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Echinacoside Protects against 6-Hydroxydopamine-Induced Mitochondrial Dysfunction and Inflammatory Responses in PC12 Cells via Reducing ROS Production.

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 may be involve in attenuating mitochondrial dysfunction and inflammatory responses by reducing ROS production.


Introduction
Parkinson's disease (PD) is a neurodegenerative disorder characterized by 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 the neuronal loss in PD [5]. 6-Hydroxydopamine (6-OHDA) is specific to dopaminergic neurons in intrastriatal rodent models and in some cells [6][7][8][9][10][11]. PC12 cell has become a popular cell model for PD research because this cell line posses many characateristics 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 stems of Cistanche salsa [12], a parasitic plant native to northwest China, which is used as a traditional Chinese herbal medicine with antisenile and antifatigue effects [13][14][15]. Several sets of data have also proved that echinacoside may be useful in the treatment of PD [16][17][18]. However, the cellular and molecular mechanisms are not fully understood. The present study was to investigate the underlying mechanisms of echinacoside against 6-OHDAinduced damage in PC12 cells.

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% CO 2 [19]. The cells were plated into 96-well plates at a density of 2 × 10 5 cells/mL. After 80% confluence, the cells were preincubated with different concentration 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 24 h at 37 ∘ C.

Morphological Changes and Viability Assay of PC12 Cells.
After treatment, morphological changes were observed under 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.

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 100 L of luciferin and luciferase [20].

Measurement of Mitochondria
Oxidation-Reduction Activity by Resazurin. After treatment, resazurin at final concentration 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].

Measurement of Mitochondrial Membrane Potential by
JC-1. The mitochondrial membrane potential was measured using 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].

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 cytoplasmic fraction and used for cytochrome C release measurements.

Determination of Caspase-3 Activity.
Caspase-3 activity was performed according to the references with modification [24]. Briefly, after treatment, the lysis buffer was added to each 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-DEVD-AMC with an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

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].

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 manufacturer's protocol.

Determination of Intracellular Reactive Oxygen Species
Level. After incubation with 6-OHDA, cells were loaded with 10 M H 2 DCFH-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 3 wavelength of 495 nm and emission wavelength of 530 nm on a microplate reader [25].

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.
Resazurin was used here to detect mitochondrial metabolic changes in PC12 cells. The results showed that cotreatment with 1 M and 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 significant decrease of mitochondrial membrane potential compared with 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).
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)).

Discussion
Echinacoside is one of the principal constituents of phenylethanoid glycosides extracted from a famous traditional Chinese medicine, Herba Cistanches. Several studies have reported echinacoside has antiapoptotic and neuroprotective properties in neurodegenerative disease [18]. In vivo study reported that echinacoside had neuroprotective effects and behavioral improvement in the mouse model of MPTPinduced 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 Parkinson'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 analogue, causes specific degeneration of substantia nigra neurons. Neurotoxicity induced by 6-OHDA is, in part, due to the production of ROS [28][29][30][31][32]. Previous studies have determined that treatment with 6-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-OHDAinduced dopaminergic neurotoxicity. Our result also showed that echinacoside alleviated 6-OHDA-induced decreases of mitochondrial oxidative-reduction activity and mitochondrial membrane potential. These results provide that 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 exposure to 100 M of 6-OHDA enhanced cytochrome C release and caspase-3 activation. However, treatment with echinacoside dose dependently inhibited cytochrome C release and caspase-3 activation. These results suggest that echinacoside could inhibit mitochondria-mediated apoptosis through 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 these 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 a proinflammatory response [38,39]. In the present study, the results showed that exposure to 100 M of 6-OHDA induced 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.