Echinacoside Protects Against 6-Hydroxydopamine-Induced Mitochondrial Dysfunction And Inflammatory Responses in PC12 Cells Via Reducing ROS Production

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Yue-Hua Wang,1,2 Zhao-Hong Xuan,3 Shuo Tian,1 and Guan-Hua Du1,2

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons at the substantia nigra. Mitochondrial dysfunction and inflammatory responses are involved in the mechanism of cell damage in PD. 6-Hydroxydopamine (6-OHDA), a dopamine analog, specifically damages dopaminergic neurons. Echinacoside (ECH) is a phenylethanoid glycoside isolated from the stems of Cistanche salsa, showing a variety of neuroprotective effects in previous studies. The present study was to investigate its effect against 6-OHDA-induced neurotoxicity and possible mechanisms in PC12 cells. The results showed that 6-OHDA reduced cell viability, decreased oxidation-reduction activity, decreased mitochondrial membrane potential, and induced mitochondria-mediated apoptosis compared with untreated PC12 cells. However, echinacoside treatment significantly attenuated these changes induced by 6-OHDA. In addition, echinacoside also could significantly alleviate the inflammatory responses induced by 6-OHDA. Further research showed that echinacoside could reduce 6-OHDA-induced ROS production in PC12 cells. These results suggest that the underlying mechanism of echinacoside against 6-OHDA-induced neurotoxicity maybe involve in attenuating mitochondrial dysfunction and inflammatory responses by reducing ROS production.

1. Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the slowly progressive degeneration of dopamine neurons in the substantia nigra pars compacta [1]. Although the neuropathological hallmarks of PD are well described, the etiology remains still undefined. Notably, several observations suggest that mitochondrial dysfunction is involved in the pathogenesis of PD and the degeneration of dopaminergic neurons [2, 3]. Mitochondria contain many redox enzymes and naturally occurring inefficiencies of oxidative phosphorylation generate reactive oxygen species (ROS) [4]. Recently, increasing evidence from human and animal studies has suggested that neuroinflammation is also an important contributor to neuronal loss in PD [5].

6-Hydroxydopamine (6-OHDA) is specific to dopaminergic neurons in intrastriatal rodent models and in some cells [6–11]. PC12 cell has become a popular cell model for PD research because this cell line possesses many characteristics of DAergic neurons. It has been used as an in vitro model for the study of PD and to determine the effect of protective and therapeutic agents. Echinacoside (Figure 1) is a phenylethanoid glycoside isolated and purified from the stemsofCistanche salsa [12], a parasitic plant native to northwest China, which is used as a try underlying mechanisms of echinacoside against 6-OHDA- induceddamageinPC12cells.

2. Materials and Methods

2.1. Materials. Echinacoside was obtained from National Institutes for Food and Drug Control (Beijing, China). RPMI-1640 and fetal bovine serum were obtained from Gibco

(Grand Island, NY, USA). 6-OHDA, MTT, 2,7-dichlorodihydrofluorescein diacetate, propidium iodide (PI), and resazurin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ATP bioluminescent assay kit was obtained from Promega (Madison, USA). 5,5,6,6 -Tetrachloro-1,1,3,3 -tet- raethyl benzimidazolyl carbocyanine iodide (JC-1) assay kit was obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Cytochrome C ELISA kit and fluorogenic substrate Ac-DVD-AMC were obtained from Invitrogen (Carlsbad, CA, USA). IL-1 and IL-6 ELISA kits were obtained from Boster Bio-engineer limited company (Wuhan, China). Spectramax M5 microplate reader was purchased from the Molecular Devices company (Sunnyvale, CA, USA).

2.2. Cell Culture and Treatment. PC12 cells were grown in RPMI-1640 supplemented with 10% horse serum and 5% fetal bovine serum. Conditions were maintained at 37∘C in a humidified atmosphere containing 5% CO2 [19]. The cells were plated into 96-well plates at a density of 2 × 105 cells/mL. After 80% confluence, the cells were preincubated with different concentrations of echinacoside in a serum-free RPMI-1640 medium for 1 h. Then, 6-OHDA was added to the wells at a final concentration of 100 M and incubated for another After treatment, morphological changes were observed under a microscope. Cell viability was evaluated by MTT assay [19]. Briefly, after treatment, MTT at the final concentration of 0.5 mg/mL was added to each well and incubated for 4 h at 37∘C.

2.3.Morphological Changes and Viability Assay of PC12 Cells. After treatment, morphological changes were observed under a microscope. Cell viability was evaluated by MTT assay [19]. Briefly, after treatment, MTT at the final concentration of 0.5 mg/mL was added to each well and incubated for 4 h at 37∘C. Then, the supernatant was removed and the formazan product was dissolved in 100 L dimethyl sulfoxide with stirring for 15 min on a microtiter plate shaker and the absorbance was read at 570 nm using a Spectramax M5 microplate reader. Evidence-Based Complementary and Alternative Medicine

2.4. Intracellular ATP Level Assay. Cellular ATP level was measured with an ATP bioluminescent assay kit according to the manufacturer’s instructions. Luminescence was monitored on a microplate reader in the luminescence mode following the addition of 100 L of luciferin and luciferase [20].

2.5. Measurement of Mitochondria Oxidation-Reduction Activity by Resazurin. After treatment, resazurin at final concen- tration of 5 M was added into the wells and fluorescence intensity was examined at an excitation of 530 nm and an emission of 590 nm [21].

2.6. Measurement of Mitochondrial Membrane Potential by JC-1. The mitochondrial membrane potential was measured using the JC-1 assay kit according to the manufacturer’s instructions. After treatment, the culture medium was removed and loaded with JC-1 solution for 15 min at 37∘C in the dark. The fluorescence intensity of the red/green ratio was determined at an excitation of 490 nm and emission of 530 nm (green fluorescent monomers) and 590 nm (red fluorescent aggregates), respectively [22].

2.7. Quantification of Cytochrome C Release. The cytosolic cytochrome C levels were measured using an ELISA kit according to the manufacturer’s instructions [23]. Briefly, after treatment, the cells were lysed and centrifugated at 1000 g for 15 min, and then the supernatant was collected as a cytoplasmic fraction and used for cytochrome C release measurements.

2.8. Determination of Caspase-3 Activity. Caspase-3 activity was performed according to the references with modification [24]. Briefly, after treatment, the lysis buffer wasaddedtoeach well and followed by centrifugation at 12 000 g for 10 min. Caspase-3 activity was tested in resulting supernatants by measuring the proteolytic cleavage of the fluorogenic substrate Ac-DVD-AMC with an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

2.9. Flow Cytometry Assay in PC12 Cells. After treatment, cells were trypsinized, washed twice with PBS, centrifuged at 1000 r/min for 5 min, and then fixed with 70% ethanol overnight at 4∘C. The cells were centrifuged and washed twice with PBS, resuspended in PBS (containing 50 g/mL RNaseA), stained with PI at the final concentration of 10 M for 30 min at room temperature in the dark, and read in a flow cytometer at 488 nm excitation [10].

2.10. Measurement of IL-1 and IL-6 Level by ELISA Assay. The cultured PC12 cells medium was collected after treatment, and the IL-1 and IL-6 concentrations were measured by commercially available ELISA kits according to the man- manufacturer's protocol.

2.11. Determination of Intracellular Reactive Oxygen Species Level. After incubation with 6-OHDA, cells were loaded with 10M H2DCFH-DA for 30 min at 37∘C in the dark. The fluorescence intensity of DCF was measured at an excitation

Evidence-Based Complementary and Alternative Medicine wavelength of 495 nm and emission wavelength of 530 nm on a microplate reader [25].

2.12. Statistical Analysis. All data are expressed as the mean ± SD. Statistical analysis of the results was carried out by one-way analysis of variance (ANOVA), followed by -test. Statistical significance was accepted at < 0.05.

3. Results

3.1. EffectofEchinacosideonMorphologyand Cell Viabilityin PC12 Cells Damaged by 6-OHDA. As shown in Figure 2(A), the viability of PC12 cells had no significant difference at the concentration of 0.1∼10 M of echinacoside treatment for

24 h. As shown in Figure 2(B), within 24 h of treatment with 6-OHDA alone, the majority of PC12 cells had undergone morphological changes such as membrane blebbing and cell shrinkage. Cotreatment with echinacoside protected the cells from 6-OHDA damage. MTT assay showed that treatment of PC12 cells with 6-OHDA alone resulted in an approximately 25% reduction in cell survival within 24 h, whereas cotreatment with 0.1 M, 1 M, and 10 M echinacoside showed a reduction of 6-OHDA-mediated cytotoxicity (all < 0.01, Figure 2(C)).

3.2. Effect of Echinacoside on Mitochondria Dysfunction Induced by 6-OHDA in PC12 Cells. Cellular ATP levels were significantly decreased following 24 h incubation with 100M 6-OHDA, whereas cotreatment with 1 Mand 10 M of echinacoside alleviated 6-OHDA-mediated ATP decrease anabolic changes in PC12 cells. The results showed that cotreatment with 1 Mand 10 M of echinacoside could significantly attenuate mitochondria oxidation-reduction activity impairment induced by 6-OHDA (both < 0.05 and Figure 3(b)).

As shown in Figure 3(c), treatment with 100 M of 6- OHDA for 24 h resulted in a significant decrease of mitochondrial membrane potential compared with the untreated group ( < 0.01). However, the decrease of mitochondrial membrane potential induced by 6-OHDA was significantly attenuated by cotreatment with 0.1 M, 1 M, and 10 M of echinacoside (all < 0.01).

3.3. Anti-Mitochondria-Mediated Apoptosis of Echinacoside in PC12 Cells against 6-OHDA Neurotoxicity. The results showed that 6-OHDA exposure resulted in an increase in cytosolic cytochrome C by 167% over the untreated group ( < 0.01). Echinacoside at the dose of 0.1 M, 1 M, and 10M could significantly reduce the cytochrome C release into cytosol induced by 6-OHDA ( < 0.05, < 0.01, and < 0.01,resp.; Figure 4(a)).

Caspase-3 activity was increased by about 3.3-fold compared with the untreated group after 6-OHDA exposure ( < 0.01). In contrast, cotreatment with 1 M and 10 M of echinacoside showed a significant decrease in caspase-3 activity compared with 6-OHDA-treated cells, respectively (both < 0.05 and Figure 4(b)).PI is membrane permeability and is generally excluded from viable cells. So it is commonly used for identifying dead cells in a population. The results showed that the apoptosis rate was significantly increased following 24 h incubations with 100 M 6-OHDA, whereas cotreatment with 1 and 10 M echinacoside significantly reduced 6-OHDA-induced apoptosis ( < 0.05, < 0.01,resp., Figure 4(c)).

3.4. Anti-Inflammatory Effect of Echinacoside in PC12 Cells Induced by 6-OHDA. The results showed that 6-OHDA exposureresultedinanincreaseinIL-1 level compared with untreated group ( < 0.05; Figure 4(a)). However, 10 M of echinacoside could significantly reduce IL-1 release induced by 6-OHDA ( < 0.05; Figure 5(a)).

IL-6 level was also significantly increased compared with untreated group ( < 0.01). However, cotreatment with 0.1, 1, and 10 M of echinacoside all significantly decreased IL-6 release compared with 6-OHDA-treated cells (both < 0.05 and Figure 5(b)).

3.5. Effect of ECH on ROS Production Induced by 6-OHDA in PC12 Cells. Cells treated with 6-OHDA showed a significant increase (about 1.4-fold) of intracellular ROS compared with untreated cells ( < 0.01). This increase was significantly attenuated by coincubation with 0.1, 1, and 10 M echinacoside ( < 0.05, < 0.01,and < 0.01,resp.; Figure 6).

4. Discussion

Echinacoside is one of the principal constituents of phenylethanoid glycosides extracted from a famous traditional reported echinacoside has anti-apoptotic and neuroprotective properties in neurodegenerative disease [18]. In vivo study reported that echinacoside had neuroprotective effects and behavioral improvement in the mouse model of MPTP- induced dopaminergic neuronal damage, while further investigation revealed that echinacoside induced downregulation of caspase-3 and caspase-8 activation in MPP+-induced apoptosis of cerebellar granule neurons [16]. Another in vivo study reported the neuroprotective effects of echinacoside on the striatal dopaminergic neurons injured by 6-OHDA [17]. However, the cellular and molecular mechanisms that underlie these actions are not fully understood, especially for their influence on mitochondrial function and inflammation responses. So our current study investigated the effects of echinacoside on mitochondrial function and inflammation responses to elucidate its mechanism against PD. The results firstly showed that echinacoside significantly alleviated 6-OHDA-induced neurotoxicity in PC12 cells through protecting mitochondrial function and anti-inflammation.

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkin- son’s disease and its experimental models [26]. Mitochondria are deeply involved in the production of reactive oxygen species (ROS) through the electron carriers of the respiratory chain in neurodegenerative diseases [27]. A wide variety of ROS are produced in the course of the normal metabolism in biological systems, but their accumulation beyond the

required amount can potentially damage lipids, proteins, and nucleic acids.

6-OHDA, a catecholamine analog, causes specific degeneration of substantia nigra neurons. Neurotoxicity induced by 6-OHDA is, in part, due to the production of ROS [28–32].

OHDA leads to neuronal death by affecting mitochondria function, including abatement in ATP production, disturbance of ROS metabolism, and the induction of apoptosis [10, 33, 34]. In the present study, our results also demonstrated that 6-OHDA damaged mitochondrial oxidative-reduction function, decreased mitochondrial membrane potential, induced mitochondria-mediated apoptosis, and increased ROS production in PC12 cells.

Resazurin is a fluorescent indicator that is intermediate only between final reductions of molecular oxygen and cytochrome oxidase. Therefore, the oxidative-reduction potential of resazurin allows the respiratory chain to function to near completion, which will provide a more accurate and sensitive indication of mitochondrial function. JC-1 is a membrane potential-sensitive probe that localizes to the inner mitochondrial membrane where it forms either monomers (green) or aggregates (red) based on mitochondrial membrane potential. Thus, resazurin and JC-1 are valuable analytical tools for examining mitochondrial function [35]. The present study indicated that decreases in mitochondrial oxidative-reduction activity and mitochondrial membrane potential may play important roles in 6-OHDA- induced dopaminergic neurotoxicity. Our result also showed mitochondrial oxidative-reduction activity and mitochondrial membrane potential. These results provide that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.

It is generally accepted that cell apoptosis induced by 6- OHDA is mediated by mitochondrial dysfunction, characterized by cytochrome C release, and caspase-3 activation [36]. An apoptotic stimulus triggers the release of cytochrome C from the mitochondria into cytosol where it then activates caspase-3 and other downstream caspases. In the present study, the results showed that PC12 cells exposed to 100 M of 6-OHDA enhanced cytochrome C release and caspase dependently inhibited cytochrome C release and caspase- 3 activations. These results suggest that echinacoside could inhibit mitochondria-mediated apoptosis by reducing cytochrome C release from mitochondria to cytosol and caspase-3 activation.

Inflammation is a neuropathological feature of Parkinsonian brains and also in experimental models of the disease. Therapeutic strategies geared toward reducing inflammation and inhibiting the production of this inflammatory factors may be a promising neuroprotective strategy for the treatment of Parkinson’s disease and related conditions [37]. Several studies also performed in the cerebrospinal fluid and during postmortem analysis in Parkinsonian patients revealed increased levels of cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), suggesting the activation of aproinflammatoryresponse[38, 39]. In the present study, the resultsshowedthatexposureto100 Mof6-OHDAinduced IL-1 and IL-6 release in PC12 cells. However, treatment with echinacoside could dose-dependently inhibit IL-1 and IL-6 release induced by 6-OHDA. The results suggest that reducing inflammatory responses are also involved in the mechanisms of echinacoside against 6-OHDA-induced neurotoxicity.

In conclusion, the results of this research showed that echinacoside was able to block 6-OHDA-induced neurotoxicity in PC12 cells, and the mechanisms may be related to mitochondrial protection and anti-inflammation via reducing ROS production. Information provided by this work will be useful to elucidate the mechanism of echinacoside for anti- PD. It is concluded that echinacoside may be a promising therapeutic option for the prevention and treatment of PD.

Conflict of Interests

The authors have declared no conflict of interests

Acknowledgments

This work was supported by Grants from the National Natural Science Foundation of China (81473383) and the National Science and Technology Major Project (2012ZX09103101-078; 2013ZX09103-001-008; and 2013ZX09102106).