Effects of myricetin against cadmium-induced neurotoxicity in PC12 cells

Abstract

Cadmium (Cd) is one of the most prevalent toxic metals widely found in the environment. Cd induces toxicity and apoptosis in various organs and cells. The nervous system is one of the primary organs targeted by Cd. Cd toxicity is correlated with induction of severe oxidative stress. Myricetin, a natural product, has been found to exert protective effects against various disease conditions. The present study aimed to evaluate the potential protective effects of myricetin on Cd-induced neurotoxicity in PC12 cells. The cells were pretreated with myricetin in the absence and presence of Cd. The viability of cells was assessed using the MTT assay. Markers of oxidative stress were investigated by the lipid peroxidation (LPO), glutathione (GSH) content, and total antioxidant capacity (TAC). Moreover, activation of caspase 3 was examined by Western blot analysis. Myricetin could significantly enhance the viability of PC12 cells. Pretreatment of the cells with myricetin, prior to Cd exposure, showed a significant decrease in the levels of LPO whereas GSH and TAC levels were increased. In addition, the activity of caspase-3 was notably prevented by myricetin. These findings revealed that myricetin has protective effects on Cd-induced neurotoxicity in PC12 cells, which can be linked to its antioxidant potential, inhibition of LPO, and prevention of caspase-3 activation.

Graphical Abstract

Graphical Abstract.

Introduction

Cadmium (Cd) is an extremely toxic heavy metal and a ubiquitous environmental pollutant. The major sources of human exposure to Cd are cigarette smoking, air pollution, and contaminated food and water [1]. Cd is manufactured as a final product during the production of other metals such as copper, lead, and zinc. Other sources include exposure via batteries, fossil fuel, fertilizers, industrial wastes, metal plating, paint pigments, pesticides, rubber and plastics stabilizers, and in lots of different products [2, 3]. Cd has serious detrimental effects on multiple organs in the human body and the most commonly affected organ systems include the kidney, brain, lung, liver, testes, and cardiovascular system. Clinical and experimental evidence has indicated that Cd, because of its long biological half-life (10–30 years), accumulates in different organs of the body [4–10]. It can cross through the blood brain barrier and accumulate in the central nervous system (CNS) thus lead to neurotoxicity with an array of clinical signs such as headache, vertigo, olfactory dysfunction, peripheral neuropathy, attention deficits, memory impairments, and learning disabilities [11–13]. Accumulated evidence has demonstrated that Cd intoxication is an etiological factor in neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, and Huntington’s disease (HD) [14–17]. It has been reported that Cd-induced neurotoxicity in cultured rat cortical neurons and rat primary midbrain neuron–glia cultures [18, 19]. Although the precise mechanisms of cadmium-induced toxicity are not fully explained, generation of reactive oxygen species (ROS) has been reported to play an important role in Cd-induced cell injuries [20].

Excessive production of ROS can react with lipids, proteins, and nucleic acids, alter their functions, thereby causing neuronal cell dysfunction and neurodegeneration. The function of cell depends on the balance between the production of ROS and antioxidant defense system. Oxidative stress is a condition that occurs when there is an imbalance between the production of ROS and antioxidant components [21]. Cd toxicity can cause apoptotic cell death through oxidative stress or excessive amounts of ROS production [22]. Mitochondria are identified as the early and primary targets in cadmium-induced apoptosis. A critical role in the regulation of these events is performed by the Bcl-2 family consisted of both proapoptotic and antiapoptotic proteins such as Bax and Bcl-2, respectively. Recently, therapeutic use of antioxidants from natural sources has been suggested as an accepted strategy in preventing or eliminating various diseases, like neurodegenerative diseases and heavy metal-induced neurotoxicity [23, 24].

Myricetin (3,3′,4′5,5′,7-hexahydroxylflavone), a polyphenolic compound, is commonly distributed in various fruits, vegetables, herbs, and many plants. Recent evidence has proposed that myricetin has anti-inflammatory, antioxidant, and antimutagenic properties. Interestingly, it has been reported that myricetin exhibited a considerable antioxidant activity, with greater free radical scavenging activity than quercetin or other flavonol rhamnosides [25]. Myricetin has also been shown to protect hepatic tissue against carbon tetrachloride-induced toxicity by its antioxidant, anti-inflammatory, and antifibrotic effects in mice [26]. Myricetin protects against H₂O₂-induced cell damage in hamster lung fibroblasts (V79-4) cells [27]. In the present study, we evaluated the protective effects of myricetin on Cd-induced oxidative stress and apoptosis in PC12 cells.

Materials and methods

Cell culture

PC12 cell line was obtained from National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). The cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated Horse Serum, 5% heat-inactivated Fetal Bovine Serum and 1% penicillin–streptomycin and the cells were cultured at 37°C with 5% CO₂.

Cell viability

In order to consider the number of viable cells in cytotoxicity and proliferation studies, cell viability assay was performed using 3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide or MTT. PC12 cells were seeded into each well of a 96-microplate at the concentration of 1 × 10⁴ cells per well. After 24 h of incubation at 37°C, the cells were pretreated with different concentrations of myricetin (0.25, 0.5, and 1 μM) for 24 h and treated with Cd for 24 h. After incubation times, 10 μl of MTT solution was added to each well and incubated for 4 h at 37°C. Then, cell culture medium was removed from wells and 100 μl of dimethyl sulfoxide was added to each well to dissolve the formazan compound. At the end, absorbance of the plate was read at 570 nm by an automatic microplate reader.

Lipid peroxidation

Lipid peroxidation (LPO) was measured by the thiobarbituric acid reactive substances (TBARS) method. TBARS is based on the conjugation of malondialdehyde (MDA) with TBA to form a red product. Briefly, the samples were mixed with TCA and the produced precipitate was added to H₂SO₄ (0.05 M). Then, thiobarbituric acid (TBA) was added and the sample was heated in a boiling water bath for 30 min. After cooling the samples, reaction mixture was extracted by n-butanol and the absorbance was read at 532 nm with a microplate reader.

GSH content

In order to evaluate the GSH levels, GSH assay was done using 5.5′-dithiobis-2-nitrobenzoic acid (DTNB) as Ellman’s reagent. The assay based on the reaction of DTNB with thiol compounds to form a yellow product. Briefly, the samples were mixed with Tris-EDTA buffer and then added to DTNB. The absorbance was measured at a wavelength of 412 nm by microplate reader.

Total antioxidant capacity

Total antioxidant capacity (TAC) was determined by ferric reducing antioxidant power (FRAP) method. This method is based on the reduction of Fe³⁺ to Fe²⁺ in the presence of 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) by the samples. Fe²⁺ reacts with TPTZ and creates a blue color that its absorbance was taken at 593 nm by microplate reader.

Western blot analysis

For the analysis of protein expression, the PC12 cells were lysed with RIPA buffer, protease inhibitor cocktail, PMSF, and sodium orthovanadate and centrifuged at 13 000 g for 20 min at 4°C. The total protein concentrations were evaluated by Bradford assay [28]. For immunoblotting, the same concentrations of proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel. After that, the proteins were transferred onto a polyvinylidene difluoride membrane which was incubated with primary and secondary antibodies. Bands were visualized using chemiluminescence detection kit (ECL, Amersham). Results were analyzed by Total Lab software (Wales, UK).

Statistical analysis

The results were presented as the mean ± standard deviation. Statistical analysis was carried out using one-way ANOVA and Tukey’s post hoc tests. A P < 0.05 was considered to be statistically significant.

Results

The effect of Cd on PC12 cell viability

We examined the effect of various concentrations of Cd (2.5–20 μM) on PC12 cells viability using MTT assay. As shown in Figure 1, Cd decreased the viability of PC12 cells in a concentration-dependent manner compared with the untreated cells. Cd showed significant cytotoxicity at 10 μM concentration for 24 h (P < 0.001), so it was chosen to induce cell injury in the subsequent experiments.

Figure 1.

Effect of cadmium (Cd) on cell viability in PC12 cells. The cells were treated with various concentrations of Cd (2.5, 5, 10, and 20 μM) and cell viability was examined by MTT assay. Results are presented as the mean ± SD (n = 5). ^*P < 0.05, ^***P < 0.001 compared with control.

The effect of myricetin on the Cd-induced cell toxicity

To investigate the protective effects of myricetin on Cd-induced cytotoxicity, PC12 cells were treated with different concentrations of myricetin for 24 h and then exposed to Cd for 24 h. As shown in Figure 2a, pretreatment of the cells with myricetin significantly decreased Cd-induced cell toxicity.

Figure 2.

Effect of myricetin on the cell viability (a) and level of MDA (b) in PC12 cells under Cd toxicity. The cells were preincubated with various concentrations of myricetin (0.25, 0.5, and 1 μM) for 24 h and then treated with Cd (10 μM) for 24 h. The results are presented as the mean ± SD (n = 3–5). ^###P < 0.001 compared with the control group. ^**P < 0.01, ^***P < 0.001 compared with the Cd group.

The effect of myricetin on the LPO levels

As shown in Figure 2b, Cd exposure increased MDA levels in PC12 cells (P < 0.01), which is an indicator of LPO or oxidative stress. Pretreatment of PC12 cells with myricetin demonstrated a considerable decline in the MDA levels in comparison with Cd group.

The effect of myricetin on the GSH content

The PC12 cells exposed to Cd displayed a markedly reduction in the GSH content compared to control group. As shown in Figure 3a, pretreatment with myricetin prevented the Cd-caused decrease of GSH content (P < 0.001).

Figure 3.

Effect of myricetin on GSH content (a) and total antioxidant capacity (TAC) (b) in PC12 cells under Cd toxicity. The cells were preincubated with myricetin for 24 h and then treated with Cd (10 μM) for 24 h. The results are reported as the mean ± SD (n = 3). ^###P < 0.001 compared with the control group. ^*P < 0.05, ^***P < 0.001 compared with the Cd group.

The effect of myricetin on the TAC

To evaluate the effect of myricetin on PC12 cells, FRAP test was performed. Our results showed that Cd exposure resulted in a significant decrease in TAC level. Myricetin pretreatment inhibited the decrease in the TAC level (P < 0.001) compared with Cd-treated PC12 cells (Figure 3b).

The effect of myricetin on the caspase 3 activation

To determine the activity of caspase-3, the cleaved form of caspase-3 evaluated by western blotting. As indicated in Figure 4, Cd increased cleaved caspase-3 level compared with control. However, pretreatment of PC12 cells with myricetin could prevent increase in cleaved caspase-3 level under Cd toxicity condition (P < 0.001).

Figure 4.

Effect of myricetin on activation of caspase-3 in PC12 cells under Cd toxicity. The cells were preincubated with myricetin for 24 h and then treated with Cd (10 μM) for 24 h (a). The density of cleaved caspase-3 was determined (b). The results are presented as the mean ± SD (n = 3). ^###P < 0.001 compared with the control group. ^*P < 0.05, ^***P < 0.001 compared with the Cd group.

Discussion

In the current study, we investigated the effects of myricetin on Cd-induced neurotoxicity in the PC12 cells. We observed that myricetin has antioxidant properties on PC12 cells. Cd is one of the most dangerous pollutants and is considered to be a serious public health threat worldwide. It is demonstrated that Cd is toxic to humans and animals even at low doses since the metal accumulates in the different tissues and has a long biological half-life in humans (10–30 years) [3].

Oxidative stress refers to an imbalance between the generation of ROS and the antioxidant capacity of the tissues and cells. Superoxide dismutase, catalase, and glutathione peroxidase are three important enzymatic antioxidant defense systems which provide protection against harmful effects of free radicals in biological systems [29]. Cd causes oxidative injury to proteins, membrane lipids, and DNA, thereby promoting pathways of apoptosis and cell damage [22].

The results of MTT assay indicated that treatment with 10 μM of Cd for 24 h reduced cell viability by about 50% in PC12 cells. There are many reports indicating that treatment with Cd (10 μM) for 24 h decreases cell viability (~50%) in several cell types, such as PC12 cells, SH-SY5Y neuroblastoma cells, and neuronal cultures [30, 31]. In addition, studies have previously shown that concentration of 10 μM of Cd for 24 h can be considered the best experimental situations that could better represent an in vivo condition, thereby mimicking chronic Cd toxicity-induced injury to tissues or body compartments [32, 33].

Our results revealed that myricetin significantly increased cell viability in a concentration-dependent manner in the presence of Cd toxicity. This result is in agreement with study showing that myricetin increased viability and also provided neuroprotection against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity in MES23.5 cells [34].

The CNS is particularly prone to oxidative stress and damage, because of its high oxygen turnover and high amount of polyunsaturated fatty acids [35].

Cd exposure has numerous effects on the CNS both in human and animals which include behavioral changes, learning disabilities, decreased equilibrium, olfactory dysfunction, and slowing of vasomotor functioning [11–13].

MDA is one of the most frequently products of LPO in the cells. Our finding demonstrated that Cd enhanced MDA levels in PC12 cells while treating with myricetin effectively reduced MDA levels. In agreement with our present findings, previous observations have also shown that myricetin reduced MDA level against brain injury and neurological deficits in a rat model of cerebral ischemia [36]. The present study showed that PC12 cells treated with Cd demonstrated a marked reduction in TAC levels. Myricetin prevented Cd-induced decrease in TAC levels. In agreement with this finding, previous studies have also shown that myricetin improved the antioxidant defense enzymes and ameliorated ochratoxins-induced oxidative stress in rat renal cortex [37].

Thiol molecules are compounds which contain a sulphydryl group. Thiols are the important members of the antioxidants that play a critical role in defense systems against free radicals [38]. Results demonstrated that PC12 cells treated with Cd showed a marked reduction in total thiol molecules. In addition, treatment with myricetin significantly prevented this reduction in Cd toxicity condition. This finding corroborates with previous studies which have shown that myricetin enhanced total thiol molecules and protected against high glucose-elicited endothelial dysfunction in human umbilical vein endothelial cells (HUVECs) [39].

Oxidative stress causes programmed cell death in tissues and cells. Apoptosis is a form of programmed cell death regulated by the Bcl-2 family of proteins. This family contains proapoptotic and prosurvival proteins such as Bax and Bcl-2, respectively. Caspases are a family of conserved protease enzymes that execute terminal stage of the apoptotic pathway and modulate upstream induction of cell destruction. Caspase-3, a key executioner in apoptosis, plays a crucial role in mitochondrial dysfunction after the release of cytochrome C [40, 41]. Moreover, we observed that Cd induced apoptosis by increasing activation of caspase-3, whereas myricetin pretreatment protects PC12 cells against Cd-induced apoptosis. This finding corroborates with previous studies which have shown that myricetin treatment inhibited the increase in the cleaved caspase-3 level and protected against high glucose-induced β-cell apoptosis [42].

Conclusion

The present study revealed that myricetin significantly ameliorated oxidative stress and apoptosis in PC12 cells. These protective effects of myricetin are mediated, at least in part, via preventing LPO, increasing TAC and GSH levels, and decreasing cleaved caspase 3 levels.

Conflicts of interest

None declared.