Neuroprotective effects of Lycium chinense Miller against rotenone-induced neurotoxicity in PC12 cells.

The American Journal of Chinese Medicine, Vol. 41, No. 6, 1343–1359 © 2013 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X13500900

Am. J. Chin. Med. 2013.41:1343-1359. Downloaded from www.worldscientific.com by 169.230.243.252 on 11/20/14. For personal use only.

Neuroprotective Effects of Lycium chinense Miller Against Rotenone-Induced Neurotoxicity in PC12 Cells A-Rang Im,* Young-Hwa Kim,* Md Romij Uddin,*,† Sungwook Chae,* Hye Won Lee,* Yeong Shik Kim‡ and Mi Young Lee* *KM-Based

Herbal Drug Research Group Korea Institute of Oriental Medicine Daejeon 305–811, South Korea †

Department of Crop Science Chungnam National University Daejeon 305–764, South Korea ‡Natural

Products Research Institute College of Pharmacy, Seoul National University Seoul 151–742, South Korea

Abstract: Rotenone, an inhibitor of mitochondrial complex I, has been widely regarded as a neurotoxin because it induces a Parkinson’s disease-like syndrome. The fruit and root bark of Lycium chinense Miller have been used as traditional medicines in Asia to treat neurodegenerative diseases. In this study, we examined the neuroprotective effects of Lycium chinense Miller extracts in rotenone-treated PC12 cells. Treatment with rotenone reduced PC12 cell viability and cellular ATP levels. Conversely, caspase 3/7 activity, the ratio of Bax: Bcl-2 expression levels, mitochondrial superoxide level, and intracellular calcium (Ca 2þ Þ concentration were elevated. Pretreatment with Lycium chinense Miller extracts significantly increased cell viability and ATP levels. Additionally, they attenuated caspase activation, mitochondrial membrane depolarization and mitochondrial superoxide production. Moreover, confocal microscopy showed that the mitochondrial staining pattern was restored from that of extracts treated cells and that the increase in intracellular Ca 2þ level was blunted by treatment with the extracts. Our results suggest that Lycium chinense Miller extracts may have the possible beneficial effects in Parkinson’s disease by attenuating rotenone induced toxicity. Keywords: Lycium chinense Miller; Neuroprotection; Rotenone; PC12 Cell; Mitochondrial Dysfunction.

Correspondence to: Dr. Mi Young Lee, KM-Based Herbal Drug Research Group, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 305–811, Korea. Tel: (þ82) 42-868-9504, Fax: (þ82) 42863-9301, E-mail: [email protected]

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Introduction As life expectancy has increased, so too has the incidence of age-related neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases (Borgesius et al., 2011). The results of many studies implicate mitochondrial dysfunction in mediating cell death in these disorders. Mitochondrial dysfunctions play an important role in the neuronal decay. Exposure to mitochondrial toxins, such as the complex I inhibitor rotenone and complex II inhibitor 3-nitropropionic acid, recapitulate some of the neuropathological features of neurodegenerative diseases (Kwong et al., 2006). The important role of mitochondrial bioenergetics and the unique trajectory of alterations in brain metabolic capacity enable a bioenergetic-centric strategy that targets disease stage specific pattern of brain metabolism for disease prevention and treatment (Moreira et al., 2010; Reddy and Reddy, 2011). Exposure of rats to rotenone, a mitochondrial complex I inhibitor, recapitulates some of the neuropathological features of Parkinson’s disease (Caboni et al., 2004; Uversky, 2004). In particular, inhibition of complex I activity results in reactive oxygen species (ROS) generation, ATP inhibition and mitochondrial membrane depolarization, leading to the death of neurons in the substantia nigra (Mizuno et al., 1995; Wang et al., 2005). Plant extracts have diverse functions and have been used throughout history to treat many diseases. In particular, both the fruit and root bark of Lycium chinense Miller have been used in traditional medicines in China, Korea and Japan, demonstrated in reports on the hypotensive, hypoglycemic, and antipyretic activities in animal models (Potterat, 2010; Lee et al., 2004). They have also been used as anti-aging therapies and as treatments for neurodegenerative disease (Ho et al., 2010; Potterat, 2010). Recently, Lycium chinense fruits have been shown to exert neuroprotective effects on trimethyltin-induced learning and memory deficits in rats (Park et al., 2011). The purpose of the study was to determine the neuroprotective effect of Lycium chinense Miller extracts against rotenone-induced neurotoxicity in PC12 cells. We evaluated the effects of Lycium chinense Miller extracts on cellular energy metabolism, mitochondrial membrane potential, mitochondrial superoxide production and changes in the level of proteins associated with caspase-mediated apoptosis.

Materials and Methods Materials Rotenone was purchased from Sigma Chemical (St. Louis, MO, USA). RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). Collagen IV-coated plates (96-well plates) were purchased from BD Biosciences (Bedford, MA). CellTiter Aqueous One Solution Cell proliferation assay kit (MTS) and Homogeneous Caspase-3/7 Assay kits were purchased from Promega Co. (Madison, USA). Luminescence ATP detection kit was purchased from PerkinElmer (Waltham, MA, USA) and JC-1 mitochondrial membrane potential detection kit from Biotium (Hayward, CA). Mitotracker, MitoSOX, Fluo-4/AM, and Annexin-V and PI double staining kit were purchased from

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Invitrogen Molecular Probes (San Diego, CA). Primary antibodies against cleaved caspase-3, caspase-3, cleaved caspase-7, caspase-7, cleaved caspase-9, caspase-9, and actin were purchased from Cell Signaling Technology (Danvers, MA, USA). p53 Primary antibody was obtained from Abcam Ltd (Cambridge, UK). Bcl-2, Bax, cytochrome c, and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).


Preparation of Lycium chinense Miller Extracts Aqueous and ethanol fruit extracts of Lycium chinense Miller were prepared by sonicating the dried ground powder (50 g, aqueous; 60 g, ethanol) suspended in distilled water and 80% ethanol, respectively, for 3 h. This procedure was repeated two times. The suspensions were lyophilized, yielding 26.8 g (water extract) and 27.6 g (ethanol extract). The extraction procedure for the root bark was the same as that described for the fruit. Aqueous and 80% ethanol extractions from 150 g and 200 g dried ground powder yielded 27.4 g and 25.2 g of the product, respectively. Individual extracts are hereafter referred to as Lycium chinense Miller root bark aqueous (LCRW), Lycium chinense Miller root bark ethanol (LCRE), Lycium chinense Miller fruit aqueous (LCFW) and Lycium chinense Miller fruit ethanol (LCFE). Cell Viability PC12 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere of 95% air and 5% CO2 at 37 C. Cells were plated at a density of 1 10 4 cells/well in 96-well collagen IV-coated plates and incubated at 37 C for 24 h. LCRW, LCRE, LCFW, and LCFW were dissolved in 5% DMSO. Cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone], 10, 50, or 100 or 200 g/ml, respectively) for 24 h, followed by treatment with 5 M rotenone for 1 h. Cell viability was determined based on the reduction of (3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) inner salt (MTS) to formazan according to the manufacturer’s instruction. After removing the medium, 200 l of RPMI containing MTS was added to each well and then incubated at 37 C for 1 h. The resulting sample was assayed at 490 nm by using a microplate fluorometer (Molecular Devices, Sunnyvale, CA, USA). Annexin V Externalization Assay for Apoptosis Cell apoptosis was determined by flow cytometry using Annexin V-FITC and propidium iodide (PI) double staining according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). PC12 cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone], 10, 50, or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. After trypsinization, cells were washed in PBS and re-suspended in 1 annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2; pH 7.4). The cell suspension was then incubated with 5 l of annexin V-conjugated fluorescein isothiocyanate (FITC) and 1 l PI

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for 15 min in the dark. Ten thousand cells were counted using a FACSCalibur flow cytometer, and data were analyzed using CellQuest software (Becton Dickinson, San Jose, CA).


Caspase-3-Like Activity PC12 cells (510 4 Þ were seeded in 96-well white plates. Cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone], 10, 50, or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Caspase-3-like activity was measured using an ApoONE TM Homogeneous Caspase 3/7 Assay Kit according to the manufacturer’s instructions. Cells were lysed in 100 l of Homogeneous Caspase-3/7 buffer containing the caspase-3 substrate, and the cell lysates were incubated for 1 h at room temperature. Caspase-3/7 was determined using a luminometer. ATP Measurement PC12 cells were cultured in 96-well white plates. Cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Total cellular ATP content was determined using a luminescence ATP detection kit and a luminometer. The ATP content was determined by running an internal standard. Mitochondrial Membrane Potential (MMP) JC-1 Mitochondrial Membrane Potential Detection Kit was used to measure the mitochondrial membrane potential in PC12 cells. Cells were seeded at a density of 110 4 cells/well in 96-well white plates for detecting fluorescence. Cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Cells were then were incubated with a 1JC-1 staining solution at 37 C for 20 min and rinsed with PBS. Red fluorescence (excitation: 550 nm; emission: 600 nm) and green fluorescence (excitation: 485 nm; emission: 535 nm) were determined using a Softmax Pro 5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). The ratio of green:red fluorescence, an index of mitochondrial membrane depolarization, was increased in dead cells and in cells undergoing apoptosis. For confocal microscopy, 110 5 cells were plated on chamber slides. Cells were imaged using an FV10i-LIV confocal microscope (Olympus, Tokyo, Japan). In live non-apoptotic cells, mitochondria appear red following aggregation of JC-1 reagent (excitation: 559 nm; emission: 570–620 nm). In apoptotic and dead cells, the dye remains in its monomeric form and appears green (excitation: 473 nm; emission: 490–540 nm). Confocal Microscopy Analysis PC12 cells were plated at 510 4 cells on a chamber slide. Cells were treated with various concentrations of LCRW, LCRE, LCFW, or LCFE for 24 h before addition 5 M of



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rotenone for 1 h. The medium was removed and cells were washed with PBS. Cells were incubated for 45 min with 10 M Mitotracker Red and 1 g/ml DAPI to detect mitochondria. Excitation/emission spectra for Mitotracker Red and DAPI are 578/598 nm and 359/461 nm, respectively. MitoSOX was used to measure mitochondrial superoxide. Cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Cells were then incubated with 5 M MitoSOX Red for 20 min at 37 C. The excitation/emission spectra for MitoSOX Red is 510/580 nm. The Ca 2þ indicator dye Fluo-4/AM was used to assess the cytoplasmic calcium concentration. After treatment with extracts and rotenone, cells were incubated with Fluo-4/AM at room temperature for 30 min. Fluo-4/AM has excitation/emission maxima of 488/520 nm used by confocal microscopes. Western Blotting PC12 cells were pretreated with LCRW, LCRE, LCFW, or LCFE (0 [rotenone alone] or 100 g/ml for 24 h, followed by 5 M rotenone for 1 h. Cells were lysed and samples (20 g protein/lane) were electrophoresed on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to PVDF membranes. Membranes were blocked for 1 h at room temperature in 5% skim milk solution. Blots were incubated overnight at 4 C with a 1:1000-dilution of monoclonal antibody. The blots were washed three times for 5 min each time with PBS-T. The membranes were then incubated for 2 h with secondary antibody and washed three times for 5 min each time with PBS-T. Proteins were detected using an enhanced chemiluminescence (ECL solution). Statistical Analysis All data are expressed as meanstandard deviation (S.D.) of at least three independent experiments. Data was normalized as a percentage of the control. Data was analyzed by one-way ANOVA, followed by multiple comparisons utlizing the Student’s t-test. A criterion of p < 0:05 was considered significant.

Results Cell Viability and MTS Assay In pilot studies, we found that 1 h treatment of PC12 cells with 5 M rotenone was sufficient to reduce cell viability by 50% (data not shown). Rotenone induced significant cytotoxicity (Fig. 1); both LCRW and LCRE (Fig. 1A) and LCFW and LCFE (Fig. 1B) extracts reduced this effect. Both fruit and root bark ethanolic extracts showed higher cell viability than that of water extracts, increasing cell viability by 200 g/ml for LCRE (81.9% viability compared to control) and 100 g/ml for LCFE (81.3% viability compared to control).


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(A)

(B) Figure 1. Neuroprotective effect of Lycium chinense extracts on rotenone-treated PC12 cells. PC12 cells were pretreated with (A) Lycium chinense Miller root bark water extract (LCRW) or Lycium chinense Miller root bark ethanol extract (LCRE) and (B) Lycium chinense Miller fruit water extract (LCFW), or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Control cells received neither Lycium chinense extract nor rotenone. Data is expressed as a percentage of control (mean S.D.). *p < 0:05.

Apoptosis of Cells Very few control cells were Annexin-V- or PI-positive cells. Conversely, rotenone treatment yielded 35.38% and 32.74% apoptotic cells (lower right and upper right quadrants representing early and late apoptosis, respectively). However, pretreatment with each of the four extracts increased the live cell populations and decreased the apoptotic and dead cell populations (Fig. 2A). Expression of the pro-apoptotic proteins p53 and Bax was increased by rotenone in PC12 cells, whereas that of the anti-apoptotic protein Bcl-2 was

Figure 2. Representative flow cytometric (A) and western blot (B) analyses. (A) Distribution of PC12 cells by using dual parameter dot plot of FITC-labeled Annexin V versus propidium iodide (PI) fluorescence, shown as logarithmic fluorescence intensity. PC12 cells were pretreated with Lycium chinense Miller root bark water extract (LCRW), Lycium chinense Miller root bark ethanol extract (LCRE), Lycium chinense Miller fruit water extract (LCFW), or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Quadrants refer to: live cells (lower left), early apoptotic cells (lower right), necrotic cells (upper left) and late apoptotic cells (upper right). (B) Left: Representative western blot of p53, Bcl-2, and Bax protein expression in PC12 cells; β-actin served as the loading control. PC12 cells were pretreated with LCFW, LCFE, LCRW, or LCRE (0 [rotenone alone] or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Right: Quantitative analysis was performed by measuring the intensity relative to the control.

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Figure 2. (Continued )

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decreased; this resulted in an increase in the ratio of Bax:Bcl-2 (1.94 relative to control) (Fig. 2B). However, Bcl-2 expression was higher following pretreatment with the extracts, thus decreasing the ratio of Bax:Bcl-2. Pretreatment with 100 g/ml LCFW was most effective, lowering the ratio of Bax:Bcl-2 to 0.48 of control.


Caspase-3-Like Activity Rotenone caused an increase of caspase-3-like activity to 145.5% of control (Fig. 3A). Pretreatment with each of the four extracts prevented the rotenone-induced increase in caspase-3-like activity. Effects with LCRW or LCRE were dose-dependent. A maximal effect with LCRW or LCRE was achieved at the lowest concentration tested (100 g/ml). In western blot analysis, rotenone-induced cells enhanced the presence of activated caspase-3, 7, and 9 while LCRE attenuated apoptosis. It was also found that rotenone caused a progressive release of cytochrome c from the mitochondria into the cytosol after 1 h treatment (Fig. 3B). The amount released from the mitochondria increased in rotenonetreated cells but weakly detected in extracts treated cells. Cellular Energy Metabolism Mitochondria provide most of the energy needed for cellular functions by the conversion of energy as fuel into ATP through oxidative phosphorylation. We investigated whether

(A) Figure 3. (A) Effect of Lycium chinense extracts on rotenone-induced caspase 3/7 activity in PC12 cells. PC12 cells were pretreated with Lycium chinense Miller root bark water extract (LCRW), Lycium chinense Miller root bark ethanol extract (LCRE), Lycium chinense Miller fruit water extract (LCFW), or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone], 10, 50, or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Caspase-3/7 activity was assessed using the luminescence-based caspase 3/7 activity kit. (B) Representative western blot of mitochondrion-dependent apoptotic proteins in rotenone-treated PC12 cells. β-actin served as the loading control. PC12 cells were pretreated with LCFW, LCFE, LCRW, or LCRE (0 [rotenone alone] or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Data in (A) are expressed as a percentage of control (mean S.D.). *p < 0:05.


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(B) Figure 3. (Continued )

protective effects of extracts impairs cellular ATP production, which might ultimately lead to cell death, by measuring ATP levels in PC12 cells. Compared with control cells, ATP levels in rotenone-treated cells were decreased (Fig. 4). Pretreatment with any of the extracts attenuated this effect. LCRE (200 g/ml) was most effective. The ATP levels were increased to 107.7% of control.

(A) Figure 4. Effects of Lycium chinense extracts on total cellular ATP levels in PC12 cells. PC12 cells were pretreated with (A) Lycium chinense Miller root bark water extract (LCRW) or Lycium chinense Miller root bark ethanol extract (LCRE) and (B) Lycium chinense Miller fruit water extract (LCFW) or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Control cells received neither Lycium chinense extract nor rotenone. Data are expressed as a percentage of control (mean S.D.). *p < 0:05.



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MMP To assess the effect of extracts on rotenone-induced oxidative stress, we determined MMP. We used JC-1 dye, since it is capable of entering selectively into mitochondria and can reversibly change its color from green to red as the membrane potentials increases, whereas cells with low MMP maintain green fluorescence. Cells treated with 5 M rotenone showed decreased red fluorescence intensity and increased green fluorescence intensity compared with untreated control. This indicates a decrease in level of MMP up to 46.1% (Fig. 5), which suggests that rotenone results in mitochondrial membrane depolarization. Pretreatment with extracts yielded significantly greater MMP, increasing MMP to near baseline levels (200 g/ml LCRW, 84.2% of control values; 200 g/ml of LCFE, 79.2% of control values).

(A) Figure 5. Effects of Lycium chinense extracts on rotenone-mediated decrease in mitochondrial membrane potential (MMP) in PC12 cells. (A) PC12 cells were pretreated with Lycium chinense Miller root bark water extract (LCRW), Lycium chinense Miller root bark ethanol extract (LCRE), Lycium chinense Miller fruit water extract (LCFW) or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone], 10, 50, 100, or 200 g/ml) for 24 h, followed by 5 M rotenone for 1 h. JC-1 Mitochondrial Membrane Potential Detection Kit. (B) Confocal images of JC-1 fluorescence (603.5). Green fluorescence (JC-1 monomers) reflects depolarized mitochondria. Red fluorescence (JC-1 aggregates) reflects polarized mitochondria. Data in (A) are expressed as a percentage of control (meanS.D.). *p < 0:05.


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MitoSOX MitoSOX Red mitochondrial superoxide indicator was used for detection of mitochondrial superoxide. Cellular MitoSOX fluorescence intensities were compared using confocal (Fig. 6). Mean intensity of MitoSOX fluorescence in rotenone-treated cells increased to

(A) Figure 6. Effects of Lycium chinense extracts on rotenone-mediated mitochondrial superoxide production in PC12 cells. (A) PC12 cells were pretreated with Lycium chinense Miller root bark water extract (LCRW), Lycium chinense Miller root bark ethanol extract (LCRE), Lycium chinense Miller fruit water extract (LCFW), or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone], 10, 50, or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Mitochondrial superoxide production was measured using MitoSOX Red. (B) Determination of mitochondrial superoxide production measured by confocal microscopy (601.0 and 603.5). PC12 cells were pretreated with LCFW, LCFE, LCRW, or LCRE (0 [rotenone alone] or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Data in (A) are expressed as a percentage of control (meanS.D.). *p < 0:05.



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185.6% of that in control cells (Figs. 6A and 6B). All foure extracts significantly reduced the effect of rotenone (Fig. 6A). Quantification of these results show that extracts protected from rotenone-induced cell death. Mitotracker and Calcium Ion Staining Confocal microscopy analysis revealed an even distribution of mitochondrial staining throughout control cells (Fig. 7A). Rotenone-treated cells exhibited reduced red fluorescence intensity in mitochondria, suggesting depolarization of the inner mitochondrial

(A) Figure 7. Confocal microscopy analysis of mitochondria (A) and intracellular calcium (Ca 2þ Þ (B) in PC12 cells. (A) PC12 cells were pretreated with Lycium chinense Miller root bark water extract (LCRW), Lycium chinense Miller root bark ethanol extract (LCRE), Lycium chinense Miller fruit water extract (LCFW), or Lycium chinense Miller fruit ethanol extract (LCFE) (0 [rotenone alone] or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Mitotracker red fluorescence indicates mitochondria (upper row). Merged images indicate colocalization with DAPI blue fluorescence (603.5) (lower row). (B) PC12 cells were pretreated with LCFW, LCFE, LCRW, or LCRE (0 [rotenone alone] or 100 g/ml) for 24 h, followed by 5 M rotenone for 1 h. Intracellular Ca 2þ level was determined using the Ca 2þ indicator dye Fluo-4/AM.


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membrane. Following pretreatment with each of the extracts, the mitochondrial staining pattern was similar to that of control cells. The extracts also increased the membrane permeability to DAPI (Fig. 7A). Rotenone caused an increase in the intracellular Ca 2þ level; pretreatment with each of the extracts attenuated the rotenone-induced increase in Fluo-4 fluorescence (Fig. 7B). Discussion Rotenone, a toxin that inhibits mitochondrial complex I, causes apoptosis via activation of the caspase-3 pathway (King et al., 2001). Rotenone-induced apoptosis in PC12 cells was associated with caspase-3 activation, cellular ATP depletion, mitochondrial membrane depolarization, and mitochondrial superoxide production. Pretreatment of cells with Lycium chinense Miller extracts attenuated or completely prevented these effects. The Bcl-2 family of proteins plays a prominent role in neuronal apoptosis by regulating mitochondrial membrane permeability. Specifically Bax and Bad promote membrane permeability transition, whereas Bcl-2 and Bcl-XL prevent it. Thus, the ratio of specific Bcl-2 family members is a critical determinant as to whether cell death or protection occurs (Mooney and Miller, 2000; Jung et al., 2007). Our data revealed an increase in the expression of Bcl-2 in PC12 cells pretreated with Lycium chinense extracts and a decrease in Bax expression, resulting in a decrease in the ratio of Bax: Bcl-2 (Fig. 2B). This suggests that the up-regulation of Bcl-2 and down-regulation of Bax is associated with the neuroprotective effects of these extracts. The execution of neuronal apoptosis involves relatively few pathways that converge on activation of the caspase. The release of mitochondrial cytochrome c and apoptosis-inducing



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factor are key events in initiating the cascade of reactions leading to apoptotic cell death. Caspase-3 is considered a key effector molecule in the apoptotic cell death cascade. Caspase may play an important role in neuronal cell death, during development as well as after neuronal injury (Cregan et al., 1999). Rotenone caused mitochondrial dysfunction as evidenced by the release of cytochrome c from mitochondria to cytosol, which in turn activates caspase-9 and caspase-3. We found that extracts may inhibit apoptotic cell death caused via the mitochondrial pathway because extracts inhibited caspase-9 and -3 activity, and the cytosolic release of cytochrome c. Pretreatment of extracts blocked the increase of cleaved caspase-3 induced by rotenone, suggesting that induction of extracts is associated with the activation of the caspase-3 signaling pathway, which contributes to neuronal cell death. Disruption of outer mitochondrial membrane integrity, a key early event in apoptosis, leads to mitochondrial membrane depolarization. In turn, the mitochondrial membrane potential is essential for ATP synthesis and maintenance of oxidative phosphorylation. This provides a causal link between the rotenone-induced increase in the ratio of Bax:Bcl-2 and ATP depletion, leading to cell death. In our study, the normalization of the Bax:Bcl-2 ratio by Lycium chinense Miller extracts was associated with preventing mitochondrial membrane depolarization and ATP depletion. Our results do not identify the specific site of action of the extracts. However, the results did demonstrate that their ability to preserve the balance of Bax and Bcl-2 expression leads to maintaining proper mitochondrial function. Otherwise, dysfunction along the mitochondrial respiratory chain will lead to reduced ATP production and increased ROS generation. Both of which are commonly seen mitochondrial pathologies in neurodegenerative diseases (Du and Yan, 2010). Plant extracts have long been used to treat disease. However, recent studies have focused on determining the mechanism by which these extracts exert their neuroprotective effects. In particular, Camellia sinensis and Erigeron breviscapus extracts reduced hydrgen peroxide-induced toxicity in PC12 cells (Lopez and Calvo, 2011; Hong and Liu, 2004). Smilacis chinae Rhizome exhibited a neuroprotective effect on N-methyl-D-aspartateinduced neurotoxicity in vitro and on focal cerebral ischemia in vivo (Ban et al., 2008). These authors also reported that resveratrol and oxyresveratrol isolated from Smilacis chinae Rhizome produced similar effects, suggesting that these might be the active components of Smilacis chinae Rhizome that are responsible for neuroprotection. The present study demonstrates that Lycium chinense Miller extracts have a neuroprotective effect against rotenone-induced apoptosis in PC12 cells. Identification of the active chemical moiety may serve as a starting point for developing neuroprotective therapeutics to treat neurodegenerative disease. Acknowledgments This research was supported by the “Study of aging-control by energy metabolism based on oriental medicine (K12101)” funded by “KM-Based Herbal Drug Research Group” of Korea Institute of Oriental Medicine.

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[Two withanolides from Lycium chinense].

Protective effects of ginsenoside Rg1 against colistin sulfate-induced neurotoxicity in PC12 cells.

Neuroprotective effects of 20(S)-protopanaxadiol against glutamate-induced mitochondrial dysfunction in PC12 cells.

Activity-guided isolation of NF-κB inhibitors and PPARγ agonists from the root bark of Lycium chinense Miller.

Supercritical fluid extract of Lycium chinense Miller root inhibition of melanin production and its potential mechanisms of action.

Quality control of Lycium chinense and Lycium barbarum cortex (Digupi) by HPLC using kukoamines as markers.

β-catenin signaling pathway against Aβ -induced tau protein over-phosphorylation in PC12 cells.

Chemically Bonding of Amantadine with Gardenamide A Enhances the Neuroprotective Effects against Corticosterone-Induced Insults in PC12 Cells.

Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves.

Discrimination of Lycium chinense and Lycium barbarum by taste pattern and betaine analysis.

Neuroprotective effects of diarylpropionitrile against β-amyloid peptide-induced neurotoxicity in rat cultured cortical neurons.

Paeonolum protects against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells.

Ligularia fischeri extract protects against oxidative-stress-induced neurotoxicity in mice and PC12 cells.

The neuritogenic and neuroprotective potential of senegenin against Aβ-induced neurotoxicity in PC 12 cells.

Neuroprotective effects of α-melanocyte-stimulating hormone against the neurotoxicity of 1-methyl-4-phenylpyridinium.

Paeoniflorin exerts neuroprotective effects against glutamate‑induced PC12 cellular cytotoxicity by inhibiting apoptosis.

Low Doses of Camptothecin Induced Hormetic and Neuroprotective Effects in PC12 Cells.

Potential neuroprotective effects of SIRT1 induced by glucose deprivation in PC12 cells.

Phytoestrogen β-Ecdysterone Protects PC12 Cells Against MPP+-Induced Neurotoxicity In Vitro: Involvement of PI3K-Nrf2-Regulated Pathway.

Effects of (-)-sesamin on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and dopaminergic neuronal cells of Parkinson's disease rat models.

The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

The Aqueous Extract of Rhizome of Gastrodia elata Protected Drosophila and PC12 Cells against Beta-Amyloid-Induced Neurotoxicity.

Rosiglitazone synergizes the neuroprotective effects of valproic acid against quinolinic acid-induced neurotoxicity in rats: targeting PPARγ and HDAC pathways.

LchERF, a novel ethylene-responsive transcription factor from Lycium chinense, confers salt tolerance in transgenic tobacco.