Carvacrol and Thymol Attenuate Cytotoxicity Induced by Amyloid β25-35 via Activating Protein Kinase C and Inhibiting Oxidative Stress in PC12 Cells

Background: Our previous findings indicated that carvacrol and thymol alleviate cognitive impairments caused by Aβ in rodent models of AD. In this study, the neuroprotective effects of carvacrol and thymol against Aβ25-35-induced cytotoxicity were evaluated, and the potential mechanisms were determined. Methods: PC12 cells were pretreated with Aβ25-35 for 2 h, followed by incubation with carvacrol or thymol for additional 48 h. Cell viability was measured by the MTT method. A flurospectrophotometer was employed to observe the intracellular ROS production. PKC activity was analyzed using ELISA. Results: Our results indicated that carvacrol and thymol could significantly protect PC12 cells against Aβ25-35-induced cytotoxicity. Furthermore, Aβ25-35 could induce intracellular ROS production, while carvacrol and thymol could reverse this effect. Moreover, our findings showed that carvacrol and thymol elevate PKC activity similar to Bryostatin-1, as a PKC activator. Conclusion: This study provided the evidence regarding the protective effects of carvacrol and thymol against Aβ25–35-induced cytotoxicity in PC12 cells. The results suggested that the neuroprotective effects of these compounds against Aβ25-35 might be through attenuating oxidative damage and increasing the activity of PKC as a memory-related protein. Thus, carvacrol and thymol were found to have therapeutic potential in preventing or modulating AD.


INTRODUCTION
lzheimer's disease is a neurodegenerative disorder characterized by progressive deterioration of brain structure and function [1] . As life expectancy is increasing in parallel with the economic development, the risk of AD along with its cost and social burden will be felt even more in the future [2] .
The excessive accumulation of senile plaque in the brain is one of the neuropathological hallmarks of AD [3] . The Aβ peptide in the plaque cores is a 39-43 amino acid sequence generated from a larger transmembrane protein, the APP [4,5] . APP can be processed by two alternative pathways: amyloidogenic and non-amyloidogenic. In the amyloidogenic pathway, β-and γ-secretase cooperatively convert APP into Aβ to form the deposits. In the non-amyloidogenic pathway, α-secretase reroutes the substrate APP toward the production of soluble APPα, which is non-toxic and prevents the formation of amyloid plaques [5] . PKC has been shown to activate α-secretase, increase the non-toxic soluble APPα release and reduce the secretion of the toxic Aβ peptide [6] . In this sense, PKC pharmacology would be an interesting area of research for development of cognition-enhancing agents and 244 Iran. Biomed. J. 24 (4): [243][244][245][246][247][248][249][250] therapeutics against memory loss [7] . On the other hand, oxidative stress has been revealed to contribute to the progression of AD. Some studies have shown that Aβ plays a crucial role in the accumulation of ROS [8,9] . High ROS levels cause oxidative stress that may result in cell death, ultimately leading to neurodegenerative diseases including AD [9,10] . Therefore, therapeutic effort for attenuating the oxidative stress could be beneficial in AD treatment [11] . Despite of extensive research on AD for more than a century, few effective treatment options have been developed [12] . Current available drugs used for the treatment of AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, there is an urgent need for the development of new drugs [13,14] . Nowadays, several medicinal plants and their constituents have been suggested as possible treatments for AD based on their effects on oxidant formation [15] and processing of APP [16] . Among the chemical compounds derived from plants, monoterpenes [2] along with phenolic compounds [17,18] exhibit diverse pharmacological activities, representing them as potential modulators of AD. In this regard, carvacrol (2-methyl-5-(1methylethyl) phenol)] and thymol (2-isopropyl-5methylphenol) as two phenolic monoterpenes are abundantly found in aromatic plants, including Thymus vulgaris and Zataria multiflora [19] . These cyclic monoterpenes exert protective activities in a variety of pathological states including oxidative stress [20,21] , which might be beneficial in reducing AD symptoms.
Previously, we investigated the effects of carvacrol and thymol in two animal models of AD and observed their effectiveness in alleviating cognitive impairments caused by Aβ [22] . In this study, carvacrol and thymol were used in an in vitro model of AD, Aβ-treated PC12 cells, and their antioxidant activity as well as their effects on PKC activity, as a memory-related enzyme, were studied.

Cell culture and treatment
PC12 cells were cultured in flasks with RPMI 1640 medium supplemented with 15% fetal bovine serum and 1% antibiotics (penicillin-streptomycin-nystatin) and incubated in a 5% CO 2 atmosphere at 37 °C. Next, 75% of culture medium was replaced with a fresh medium every 48 h. PC12 cells were passaged when the culture was 80% confluent. The cells were subcultured once a week in the ratio of 1:4. Cell culture solutions were prepared under sterile conditions. The cells in exponential growth were seeded on 96-well plates at a density of 2 × 10 4 cells per well, in 100 μl of growth medium and allowed to attach for 24 h. The cells were then exposed to the desired concentrations of Aβ, carvacrol, thymol, or Bryostatin-1 according to each experiment.

Cell viability assay
The MTT assay was used for three different experiments. First, to achieve the appropriate concentration of Aβ, the cells were treated with different concentrations of Aβ (30,50, and 100 μM) for 48 h. Second, to detect the safety of carvacrol and thymol, cells were exposed to different concentrations of carvacrol and thymol ranging from 1 μM to 1000 μM for 48 h. The third stage was an assay in which the cells were treated with Aβ 25-35 (50 μM), followed by indicated concentrations of carvacrol (10, 20, and 50 μM), thymol (10, 20, and 50 μM), or Bryostatin-1 (10 nM). The control group was treated with DMSO 1% (v/v). After each of these three experiments and at the end of incubation, cell viability was evaluated by MTT assay [23] . In Brief, 10 µl of MTT solution, diluted in sterile PBS (5 mg/ml), was added to the culture plates. The plates were incubated at 37 °C for 4 h. Then the media was carefully removed, and 100 μl of DMSO as lysis buffer was added to each well. After 60 min, the amount of MTT formazan product was determined by measuring absorbance using a 96-well ELISA microplate reader at a wavelength of 545 nm and a reference wavelength of 630 nm. The results were Iran. Biomed. J. 24 (4): 243-250 245 expressed as the percentage of MTT reduction, assuming that the absorbance of control was 100%. All experiments were performed three independent times, each in triplicate.

Measurement of ROS generation
The intracellular ROS formation levels were monitored using the fluorescent probe DCF-DA, which readily diffuses through the cell membrane [24] . Within the cells, the nonfluorescent dye reacts with the intracellular ROS and is converted into DCF, which is a green fluorescent dye. PC12 cells were treated with 50 μM of Aβ [25][26][27][28][29][30][31][32][33][34][35] , followed by the various concentrations of carvacrol (10, 20, and 50 μM), thymol (10, 20, 50 μM), or 10 nM of Bryostatin-1 for 48 h. At the end of the treatment, the plates were washed twice with PBS. Then the cells were incubated in the presence of DCF-DA at a final concentration of 10 μM at 37 °C for 50 min. After incubation, DCF intensity was determined with a fluoremeter (BioTeK, Swindon, UK). The excitation and emission wavelengths were 485 nm and 528 nm, respectively. The results were expressed in relation to ROSassociated fluorescence intensity of control cells, which was set to 100%. The control group was treated with DMSO 1% (v/v). All experiments were repeated three independent times, each in triplicate.

Sample preparation of cell lysates
PC12 cells with different treatments were cultured in a dish (100 mm) to reach 80-90% confluence. After the removal of the culture medium, cells were washed twice with cold PBS and lysed with 300 μl/dish of cool RIPA buffer solution containing 5 M of sodium chloride, 1% Triton X-100, 0.5% sodiumdeoxycholate, 1M of Tris-HCl (pH 7.4), 0.1% SDS, supplemented with protease inhibitor. After 15-min incubation period, the plates were rotated by hand to cover cells with a film of lysis buffer. The cells were then immediately dislodged with a cell scraper, and the cell lysate were collected in a pre-chilled 1.5-ml microcentrifuge tube. In the next step, the microtubes with the cell suspensions were taped vigorously five times and leaved on ice for 15 min. Afterwards, the samples were centrifuged at 16,000 ×g at 4 °C for 15 min to separate the supernatant. At the end, the clear supernatant fractions were transferred to new prechilled 1.5-ml microtubes and stored at -70 °C. The protein concentration of each sample was determined using the Bradford method, and equal amounts of proteins were analyzed for PKC activity assay.

Kinase activity assay
The non-radioactive PKC kinase activity assay kit (ENZO, ADI-EKS-420A) was based on a solid-phase ELISA that utilizes a polyclonal antibody recognizing the phosphorylated form of the substrate. The assay was designed for the analysis of PKC activity in the solution phase. In brief, the wells of the PKC substrate microtiter plate were filled with 50 μl of kinase assay dilution buffer at room temperature for 10 minutes, and then the liquid was carefully aspirated from the wells. The samples were added to the appropriate wells of the PKC substrate microtiter plate, and the reaction was initiated by adding 10 μl of diluted ATP to each well. After 90 minutes, the reaction was stopped by discarding the contents of each well. Then 40 μl of phosphospecific substrate antibody was added to each well, and the plate was incubated at room temperature for 60 minutes. After that, the wells were washed four times with 100 μl of 1 wash buffer, and 40 μl of diluted anti-rabbit IgG, horseradish peroxidase conjugate, was added to each well. After 30 minutes, the wells were washed four times with 100 μl of 1 wash buffer. Then 60 μL of 3,3',5,5'-tetramethylbenzidine substrate was added to each well, and the plate was incubated at room temperature for 30 minutes. Finally, 20 μl of acid stop solution was added to each well. The absorbance was measured at 450 nm using a Bio-Rad microtiter plate reader (USA).

Statistical analysis
All data were presented as means ± SEM. Statistical analyses were carried out using one-way ANOVA, followed by Tukey's honestly significant difference. p < 0.05 was considered to be statistically significant compared with Aβ 25-35 treatment or control group.

Safety of carvacrol and thymol
The safe doses of carvacrol and thymol were first determined by measuring the cell viability assay. Carvacrol and thymol at concentrations ranging between 1 and 1000 μM were added separately to the wells containing PC12 cells for 48 h. The results showed statistically significance difference between the control group (1% DMSO), and the groups were treated with carvacrol at concentrations up to 200 μM; however, treatment with high doses of carvacrol (500 and 1000 μM) resulted in PC12 cytotoxicity. Furthermore, exposure to concentrations up to 100 μM of thymol did not decrease the cell viability compared with the control group (Fig. 2). Our findings indicated that 1-200 μM concentrations of carvacrol along with 1-100 μM concentrations of thymol revealed no cytotoxicity against PC12 cells and those used for our further experiments.

DISCUSSION
In our previous work, we demonstrated the potential neuroprotective effects of carvacrol and thymol in AD animal models [22] . Our present results indicated that, applying carvacrol and thymol attenuated cell death induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] , and prevented Aβ 25-35 -induced oxidative stress in PC12 cells. In addition, Aβ [25][26][27][28][29][30][31][32][33][34][35] inhibited PKC activation, whereas carvacrol and thymol could reverse this effect similar to Bryostatin-1, as a potent PKC activator. Aβ deposition and subsequent formation of senile plaques in the brain have well been recognized as key events during neuropathogenesis of AD. Therefore, studies on the discovery of drugs inhibiting Aβ formation and toxicity are of great significance for AD treatment [25] . Studies have suggested that the neurobiological effects of phytochemicals derived from medicinal plants may contribute to clinical benefits in AD [14,26] . Carvacrol and thymol, natural monoterpenoid phenols, are obtained from many plants belonging to Lamiaceae family and possess diverse pharmacologic properties including antibacterial [20] , cardioprotective [21] , and anticancer [27] effects. Neuroprotective effects of these compounds have been reported in a variety of animal models, including AD [22,28,29] . Herein, the effects of carvacrol and thymol on Aβ-induced cytotoxicity were evaluated in PC12 cells. The rats adrenal pheochromocytoma cells line, namely PC12, appears to be an in vitro cell model for neurobiology and neurochemistry studies [30] . PC12 cells were found to respond specifically to the 25-35 fragment of Aβ as a neurodegenerative factor in AD, stimulating the production of cellular ROS and Aβmediated cell death [31,32] . The results of MTT assay revealed that treatment with carvacrol or thymol significantly increased the cell viability compared to that of the Aβ 25-35 group.
Intracellular oxidative stress resulting from imbalance in ROS production and cellular antioxidant defense mechanisms mediates the damage to proteins and nucleic acids, which has direct and deleterious consequences in AD [33] . Evidence has confirmed that the increased amounts of Aβ peptide elevate the production of ROS [34,35] ; therefore, antioxidants may be merged as therapeutic strategies to attenuate Aβ-induced neurotoxicity and improve neurological outcomes in AD [11] . In this study, when the PC12 cells were incubated with Aβ 25-35 , the contents of ROS significantly increased. Meanwhile, co-treatment with carvacrol or thymol attenuated the Aβ 25-35 -induced increase of ROS levels. Therefore, the ability of these compounds to protect PC12 cells could be partially attributed to their antioxidant property. Our results are in accordance with other studies denoting that carvacrol and thymol possess strong anti-oxidative activity [20,21] . Many investigations have reported that antioxidant agents may slow the progression of AD [10] . For instance, Asadbegi et al. [36] have disclosed that thymol, a natural antioxidant, can be therapeutic against high risk factors for AD. In the present study, administrating carvacrol and thymol could effectively ameliorate the induction of oxidative stress in PC12 cells. As oxidative stress is a process always associated with AD, carvacrol and thymol could be considered as potential candidates for AD treatment.
PKC as a phospholipid-dependent protein kinase is a crucial player in various cellular functions either in neuronal and non-neuronal cells [37] . It is well established that signaling deficits of PKC pathway have an important role in the pathophysiology of neurodegenerative disorders including AD [38,39] . On the other hand, PKC activators are regarded as a potential candidate for the treatment of AD because of APP processing activation through α-secretase [6,40] . In this regard, it has been shown that Bryostatin-1 reduces amyloid plaques in the brain of transgenic AD mice, improves behavioral outcomes and consequently decreases the death rate [7] . Consistent with these data, our findings obtained from PKC activity assay revealed that carvacrol and thymol reversed Aβ-triggered PKC inhibition, representing a new mechanism of action for carvacrol and thymol in reducing Aβ-induced neurotoxicity. Interestingly, Bryostatin-1 acted in the similar way, suggesting the possible modulating role for carvacrol and thymol in PKC activity.
The results of the current study revealed that the protective effects of carvacrol and thymol against Aβinduced cytotoxicity are not only attributable to their antioxidant properties but also they may occur through other mechanisms such as PKC stimulation. In accordance with our results, Chen et al. [41] have displayed that the cardioprotective effects of carvacrol may be attributed to its antioxidant property through the activation of the MAPK/ERK signaling pathway, as the common downstream effectors of PKC [40] . Therefore, considering the possible neuroprotective role of PKC in AD, it is reasonable to suggest administration of carvacrol and thymol as potential disease-modifying agents.
Given the involvement of multiple factors in the pathogenesis of AD as well as the aberrant processing of APP, AD treatment requires multiple drug therapies to address the varied pathological aspects. New potential approaches have been developed to target multiple sites in the brain with single molecular entities for treatment of AD [42,43] . The results of the present study showed that carvacrol and thymol may present multi-functional properties, including antioxidant Iran. Biomed. J. 24 (4): 243-250 249 activity and PKC activity modulation. Our results are consistent with a number of previous studies, introduced numerous bio-molecules belonging to terpenoid [44] or phenolic structures [45,46] as novel therapeutic candidates with the properties such as radical scavenging and PKC activation. Our results provide evidence for the neuroprotective effect of carvacrol and thymol against cytotoxicity induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] . The underlying mechanism might be through decreasing ROS formation as well as stimulating PKC activity. Taken together, these two compounds may have therapeutic potential in preventing or modulating AD. However, further studies are required to clarify all possible neuroprotective mechanisms of carvacrol and thymol in AD models.