Neuroprotective Effects of SG-168 Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Abstract

Postmortem examinations of tissues of humans and rodents with a host of neurodegenerative conditions, including Alzheimer's and Parkinson's diseases, have identified oxidative damage in proteins, lipids, and DNA. The aim of this study was to better understand the cellular mechanisms of neuronal cell degeneration induced via oxidative stress and the protective roles of bioactive substance. In order to achieve this aim, we established a screening program to discover therapeutic agents that exhibit preferential neuroprotective activity in H₂O₂-treated PC12 cells. During the course of our screening program, we isolated an active compound, SG-168, from Dendrobium nobile Lindley and identified it as a neuroprotective agent. SG-168 was identified as a compound with an acetal skeleton, a prototypical compound, by electrospray ionization-mass spectrometry analysis and various nuclear magnetic resonance spectroscopic methods. The protective effect of SG-168 in PC12 cells with H₂O₂-induced oxidative damage was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. As expected, incubation with H₂O₂ for 2 hours resulted in cell viability of 31.8% compared to the control, while pretreatment of SG-168 increased cell viability by 15–50% compared to the H₂O₂-stressed control cells. These results showed that SG-168 inhibits H₂O₂-induced apoptotic cell death. Interestingly, flow cytometric analysis showed that H₂O₂-treated PC12 cells incubated with SG-168 exhibited greatly suppressed apoptosis. In summation, the results of this study suggest that SG-168 has potential as a new antioxidant agent against neuronal diseases.

Introduction

T he human body cannot survive without oxygen, requiring its properties for vital physiological processes.^1,2 For example, the brain requires energy in the form of ATP, which is generated by reaction between oxygen and glucose.^3,4 However, paradoxically, it is also potentially dangerous as a cause of oxidative stress, this is, when a cell is subjected to an imbalance between cellular production of free radical species and its ability to eliminate them by using endogenous antioxidant defense mechanisms.^5
–7 Oxidative stress is mediated by excess reactive oxygen species, which include hydroxyl radicals. Specifically, superoxide anion and hydrogen peroxide (H₂O₂) cause oxidative damage to various biological macromolecules, including lipids, protein, and DNA, and ultimately cell death via apoptosis or necrosis, especially in organs such as the brain.^8
–10 The brain is particularly vulnerable to free radical damage for a number of reasons. Membrane lipids in the brain contain high levels of polyunsaturated fatty acid side chains, which are prone to free radical attack.^11,12 Moreover, the brain also has been shown to contain low to moderate levels of enzymes such as catalase, superoxide dismutase (SOD), and glutathione peroxidase, which play an important role in metabolism of reactive oxygen species.^13

–16 As a result, oxidative stress has long been implicated in the etiology and progression of many neurodegenerative diseases. Currently, there is no effective cure for neurodegenerative diseases. Many synthetic chemicals such as phenolic compounds have been proven to be strong radical scavengers, but they usually induce severe adverse effects.^17,18 As a result, investigations in the neurodegenerative field have been focused on modulating or emulating the protective effects of key enzymatic components that regulate oxidative stress, with the aim of developing rational drug or genetic therapies.^19,20

With the aim of adding to this knowledge base, we established a screening system to discover therapeutic agents that can scavenge free radicals and protect cells from H₂O₂. Using this system, we isolated an active compound, SG-168, from Dendrobium nobile Lindley, which can protect PC12 cells against H₂O₂-induced apoptosis. By examining its chemical structure and physicochemical properties through liquid chromatography–mass spectrometry analyses and various nuclear magnetic resonance spectroscopic methods, we were able to identify SG-168 as a 3-[[(6-methoxy-10-methyl-1H,3H-benzo[h]furo[4,3,2-de]-2-benzopyran-1-yl)oxy]methyl]-5-methylnaphtho[2,3-b]furan-4,9-dione skeleton as an acetal, a prototypical compound. However, the neuroprotective activity of SG-168 has not been previously reported.

D. nobile Lindley (known as Seokgok in Korea and Shi Hu in mainland China) is widely used in both traditional Chinese and Korean folk medicine as an analgesic, an antipyretic, a tonic to nourish the stomach, and an anti-skin-aging agent and for treating diabetes and cardiovascular disease.²¹ Pharmacological studies have revealed that the phenanthrenes isolated from D. nobile Lindley exhibit antifibrotic activity.²² Besides containing bibenzyl derivatives and fluorenones, D. nobile Lindley extracts have been shown to possess antioxidant activity.²³ This suggests that crude extracts of D. nobile Lindely are nontoxic or only slightly toxic to humans and animals. Considering its low toxicity and potent neuroprotective effect, the crude extract of D. nobile Lindely, which contains SG-168, might prove to be a valuable therapeutic agent for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

In this study, we present results on the antioxidant and neuroprotective effect of SG-168 against H₂O₂-induced cell death in PC12 cells. Furthermore, we suggest that SG-168 may possess potential as an antioxidative therapeutic agent for neuronal diseases.

Materials and Methods

Materials

Dried D. nobile Lindley was obtained from Kumkang Pharm. Co. Ltd. (Masan, Republic of Korea). SG-168 isolated from D. nobile Lindley was prepared as a stock solution of 10 mg/mL in dimethyl sulfoxide (DMSO) and stored at −20°C. SG-168 was added to cell culture medium such that the DMSO made up less than 1% of the volume of the culture medium. H₂O₂ solution was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in sterilized phosphate-buffered saline (PBS) at a stock concentration of 50 m M . Other chemicals were all commercially available reagent-grade products.

Purification of SG-168 from D. nobile Lindley

The neuroprotective compound was isolated and purified from D. nobile Lindley as follows. The dried D. Lindley (250 g) was percolated overnight with methanol (5 L). The extracts were concentrated in vacuo to eliminate the methanol. The resulting aqueous solution was extracted with diethyl ether. The organic layer was concentrated, applied to a silica gel column, and eluted with chloroform–methanol (50:1 to 1:1 [vol/vol]). Finally, the pure compound was obtained by reverse-phase column chromatography and eluted with 60% methanol to give a dark green powder (4 mg).

The active compound, SG-168, prevented H₂O₂-induced cytotoxicity in PC12 cells. Using liquid chromatography–mass spectrometry spectroscopy, the molecular formula of SG-168 was found to be C₃₀H₂₂O₇. The structure of SG-168 was determined from its physicochemical properties and with various nuclear magnetic resonance spectroscopic methods. Using background literature research, SG-168 was determined to have a 3-[[(6-methoxy-10-methyl-1H,3H-benzo[h]furo[4,3,2-de]-2-benzopyran-1-yl)oxy]methyl]-5-methylnaphtho[2,3-b]furan-4,9-dione skeleton as an acetal, which had previously been reported as a prototypical compound (Fig. 1).²⁴

FIG. 1.

Chemical structure of SG-168.

Measurement of 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability

Eighty microliters of 0.2 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) ethanol solution was added to 20 μL of sample solution of different concentrations and allowed to react at room temperature. A control, containing 20 μL of DMSO and 80 μL of 0.2 mM DPPH, was prepared. The mixture was held at room temperature for 10 minutes, and then the absorbance was measured at 492 nm. The ability to scavenge the DPPH was calculated as percentage radical scavenging activity (RSA) using the following equation: RSA (%) = (1 −A/B) × 100, where A is the absorbance of the sample and B is the absorbance of the control. Vitamin C was used as the positive control.

Measurement of SOD-like activity

The reaction mixture contained 50 μL of Tris-HCl buffer and 7.2 mM pyrogallol, with or without sample. After incubation at 25°C for 45 minutes, the absorbance at 405 nm was determined against a blank. Vitamin C was used as the positive control in this assay. The SOD-like activity was calculated using the following equation: SOD-like activity (%) = (1 − A/B) × 100, where A is the absorbance of sample and B is the absorbance of the control.

Cell culture

Rat pheochromocytoma PC12 cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and were routinely grown in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 5% horse serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and 3.7 g/mL NaHCO₃ and were maintained in a 37°C humidified 5% CO₂ atmosphere. In all experiments, cells were treated with SG-168 before treatment with H₂O₂ for the indicated times. All in vitro experiments were performed using exponential growth-phase cells and were repeated at least twice.

Morphological analysis

PC12 cells in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% horse serum were seeded into six-well plates (2 × 10⁵ cells/mL) and incubated overnight in a humidified atmosphere of 5% CO₂ at 37°C. The cells were pretreated with SG-168. After incubation for 30 minutes, the cells were treated with 0.5 mM H₂O₂ for 2 hours. The cellular morphology was observed by using a phase-contrast microscope (Nikon, Tokyo, Japan) at × 100 magnification.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays

Cell viability was measured with blue formazan that was metabolized from colorless tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] (Sigma-Aldrich) by mitochondrial dehydrogenase, which is active only in live cells. PC12 cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/mL and allowed to attach overnight at 37°C. H₂O₂ was used to induce apoptosis at the doses indicated. Pretreatment with SG-168 was done for 30 minutes, and then the cells were treated with 0.5 m M H₂O₂ for 2 hours. Following the insult, MTT was added to wells at a final concentration of 5 mg/mL, and the plates were incubated at 37°C for 1 hour. Supernatants were then aspirated off, and formazan crystals were dissolved with 100 μL of anhydrous DMSO. The absorbency of each well was then measured at 540 nm using the ELISA reader (model 680, Bio-Rad, Hercules, CA, USA), and the percentage viability was calculated.

Lactate dehydrogenase release assay

The cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH). An LDH release assay kit was purchased from Wako Pure Chemical Industries (Osaka, Japan). PC12 cells were seeded in 96-well plates (1 × 10⁵ cells/mL), and the cells were pretreated with SG-168. After incubation for 30 minutes, the cells were treated with 0.5 m M H₂O₂ for 2 hours. The supernatant was used to assay the LDH activity. The reaction was initiated by mixing 50 μL of the cell-free supernatant with a potassium phosphate buffer containing NADH and sodium pyruvate to a final volume of 100 μL in a 96-well plate. The absorbance of the sample was read at 540 nm. Data were normalized to the activity of LDH released from vehicle-treated cells (100%) and are expressed as a percentage of the control value.

Cell staining

Apoptosis was investigated by staining the cells with Hoechst 33342 (Sigma-Aldrich). PC12 cells were washed twice with PBS and then fixed in PBS containing 10% formaldehyde for 4 hours at room temperature. Fixed cells were washed with PBS and stained with Hoechst 33342 for 30 minutes at room temperature. PC12 cells were examined under a fluorescence microscope (Nikon) for nuclei showing typical apoptotic features such as chromatin condensation and fragmentation. Photographs were taken at a magnification of × 400.

Flow cytometric analysis

PC12 cells were harvested and fixed with ice-cold 70% ethanol. The fixed cells were stained with 50 μg/mL propidium iodide at room temperature in the dark for 30 minutes. PC12 cells were analyzed by cytometry (Beckman Coulter, Fullerton, CA, USA) with a laser excitation wavelength of 488 nm and an emission wavelength of 620 nm (FL3) to quantify the red propidium iodide fluorescence. The percentages of cells in various phases of the cell cycle—namely, sub-G₁, G₁, S, and G₂/M—were assessed using WINCYCLE 32 software (Beckman Coulter). Every measurement typically counted at least 10,000 events. Apoptotic rates were determined by the percentage of nuclei accumulated at the peak of the sub-G₁ phase.

Statistical analysis

All data are expressed as the means of three determinations, and the differences were analyzed for significance using the SPSS Package for Windows (Version 14.0; SPSS Inc., Chicago, IL, USA). The data were evaluated by one-way analysis of variance followed by Scheffé's test. The differences were considered significant at P < .05.

Results

H₂O₂-induced cytotoxicity in PC12 cells

Neuronal cell death has been shown to be elicited by increases in various kinds of reactive oxygen species.²⁵ In order to determine the cytotoxicity underlying neuronal loss we established a model of oxidative stress by exposing PC12 cells to H₂O₂. PC12 cells were incubated with various concentrations of H₂O₂ for 2 hours. Morphological alteration was assessed by phase-contrast microscopy. As expected, the control group exhibited round cell bodies with clear edges and a fine dendritic network. However, incubation with 0.1, 0.5, and 1 m M H₂O₂ for 2 hours decreased the number of viable cells and induced shrinkage of cell bodies and disruption of the dendritic networks (Fig. 2A). Apoptotic death is characterized by a series of biochemical changes that include nuclear and chromatin condensation.²⁶ Chromatin condensations were assessed in H₂O₂-treated PC12 cells by Hoechst 33342 staining. As shown in Figure 2B, control cells exhibited uniformly dispersed chromatin, normal organelles, and intact cell membranes. However, H₂O₂-treated cells showed typical apoptotic characteristics, including condensation of chromatin, shrinkage of nuclear material, and appearance of a few apoptotic bodies. Moreover, we investigated the effect of H₂O₂ on cell viability. PC12 cells were exposed to 0.5 m M H₂O₂ for 1, 2, 3, and 6 hours, after which cell viability was measured by the MTT reduction assay. Figure 2C shows the relationship between H₂O₂ treatment and percentage of cell survival relative to the control. Exposure of cells to H₂O₂ resulted in a time-dependent decrease in cell viability.

FIG. 2.

Hydrogen peroxide (H₂O₂)-induced cell death in PC12 cells. (A) Morphological analysis in H₂O₂-treated PC12 cells for 2 hours: (a) control, (b) 0.1 m M H₂O₂, (c) 0.5 m M H₂O₂, and (d) 1 m M H₂O₂. Photographs were taken with a phase-contrast microscope at ×100 magnification. (B) Apoptosis analysis in H₂O₂-treated PC12 cells for 2 hours: (a) control, (b) 0.1 m M H₂O₂, (c) 0.5 m M H₂O₂, and (d) 1 m M H₂O₂. Photographs were taken with a fluorescence microscope at ×400 magnification. Fixed cells were stained with Hoechst 33342 (10 μM). Arrows indicate apoptotic cells. (C) Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay with 0.5 m M H₂O₂ for 1, 2, 3, and 6 hours. ^*** P < .001 compared with the control.

These results indicated that H₂O₂ induced cytotoxicity and apoptosis in the PC12 cells.

Antioxidant activity of SG-168

The DPPH RSA and SOD-like activity of SG-168 were determined, and the results are shown in Table 1. SG-168 exhibited good scavenging activity by its capacity to reduce the stable radical DPPH to yellow-colored diphenylpicrylhydrazine in a dose-dependent manner, and it reached up to 30.8% at the concentration of 2,000 μg/mL. The SOD-like activity of SG-168 showed trends similar to the DPPH RSA. The effective concentration at which radicals were scavenged by 50% for DPPH RSA (3.16 mg/mL) and SOD-like activity (2.39 mg/mL) of SG-168 was relatively lower than those of vitamin C (0.66 and 0.61 mg/mL, respectively), a known antioxidant used as the positive control (data not shown).

Table 1.

Antioxidant Activity of SG-168

	Concentration (μg/mL)
	50	100	500	1,000	2,000
RSA (%)^*	10.53 ± 0.19^d	14.01 ± 0.98^c	23.89 ± 1.96^b	27.69 ± 3.40^a	30.84 ± 1.23^a
SOD-like activity (%)^**	9.05 ± 0.19^e	12.58 ± 0.99^d	20.09 ± 2.49^c	33.44 ± 2.50^b	39.07 ± 0.33^a

Data are mean ± SE values of triplicate experiments.

Radical scavenging activity (RSA) (%) = (1 – absorbance of sample/absorbance of the control) × 100.

Superoxide dismutase (SOD)-like activity (%) = (1 – absorbance of sample/absorbance of the control) × 100.

abcd

Values not sharing the same letter are significantly different from one another by Duncan multiple range test (P < .05).

Protective effect of SG-168 against H₂O₂-induced cytotoxicity and apoptosis of PC12 cells

To assess the neuroprotective effect of SG-168, we investigated for morphological alterations such as cell shrinkage and membrane blebbings that are normally associated with apoptotic cell death. PC12 cells treated with 0.5 m M H₂O₂ became round, detached from the bottom, and aggregated as assessed by phase-contrast microscopy (Fig. 3). In contrast, cultures exposed to the same amount of H₂O₂ in the presence of SG-168 appeared remarkably preserved, indicating that SG-168 protects against H₂O₂. The protective effect of SG-168 can be further investigated by active mitochondria of living cells, which can cleave MTT to produce formazan. The amount of formazan is directly related to the number of living cells.

FIG. 3.

Effect of SG-168 on H₂O₂-induced morphological alterations of PC12 cells for 2 hours: (a) control, (b) 5 μg/mL SG-168, (c) 10 μg/mL SG-168, (d) 0.5 m M H₂O₂, (e) 0.5 m M H₂O₂ + 5 μg/mL SG-168, and (f) 0.5 m M H₂O₂ + 10 μg/mL SG-168. Photographs were taken with a phase-contrast microscope at ×100 magnification.

As shown in Figure 4A, incubation with 0.5 m M H₂O₂ for 2 hours resulted in a cell viability rate of 31.8% compared to the control. However, by pretreating the cells with various concentrations of SG-168, cell viability was increased by 15–44% compared to the viability of the H₂O₂-stressed PC12 cells. Further analysis of cell toxicity was performed with the LDH release assay. LDH is a stable cytoplasmic enzyme present in all cells, and it is rapidly released into the cell culture supernatant upon damage of the plasma membrane. An increase in the number of dead or plasma membrane-damaged cells results in an increase in LDH activity in the culture supernatant. Here, the PC12 cells were exposed to various concentrations of SG-168 and H₂O₂ for 2 hours. As expected, SG-168 reduced cell damage in a dose-dependent manner, shown by a 14–35% decrease in LDH release from the H₂O₂-stressed PC12 cells (Fig. 4B). SG-168 offered almost complete protection against H₂O₂-induced cell death, and viability was restored almost to control levels. Furthermore, exposure of PC12 cells to 0.5 m M H₂O₂ caused apparent apoptotic nuclei changes at 2 hours. These changes in nuclear characteristics of apoptosis were decreased significantly in cells pretreated with 5 and 10 μg/mL SG-168 (Fig. 5A). In order to analyze the protective effect of SG-168 on H₂O₂-induced cell injury, we investigated nuclear changes by flow cytometry. Flow cytometry detects apoptotic dead cells as well as those with fragmented nuclei, which are also called sub-G₁ cells. As shown in Figure 5B, analysis of PC12 cells DNA contents following 0.5 m M H₂O₂ treatment revealed an increase in the proportion of cells with sub-G₁ DNA content to 29.7%. Treatment with 0.5 m M H₂O₂ in the presence with 5 and 10 μg/mL SG-168 reduced the apoptotic sub-G₁ population to 0.8% and 0.5%, respectively (Fig. 5B).

FIG. 4.

Effects of SG-168 on cell viability of H₂O₂-challenged PC12 cells: (A) MTT reduction assay and (B) lactate dehydrogenase (LDH) release assay. PC12 cells were pretreated for 30 minutes with various concentrations of SG-168. The cells were then treated with 0.5 m M H₂O₂ for 2 hours. The normal growth condition was untreated with 0.5 mM H₂O₂. After the MTT assay, the MTT reduction rates were calculated by comparing with control survivals. As for the results of LDH release assay, data were normalized to the activity of LDH released from vehicle-treated cells (100%) and expressed as percentages of the control. Data are mean ± SD values of triplicate determinations and are representative of at least three independent experiments. ^*** P < .001 compared with the H₂O₂-treated control.

FIG. 5.

SG-168 inhibition of H₂O₂-induced apoptosis in PC12 cells. (A) Hoechst 33342 staining: (a) control, (b) 5 μg/mL SG-168, (c) 10 μg/mL SG-168, (d) 0.5 m M H₂O₂, (e) 0.5 m M H₂O₂ + 5 μg/mL SG-168, and (f) 0.5 m M H₂O₂ + 10 μg/mL SG-168. Fixed cells were stained with Hoechst 33342 (10 μM) and examined by fluorescence microscopy at × 400 magnification. The arrows indicate apoptotic cells. (B) Flow cytometric analysis in H₂O₂-treated PC12 cells for 2 hours. The cell cycle distribution was determined by a flow cytometric analysis of the DNA content after staining with propidium iodide. Data are relative values ± SD of three independent experiments. ^*** P < .001 compared with the H₂O₂-treated control.

Taken together, these results lead to the conclusion that SG-168 with its acetal skeleton was effective for the protection from H₂O₂-induced cytotoxicity in PC12 cells.

Discussion

Oxidative stress-induced cell damage has long been implicated in the physiological process of aging and in a variety of neurodegenerative disorders such as Parkinson's and Alzheimer's diseases.²⁷ However, the exact role of oxidative species in these disorders remains unclear, and little is known regarding the complex series of events that lead to neuronal death.²⁸ In this study, we used the rat pheochromocytoma PC12 cell line to examine oxidative stress-induced cell death. Initially, we verified that treatment of PC12 cells with H₂O₂ induces oxidative stress through morphological assessment. We confirmed that treating cells with H₂O₂ resulted in a dose-dependent viability loss (Fig. 2). The detailed mechanisms by which H₂O₂ initiates apoptosis are not fully understood. In this regard, H₂O₂ effects may be either direct or indirect, or both. We also explored whether H₂O₂ induces neuronal cell apoptosis. H₂O₂-treated cells stained with the fluorescent DNA-binding dye Hoechst 33342 displayed typical morphological features of apoptosis with sickle-shaped nuclei. In order to explore the role of H₂O₂-induced cytotoxicity in PC12 cell apoptosis, we observed changes in the MTT reduction assay. Our data showed that H₂O₂ at 1, 2, 3, and 6 hours significantly induces a decrease in MTT reduction.

One of the markers of neurodegenerative disorders in neurons is impaired antioxidant potential due to damage to the defense mechanisms responsible for maintaining intracellular redox homeostasis.²⁹ More recently, the search for natural antioxidants with neuroprotective potential has gained significant momentum. SG-168, a major active ingredient occurring naturally in D. nobile Lindley, has been reported to be a prototypical compound.²⁴ The free radical scavenging activities of SG-168 were measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH.³⁰ The DPPH assay has been widely used to provide basic information on the antioxidant activity of plant extracts or of single compounds because this method is a simple, rapid assay that has been shown to be accurate and reliable.³¹ The SOD-like activity reveals the cumulative capacity of antioxidants to scavenge superoxide anion radicals by nonenzymatic and by SOD action.³² The latter catalyzes the dismutation of two superoxide radical anions into H₂O₂ and oxygen, and its essential role is connected with scavenging damaging reactive oxygen species from the cellular environment.³³ Hirayama et al.³⁴ investigated the singlet oxygen quenching abilities of 18 carotenoids and reported that the conjugated keto group enhanced quenching. Therefore, the radical scavenging effect of SG-168 could be attributable to the chemical structure of its conjugated keto group and its electron transfer/hydrogen donating ability. The results suggested that the SG-168 displays DPPH RSA and SOD-like activity that could help prevent or ameliorate oxidative damage. However, its antioxidant activity was much lower than that of plant-derived compounds with hydroxyl groups. The 50% inhibitory concentrations of single compounds with hydroxyl groups such as quercitrin, caffeic acid, and rosmarinic acid for DPPH RSA were 12.9 μg/mL, 5.3 μg/mL, and 10.0 μg/mL, respectively.^35,36 Wolfe and Liu³⁷ reported that single non-phenolic compounds with a keto group have lower antioxidant activity than single compounds with a hydroxyl group.

The protective effect of SG-168 on H₂O₂-reduced cell viability was determined in PC12 cultured cells by morphological observation. Our investigations confirmed that treating cells with H₂O₂ induced significant cell death. However, pretreatment with 5 and 10 μg/mL SG-168 greatly increased cell viability, which was further confirmed by the MTT reduction assay (Figs. 3 and 4A). In agreement with theses findings, we also found that its cytotoxic effects were attenuated in the presence of SG-168 in the PC12 cells. These results indicate that SG-168 protects PC12 cells from H₂O₂-induced cytotoxicity (Fig. 4B). Excessive reactive oxygen species ultimately lead to apoptotic or necrotic cell death. Therefore, we further explored whether SG-168 has a protective effect against neuronal cell apoptosis. H₂O₂-treated cells stained with Hoechst 33342 displayed morphological features of apoptosis such as chromatin condensation, whereas SG-168 pretreatment mitigated these morphological changes (Fig. 5A). Apoptosis was also quantitatively analyzed by flow cytometry, which detects apoptotic cells with fragmented nuclei. As shown in Figure 5B, SG-168 treatment significantly reduced the numbers of apoptotic cells induced by H₂O₂. Based on this finding, we propose that SG-168 protects cells from H₂O₂-induced apoptosis. SG-168 is a furonaphthalene derivative, structurally similar to cacalol, cacalone, and maturone. The potent antioxidative sesquiterpene cacalol was isolated from Cacalia delphiniifolia and has been reported to exhibit antioxidative activity in a rat brain homogenate model (50% inhibitory concentration = 40 nM) and also proved to be a potent neuroprotective substance by protecting neuronal hybridoma N18-RE-105 cells from l-glutamate toxicity.³⁸ Therefore, the similar neuroprotective activity of SG-168 against H₂O₂-induced apoptosis may be attributed to the presence of the phenol and furan rings.

In summary, the results of the current study suggest that SG-168 is able to protect cultured PC12 cells against damage in a challenge by H₂O₂. Taken together, these data suggest that SG-168 has potential for protecting against free radical and oxidative damage, thus helping to treat or delay the progression of neurodegenerative diseases.

Footnotes

Acknowledgment

This work was supported by a grant in 2010 from the Kyungnam University Foundation.

Author Disclosure Statement

J.-H.H. is an employee of Advanced Industrial Science & Technology. No competing financial interests exist for M.-Y.Y., J.-H.P., M.-R.L., H.-J.K., E.P., and H.-R.P.

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Neuroprotective Effects of SG-168 Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Abstract

Introduction

Materials and Methods

Materials

Purification of SG-168 from D. nobile Lindley

Measurement of 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability

Measurement of SOD-like activity

Cell culture

Morphological analysis

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays

Lactate dehydrogenase release assay

Cell staining

Flow cytometric analysis

Statistical analysis

Results

H2O2-induced cytotoxicity in PC12 cells

Antioxidant activity of SG-168

Protective effect of SG-168 against H2O2-induced cytotoxicity and apoptosis of PC12 cells

Discussion

Footnotes

Acknowledgment

Author Disclosure Statement

References

H₂O₂-induced cytotoxicity in PC12 cells

Protective effect of SG-168 against H₂O₂-induced cytotoxicity and apoptosis of PC12 cells