RETRACTED ARTICLE: Chinese medicine Di-Huang-Yi-Zhi protects PC12 cells from H2O2-induced apoptosis by regulating ROS-ASK1-JNK/p38 MAPK signaling

Background Oxidative stress mediates the nerve injury during the pathogenesis of Alzheimer’s disease (AD). Protecting against oxidative stress damage is an important strategy to prevent and treat AD. Di-Huang-Yi-Zhi (DHYZ) is a Chinese medicine used for the treatment of AD, but its mechanism remains unknown. This study is aimed to investigate the effect of DHYZ on H2O2 induced oxidative damage in PC12 cells. Methods PC12 cells were treated with H2O2 and DHYZ. Cell proliferation was detected by Cell counting kit-8 (CCK-8) assay. Cytotoxicity of H2O2 was measured by lactate dehydrogenase (LDH) release assay. Apoptosis were identified by Annexin V-FITC/PI staining. Caspase 3 activity was detected by commercial kit. Mitochondrial membrane potential (MMP) was detected by JC-1 staining. Reactive oxygen species (ROS) was 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Protein expression and phosphorylation was identified by western blot. Results The results showed that DHYZ antagonized H2O2-mediated cytotoxicity and proliferation inhibition. DHYZ reduced ROS production, stabilize mitochondrial membrane potential, inhibit Caspase-3 activity and apoptosis induced by H2O2. In addition, DHYZ inhibited the phosphorylation of ASK1, JNK1/2/3 and p38 MAPK which were up-regulated by H2O2. Conclusions The present study suggested that DHYZ protected PC12 cells from H2O2-induced oxidative stress damage and was related to inhibition of ROS production and ASK1-JNK/p38 MAPK signaling. The present study provides experimental evidence for the application of DHYZ for the management of oxidative stress damage and AD.


Background
Alzheimer's disease (AD) is an age-related degenerative disease of the central nervous system. AD presents as progressive cognitive impairment, and is closely related to β-amyloid (Aβ) and Tau pathology [1][2][3]. Both Aβ and Tau can cause oxidative stress (OS) [4][5][6]. OS can mediate nerve injury and participate in the pathogenesis of AD [7,8]. Reactive oxygen species (ROS) are the main effectors in the OS process, and can oxidize proteins, lipids and DNA, affect mitochondrial function, activate Caspase-3, promote neuronal apoptosis, and thus participate in the pathogenesis of AD [9,10]. Intervention of OS damage is an important strategy for the prevention and treatment of AD [11,12].
Traditional Chinese medicine (TCM) is known to play an important role in the prevention and treatment of AD. Based on the theory of TCM, clinical medication and related studies, we established a Chinese herbal formula Di-Huang-Yi-Zhi (DHYZ). DHYZ consists of Shu-Di (prepared root of Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey.), Yi-Zhi-Ren (fruits of Alpinia oxyphylla Miq.), Shi-Chang-Pu (root of Acorus tatarinowii Schott), Fu-Shen (Poria with hostwood) and Dan-Shen (root of Salvia miltiorrhiza Bunge) (Chinese patent ZL2008102047153.3). All these herbs are effective for the prevention and treatment of AD.
Previous studies have shown that DHYZ can antagonize Aβ-mediated neurotoxicity and inhibit Aβ-induced neurocyte apoptosis in vitro [13]. DHYZ can also reduce synaptic loss, antagonize Aβ-mediated nerve injury and inhibit phosphorylation of Tau protein, thereby improving the learning and memory abilities of AD mice and rats [14,15]. DHYZ can enhance the therapeutic effect of Donepezil on AD and Parkinson's disease dementia, improve clinical symptoms, cognitive ability and daily lives of patients [16,17]. The effect of DHYZ on OS damage remains unknown. The present study explored the protective effect of DHYZ on OS mediated by H 2 O 2 in PC12 cells.

DHYZ extraction
Cell culture PC12 cells were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. PC12 cells were cultured in RPMI-1640 containing 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, and maintained at 37°C in a humidified incubator with 5% CO 2 . PC12 cells in logarithmic growth phase were used for subsequent experiments.
Cell proliferation assay PC12 cells were seeded in a 96-well plate (1 × 10 4 /well). After 24 h of culture, PC12 cells were treated with different concentrations of DHYZ or the same volume of serum-free RPMI-1640 for 24 h, or 400 μM of H 2 O 2 for 4 h. CCK-8 reagents were used to detect cell proliferation according to the manufacturer's instructions. Cell survival rate was calculated by the following formula: Cell survival (%) = (experimental OD value/control OD value) × 100%.
LDH cytotoxicity assay PC12 cells were plated in a 96-well plate (1 × 10 4 /well). After 24 h of culture, DHYZ (50-200 μg/mL) or the same volume of serum-free RPMI-1640 were added. After 24 h of treatment, the cells were treated with H 2 O 2 (400 μM) for 4 h. The amount of LDH released in each group was measured according to the manufacturer's instructions. The results were expressed as fold of nontreated normal group.

Detection of apoptosis
PC12 cells treated with DHYZ and H 2 O 2 were collected and washed with PBS. The cells were suspended in 100 μl Binding Buffer. Annexin V-FITC and propidium iodide (PI) (5 μl each) were added, mixed and incubated for 15 min at room temperature, and then 400 μl Binding Buffer was added and mixed. Cell apoptosis was detected by flow cytometry (BD Biosciences, San Jose, CA).

MMP detection
PC12 cells were incubated in a 24-well plate (5 × 10 4 / well). After 24 h of culture, different concentrations of DHYZ or the same volume of serum-free RPMI-1640 were added. After 24 h, PC12 cells were treated with 400 μM H 2 O 2 for 4 h. JC-1 staining was performed according to the manufacturer's instructions. Fluorescence microscopy and flow cytometry were used to identify positive JC-1 staining.

Detection of Caspase-3 activity
PC12 cells treated with DHYZ and H 2 O 2 were collected, and the activity of Caspase-3 was detected according to the manufacturer's manual.

ROS detection
The ROS production was detected according to the manufacturer's instructions. Briefly, PC12 cells were cultured in a 6-well plate (2.5 × 10 5 cells/well). After 24 h of culture, DHYZ (50-200 μg/mL) or the same volume of serum-free RPMI-1640 were added. After 24 h, the cells were treated with 400 μM of H 2 O 2 for 4 h. For ROS detection, the cells were incubated with 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (10 μmol/L) for 30 min at 37°C, observed under a fluorescence microscope and quantified with a fluorescence microplate reader (Thermo Fischer Scientific, Waltham, MA). For ROS inhibition, DHYZ-treated PC12 cells were incubated with NAC (500 μM) for 2 h followed by H 2 O 2 treatment.

Western blot
PC12 cells treated with DHYZ and H 2 O 2 were collected, lysed in RIPA buffer and quantified using the BCA kit. Proteins were separated by 8-10% SDS-PAGE electrophoresis and transferred to PVDF membrane on a semidry transfer unit. The membranes were blocked with 5% non-fat milk for 2 h, incubated with antibodies against ASK1, p-ASK1, JNK1/2/3, p-JNK1/2/3, p38 MAPK and p-p38 MAPK (1:800) or GAPDH (1:2000) at 4°C overnight. The blots were washed with TBST and incubated with secondary antibody (1:5000) at 37°C for 2 h. The bands were visualized by the ECL method. Proteins expression were quantified by Image J software.

Statistical analysis
All data were analyzed by SPSS 21.0 software, and the results were expressed as mean ± standard deviation (SD). Intergroup differences were analyzed by one-way analysis of variance (ANOVA) and LSD-t or Dunnett's test. P < 0.05 was considered as significant difference.

DHYZ attenuates H 2 O 2 -mediated cytotoxicity
We first observed the effects of H 2 O 2 and DHYZ on PC12 cell proliferation. As shown in Fig. 1, 100-600 μM of H 2 O 2 significantly inhibited the proliferation of PC12 cells (P < 0.01), and the 50% inhibitory concentration (IC50) was about 400 μM. Low doses of DHYZ (25-400 μg/mL) showed no significant effect on the proliferation of PC12 cells. Based on these observations, 400 μM of H 2 O 2 and 50-200 μg/mL of DHYZ were selected for subsequent experiments. Further study revealed that DHYZ could antagonize the inhibitory effects of H 2 O 2 on proliferation of PC12 cells (P < 0.05).
The cytotoxicity of H 2 O 2 on PC12 cells was detected by the LDH release assay. LDH is released from cells when cell membrane is damaged, and thus can reflect cell damage and cytotoxicity. The results showed that the LDH release from PC12 cells increased after H 2 O 2 treatment (P < 0.01). After DHYZ treatment, the H 2 O 2induced LDH release was reduced (P < 0.05). These results suggested that DHYZ could antagonize H 2 O 2mediated cytotoxicity.

DHYZ antagonizes H 2 O 2 -induced apoptosis
Apoptosis contributes to oxidative stress-mediated neuronal damage [21]. In the present study, Annexin V-FITC and PI double staining were used to detect apoptosis by flow cytometry. The results showed that H 2 O 2 could promote apoptosis in PC12 cells (P < 0.01). Apoptosis of PC12 cells was significantly decreased after DHYZ treatment in a dose-dependent manner (P < 0.01) (Fig. 2).

DHYZ undermines H 2 O 2 -induced reduction of MMP
Mitochondria are important organelles that regulate apoptosis. In this study, MMP was detected by JC-1 staining. JC-1 accumulates in the mitochondrial matrix, forms J-aggregates and produces red fluorescence when the MMP is high, while JC-1 exists as a monomer and emits green fluorescence when the MMP is low. The PC12 cells showed green fluorescence after H 2 O 2 treatment. Upon DHYZ treatment, the red fluorescence intensity of PC12 cells increased and the green fluorescence decreased (Fig. 3) (P < 0.05). These results suggested that DHYZ could reverse H 2 O 2 -induced reduction of MMP.

DHYZ inhibits H 2 O 2 -activated Caspase-3
Caspase-3 is an executive protease in the apoptotic process, which is regulated by mitochondria and death receptor pathway [22]. In this study, a specific enzyme substrate was used to detect the activity of Caspase-3. The results showed that the activity of Caspase-3 increased significantly in PC12 cells after H 2 O 2 treatment (P < 0.01). DHYZ inhibited H 2 O 2 -activated Caspase-3 in a dose-dependent manner (Fig. 4) (P < 0.05).

Effect of DHYZ on H 2 O 2 -induced ROS production
H 2 O 2 can produce HO• to damage cells [23]. We observed ROS production by DCFH-DA staining. DCFH-DA enters cells and generates DCFH under the action of esterases. DCFH generates fluorescent DCF under the action of ROS. The fluorescence intensity of DCF can reflect ROS level. The results showed that the green fluorescence of PC12 cells was enhanced after H 2 O 2 treatment (P < 0.01), but DHYZ treatment decreased the green fluorescence in a dose-dependent manner (P < 0.05) (Fig. 5A and B) suggested that DHYZ could reduce H 2 O 2 -induced ROS production.
NAC was further used to block the ROS production. The results showed that 500 μM of NAC significantly inhibited ROS production, and also antagonized the effects of H 2 O 2 on Caspase-3 activity and apoptosis (P < 0.01). NAC also attenuated the effects of DHYZ on Caspase-3 activity and apoptosis (P < 0.05) (Fig. 5C-F). These observations suggested that ROS participated in the effects of H 2 O 2 and DHYZ.

Fig. 1 Effects of DHYZ on H 2 O 2 -mediated cytotoxicity. PC12 cells were treated with H 2 O 2 for 4 h (a) or DHYZ for 24 h (b) and cell proliferation
was detected by CCK-8 assay. PC12 cells were pre-treated with DHYZ for 24 h, followed by H 2 O 2 treatment for 4 h and subjected to CCK-8 assay (c) and LDH release assay (d). △ P < 0.01, versus normal group; * P < 0.05, ** P < 0.01, *** P < 0.001, versus control group. The data are mean ± SD of three independent experiment each in triplicate

Discussion
Brain, as an organ with high oxygen consumption, is prone to accumulate ROS during the metabolic process.
In addition, fewer antioxidant enzymes can penetrate the blood-brain barrier, and the brain contains more unsaturated fatty acids and transition metals, which also lead to ROS accumulation, promote synapse and neuronal loss, and accelerate the progress of AD [11,26] Antioxidative damage and maintenance of normal neuronal function are important strategies to prevent and treat AD [12,26]. The present study showed that H 2 O 2 had cytotoxic effect on PC12 cells and inhibited cell proliferation. DHYZ could antagonize the cytotoxicity and proliferation inhibition of H 2 O 2 , suggesting that DHYZ had neuroprotective effect. Under physiological condition, the body is in an equilibrium state of redox. During oxidative stress, excessive ROS accumulation in cells lead to irreversible mitochondrial permeability, transition pore opening, decreased MMP, release of cytochrome C from mitochondria, successive activation of Caspase-9 and Caspase-3, and apoptosis initiation [27][28][29]. The present study showed that apoptosis of PC12 cells was observed after H 2 O 2 treatment, accompanied by decrease of MMP and increase of Caspase-3 activity, suggesting that H 2 O 2 could induce apoptosis through the mitochondrial pathway. Upon DHYZ treatment, the effect of H 2 O 2 on apoptosis was antagonized.
ROS is an effector of oxidative stress in cells. The present study showed that H 2 O 2 increased the ROS level in PC12 cells. NAC-mediated inhibition of ROS could antagonize the effects of H 2 O 2 on apoptosis and Caspase-3, suggesting that H 2 O 2 mediated oxidative    [24,25,30,31]. ROS can promote ASK1 phosphorylation, activate JNK or p38 MAPK, regulate downstream apoptosis-related proteins, and induce neuronal apoptosis. Inhibiting JNK and MAPK can protect against nerve damage [32,33]. The present study showed that phosphorylation of ASK1, JNK and p38 MAPK were up-regulated after H 2 O 2 treatment. DHYZ could also inhibit the phosphorylation of ASK1, JNK and p38 MAPK. These obversion suggested that ASK1, JNK and p38 MAPK participated in the effect of DHYZ.
DHYZ is established according to the TCM principles. Shu-Di (prepared root of R. glutinosa (Gaert.) Libosch. ex Fisch. et Mey.) is the Monarch (Jun) herb and used for tonifying kidney. Shi-Chang-Pu (root of A. tatarinowii Schott) and Dan-Shen (root of S. miltiorrhiza Bunge) are the Minister (Chen) herbs and used for dissolving stasis and phlegm, and calming mental-state. Fu-Shen (Poria with hostwood) is the Assistant (Zuo) herb and used for calming spirit. Yi-Zhi-Ren (fruits of A. oxyphylla Miq.) is the Guide (Shi) herb and used as herb for warming kidney. All those herbs are synergistically acted in DHYZ from the perspective of TCM and have showed beneficial for AD treatment [13,[34][35][36][37][38].

Conclusions
In summary, the present study suggested that DHYZ protected PC12 cells from H 2 O 2 -induced oxidative stress damage, and its mechanism was related to inhibition of ROS production and ASK1-JNK/p38 MAPK phosphorylation. The present study provided experimental evidence for alleviating oxidative stress damage, preventing and treating AD and neurodegenerative diseases by TCM.