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 alexia (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 TGF-β, 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 H₂O₂ 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.

 

Chemicals and instruments

Eerdun Wurile (internal medicine number M14010080, batch number 20180225) was obtained from National Mongolian Pharmaceutical Preparation Center, International Mongolian Hospital, Inner Mongolia, China. Voucher specimens have been deposited in the Virtual Herbarium of Inner Mongolia Medical University, Hohhot, China. TRIZOL reagent was purchased from Invitrogen (Carlsbad, CA, USA). PrimeScript^TM RT Master Mix (Perfect Real Time) and TB Green^TM Premix Ex Taq^TM â…¡ (Tli RNaseH Plus) were purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). Absolute ethanol, n-butanol, ethyl acetate and petroleum ether were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd. (Tianjin, China). HPLC grade water was prepared by Milli-Q® Direct 8 Water Purification System from Millipore (Burlington, MA, USA).

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% CO₂ 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 5×10⁴ 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-2-yl)-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 ml 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 ml 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 mL 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.

 

 

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 mg/mL. The water extract of EW showed negligible cytotoxicity on PC-12 cells (Figure 1). The IC₅₀ of the extracts were as following: water extract (400 mg/ml), ethanol extract (160 mg/mL), n-butanol extract (220 mg/mL), ethyl acetate extract (200 mg/mL), and petroleum either extract (80 mg/ml).

 

 

Treatment of PC-12 cells with hydrogen peroxide (H₂O₂) induced serious damage and cell death, with only 40% cell  viability  24 h  after  treatment  (250   mM H₂O₂). Subsequent treatment of the H₂O₂-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 H₂O₂ treatment, WE and EE showed slightly higher potential for quenching the oxidative stress in neurons (Figure 2).

 

 

H₂O₂ 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  H₂O₂  and subsequently exposed the cells to different extracts of EW at different concentrations. As shown in Figure 3, treatment of PC-12 cells with H₂O₂ 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 by-product  of  oxygen  metabolism  that causes cell damage (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 H₂O₂. Then, the cells were treated with various EW extracts, and the SOD activity measured. Treatment with H₂O₂ 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 H₂O₂.

 

 

Catalase catalyzes the decomposition of H₂O₂ to water and oxygen, which protects cells from oxidative damage by ROS. CAT activity in the cell decreases when the cell is exposed to H₂O₂. To measure the influence of EW extracts on CAT activity, PC-12 cells were sequentially treated with H₂O₂ and various EW extracts, respectively, and after 24 h, CAT activity was measured. EW extracts rescued the decrease of CAT activity induced by H₂O₂. 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 H₂O₂ 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 H₂O₂ 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 H₂O₂ 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 H₂O₂ treated PC-12 cells (Figure 8), while upregulating Akt and p38, implicating potential anti-inflammatory and anti-apoptotic effect of EW. Neurite outgrowth from PC12 cells is a well characterized model of neuron differentiation (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.

 

 

 

 

 

 

 

 

 

 

 

This study shows that the extracts of EW reverse hydrogen peroxide (H₂O₂)-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.

 

The authors have not declared any conflict of interests.

 

 

This research was kindly supported by Inner Mongolia Plan of Science and Technology (2018), Internal Funding from Research Institute of Mongolian Medicine of Inner Mongolia Autonomous Region (2016YJS31), Natural Science Foundation of Inner Mongolia Autonomous Region (2017MS0821), and funding from Department of Finance of Inner Mongolia Autonomous Region (CZT_201701), China.