When a stressor (psychological or physical) is sensed in the body, a signal is sent to the hypothalamus, activating the hypothalamic–pituitary–adrenal (HPA) axis [1
]. This leads to the synthesis of glucocorticoids in the adrenal cortex [2
]. Glucocorticoids play an essential role in the regulation of a variety of processes, including metabolism and immune function [3
]. Glucocorticoid secretion and the level of glucocorticoids in the blood are regulated via the negative feedback suppression of the HPA axis [4
]. However, sustained exposure to high concentrations of glucocorticoids in the blood, due to extreme or chronic stress, can cause dysfunction of the HPA axis and negative feedback mechanisms, resulting in the excessive secretion of glucocorticoids, which causes damage to the nervous system [6
It is reported that continuous exposure to high concentrations of glucocorticoids causes DNA damage in hippocampal nerve cells that encode and recall memories, eventually leading to apoptosis in these cells [9
]. Glucocorticoid-induced apoptosis is a cause of neurological dysfunction, including memory loss, learning disabilities, and cognitive impairment, and can also trigger mental disorders such as anxiety and depression [12
]. Thus, if the neurons can be protected from glucocorticoid-induced damage, neurological dysfunction due to chronic stress may be reduced.
Although benzodiazepines are used to treat stress-related nervous system disorders, the long-term use or combination of drugs can cause severe side effects, including drug addiction, drowsiness, vertigo, mental confusion, muscle weakness, and movement disorders [17
]. Therefore, it is necessary to find natural substances that are safe for long-term use without causing severe side effects.
is a large genus in the family Crassulaceae, and its phytochemical composition has been widely reported [18
plants have been used in traditional medicine for their antitumor, anti-inflammation, vasodilation, and wound-healing properties in various countries for a long time [18
]. S. takesimense
is a medicinal plant species endemic to Ulleung Island. Cell studies have demonstrated anti-inflammatory effects for S. takesimense
], and Vu et al. (2013) identified antibacterial compounds from S. takesimense
, including eight antibacterial gallotannins such as gallic acid, methyl gallate, and 4,6-di-O
-galloylarbutin by electrospray ionization mass spectrometry and proton nuclear magnetic resonance spectroscopy [22
]. Antioxidant compounds have also been identified in S. takesimense
, including phenolic constituents such as 2,6-di-O
-beta-d-xylopyranoside, and 1-(4-hydroxyphenyl)-2-(3,5-dihydroxyphenyl)-2-hydroxyethanone, by spectroscopic analyses (IR, UV, NMR, and HR-MS) and chemical degradation [24
] (Table 1
). However, there have been no studies regarding its neuroprotective effect on glucocorticoid-induced neuronal damage and the potential underlying mechanisms.
In the present study, we investigated the neuroprotective effect of water extract of S. takesimense
(WEST) against stress inducers and their underlying mechanisms. We used the PC12 cell line, which is derived from a rat pheochromocytoma and expresses high levels of glucocorticoid receptors, and which is widely used in nerve injury studies, among others [25
]. Corticosterone, the major glucocorticoid in rodents [9
], was used to induce cellular stress in this study.
We demonstrated the neuroprotective effect of WEST against corticosterone-induced apoptosis in PC12 cells and discuss whether the cytoprotective effects of WEST occur via the inhibition of ER stress and improved mitochondrial dysfunction.
2. Materials and Methods
2.1. Preparation of WEST
S. takesimense was obtained from Ulleung County, North Gyeongsang Province, Korea, in January 2018. Leaves of S. takesimense were dried, and the dried leaves (10 g) were extracted with distilled water (1:10 (wt/vol)) twice (each time for 90 min) in a reflux extractor. The water extracts were combined and filtered through an 8 µm-pore-size filter paper (Whatman™ Grade 2, GE Healthcare, Sheffield, UK) and then evaporated under reduced pressure using a rotary evaporator (N-1000S, Tokyo rikakikai Co., Tokyo, Japan). The concentrated extracts were freeze-dried using a freeze dryer, FD8505 (Ilshin Lab Co., Ltd., Seoul, Korea).
2.2. Cell Culture and Treatment
PC12 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 µg/mL of streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Twenty-four hours after the cells were seeded, the medium was refreshed with RPMI 1640 containing 1% (v/v) FBS and the same antibiotics as described above.
To investigate the cytoprotective effect of WEST, the cells were divided into four groups: non-treated control (CON), 200 µM corticosterone treatment only (CORT), 50 µg/mL WEST pretreatment plus 200 µM corticosterone (WEST + CORT), and 50 µg/mL WEST treatment only (WEST). Twenty-four hours after seeding the cells, WEST was incubated for 2 h before the treatment with corticosterone, and then, the cells were co-incubated with WEST and corticosterone for 24 h.
2.3. Cell Viability Assay
Cell viability was evaluated using the EZ-CYTOX kit (Daeil Lab, Seoul, Korea) according to the manufacturer’s instructions. Briefly, cells (5 × 104 cells/well) were seeded in 96-well plates, and after 24 h of treatment, the cells were co-incubated with 20 µL of EZ-Cytox solution in 200 µL of total cell volume for 2 h at 37 °C in darkness. The absorbance was measured at a wavelength of 450 nm using a spectrophotometer (SpectraMax M2 microplate reader, Molecular Devices, Sunnyvale, CA, USA).
2.4. LDH Leakage Assay
To determine the intensity of cell injury, the activity of lactate dehydrogenase (LDH) released from cells was measured using an EZ-LDH kit (Daeil Lab, Seoul, Korea). Cells were seeded in 96-well plates. At the end of the treatment, the supernatant was collected and reacted with the LDH reaction mixture following the manufacturer’s protocol. After 1 h at room temperature in darkness, the absorbance of the samples was measured at 450 nm using a spectrophotometer (SpectraMax M2 microplate reader, Molecular Devices, Sunnyvale, CA, USA).
2.5. Hoechst 33342 and PI Double Staining
Chromatin condensation was analyzed by nucleus staining with Hoechst 33342 and PI double staining. Cells were plated on 18 mm coverslips. After the indicated treatment, the fixed cells were stained with 8.1 µM Hoechst 33342 solution (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at room temperature and then co-stained with 1.5 µM PI solution (Thermo Fisher Scientific, Waltham, MA, USA) for another 15 min. The cells were visualized by fluorescence microscopy (ZEISS, Jena, Germany), and the apoptotic nuclei were counted in five randomly selected fields in each group in three experiments. The data are expressed as a percentage of the total number of nuclei counted.
2.6. TUNEL Staining
Internucleosomal DNA fragmentation was detected using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) kit (Promega, Madison, WI, USA). In short, cells were cultured on 18 mm coverslips. After the indicated treatment, the fixed cells were incubated with TUNEL reaction mixture for 1 h at 37 °C according to the manufacturer’s protocol and then viewed under a fluorescence microscope (ZEISS, Jena, Germany). TUNEL-positive nuclei were counted in five randomly chosen fields per coverslip three times; then, the apoptotic percentage was calculated by comparing the TUNEL-positive counts with the total cell nuclei, determined by Hoechst 33342 counterstaining.
2.7. Intracellular ROS Level Assay
Intracellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescein diacetate (DCF-DA) (Sigma-Aldrich, Inc., St. Louis, MO, USA). Briefly, cells were seeded into a 96-well black plate. At the end of treatment, the culture medium was removed, and the cells were incubated with 10 µM DCF-DA for 1 h at 37 °C. The fluorescence of the DCF was measured with a fluorescence spectrophotometer (SpectraMax M2 microplate reader, Molecular Devices, Sunnyvale, CA, USA) at excitation and emission wavelengths of 485 and 535 nm, respectively.
2.8. Intracellular Ca2+ Level Assay
Intracellular Ca2+ levels were measured using the Fura-2/AM (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells were seeded in a 96-well black plate. After the indicated treatment, the cells were collected and co-incubated with a 5 µM Fura-2/AM working solution for 1 h at 37 °C. The fluorescence intensity was measured using a fluorescence spectrophotometer at excitation wavelengths of 340 nm (Ca2+-bound form) and 380 nm (Ca2+-unbound form), with an emission wavelength of 510 nm. The intracellular Ca2+ level was reflected by the ratio of fluorescence (F340/F380).
2.9. Detection of mPTP Opening
The mitochondrial permeability transition pore (mPTP) opening of mitochondria was detected using a mitochondrial transition pore assay kit using the calcein–cobalt quenching method (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells were plated on 18 mm coverslips. After treatment, the fixed cells were labeled with the labeling solution (including 1 µM calcein AM and 1 mM cobalt chloride) for 15 min at 37 °C according to the kit manufacturer’s instructions, and visualized by fluorescence microscopy (ZEISS, Jena, Germany).
2.10. Measurement of MMP
Changes in the mitochondrial membrane potential (MMP) were assessed with a mitochondrial membrane potential kit using the JC-10 dye (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were seeded into a 96-well black plate. After the indicated treatment, the cells were co-incubated with the JC-10 solution for 1 h at 37 °C according to the kit manufacturer’s instructions. Monomeric JC-10 green fluorescence (λex = 490/λem = 525 nm) and aggregate JC-10 red fluorescence (λex = 540/λem = 590 nm) were measured using a fluorescence spectrophotometer (SpectraMax M2 microplate reader, Molecular Devices, Sunnyvale, CA, USA), and the ratio of red to green fluorescence was calculated.
For monitoring apoptosis, cells were plated on 18 mm coverslips. After the end of treatment, the fixed cells were reacted with JC-10 solution for 1 h at 37 °C according to the manufacturer’s instructions, and visualized by fluorescence microscopy (ZEISS, Jena, Germany).
2.11. ATP Detection Assay
Cellular ATP levels were measured using a luminescent ATP detection assay kit (Abcam, Cambridge, UK) based on firefly luciferase in accordance with the manufacturer’s instructions. Briefly, cells were seeded into a 96-well white plate. At the end of treatment, the cells were lysed with detergent and reacted with substrate solution on a shaker at room temperature in the dark. The plate was dark-adapted by covering it for 10 min. The luminescence was measured using a luminescence spectrophotometer (SpectraMax M2 microplate reader, Molecular Devices, Sunnyvale, CA, USA).
2.12. Western Blot Analysis
Twenty micrograms of total protein was separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) for immunoblot analysis using the following primary antibodies: GRP78 (1:1000), GADD153 (1:1000), Bax (1:2000), Bcl-2 (1:1000), cytochrome c (1:500), caspase-9 (1:1000), caspase-3 (1:1000), and GAPDH (1:1000). The primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), except for the Bcl-2 and GAPDH antibodies, which were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The PVDF membranes were incubated overnight at 4 °C with the primary antibodies, and subsequently incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit immunoglobulin G (IgG) or horse anti-mouse IgG (Cell Signaling Technologies, Danvers, MA, USA). The target protein bands were detected using a chemiluminescence image analyzer (CAS-400SM, Davinch-K, Seoul, Korea) and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The protein levels were normalized to the GAPDH protein levels.
2.13. Statistical Analysis
The results are presented as means ± standard deviations (SDs). All statistical analyses were performed with one-way ANOVA using the JMP 5 software (SAS Campus Drive, Cary, NC, USA). The values were considered significantly different at p < 0.01 or 0.05. All experiments were performed a minimum of three times.
The ER plays a central role in the biosynthesis of proteins and lipids, and in the storage of calcium in cells, while mitochondria are organelles central to the production of ATP as well as to the synthesis and processing of various metabolites, and regulation of cell death [28
]. These organelles play essential roles in sensing and responding to cell stress, and their interactions affect the functional organization of organelles and ultimately regulate cell survival [29
]. ER stress is a crucial trigger in the apoptotic process, which can lead to mitochondrial dysfunction via various mechanisms, inducing apoptosis [30
It has been reported that the expression of GRP78 and GADD153 is significantly enhanced during ER stress [32
]. GRP78 is an ER chaperone, important for ER function as a master regulator of the unfolded protein response (UPR) [34
]. GADD153 is a transcription factor encoded by the DNA damage inducible transcript 3 (DDIT3
) gene, which is highly upregulated during ER stress [35
Our study demonstrated that the ER stress markers GRP78 and GADD153 were significantly increased in corticosterone-stressed PC12 cells. However, the expression of these proteins was reduced in PC12 cells pretreated with WEST. This study also found that the concentration of intracellular Ca2+, another major indicator of ER stress, was upregulated in corticosterone-treated PC12 cells but downregulated in cells pretreated with WEST. These results indicate that the neuroprotective effect of WEST against corticosterone-induced apoptosis was mediated by the inhibition of ER stress.
signaling is crucial for physiological and functional interactions between the ER and mitochondria [36
]. The unique juxtaposition of these organelles plays a crucial role in the pathogenesis of metabolic diseases [37
]. During chronic ER stress, a resistive response occurs, and abnormal calcium signals are transmitted from the ER to the mitochondria, leading to cell death [38
The overloading of Ca2+
released from the ER due to intracellular stress induces mitochondrial dysfunction by inducing the depolarization of the inner mitochondrial membrane (IMM), mPTP opening, and ROS generation, ultimately promoting the activation of the caspase-regulated apoptosis pathway [39
]. The magnitude and outcome of Ca2+
responses during apoptosis can be regulated by various Bcl-2 family members [29
]. The Bcl-2 family members contain several homologs, including anti-apoptotic proteins and pro-apoptotic proteins [42
]. The anti-apoptotic proteins Bcl-2 and Bcl-XL inhibit Ca2+
delivery, while the pro-apoptotic proteins Bax and Bak stimulate Ca2+
]. The resulting balance between anti- and pro-apoptotic members has been proven to influence the mitochondrial apoptosis pathway. Nutt et al. reported that the overexpression of Bax and Bak promotes apoptosis by increasing Ca2+
]. In the current study, the intracellular Ca2+
concentration was increased, the protein expression of Bax was enhanced, and that of Bcl-2 was attenuated in PC12 cells treated with corticosterone. These results are consistent with those of other studies [8
]. By contrast, we confirmed that these effects were reversed in PC12 cells pretreated with WEST.
The overaccumulation of Ca2+
in mitochondria causes the transient depolarization of the IMM [47
], leading to the opening of mPTPs, which are voltage-dependent, high-conductance channels [48
]. The uncontrolled opening of mPTPs leads to MMP collapse, and several pro-apoptotic proteins, such as cytochrome c and apoptosis inducing factor (AIF), are released into the cytosol from the intermembrane space through mPTPs, where they modulate the final steps of the apoptotic cascade [49
]. Thus, the IMM potential is also essential for signaling, especially in cell survival decisions and mitophagy [29
]. In the present study, corticosterone treatment led to abnormal mPTP opening and MMP collapse in PC12 cells. By contrast, pretreatment with WEST inhibited mPTP opening and stabilized the MMP.
The IMM includes the electron transport chain (ETC; complexes I–IV) of the oxidative phosphorylation system, and the electrons are passed from the electron donors to electron acceptors through the ETC [51
]. This electron transfer couples with the transfer of protons across the IMM, creating an electrochemical proton gradient that drives the synthesis of ATP by F1
-ATPase in the IMM [48
]. Most of the proton motive force is contained in the MMP; thus, decreasing the MMP reduces the amount of free energy available to produce ATP [48
]. In this study, we confirmed that the generation of ATP decreased with the loss of MMP upon corticosterone treatment and found that these effects were reversed in PC12 cells pretreated with WEST. Thus, the stability of the MMP is vital for maintaining the function of mitochondria, and these findings prove that WEST prevents the mitochondrial dysfunction induced by corticosterone.
We next assessed the expression of the mitochondrial apoptotic pathway-related proteins cytochrome c, caspase-9, and caspase-3. Corticosterone treatment upregulated cytochrome c, caspase-9, and caspase-3 protein expression, consistent with other studies [8
]. By contrast, pretreatment with WEST downregulated the expression of these proteins. Our data demonstrate that WEST markedly attenuated the overexpression of cytochrome c, caspase-9, and caspase-3 induced by corticosterone. In the final step of apoptosis, caspase-3 cleaves the inhibitor of caspase-activated DNase (ICAD), which leads to the activation of caspase-activated DNase (CAD), generating DNA fragments in the nucleus [53
]. We determined that DNA fragmentation was increased by corticosterone-induced apoptosis in PC12 cells pretreated with WEST. We confirmed that WEST can inhibit apoptosis by effectively reducing apoptotic nuclei (Figure 8
We also measured intracellular ROS levels in corticosterone-induced injury. Several studies have shown that glucocorticoid treatment alters the activity of antioxidant enzymes and thus increases the ROS level [54
]. Excessively elevated ROS levels alter the cellular redox balance and cause damage to major macromolecules [54
]. In particular, neuronal cells are susceptible to ROS-induced damage, partly due to their low levels of antioxidant enzymes, resulting in a deficient antioxidant defense system [54
]. In the present study, PC12 cells exposed to corticosterone significantly increased the intracellular ROS level, consistent with the results of other studies [52
]. However, pretreatment with WEST reduced ROS levels.
Several studies have reported that S. takesimense
has antioxidant constituents [24
]; our study also showed that the ROS increased by corticosterone were reduced by treatment with WEST. As many studies have shown that ER stress and the mitochondrial apoptosis pathway are attenuated by antioxidants [57
], the WEST-mediated cytoprotective effect might have been partly due to the antioxidant effects that inhibit apoptosis.
We have demonstrated that WEST exerts a neuroprotective effect by inhibiting the apoptotic pathway involved in corticosterone-induced ER stress and mitochondrial dysfunction. Although the cellular mechanisms of action of WEST have not yet been fully elucidated, our results provide novel evidence that WEST reduces neuronal damage due to the neurotoxicity caused by chronic stress. However, the use of plant extracts is limited by various factors. Firstly, it is difficult to define the composition and role of the active molecules. In addition, it is difficult to reproduce the action of the extract, due to the variability in its composition, which is caused by various factors (season, humidity, temperature, etc.). Considering these limitations, further studies on the neuroprotective effects of WEST using animal models and clinical samples are warranted. Further studies may be used to develop new therapeutics that protect against diseases caused by neurological dysfunction.