 1,8-cineol on Aβ 25-35 induced neuron injury through inhibition of IL-6, IL-8 production and NF-κB expression.


INTRODUCTION
Amyloid beta (Aβ), which aggregate into oligomers in neurons, play a critical role in the pathogenesis of Alzheimer's disease (AD). Increasing evidence demonstrated that Aβ toxicity induced neurotoxicity in the cerebral cortex and hippocampus in vitro and in vivo, resulting in neuronal apoptosis and cognitive dysfunction (Sowade et al. 2017; Kim et al. 2014; Robert et al. 2015). Abundant senile plaques, mainly composed of Aβ peptide, were found in AD patients brains, contributing to inflammatory responses (Vukic et al. 2009). Though mechanisms are not fully understood, lots of studies tried to find the way to inhibit the Aβ neurotoxicity in the brain.
Deposits of Aβ in AD patients brain induced inflammation, leading to secretion of proinflammatory cytokines such as TNF-α and IL-8 (Hanzel et al. 2014). The inflammatory pathway was further accelerated by nuclear factor-κB (NF-κB) activation, which translocated from the cytoplasm to the nucleus binding to its specific target genes including those involved in the inflammatory response (Srinivasa et al. 2015).
1,8-cineol is a major monoterpene principally from Eucalyptus essential oils, which has been used to treat bronchitis and asthma (Worth and Dethlefsen 2012). It strongly inhibited the production of cytokines, leukotriene, and prostaglandins in asthma (Juergens et al. 2003). Recent study demonstrated that 1,8cineol protected rat pheochromocytoma PC12 cells from Aβ toxicity (Khan et al. 2014). Our previous study indicated the role of 1,8-cineol as a strong inhibitor of TNF-α and IL-1β cytokines by inhibiting TLR4 expression (Zhao et al. 2014). However, the effect of 1,8-cineol on Aβ toxicity in cortical neurons are still unknown. Thus, the present study was performed to investigate the protection of 1,8cineol against Aβ 25-35 -induced cell injury in primary cultured rat cortical neurons.

Primary culture of cortical neurons
Primary cortical neurons were obtained from the cortical cortex of neonatal Sprague-Dawley rats within 24 h of birth (Zhang et al. 2013;Meloni et al. 2001). Briefly, rats were decaptitated and the brains were rapidly removed with ice-cold Hank solution. The cerebral cortices were dissected and digested with 0.25% trypsin for 10 minutes at 37 C. The dissociated cells were then immediately seeded onto 24 or 6-well plates pre-coated with poly-L-lysine (0.1 mg/mL). Cells were incubated in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 10% horse serum, 1% penicillin and streptomycin, 2 mM glutamine, 0.01% N 2 , and 0.04% B 27 . Three days after plating, proliferation of non-neuronal cells were inhibited by adding cytosine arabinoside (10 μM) for 24 h. Cultures were maintained at 37 C under a humidified atmosphere with 5% CO 2 . On day 10 in vitro, neuronal cells comprised about 95% of the primary cultured cells, were used for experiments.
All the animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Ethics Committee of Laboratory Animal Care and Welfare, Weifang Medical University.

Drug administration
The neuronal cells were randomly divided into six groups, then were treated with Aβ 25-35 oligomers at concentrations of 0, 2.5, 5, 10, 20, 40 μM for 24 h. The optimal concentration was used for subsequent experiments. Soluble oligomeric forms of Aβ 25-35 were prepared as reported previously. Aβ 25-35 was firstly dissolved into sterile ddH 2 O to make a stock solution of 1 mM and stored at -20 C. To prepare oligomers, the peptide was dissolved to a final concentration of 100 μΜ, followed by incubation at 37 C for 24 h to allow aggregation.
To investigate the effect of 1,8-cineol against Aβ 25-35 toxicity on neuronal cells, primary cultured cortical neurons were pretreated with 1,8-cineol at concentrations of 1, 3, 10 μM for 1 h, then co-treated with Aβ 25-35 for 24 h. 1,8cineol was diluted with sterile saline. The medium was collected and LDH release was detected. Plates were washed carefully with phosphate-buffered sline (PBS), and cell viability was measured by MTT reduction assay.

Assessment of cell viability and LDH assay
Cell viability was assessed by MTT reduction assay, performed as described previously. Briefly, MTT ( 3-( 4,5-dimethylthiazol-2-yl )-2,5diphenyl-tetrazolium bromide ) was dissolved in dimethyl sulfoxide ( DMSO ) at 5 mg/ml as a stock solution. At the end of experiments, MTT was added in culture medium to reach a final concentration of 0.5 mg/ml at 37 C for 4 h. Then, the medium was removed and 100 μl DMSO was added to dissolve the formazan precipitates. The absorbance was measured at a wavelength of 570 nm in a ELISA microplate recorder.
LDH activity in the medium was determined according to the protocols of the LDH ELISA kit designed by manufacturer. An aliquot of media was mixed with NAD and lactate solution, and then measured at a wavelength of 450 nm in the ELISA recorder. All the results are expressed as percentages of the control group.

Immunoblot examination
The primary neurons were cultured in 6-well plates and protein lysates were extracted after treatment. The neuronal cells were washed three times with 0.01 mM PBS, then radio immunoprecipitation assay (RIPA) buffer containing pepstatin 1, leupeptin 2, phenylmethyl -sulfonyl fluoride 1, aprotinin 80 were added into each well and cells were harvested. The lysates were centrifuged at 12,000 g for 30 min at 4C. The protein samples diluted with loading buffer and separated by 10% SDS-PAGE then transferred onto nitrocellulose membranes (Invitrogen, USA). The membranes were then incubated with blocking buffer (Tris-bufferred saline containing 7.5% defatted milk powder). Afterward the membranes were incubated with the rabbit polyclonal anti-NF-κB (1:200) or mouse monoclonal anti-GAPDH antibody (1:5000) overnight, and then incubated with anti-rabbit IRDye700DX®-conjugated antibody or anti-mouse IRDye800DX®-conjugated antibody (1:5000, Rockland, USA). The signals were detected by an Odyssey infrared imaging system. Protein bands were quantitatively evaluated by densitometry using Quantity One® analysis software.

Determination of cytokine production
After drug treatment, the media were collected from neurons cultures with 1,8-cineol and Aβ 25-35 administration for 24 h. The media were centrifuged at 2000 rpm for 10 min to remove cells and debris. The interleukin-6 and interleukin-8 in the media were examined using ELISA kits for IL-6 and IL-8 according to manufacturers' instructions.

Statistical Analysis
All data are expressed as means ± SEM. Statistical comparisons of viability between different treatment groups were performed with One-way ANOVA, followed by Turkey's post hoc test (SPSS 15.0 for Windows, SPSS inc., USA). P < 0.05 was considered statistically significant.

Aβ 25-35 induced toxicity on rat cortical neurons
To investigate the effect of Aβ 25-35 on neuron viability, primary cultured cortical neurons were treated with various concentrations of Aβ 25-35 (0 , 2.5 , 5 , 10 , 20 , 40 μM) . Untreated group was added into same volume of vehicle as control. After 24 h, cell viability was measured using the MTT reduction assay. As shown in Fig.1A, the viability of cortical neurons was significantly dose-dependently reduced after treated with Aβ 25-35 for 24 h. Compared with control group, the viability treated with 2.5 μM of Aβ 25-35 was lightly reduced but no significant difference was found. However, Aβ 25-35 treatment also induced significant increase on LDH activities in a concentration -dependant manner in cultured cortical neurons (Fig.1B). Combined with the above results, 20 μM of Aβ 25-35 was selected as an optimal concentration for subsequent experiments, since the cell viability was 60-70% at the concentration (66.5 ± 6.7 %, P < 0.01) compared with untreated neuronal cells.

Anti-inflammatory effect of 1,8-cineol on cytokine production
Biochemical and neuropathological studies highlighted that Neuroinflammation played an important role in the progression of Alzheimer's disease. Inflammation process once initiated, may contributed to neuronal dysfunction and cell death, establishing a vicious cycle. In the present study, Aβ 25-35 20 μM caused a higher level of IL-6 and IL-8 production in the supernatant of cultured cortical neurons as compared with untreated neuronal cells (P < 0.01). 1,8-cineol pretreatment with concentrations of 3 and 10 μM significantly decreased the level of IL-6 ( Fig.2A, P < 0.01 ) and IL-8 ( Fig.2B, P < 0.05 ) release in cortical neurons compared with neuronal cells treated with Aβ 25-35 only.

1,8-cineol inhibited NF-κB activation
To study the anti-inflammatory mechanism of 1,8-cineol, we determined and analyzed the protein level of NF-κB p65 in cortical neurons. It has been reported that Aβ can activate NF-κB in neurons cells, indicating an important role of this molecular pathway in the pathogenesis of AD. As previously demonstrated, 1,8-cineol regulated the function of NF-κB in inflammatory conditions. In the present study, we verified whether in our experimental conditions, 1,8-cineol modulated Aβ 25-35 induced NF-κB activation. Western blot analysis showed a significant increase of NF-κB p65 expression with Aβ 25-35 incubation. Pretreatment with 1,8-cineol 10 μM in cortical neurons followed by Aβ 25-35 exposure led to a significant reduction of NF-κB p65 expression after 24 h ( Fig.3A and B, P < 0.05 ).   In the present study, we assessed Aβ 25-35induced toxicity in rat cortical neurons by MTT and LDH assay, and confirmed that Aβ 25-35 treatment at 20 μM for 24 h significantly reduced cell viability. Pretreatment with different concentrations of 1,8-cineol markedly attenuated decreased cell viability. The protective effect of 1,8-cineol was further confirmed by LDH release detection. We also found that 1,8-cineol effectively reduced the production of pro-inflammatory cytokines IL-6 and IL-8 and regulated NF-κB activation induced by Aβ 25-35 treatment. All these findings demonstrated that 1,8-cienol could protect neuronal cells against Aβ 25-35 -induced toxicity.
Multiple studies suggests that Aβ peptides play a pivotal role in inducing neuroinflammation in AD (Pimplikar 2014). Aβ-burdened neurons might be the initial cells triggering inflammation, resulting in the progression of the disease (Hanzel et al. 2014). Inflammatory cytokines like TNF-α, IL-8, IL-6 were significantly increased in the cortex and hippocampus in AD, which can also promote Aβ production by modulating γ-secretase activity (Liao et al. 2004). Consistent with these findings, in the present study, cortical neurons treated with Aβ displayed increased levels of TNF-α and IL-1β, which was restored by 1,8cineol pretreatment. The mechanism underlying Aβ-induced inflammation is involved in several signal pathways. It has been reported that NF-κB activation and translocation was closely related with cytokine production induced by Aβ (Yu et  In summary, we demonstrated that 1,8-cineol exerted neuroprotection against Aβ-induced neurotoxicity by inhibiting IL--6, IL-8 production and regulating NF-κB activation in neuronal cells. Future study are needed to focus on neuroprotective effect of 1,8-cineol in vivo. The more attention paid to clarify the further mechanisms of 1,8-cineol will help us to discover more potent candidates for the treatment of AD.