Eerdun Wurile protects neuron and promotes neurite outgrowth through regulation of apoptotic gene expression in PC-12 cells

Eerdun Wurile (EW) is one of the most widely used traditional Mongolian medicines for stroke recovery. Previous studies revealed that EW regulates brain gene expression in a rat model of middle cerebral artery occlusion (MCAO). However, the fraction of active components and the specific genes regulated by such fractions have not been elucidated clearly. The study shows that the extracts of EW regulate the expression of genes involved in oxidative stress and apoptosis in rat pheochromocytoma (PC-12) cells. Hydrogen peroxide (H2O2)-induced cell death was reversed by EW extracts, and reactive oxygen species (ROS) production was reduced, while superoxide dismutase (SOD) activity as well as catalase (CAT) activity increased significantly. Moreover, the expression of Bcl-2, PARP and NF-kB p65 was upregulated by EW extract, while Bax was downregulated. Similarly, caspase 9 and Jnk was remarkably downregulated by EW extracts. Significantly, EW extracts promoted the neurite outgrowth of PC-12 cells. Our data collectively suggested that EW contains active fractions that regulate the expression of genes involved in oxidative stress and cell apoptosis, which may contribute to the neural protection effect of EW.


INTRODUCTION
Stroke is the second major cause of death worldwide (Donnan et al., 2008;Wang et al., 2017). Stroke related disability rate is considerably high, which can lead to numbness, incontinence, as well as speech and vision loss. Prompt restoration of blood supply is the major principle for stroke therapy (Wang et al., 2017), and the thrombolysis medicine (tissue plasminogen, tPA) remarkably decreases the rate of disability (Saver, 2006). Eerdun Wurile (EW) is one the most effective traditional Mongolian medicine used in clinic for stroke recovery. Treatment of stroke with EW significantly alleviates stroke symptoms including limb numbness, slurred speech, and  (Hua et al., 2014;Tian, 2011). The remarkable therapeutic effects and negligible side effects of EW are due to the natural products used for the formulation of EW, including fruits of Terminalia chebula Retz, Amomum tsaoko, Gardenia jasminoides Ellis, seeds of Myristica fragrans, Abutilon theophrasti, Melia toosendan, Cassia obtusifolia, flowers of Sieb Carthamus tinctorius, and roots of Glycyrrhiza uralensis Fis, and Saussurea costus. These plants contain bioactive molecules such as isoliquiritigenin and diphenylheptanes, which have been proven to have protective effects in rat middle cerebral artery occlusion (MCAO)-induced ischemic stroke model (Zhan and Yang, 2006) through protection from nerve injury and post-stroke recovery (Zhang et al., 2016;Han et al., 2017).
It was discovered that EW can regulate the gene expression in the peri-ischemic center, resulting in significant upregulation of growth factors including TGFβ, Igf1, and Igf2, which may contribute to nerve tissue repair and growth. EW treatment in a rat middle cerebral artery occlusion (MCAO) model also induced significant upregulation of microglia markers, including Iba-1, CX3CR1, CD68 and CSF1R, which may have resulted from anti-inflammatory polarization of microglia upon EW treatment (Gaowa et al., 2018;Qiburi et al., 2020). We hypothesize that the active molecules in EW also protect nerve tissue through direct intervention with neurons. To prove this, we treated neuron cells with various extracts of EW, and investigated the protection effects of the extracts on the cells treated with H 2 O 2 as an oxidative inducer. This data suggest that EW extracts reduce oxidative stress induced cell death and ROS production, and increase SOD activity and CAT activity, and lead to the protection of rat pheochromocytoma (PC-12) cells from oxidative stress-induced cell death.

Extraction of samples
EW powder (0.2 g per flask) were extracted under reflux with 20 ml of either distilled water (WA), absolute ethanol (EE), n-butanol (BE), ethyl acetate (EAE) or petroleum ether (PE) for 24 h at 37°C. The extracts were evaporated with a rotary evaporator under reduced pressure to remove the solvent which was dissolved in DMSO and stored at 4°C.

Cell culture and cytotoxicity assay
PC-12 (rat pheochromocytoma cell line) was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Pudong, Shanghai, China). PC-12 cells were cultured at 37°C, 5% CO 2 in a DMEM supplemented with 10% FBS (HyClone) and 1% 100 units/ml penicillin and 100 µg/ml streptomycin. PC-12 cells were seeded in 24-well plates at 5×10 4 cells per well in 500 µl of culture medium and reached 70-80% for experimental use. To evaluate cellular toxicity of EW extracts, the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was carried out. PC-12 cells were seeded in a 96-well plate (Corning-Coaster, Tokyo, Japan) at a density of 10000 cells/well in 100 l DMEM medium and the cells were incubated for 24 h. The cells were then treated with various concentrations of EW extracts in 100 ml of DMEM medium. After the treated cells were incubated for 24 h, 50 l of 1X MTT solution was added to each well, and incubated at 37°C for 4 h. After the viable cells with active metabolism convert MTT into a purple colored formazan product, the cell medium was removed, and 100 L of DMSO was added to dissolve the formazan product. The quantity of formazan is measured by recording changes in absorbance at 570 nm, using a plate reading spectrophotometer (FilterMax F5, Molecular Devices, USA).

ROS production, CAT activity and SOD activity
Reactive oxygen species (ROS) production, Catalase (CAT) activity and Superoxide dismutase (SOD) activity were measured using kits (Nanjing Jiancheng, Nanjing, China) according to the manufacturer's instruction.

Real-time polymerase chain reaction (PCR)
The total RNA was extracted from PC-12 cells, using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The expression of mRNA was measured using PrimeScript TM RT Master Mix (Perfect Real Time) for RT-qPCR and TB Green TM Premix Ex Taq TM (Tli RNaseH Plus) for quantitative PCR. Primers were purchased from Takara Biotechnology Co., Ltd (Beijing, China). The sequences of primers used for RT-qPCR were listed in Table 1.

Statistical analysis
Statistical significance was determined using one-way analysis of variance (ANOVA) with a Dunnett's multiple comparisons test. P values of < 0.05 were considered statistically significant. All the results were expressed as mean ± SEM. The analysis was performed using GraphPad Prism 7.0.

RESULTS AND DISCUSSION
To determine neuroprotective effects of EW, the components of EW were extracted according to the polarity of the chemicals using five different solvents. The  yield of the extracts increased with the increase of the polarity of the solvent used in the extraction process, with water yielding the highest amount of the chemical mixture and petroleum ether providing the least amount of solid products. Meanwhile, the PE extracts showed considerable cytotoxicity on PC-12 cells, with 30% cell viability at a concentration of 150 g/mL. The water extract of EW showed negligible cytotoxicity on PC-12 cells (Figure 1). The IC 50 of the extracts were as following: water extract (400 g/ml), ethanol extract (160 g/mL), n-butanol extract (220 g/mL), ethyl acetate extract (200 g/mL), and petroleum either extract (80 g/ml). Treatment of PC-12 cells with hydrogen peroxide (H 2 O 2 ) induced serious damage and cell death, with only 40% cell viability 24 h after treatment (250 M H 2 O 2 ). Subsequent treatment of the H 2 O 2 -treated cells with EW extracts remarkably increased the cell viability, demonstrating that EW extracts can rescue the neurons undergoing oxidative stress. While all extracts promoted cell survival after H 2 O 2 treatment, WE and EE showed slightly higher potential for quenching the oxidative stress in neurons (Figure 2). H 2 O 2 treatment induces upregulation of reactive oxygen species (ROS) in neurons, which mimics the oxygen glucose depravation (OGD) condition. After acute ischemic stroke, the production of ROS rapidly increases in the infarct center, which immediately overwhelms defensive antioxidant, leading to autophagy, apoptosis, and necrosis (Rodrigo et al., 2013). To assess whether or not EW extracts quench ROS or downregulate ROS production, we first treated PC-12 cells with H 2 O 2 and Figure 2. EW extracts rescue hydrogen peroxide (H 2 O 2 )-induced oxidative injury and cell death. PC-12 cells pre-treated with H 2 O 2 (250 M) were treated with positive control (quercetin, QT) or various EW extracts. Cell viability was measured 24 h after the treatment. The symbol "*" (p < 0.05), "**" (p < 0.01), "***" (p < 0.005) and "****" (p < 0.0001) indicates significant differences compared with the NT group. Figure 3. EW extracts reduce hydrogen peroxide (H 2 O 2 )-induced ROS production. PC-12 cells pre-treated with H 2 O 2 (250 M) were treated with positive control (quercetin, QT) or various EW extracts. ROS production was measured 24 h after the treatment. The symbol "*" (p < 0.05), "**" (p < 0.01), "***" (p < 0.005) and "****" (p < 0.0001) indicates significant differences compared with the NT group. subsequently exposed the cells to different extracts of EW at different concentrations. As shown in Figure 3, treatment of PC-12 cells with H 2 O 2 significantly upregulated the ROS production. The positive control (QT) reduced ROS dose dependently. All EW extracts decreased ROS production, with PE extract showing most potent inhibitory zeffect of ROS production ( Figure 3), indicating that EW contains active chemicals that ether downregulate the expression of genes involved in ROS production, or directly quench ROS, which is beneficial for neural protection after stroke hit.
Superoxide dismutase (SOD) catalyzes the dismutation of the superoxide (O2−), which is a byproduct of oxygen metabolism that causes cell damage h after the treatment. The symbol "*" (p < 0.05), "**" (p < 0.01), "***" (p < 0.005) and "****" (p < 0.0001) indicates significant differences compared with the NT group. (Hayyan et al., 2016). SOD is an important antioxidant defense by being a major free radical scavenging system, and it has powerful anti-inflammatory activity. The SOD activity is decreased in the serum of acute cerebral ischemic injury, and replacement of antioxidant activity is beneficial for stroke patients (Spranger et al., 1997). To assess the impact of EW on SOD activity in neurons, SOD deficient cell model was first established by treating PC-12 cells with H 2 O 2 . Then, the cells were treated with various EW extracts, and the SOD activity measured. Treatment with H 2 O 2 significantly decreased the SOD activity, which is rescued by QT. All EW extracts exhibited the ability to enhance SOD activity. Cells treated with WE, EE and PE extracts showed the best SOD activity (Figure 4), demonstrating that EW can replace SOD activity decreased by H 2 O 2 .
Catalase catalyzes the decomposition of H 2 O 2 to water and oxygen, which protects cells from oxidative damage by ROS. CAT activity in the cell decreases when the cell is exposed to H 2 O 2 . To measure the influence of EW extracts on CAT activity, PC-12 cells were sequentially treated with H 2 O 2 and various EW extracts, respectively, and after 24 h, CAT activity was measured. EW extracts rescued the decrease of CAT activity induced by H 2 O 2 . PC-12 cells treated with PE extract showed highest CAT activity ( Figure 5).
Since EW extracts can efficiently reduce ROS production, and increase SOD and CAT activity, we next analyzed the viability of H 2 O 2 treated PC-12 cells using live/dead cell double staining using calcein AM and propidium iodide staining kit. The kit which contains Calcein-AM (stain viable cells) and Propidium Iodide (PI) (stain dead cells) solutions can stain viable and dead cells simultaneously. As shown in Figure 6, PC-12 cells treated with EW extracts efficiently maintained the cell viability (green channels) after H 2 O 2 treatment, which showed red signal in PI channel. To investigate the underlining mechanism for antioxidant effect of EW extracts, the expression of genes involved in cell apoptosis were measured. As shown in Figure 7, the decreased expression of Bcl-2, PARP and NF-kB (p65) upon H 2 O 2 treatment was reversed by the treatment of EW extracts, with EE and PE extracts showing the best upregulating effects of these genes. The upregulation of PARP can significantly facilitate DNA damage repair pathway (Pascal, 2018). On the other hand, the Bax was significantly downregulated by BE, EAE and PE extracts. Bax interacts with the mitochondrial voltage-dependent anion channel (VDAC), and increases the opening of VDAC, leading to the loss in membrane potential and the release of cytochrome c (Oltvai et al., 1993).
Caspase 9 is crucial to the apoptotic pathway in many tissues including in the nervous system. During ischemic stroke, neuronal apoptosis leads to the release of Caspase 9 from the mitochondria and accumulating of Caspase 9 in the nuclei of neurons in hippocampus (Krajewski et al., 1999). PE extract of EW significantly downregulates the expression of Caspase 9 in H 2 O 2 treated PC-12 cells (Figure 8), while upregulating Akt and p38, implicating potential anti-inflammatory and antiapoptotic effect of EW. Neurite outgrowth from PC12 cells is a well characterized model of neuron differentiation Figure 5. EW extracts enhance CAT activity after hydrogen peroxide (H 2 O 2 ) treatment. PC-12 cells pretreated with H 2 O 2 activity was measured 24 h after the treatment. The symbol "*" (p < 0.05), "**" (p < 0.01), "***" (p < 0.005) and "****" (p < 0.0001) indicates significant differences compared with the NT group. Figure 6. EW extracts regulate the expression of genes involved in apoptosis. PC-12 cells pre-treated with H 2 O 2 were treated with positive control (quercetin, QT) or various EW extracts. Gene expression level was measured by RT-qPCR 24 hours after the treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0001. Figure 7. EW extracts regulate the expression of genes involved in apoptosis. PC-12 cells pre-treated with H 2 O 2 were treated with positive control (quercetin, QT) or various EW extracts. Gene expression level was measured by RT-qPCR 24 h after the treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0001. Figure 8. EW extracts regulate the expression of genes involved in apoptosis. PC-12 cells pre-treated with H2O2 were treated with positive control (quercetin, QT) or various EW extracts. Gene expression level was measured by RT-qPCR 24 hours after the treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0001.  (Burstein et al., 1982). To assess whether or not EW extracts stimulate neurite outgrowth, PC-12 cells were treated with various EW extracts at two different concentrations, and observed the cell morphology under microscope. At a concentration of 100 µg/ml, PE extract significantly enhanced neurite outgrowth of PC-12 cells (Figure 9). The growth of neurite is stimulated by neurotrophic factors, such as nerve growth factor (NGF). Our previous studies evidenced that EW upregulates the expression of neurotrophic factors such as IGF1 and IGF2. PE extract has the most potent stimulatory effect for neurite growth, which implicates that PE extract may contain the growth stimulating bioactive molecules.

Conclusion
This study shows that the extracts of EW reverse hydrogen peroxide (H 2 O 2 )-induced cell death, reduce ROS production, and increase SOD as well as CAT activity. EW extracts upregulated the expression of Bcl-2, PARP and NF-kB p65, and downregulated Bax, Caspase 9 and Jnk. Moreover, EW extracts enhanced the neurite outgrowth of PC-12 cells. Collectively, the data suggests that the active fractions of EW may contribute to the neural protection effect of EW through regulating the expression of genes involved in apoptosis.