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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 64  |  Issue : 1  |  Page : 24-31

Astragalus protects PC12 cells from 6-hydroxydopamine-induced neuronal damage: A serum pharmacological study


1 Department of Anatomy and Neurobiology, Shandong University School of Basic Medicine, Shandong, China
2 Department of Orthopaedics, Cheelee College of Medicine, Qilu Hospital (Qingdao), Shandong University, Shandong, China
3 Department of Anesthesiology, Second Hospital of Shandong University, Shandong, China
4 Department of Psychiatry, Shandong Mental Health Center, Shandong, China

Date of Submission27-Jun-2020
Date of Decision26-Nov-2020
Date of Acceptance21-Dec-2020
Date of Web Publication05-Feb-2021

Correspondence Address:
Dr. Chuan-Gang Li
Department of Anesthesiology, Second Hospital of Shandong University, Jinan 250033, Shandong
China
Dr. Zeng-Xun Liu
Department of Psychiatry, Shandong Mental Health Center, Jinan 250014, Shandong
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_50_20

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  Abstract 


Accumulating evidence has already indicated that traditional Chinese medicine (TCM) possesses tremendous potential for treating neurodegenerative diseases. Astragalus, also named Huangqi, is a famous traditional medical herb that can be applied to treat cerebral ischemia and prevent neuronal degeneration. Nevertheless, the underlying mechanisms remain largely unexplored. In the present study, Astragalus-containing serum (ASMES) was prepared and added into the culture medium of PC12 cells to explore its neuroprotective effect on 6-hydroxydopamine (6-OHDA)-caused neuronal toxicity. Our data showed that ASMES significantly ameliorated the cellular viability of cultured PC12 cells against the neurotoxicity induced by 6-OHDA (P < 0.05). Moreover, ASMES significantly decreased the cell apoptosis triggered by 6-OHDA (P < 0.01). Furthermore, 2′,7′-dichlorofluorescin diacetate assay was performed to detect the changes in oxidative stress, and we showed that 6-OHDA elevated the production of reactive oxygen species (ROS), whereas ASMES significantly reversed these changes (P < 0.01). Besides, mitochondrial membrane potential (MMP) assay showed that ASMES could restore 6-OHDA-damaged MMP in cultured PC12 cells (P < 0.05). In conclusion, Astragalus could protect PC12 cells from 6-OHDA-caused neuronal toxicity, and possibly, the ROS-mediated apoptotic pathway participated in this process. Collectively, our findings provided valuable insights into the potential in treatment of neurodegenerative diseases.

Keywords: 6-hydroxydopamine, apoptosis, Astragalus, neuroprotection, serum pharmacology


How to cite this article:
Guo LY, Shi FL, Li M, Sun JH, Li CG, Liu ZX. Astragalus protects PC12 cells from 6-hydroxydopamine-induced neuronal damage: A serum pharmacological study. Chin J Physiol 2021;64:24-31

How to cite this URL:
Guo LY, Shi FL, Li M, Sun JH, Li CG, Liu ZX. Astragalus protects PC12 cells from 6-hydroxydopamine-induced neuronal damage: A serum pharmacological study. Chin J Physiol [serial online] 2021 [cited 2021 Dec 2];64:24-31. Available from: https://www.cjphysiology.org/text.asp?2021/64/1/24/308745

The authors Li-Ying Guo and Feng-Lei Shi contributed equally to this work.





  Introduction Top


As an age-related disorder, neurodegenerative disease is characterized by progressive neuronal dysfunction and loss in the central nervous system.[1] Many mechanisms contribute to the pathogenesis of the neurodegenerative disease, including oxidative stress, inflammatory response, mitochondrial dysfunction, deposition of aggregated proteins, and initiation of apoptosis pathway.[2],[3] Currently, alternative therapy can only retard but not reverse the progression of the degenerative disease, and there is no cure available until now.[4] Traditional Chinese medicine (TCM) has been shown tremendous potential in drug discovery.[5] Preceding studies have demonstrated that some TCM formulae, such as Bushen-Yizhi formula and modified Chunsimyeolda-tang, exert neuroprotective effects through inhibition of cellular apoptosis.[6],[7] A current report has also demonstrated that the extract of traditional medicine Paeonia moutan can attenuate neuroinflammatory response and improve the movement ability in the mouse model suffering from Parkinson's disease.[8] Therefore, TCM may provide a novel insight into a therapeutic strategy for neurodegenerative diseases.

Astragalus, also named Huangqi, is a famous traditional medical herb that is commonly used for treating ischemic disease and myocardial injury.[9],[10],[11] Pharmacological studies have reported that the extracts of Astragalus have pleiotropic effects including anti-oxidative,[11] anti-inflammatory,[12] hepatoprotective,[13] and neuroprotective properties.[14] Among these effects, the anti-oxidative effect is prominent and can explain many other actions of Astragalus.[15] Moreover, a recent study has also suggested that Astragalus attenuates the intestinal ischemia-reperfusion injury in rats through an anti-oxidative mechanism.[11] Importantly, several chemical contents of Astragalus, including flavonoids, possess antioxidant activities. This anti-oxidative effect exactly caters to the critical step of oxidative damage caused by 6-hydroxydopamine (6-OHDA), which is normally applied as a neurotoxin to construct pharmacodynamic models in animals and cultured neurons. Besides, Buyang Huanwu decoction, in which Astragalus is the major component, has been found to promote the survival of PC12 cells insulted by 6-OHDA.[16] Therefore, we hypothesized that Astragalus, possibly, could prevent neuronal cytotoxicity induced by 6-OHDA.

Oxidative stress is a pathophysiological condition arising from the overproduction and large accumulation of reactive oxygen species (ROS).[17] ROS contains free radicals which are produced during metabolic processes and derived from multiple sources, such as mitochondrial dysfunction,[18] neuroinflammation,[19] and dopamine metabolism.[20] The imbalance of redox homeostasis caused by oxidative stress can damage bio-macromolecules and initiate the apoptosis pathways.[17] Therefore, oxidative stress is closely associated with pathogenesis of ischemic and neurodegenerative diseases.[21],[22] Besides, studies on SH-SY5Y cells and PC12 cells have demonstrated that oxidative stress plays a vital role in the detrimental effects of 6-OHDA.[23],[24] Collectively, the abovementioned evidence indicates that the neuroprotective effects of Astragalus may be linked to the mechanism of oxidative stress.

In the traditional pharmacology of TCM, crude extracts of drugs, such as aqueous extract and ethanol extract, are widely used in the studies of pharmacological actions and mechanisms. However, the results of these in vitro studies are often inconsistent with those in vivo investigations.[25] The invalidity of methodology makes the results dubitable. Serum pharmacology, proposed by Tashino,[26] has been accepted as a more scientific method in which the drug-containing serum is prepared from animals given drugs orally. TCM consists of complex components, and most of their pharmacokinetics remain unknown. Some herbs do not exert pharmacological effects until a series of biotransformation is accomplished[27] and some ingredients even cannot be absorbed into circulation under normal physiological conditions.[28] The methodology of serum pharmacology not only emphasizes the interactions between TCM and organism but also avoids the biases due to the complexity of herbs. Especially, it is suitable for pharmacological research on cultured cells. Therefore, serum pharmacology has incomparable advantages over traditional pharmacology in exploring the effect of TCM, including Astragalus on cultured neurons.

In the present study, we damaged PC12 cells using 6-OHDA and established a neurotoxic model in vitro. Astragalus-containing serum (ASMES) was prepared from rats orally administered with Astragalus decoction. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to examine cell viability. Hoechst 33,342/propidium iodide (PI) staining and flow cytometry were applied to determine cellular apoptosis. Moreover, ROS assay and JC-1 staining were conducted to explore its mechanism of ASMES against 6-OHDA-induced neuronal damages in cultured PC12 cells.


  Materials and Methods Top


Reagents and drugs

Astragalus was bought from Jianlian Pharmacy (Jinan, China). 6-OHDA was obtained from Sigma-Aldrich, USA (Lot No. MKCC1473). Dulbecco's modified Eagle's medium (DMEM) (Lot No. 8118299, Thermo Fisher Scientific, China) and fetal bovine serum (FBS) (04-001-1ACS, Biological Industries, Israel) were used for daily cell culture. Ethylenediaminetetraacetic acid (EDTA)-free trypsin (Cat. No. T1350) and MTT assay kit were provided by Solarbio (Beijing, China). The assay kits for measuring ROS and mitochondrial membrane potential (MMP), and Hoechst 33342/PI regent were obtained from a biotechnological company of Beyotime (Shanghai, China). Annexin V-FITC/PI Apoptosis Detection Kit (Cat No. A211-02) was bought from Vazyme, China. All the other chemical reagents in this experiment were commercially provided separately.

Animals

Adult Wistar rats (male, weighed 250 ± 30 g) were supplied by Shandong University Animal Center for laboratory research. The rats for experiment were housed in a standard reversed light cycle (12 h light/12 h dark) with ad libitum of food and water. All processes concerning animals were ratified by the Committee on Animal Ethics of Shandong Mental Health Center (2019R41). The experiment was conducted according to the guidelines for the care and operation of laboratory animal center.

Preparation of drug containing serum

Procedures in detail were performed according to a previous report.[16] In brief, the powdered components of Astragalus were decocted with distilled water. Wistar rats under identical conditions were treated with Astragalus decoction and distilled water, which named as ASMES group and control serum (CS) group, respectively. There were 15 male Wistar rats in each group. The drugs were intragastrically administrated to rats twice a day for 3 days. The dosage for one time of Astragalus administration decoction was 6 mL/100 g, and the final concentrations was 0.5 g/mL. On the 4th day of administration, double dosages were given to rats of each group to obtain enough drugs in blood. Two hours later after the last administration, the rats were conducted a surgery and heart blood was collected and then put the blood in 4°C overnight. In the next day, the blood was carried on centrifugation at 3000 r/min for 20 min, and then, the supernatant was separated. Subsequently, the supernatant was put in 56°C for 30 min to inactive complement. Sterile tips and tubes were used throughout the whole procedure. At last, the serum was defined as 100% ASMES stored at −20°C before using.

Cell culture

The differentiated rat pheochromocytoma cell (PC12) were planted in culture plates and grown in DMEM which contained 10% FBS. The cells were cultured at an environment of 37°C with 5% CO2 and 95% humidity constantly and were classified into three groups if no other instructions: control group (10% CS), 6-OHDA damage group (10% CS + 100 μM 6-OHDA), and ASMES protection group (8% ASMES + 2% CS + 100 μM 6-OHDA). The cells of all the groups were cultured with 10% blended serum for 1 h. Subsequently, 100 μM 6-OHDA was added into the culture medium for additional 24 h incubation before the subsequent experiments.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide detection

As a popular colorimetric assay, MTT is frequently carried out to examine the cellular viability and cytotoxicity. Briefly, planting PC12 cells on 96-well culture plates with DMEM medium containing 10% FBS at a density of 1 × 104/well after passaging. Then, 6-OHDA of various concentrations with or without ASMES was used to treat the cells for another 24 h. MTT was added dropwise into the culture plates at the final concentration of 0.5 mg/mL. After the cells had been incubated at 37°C for 4 h, the medium was aspirated and 100 μL dimethyl sulfoxide was then added into the well to make the formazan granules dissolved. The colorimetric value of formazan which indicates the cell viability was detected by a spectrophotometer at 570 nm (Multiskan, USA).

Hoechst/propidium iodide assay

The nuclei of living and dead cells were stained by Hoechst 33342 and PI, respectively. After treated with 6-OHDA together with or without ASMES, the PC12 cells were incubated in solutions containing Hoechst 33342 and PI at the final concentration of 10 μg/mL. After 15-min incubation and keeping in dark, the staining solution was removed and the morphological changes were observed and the fluorescent alternations were captured under a Zeiss microscope (Carl Zeiss, Germany). Hoechst is a DNA stain that can cross the functional normal membrane, so cells manifest blue fluorescence. In contrast, PI can only cross injured and dead cell membrane and thus the cells manifest red fluorescence. The apoptotic rate and necrotic rate were represented as the percentage of apoptotic cell nuclei and necrotic cell nuclei in three randomly fields of different groups.

Flow cytometry

6-OHDA-caused apoptosis of PC12 cells was detected by Annexin V-FITC/PI flow cytometry more accurately; the process was conducted on the basis of description in the manufacture (Vazyme, China). Briefly, cells were cultured in a 6-well plate at a density of 2 × 105 cells per well and then treated with 6-OHDA together with or without ASMES for 24 h. After collected the supernatant and washed the cells lightly with phosphate-buffered saline (PBS), the cells were digested with EDTA-free trypsin for 3 min at 37°C and washed twice with PBS at 4°C. Subsequently, cells were resuspended with 100 μL 1X binding buffer, and then, the mixture of 5 μL Annexin V-FITC and 5 μL PI was added to stain for 10 min in the dark. Finally, 400 μL 1X binding buffer was added to each sample. The data were obtained and analyzed by CytoFLEX Flow Cytometer (Beckman, USA).

Reactive oxygen species measurement

2′,7′-dichlorofluorescin diacetate (DCFH-DA) can be used to detect oxidative stress and ROS production. DCFH-DA can enzymatically transform to the strong green fluorescent substance DCF under the presence of ROS. According to this reaction, the PC12 cells treated with 6-OHDA were then incubated with different mixed serums for additional 24 h. After that, the cells were incubated in 10 μM DCFH-DA solution keep in the dark for 20 min at a temperature of 37°C. Finally, the cells were washed with culture medium and the fluorescent intensity was analyzed under a microscope. Once oxidation by ROS, DCFH changes to de-esterified DCF which is a fluorescent compound. Thus, the signal of fluorescence can represent ROS level.

Detection of mitochondrial membrane potential

JC-1 is a cationic dye and can accumulate in mitochondria of the cell, which is used to detect the changes of variation of MMP and mitochondrial integrity. The PC12 cells were planted at 2.5 × 104/cm2 in 24-well culture plates and cultured for 24 h prior to 6-OHDA or ASMES treatment. Next, 10 μM JC-1 solution was added to the wells and incubated cells for 20 min and then rinsed with cold PBS buffer. Then, 250 μL DMEM was added in the well and the fluorescence of each well was detected by fluorescent microscopy. The monomer and aggregate of JC-1 yield green and red fluorescence, respectively, which can reflect the changes of MMP.

Statistical analysis

All values in this experiment were recorded as the form of mean ± standard error of the mean. Statistical analysis was conducted with one-way ANOVA (not repeated measures) and further analyzed with Tukey's post hoc test. P < 0.05 was regarded as statistically significant. The statistical analysis was carried out by GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA).


  Results Top


Astragalus-containing serum improves cell viability against 6-hydroxydopamine-induced neurotoxicity

PC12 cells grew well in the control group, whereas 6-OHDA significantly destroyed the cultured cells, The cell bodies became bright-round vacuolar and the cell processes disappeared. [Figure 1]a and [Figure 1]b. When incubated with different concentrations of ASMES plus CS, the morphological damage of cells was significantly ameliorated, exhibiting smooth cell membrane and long neurites [Figure 1]c, [Figure 1]d, [Figure 1]e. When 6-OHDA was added into the culture medium of PC12 cells at a series of concentrations of 50, 75, 100, 125, and 150 μM, the cellular viability was gradually reduced with the increase of concentration [Figure 1]f. In the circumstance of 100 μM 6-OHDA, the cell viability of PC12 cells was lower compared with the control group (P < 0.001). Therefore, 100 μM was chosen in the subsequent experiments. The MTT data also showed that ASMES exerted its neuroprotective effect on cell viability through a concentration-dependent manner and 8% ASMES significantly increased the cell viability compared with the 6-OHDA group [Figure 1]g, P < 0.05]. In contrast, no obvious growth-promoting effect was observed in cells only treated with ASMES [P > 0.05, [Figure 1]h.
Figure 1: Neuroprotection of Astragalus-containing serum on 6-hydroxydopamine-induced neurotoxicity. (a) Cells were cultured with 10% control serum. (b-e) Cells were cultured with 10% control serum, 2% Astragalus-containing serum + 8% control serum, 4% Astragalus-containing serum + 6% control serum, and 8% Astragalus-containing serum + 2% control serum, respectively, for 1 h and then cultured with 100 μM 6-hydroxydopamine for extra 24 h. Astragalus-containing serum promoted the cell growth with larger number and longer neurites. (f) PC12 cells were incubated with 10% control serum for 1 h and then cultured with 6-hydroxydopamine in various concentration for 24 h. When the concentration of 6-hydroxydopamine increased, the cell viability was decreased gradually. Thus, 100 μM 6-hydroxydopamine was selected to establish neurotoxic model. (g) The effects of Astragalus-containing serum and 6-hydroxydopamine on cell viability. (h) The cells were cultured with Dulbecco's modified Eagle's medium containing 2%, 4%, and 8% Astragalus-containing serum for 24 h. *P < 0.05 (g, vs. 10% control serum + 100 μM 6-hydroxydopamine group); **P < 0.01 (f and g, vs. 10% control serum group); ***P < 0.001 (f, vs. 10% control serum group). 2%, 4%, 8%, and 10% referred to the ultimate concentrations of different serums in culture medium. The scale bar = 100 μm (n = 3).

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Anti-apoptotic effects of Astragalus-containing serum on cellular damage caused by 6-hydroxydopamine

We detected the effects of ASMES on cell apoptosis using Hoechst 33342/PI staining; PC12 cells displayed dull blue fluorescence, indicating viable and functional cells, whereas the apoptotic cells exhibited bright blue fluorescence due to nuclear condensation and chromatin shrinkage. Red fluorescence represented dead cells. The results demonstrated that 6-OHDA remarkably increased the apoptotic rate of cells [P < 0.001, [Figure 2]a and d], while ASMES effectively reversed the elevation of apoptosis compared with the 6-OHDA group [P < 0.01, [Figure 2]g. Moreover, ASMES also decreased the proportion of necrotic cells compared with the 6-OHDA group [P < 0.01, [Figure 2]b, [Figure 2]e and [Figure 2]h. [Figure 2]c, [Figure 2]f and [Figure 2]i are merge figures of Hoechst 33342/PI staining. White arrow indicates apoptotic cell, and yellow arrow indicates necrotic cell. Statistical analysis confirmed the morphological changes [Figure 2]j and [Figure 2]k. Flow cytometry was used to further determine the effects of ASMES on cell apoptosis. The data showed that 6-OHDA apparently induced cell apoptosis compared with the control [P < 0.001, [Figure 3]a, [Figure 3]b and [Figure 3]d, whereas ASMES obviously decreased cell apoptosis, especially inhibiting the early apoptotic rate of injured PC12 cells [P < 0.01, [Figure 3]b, [Figure 3]c, [Figure 3]d.
Figure 2: Anti-apoptosis and anti-necrosis effects of Astragalus-containing serum. PC12 cells were cultured with 10% blended serum containing 8% Astragalus-containing serum and 2% control serum for 1 h and then added 100 μM 6-hydroxydopamine for another 24 h. (a-i) After Hoechst 33342/propidium iodide staining, fluorescent images were capture by virtue of fluorescence microscope. PC12 cells which display dull blue fluorescence indicate viable cells, whereas the apoptotic cells display bright blue fluorescence. Red fluorescence represents dead cells. (j and k) Apoptosis and necrosis of cultured cells were detected. The percentage of apoptotic cells and necrotic cells was significantly decreased when co-incubated with Astragalus-containing serum. **P < 0.01 (j and k, vs. 10% control serum + 100 μM 6-hydroxydopamine group); ***P < 0.001 (j and k, vs. 10% control serum group). White arrows indicate apoptotic cell, and yellow arrows indicate necrotic cell. The scale bar in a indicates 100 μm (n = 3).

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Figure 3: Apoptotic rate determined by flow cytometry. PC12 cells were cultured with 10% control serum (a and b) and 8% Astragalus-containing serum + 2% control serum (c) for 1 h, respectively, and then added 100 μM 6-hydroxydopamine to b and c group for another 24 h; then, flow cytometry was used for the detection of the apoptosis rate (a-c). The early apoptosis rate of cells was significantly decreased in the Astragalus-containing serum group (d). ***P < 0.001 (d, vs. 10% control serum group); ##P < 0.01 (d, vs. 10% control serum + 100 μM 6-hydroxydopamine group) (n = 4).

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Astragalus-containing serum reverses the increased reactive oxygen species production evoked by 6-hydroxydopamine

By assessing oxidative stress, we could measure the level of intracellular ROS production. When intracellular ROS was produced, it oxidized DCFH-DA to DCF with fluorescence. Therefore, there was a weak green fluorescence signal in the control group with low ROS production [Figure 4]a. When treated with 100 μM 6-OHDA, the bright green fluorescence was visible [Figure 4]b. Moreover, ASMES significantly reversed the change of fluorescence [Figure 4]c. Statistical analysis showed that the intracellular ROS production of PC12 cells was strikingly escalated when 100 μM 6-OHDA was applied compared with the control group (P < 0.01). However, ASMES remarkably reversed the augmentation of intracellular ROS level caused by 6-OHDA [P < 0.01, [Figure 4]d.
Figure 4: Astragalus-containing serum inhibits the elevation of reactive oxygen species production. PC12 cells were incubated in Dulbecco’s modified Eagle’s medium containing 8% Astragalus-containing serum and 2% control serum for 1 h and then using 100 μM 6-hydroxydopamine to treat for additional 24 h. (a-c) Reactive oxygen species production was measured by 2',7'-dichlorofluorescin diacetate dye. (d) The changes of fluorescent intensity of the cultured cells were analyzed. 6-hydroxydopamine obviously elevated reactive oxygen species production, whereas Astragalus-containing serum reduced the accumulation of reactive oxygen species. **P < 0.01 (d, vs. 10% control serum group); ##P < 0.01 (d, vs. 10% control serum + 100 μM 6-hydroxydopamine group). The scale bar in a indicates 100 μm (n = 3).

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Astragalus-containing serum rescues the depolarization of mitochondrial membrane potential caused by 6-hydroxydopamine

MMP (ΔΨm) was measured to investigate early apoptosis. The existence of JC-1 fluorescent probe was alternated in a potential-dependent manner [Figure 5]a. When there was a high level of MMP, JC-1 was aggregated, and the cells showed red fluorescence. When the level of MMP was reduced, JC-1 was depolymerized to monomer and the cells displayed green fluorescence. Exposure to 6-OHDA reduced the MMP level of PC12 cells, showing obvious green fluorescence [Figure 5]b. In contrast, ASMES reversed these changes [Figure 5]c. Statistical analysis indicated that 6-OHDA remarkably disrupted the MMP (P < 0.01), while incubation of ASMES significantly depolarized and increased the MMP [P < 0.05, [Figure 5]d.
Figure 5: Astragalus-containing serum rescued the depolarization of mitochondrial membrane potential after 6-hydroxydopamine treatment. (a-c) The mitochondrial membrane potential of cultured PC12 cells was detected using JC-1 dye. (a) The healthy cells of the control showed that yellow fluorescence indicated high mitochondrial membrane potential with aggregate of JC-1. (b) In the 6-hydroxydopamine group, some cells showed that green fluorescence indicated low mitochondrial membrane potential with JC-1 of monomer. (c) Astragalus-containing serum partly reversed the changes caused by 6-hydroxydopamine. (d) The Astragalus-containing serum protection group showed more yellow fluorescence and less green fluorescence compared to the 6-hydroxydopamine damage group. **P < 0.01 (d, vs. 10% control serum group); #P < 0.05 (d, vs. 10% control serum + 100 μM 6-hydroxydopamine group). The scale bar = 100 μm (n = 3).

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  Discussion Top


In our current study, we established the in vitro neurotoxic cell model in PC12 cells through 6-OHDA exposure. ASMES was prepared based on the serum pharmacological method to explore its neuroprotective effects and the underlying mechanism. The results showed that ASMES significantly ameliorated the cellular viability of PC12 cells, attenuated the morphological injuries, and decreased the cell apoptosis caused by 6-OHDA. Moreover, the intracellular ROS level of PC12 cells was remarkably reduced after incubation with ASMES. Furthermore, ASMES dramatically rescued the depolarization of MMP. All of the abovementioned findings indicated that ASMES might exert a neuroprotective effect through the anti-oxidative and anti-apoptotic properties.

Astragalus, the root of Huangqi, is a well-known traditional Chinese herb. Extensive pharmacological effects have been declared including anti-inflammation,[12] anti-oxidation,[15] and anti-carcinoma,[29] and some effects have even been applied maturely in clinical practice, like myocardial preservation.[30] Calycosin, the extract of Astragalus, has been reported to possess neuroprotective effects on cerebral ischemia and reperfusion damage in rats.[14] In the previous study, the drug-containing serum of Buyang Huanwu decoction shows obvious neuroprotective effects on cultured neurons damaged by 6-OHDA.[16] Astragalus is the prominent ingredient that accounts for 50% of the total weight of Buyang Huanwu decoction. Therefore, we prepared ASMES to investigate its neuroprotective effects. Our MTT assay demonstrated that ASMES could significantly increase the cell viability against 6-OHDA damage, which was consistent with our expectation. As the concentration of ASMES was increased, the cell viability was gradually elevated in a concentration-dependent manner.

ROS is a sensitive indicator of oxidative stress of cells. The imbalance between the anti-oxidative mechanism and the production of oxidant causes the abnormal accumulation of ROS. Accumulating evidences have demonstrated that 6-OHDA can induce the overproduction of ROS via various signaling pathways.[31] The oxidability of ROS damages DNA strands, affects cellular membrane structures, and impairs the enzymatic viability.[32] Enormous studies have demonstrated that ROS is closely associated with aging and neurodegeneration.[32],[33] Mitochondrion, as an active energy producer, is one of the main sources of oxidative stress. The inefficiency of redox enzymes and irreversible DNA damage lead to the overproduction of ROS. Therefore, the dysfunction of mitochondria has a reciprocal causation relationship with ROS.[34] The results demonstrated that ASMES remarkably restored the disrupted MMP. Besides, the intracellular ROS level was reduced after incubation with ASMES, indicating that the neuroprotective effect of ASMES was associated with the anti-oxidative property.

As a form of programmed cell death, apoptosis can be activated under the alternations of MMP and the generation of ROS.[35] Early apoptosis can reflect the depolarization of MMP.[36],[37] A previous study has demonstrated that apoptosis promotion plays a significant role in 6-OHDA induced cytotoxicity.[38] In this study, we found that the apoptotic rate was dramatically elevated after 6-OHDA exposure, whereas ASMES significantly decreased the apoptosis. Moreover, the proportion of necrotic cells was significantly decreased in the presence of ASMES also suggesting that the mechanism of necrosis was involved in this phenomenon. Previous reports demonstrated that Astragalus inhibits cellular apoptosis through decreasing JNK3 expression in cerebral neuron injury.[39] Moreover, as a bioactive compound of Astragalus, astragaloside IV significantly protects cortical neurons from oxidative damage by increasing protein kinase A and cyclic adenosine 3′,5′-monophosphate-response element binding phosphorylation.[40] The molecular mechanisms of ASMES neuroprotection need to be further explored.

The method of serum pharmacology can mimic the in vivo biotransformation and partly eliminates the deviation step from the complex of crude extraction of herbs. This study of ASMES provided direct evidence of the neuroprotective effects of Astragalus. The effective ingredients in ASMES are still unclear, which is also beyond the range of our present study. Active constituents of Astragalus root have been identified and characterized. These components include: polysaccharide cycloartane glycoside fractions (astragalosides I–IV and trigonosides I–III), four major isoflavonoids (formononetin, ononin, calycosin, and its glycoside), saponins, several minor isoflavonoids, and other biogenic amines. Previous studies have declared that ASMES contains many bioactive saponins and flavonoids, such as astragaloside IV, ononin, and formononetin.[41] Some of these bioactive substances show potential biological effects, including neuroprotection. An earlier study has reported that formononetin can protect traumatic brain injury rats from neurological damage.[42] Astragaloside IV shows neuroprotective effects on ischemia/reperfusion-induced lesions in cortical neurons.[43] Possibly, ASMES contained the above ingredients. The complexity and diversity of TCM endow its tremendous potential in drug discovery. Therefore, further studies remain to be necessary.


  Conclusions Top


ASMES protected PC12 cells against 6-OHDA-induced neurotoxicity. Moreover, ASMES significantly attenuated the oxidative stress and decreased the apoptotic rate, which could be responsible for Astragalus neuronal protection.

Acknowledgment

The authors wish to thank all the participants and their companions involved in the study.

Financial support and sponsorship

The authors thank the supporting by a grant of Shandong Provincial Natural Science Foundation (No. ZR2012HM026).

Conflicts of interest

There are no conflicts of interest.



 
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