Neuroprotective Effects of Chrysin on Diclofenac‑Induced Apoptosis in SH‑SY5Y Cells Ekrem Darendelioglu1


Accumulating evidences demonstrated that Reactive Oxygen Species (ROS) may lead to serious damages to numerous cel- lular biomolecules, consequently resulting in the development of several neurological diseases. Diclofenac (Dic), the most widely preferred non-steroidal anti-inflammatory drug (NSAID) induces apoptosis by an alteration in function of mitochon- dria and creation of ROS. Chrysin (Chr) is a naturally active component that is found in numerous plants and bee products and retains strong neuroprotective and antioxidant properties. However its effect of Dic induced injury on SH-SY5Y neuron cells have not been investigated to date. The goal of present research was to study the molecular mechanisms of Chr protec- tion from oxidative injury caused by Dic in SH-SY5Y cells. Dic induced significant toxicity on the cells and this effect was reversed by pre-treatment with Chr. Dic triggered a noteworthy increase in the cellular ROS and Lipid peroxidation (LPO) levels and decrease in Total antioxidant status (TAS) level while pre-treatment with Chr reversed these effects. Dic induc- tion increased the Bax, cytochrome c, cas-3, cas-8 and p53 expression at gene transcription level. Elevated levels of these genes considerably decreased by Chr pre-treatment revealing the defensive effects of Chr. The results obviously presented that exposure of SH-SY5Y with Dic resulted in oxidative stress and apoptosis while pre-treatment of neuron cells with Chr protects the cells against apoptosis triggered by Dic induction.

Keywords Antioxidant · Oxidative stress · Apoptosis · Diclofenac · Chrysin · Caspase-3

Reactive oxygen species (ROS), generally generated in cel- lular metabolic pathway, act as second messengers partici- pated in various intracellular events. When the levels of ROS increase due to oxidant usage and defects in anti-oxidant systems, these radicals turn out to be harmful toxic agents for organism. Accumulating evidences demonstrated that ROS may lead to serious damages to numerous cellular bio- molecules such as proteins, lipids, and DNA, consequently resulting in the development of several disorders. Addition- ally, the increase in ROS levels results in lipid peroxidation (LPO) that can be defined as a process in which ROS attack double bond(s) of carbon atoms in lipids. As mitochondrial membrane is made up by lipids, ROS is associated with loss of its potential which is vital for opening the mitochondrial membrane permeability transition pore [1–4]. Several research have confirmed the effect of ROS in neu- rological disorders such as Alzheimer’s and Parkinson’s dis- eases. Lately, the antioxidant approaches have shown poten- tial in the treatment of neurodegenerative disorders [5–7]. Diclofenac (Dic), the most widely preferred non-steroidal anti-inflammatory drug (NSAID) induces apoptosis through generation of ROS [8, 9]. It has been reported to have cyto- toxicity and provokes apoptosis in a range of cell culture lines that include HepG2, SH-SY5Y and gastric mucosal cells [9, 10]. ROS and mitochondria have a crucial action in trigger- ing apoptosis under pathologic and physiologic conditions. Proof from current researches demonstrate that mitochondria is both source and target of ROS.

The release of cytochrome of the Bcl-2 [12]. Moreover, one of the central apoptotic pathways is the intrinsic pathway which is linked with an increase in levels of cytochrome c which provokes apoptotic pathway, resulting activation of caspase-3. Therefore, the active caspase-3 is increased and triggers apoptosis through mitochondrial pathway [1]. Flavonoids are phenolic compounds obtained from plant and bee products such as royal jelly, propolis and honey. Numerous research have demonstrated that flavonoids show molecular activities such as anti-allergenic, -viral, -inflam- matory and -oxidant actions. Among those functions, great- est attention has been given to the understanding of the antioxidant property of flavonoids, as they are capable of reducing free radical generation and scavenging free radi- cals. The capability of flavonoids to be used as antioxidants in vitro has been the focus of a number of research in the preceding years, and essential structure–activity relation- ships of the antioxidant capacity have been recognized [13–16]. Chrysin (Chr) is a naturally active component several bee products and plants with strong anti-inflammatory, -cancer -oxidation and neuroprotective activities [17–20]. Its poten- tial for medical applications towards biomolecular effects of aging have also been well documented [21].The goal of current study is to explore the molecular and anti-oxidant mechanisms of Chr protection from oxidative injury caused by Dic in SH-SY5Y cells. It was speculated that Chr would diminish Dic induced SH-SY5Y cell apop- tosis by regulating the expression levels of anti-apoptotic, apoptotic and pro-apoptotic genes.

Materials and Methods

Cell Culture

SH-SY5Y (ATCC® CRL-2266™) cells were generously received from Ataturk University and seeded in DMEM including 10% FBS and 1% antibiotics. The cells were grown in humidified incubator with 5% CO2. The culture was verified for mycoplasma contamination by use of myco- plasma test kit.

Experimental Groups

The neuron cells were separated in four different groups. In control, SH-SY5Y cells were grown in complete medium. In Dic group, neuron cells were grown in the presence of0.5 mM Dic for 24 h. In Chr + Dic pre-treatment group, SH- SY5Y cells were pre- incubated with 0.05 mM
Chr for 3 h and 0.5 mM Dic added. In Chr group, cells were treated with 0.05 mM Chr in complete medium for 24 h.

Cell Viability Assay

To determine the concentration and time of Dic induction to generate toxic effects on neuroblastoma cells, they were treated with 100, 200, 300 and 500 µM Dic for 12 and 24 h. The cytotoxicity of Dic, Chr + Dic and Chr was assessed by Water Soluble Tetrazolium-1 (WST-1) cell proliferation assay kit following the instructions supplied with the kit. The cells were maintained in 96-well plate with each well containing 5 × 103 cells. Cells were preserved as indicated in experimental groups. Following that, 3 μl of WST-1 was included into each well. After incubating 2 h with medium, the absorbance was measured at a wavelength of 450 nm (reference: 630 nm).

Intracellular ROS Assay

Cellular ROS generations were analysed by measuring 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) activity. Cells were maintained as described in experimen- tal groups. Then, the cells (1 × 106) were collected and pre- incubated in the presence of 2 μM DCFH-DA at 37 °C for 60 min. Fluorescence measurement was performed by spec- trofluorometer as described in Darendelioglu et al. (2016) [1].

Lipid Peroxidation (LPO) Assay

LPO production was examined by the thiobarbituric acid reactive substances (TBARS) assay, which detects malondi- aldehyde (MDA). MDA level was measured by the method described by Ohkawa et al. (1979) [22]. Neuron cells were collected and centrifuged for 2 min at 2.5 krpm. Following that, the neuron cells were incubated with 70% w/v trichlo- roacetic acid and 1 mL of 0.8% w/v thiobarbituric acid. After that, they were left at 95 °C for half an hour. Following that the suspension was left on ice for 5 min, and the SH-SY5Y were centrifuged at 15 krpm for 10 min. The absorbance of samples was measured at 532 nm.

Total Antioxidant Status (TAS)

The cells were treated as described in experimental groups. The experiments were carried out following the protocols supplied with Human TAS ELISA kit (SunRed).


The assay was performed following the procedure supplied with the ApopTag kit (Milipore). The SH-SY5Y cells were grown in a 6-well plate containing approximately 5 × 105 cells/well. The cells were maintained as described in experimental groups. Later, the cells were observed under an inverted light microscope. At least three images were obtained for each treated group and Apoptotic Index (AI) was calculated as described in Darendelioglu et al. [1].

QRT‑PCR Analysis

The expression levels of the genes studied within this research were investigated by quantitative real time gene expression assay kit (Jena Bioscience) within Rotor-Gene Q (Qiagen). mRNA isolation was performed by Jena Biosci- ence Isolation Kit. cDNA synthesis was carried out accord- ing to kit procedure (Jena Bioscience). The amount and ratio of the mRNA was measured by nano-drop. The expression levels of p53, Bax, Bcl-2, cytochrome c, cas-8 and cas-3 and GAPDH as a housekeeping gene were analysed as explained in Darendelioglu et al. [1]. Normalisation of mRNA levels of apoptosis related genes was carried out by use of GAPDH.

Caspase‑3 Staining

Pro-caspase-3 positive cells were immunohistochemically stained by UltraVision LP Large Volume Detection Sys- tem, HRP polymer kit, H2O2 block and DAB substrate kit (Thermo Scientific, USA) with pro-caspase-3 antibody (sc- 271759, Santa Cruz Biotechnology, USA) that identifies pro- caspase-3 which is a key marker of apoptosis. SH-SY5Y cells were grown as explained in cell culture. 1% paraform- aldehyde was used to fix the cells, washed with phosphate buffered saline (PBS) and permeabilized with ethanol:acetic acid (2:1 v/v). In this method, pro-caspase-3 is targeted by an antibody which is conjugated with a peroxidase enzyme, and in the presence of H2O2, DAB is converted to its oxidized form, developing a brown precipitate. Following that, the nuclei of the cells were counter-stained with haematoxylin and they were imaged with an inverted light microscope.


All experiments were repeated at least three times. Statistical analysis and comparable data groups were assessed using GraphPad Prism 5 by one-way ANOVA Newman-Keuls Post-Hoc Test; p < 0.05 was considered as significant. Results Cell Viability Assay To determine the concentration and time of Dic induction to generate toxic effects on neuroblastoma cells, they were treated with 100, 200, 300 and 500 µM Dic for 12 and 24 h. Cell toxicity experiments were performed by WST-1 assay. Dic exposure for 12 or 24 h reduced cell proliferation in a dose-dependent and timely manner. As given in Fig. 1a 500 μM Dic induced remarkable decreases in cell survival while Chr treatment (0.05 mM) augmented cell viability of Dic-induced SH-SY5Y cells in a concentration-dependent manner (Fig. 1b). Detection of Intracellular ROS, LPO and TAS Exogenously added Dic exposed intracellular ROS produc- tion and influenced the accompanying cellular metabolic response. The effects of Chr treatment on Dic-induced ROS production in SH-SY5Y cells were evaluated. The results showed that Dic treatment resulted in a considerable increase in ROS production; however, 0.05 mM Chr pre- treatment remarkably decreased ROS generation (Fig. 2a). To investigate whether Chr contributed to the defence of SH-SY5Y cells from Dic-induced injury, LPO assay was performed using 0.05 mM Chr. As shown in Fig. 2b, the level of MDA in Dic-triggered SH-SY5Y cells remarkably increased while Chr treatment reduced MDA levels com- pared with Dic-stimulated SH-SY5Y cells. TAS was notice- ably reduced in Dic-triggered SH-SY5Y cells however Chr pre-treatment reversed this effect (Fig. 2c). TUNEL Assay Effect of Chr on preventing apoptosis induced by Dic in SH-SY5Y cells was also examined. The results revealed that Dic induced apoptotic pathway by forming DNA strand breaks and increase in AI, as calculated by TUNEL assay. Pre-treatment of Chr considerably inhibited the Dic-induced apoptosis (Fig. 3). To confirm whether Dic-induced cell death was due to apop- tosis, activation of caspase-3, a key mediator of apoptosis, was studied. As shown in Fig. 4, caspase-3 expression was significantly activated by the incubation with Dic-induced SH-SY5Y cells. Chr remarkably attenuated caspase-3 acti- vation (Fig. 4). QRT‑PCR Analysis To test whether mRNA levels of apoptotic and anti-apop- totic genes were affected by Dic and Chr, mRNA levels of p53, Bax, Bcl-2, cytochrome c, cas-8 and cas-3 were evalu- ated by qRT-PCR. As shown in Fig. 5, the mRNA levels of Bax, cytochrome c, p53, cas-8 and cas-3 were dramati- cally increased while expression of anti-apoptotic Bcl-2 was downregulated by Dic treatment. However, pre-treatment with Chr reversed the expressions of the corresponding genes. Discussion Herein the effects of NSAID (Dic) and potential protective effect of Chr on SH-SY5Y neuroblastoma cell line were investigated. The toxicity of the Dic, already reported for a range of cell culture lines that include HepG2 and gas- tric mucosal cells [9, 10]. In this study, the proof that Dic induces cytotoxicity and apoptosis through an alteration of the redox homeostasis and expression of the genes found in intrinsic and extrinsic pathway of apoptosis were presented while Chr reversed these effects. Dic was found to induce cell death in neuron cells in a dose dependent and timely manner as shown in Fig. 1a high- lighting that it can be toxic for neuron cell. Previous research revealed that the safe therapeutic doses of Dic in patient’s blood is around 10–30 μM [23]. Nevertheless, blood con- centration of the drug may increase in some situations par- ticularly as a result of long-term treatment and overdose use of the drug [24, 25]. Chr, a flavonoid with several significant molecular activities, was also explored in literature for its potential neuroprotective properties [26]. Due to these rea- sons, dose and time dependent experiments on the effect of Dic were performed and the results demonstrated that Dic induced significant toxicity on the cells and this effect was reversed revealing the efficiency of Chr in protection of neuronal cells from Dic induced injury. Accumulating evidences demonstrate that apoptotic path- way is controlled by numerous signals and metabolic path- ways. Involvement of ROS in apoptosis via inducing the oxidative stress, have been shown in previous study [1]. A rise in ROS and LPO levels trigger mitochondrial membrane depolarization, cytochrome c release, activation of caspases and nuclear fragmentation [1, 27]. Dic-induced apoptosis arisen from a change in ROS levels was also shown in renal and gastric cells [28, 29]. Chr was shown to inhibit LPO level, glutathione depletion and decreased level of c-kit [30]. Comparable data were also obtained in this study for which Dic triggers a noteworthy increase in the cellular ROS and LPO levels. Pre-treatment with Chr attenuated level of ROS and LPO. In this study, AI was evaluated by TUNEL assay and the results demonstrated that Dic exposure significantly increased AI in neuron cells. The obtained data further reveal that oxidative damage is probably participated in Dic-triggered apoptosis. Notably, pre-treatment with Chr reversed increase in AI in Dic-induced SH-SY5Y neuron cells. Overall, these findings strongly demonstrate the capa- bility of Chr to decrease ROS level as it plays a protecting role in Dic-triggered apoptosis. Apoptosis is a well-studied molecular pathway which is characterised extrinsic and intrinsic pathways, both of which result in the caspase activation. In the intrinsic path- way, the pro-apoptotic Bax, translocates to mitochondria after death signalling and induces cytochrome c release. The disturbance of the mitochondrial outer membrane and the release of cytochrome c into the cytoplasm stimulates the assembly of apoptosomes, where caspase-9 activates caspase-3 [31]. In extrinsic pathway, caspase-8 has been reported to activate pro-caspase-3 [32]. Being a powerful inductor of apoptosis, expression level of p53 is low in normal cells. In response to diverse stress conditions, its expression level increases [12]. Chr causes reduction in expression level of p53, cytochrome c and cas-3, upregula- tion in level of Bcl-2, inactivation of MAPK; p38 and JNK, decline level of NF-κB, expression of PTEN and increase of VEGF/AKT pathway [33]. In the current study, it was shown that Dic induction increased the Bax, cytochrome c, cas-3, cas-8 and p53 and decreased anti-apoptotic Bcl- 2 expression at gene transcription level. mRNA levels of these genes considerably reversed by Chr pre-treatment. Caspase-3 was reported as a crucial protease in both mitochondrium and death receptor dependent pathways, besides its vital role in free radical- mediated apoptosis [34, 35]. To further confirm whether Dic-induced cell death was due to apoptosis, activation of caspase-3, a key mediator of apoptosis, was studied through immune-his- tochemical staining. As represented in Fig. 4, caspase-3 expression was significantly activated by the incuba- tion with Dic-induced SH-SY5Y cells. Chr remarkably decreased caspase-3 activation (Fig. 4). Different studies have confirmed role of increase in active and/or pro-cas- pase-3 expression in apoptosis [36, 37]. Our results also demonstrated that, Chr significantly decreased cell viability and increased MDA level compared to control. Depending on their dose, flavonoids may act as prooxidants or antioxidants and accordingly, stimulate the oxidation of other compounds [38]. Among flavonoids, Chr is a natural flavone found in several plant extracts, honey, and propolis that has multiple biological activities [39]. Previous studies have indicated the effectiveness of Chr in decreasing the viability of cancer cells [40]. The cytotoxicity owing to Chr has been credited to the presence of peroxi- dase-like activity in hepatocytes, causing oxidation of Chr to produce toxic compounds [41]. In light of this information, the decrease in cell viability and increase in MDA level after Chr treatment can be attributed to prooxidant activity of Chr. The mRNA expression data in the present study showed a reduced expression of Bcl-2 and an increased expression of Bax after Dic induction. Further treatment with Chr could significantly attenuate these abnormal genes expression to normal, thereby indicating the pharmacologically active compound Chr plays a central role in inhibiting apoptosis by regulations in the mRNA expression of Bcl-2 and Bax in Dic induced condition to promote neuronal survival. In differ- ent study carried out by Kandhare et al., 2014 demonstrated that Chr exerts its neuroprotective effect via its antiapop- totic potential. It shows its effect by interacting with tumor necrosis factor α (TNF-α) and IL-β, and is linked with cas- pase and bcl-2 expressions at gene level [42]. Additionally, in another study treating the human lung adenocarcinoma epithelial cells with Chr resulted in a rise in the Bax/Bcl-2 ratio where Bax protein expression was increased but Bcl-2 protein expression decreased after Chr treatment [43]. Under certain conditions flavonoids may behave as pro-oxidants and thus, promote the oxidation of other compounds. Pro- oxidant properties of flavonoids appear to be dose-depend- ent [38]. In this study, concentration dependent Chr effect, prooxidant activity or other effect possibly could explain the reason behind Chr treatment decreases bcl-2 expression. In nutshell, the results obviously presented that expo- sure of SH-SY5Y with Dic resulted in oxidative stress via a decrease in CAT and TAS activity, an increase in ROS and LPO level, increase in expression level of cas-3, cas-8, bax, cytochrome c and p53, decrease in mRNA level of anti- apoptotic Bcl-2. The pre-treatment of neuron cells with Chr reversed the effects against Dic induced apoptosis events essentially via mitigation of oxidative stress and apoptosis. Acknowledgements The author would like to thank the head of the Molecular Biology and Genetics department and the directorate of Central Research Laboratory of the Bingol University for their labora- tory facilities. 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