International Immunopharmacology 22 (2014) 151–159
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Notoginsenoside R1 attenuates amyloid-β-induced damage in neurons by inhibiting reactive oxygen species and modulating MAPK activation Bo Ma a, Xiangbao Meng a, Jing Wang a, Jing Sun a, Xiaoyu Ren a,b, Meng Qin a, Jie Sun a, Guibo Sun a,⁎, Xiaobo Sun a,⁎ a b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 151, Malianwa North Road, Haidian District, Beijing 100193, PR China Eastern Liaoning University, No. 325 Wenhua Street, Yuanbao District, Dandong Liaoning 118003, PR China
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Article history: Received 11 June 2013 Received in revised form 7 June 2014 Accepted 11 June 2014 Available online 24 June 2014 Keywords: Amyloid-β Alzheimer's disease PC12 Apoptosis MAPK Notoginsenoside R1
a b s t r a c t Progressive accumulation of amyloid-β (Aβ) is a pathological hallmark of Alzheimer's disease (AD). Aβ increases free radical production in neuronal cells, leading to oxidative stress and cell death. An intervention that would reduce Aβ-related neurotoxicity through free radical reduction could advance the treatment of AD. Notoginsenoside R1 (NR1), the major and most active ingredient in the herb Panax notoginseng, can reduce reactive oxygen species and confer some neuroprotective effects. Here, NR1 was applied in a cell-based model of Alzheimer's disease. Cell viability, cell death, reactive oxygen species generation, and mitochondrial membrane potential were assessed in cultured PC12 neuronal cells incubated with Aβ25–35. In this model, Aβ was neurotoxic and induced necrosis and apoptosis; however, NR1 significantly counteracted the effects of Aβ by increasing cell viability, reducing oxidative damage (including apoptosis), restoring mitochondrial membrane potential, and suppressing stress-activated MAPK signaling pathways. These results promise a great potential agent for Alzheimer's disease and other Aβ pathology-related neuronal degenerative disease. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer's disease (AD) is the most common human age-related neurodegenerative disorder. AD pathology is characterized by deposition of extracellular senile plaques and intracellular neurofibrillary tangles and selective loss of synapses and neurons [1]. A common feature of AD is the accumulation of amyloid-β (Aβ) [2], 39–43 amino acid peptides produced by cleavage of the amyloid precursor protein by β- and γ-secretases [3]. Aβ peptides aggregate to form fibrillar deposits that are the principal component of senile plaques. Numerous studies have demonstrated that fibrillar Aβ aggregates, but not soluble Aβ, are neurotoxic [4]. Therefore, Aβ aggregates are thought to be the central molecules in the pathological development of AD. Aβ-induced toxicity involves oxidative stress, inflammation, and perturbation of calcium homeostasis [5]. In addition, both necrotic and apoptotic processes occur in primary neurons and neuronal cell lines after exposure to micromolar
Abbreviations: AD, Alzheimer's disease; ANOVA, analysis of variance; Aβ, amyloid-β; DA, diacetate; DCF, 2′,7′-dichlorofluorescein; DMSO, dimethyl sulfoxide; ERK 1/2, extracellular signal-regulated kinases 1/2; H2DCF, reduced DCF; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinase; NR1, notoginsenoside R1; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species. ⁎ Corresponding authors. Tel./fax: +86 10 62898496. E-mail addresses: [email protected] (G. Sun), [email protected] (X. Sun).
http://dx.doi.org/10.1016/j.intimp.2014.06.018 1567-5769/© 2014 Elsevier B.V. All rights reserved.
concentrations of Aβ [6,7]. However, the underlying mechanisms of toxicity and the neuronal cellular signaling cascades activated by Aβ are not fully understood. Between its two predominant forms, Aβ1–42 is dominant in senile plaques and has stronger aggregation propensity and increased toxicity than Aβ1–40. Aβ25–35 exerts neurotoxic effects similar to those produced by parent Aβ1–40/42 peptides, such as learning and memory impairment, neuronal apoptosis, and oxidative stress; therefore, Aβ25–35 is used to establish AD models for studying the neurotoxic properties of Aβ and for drug screening [5]. Because memory loss and cognitive dysfunction are the main clinical symptoms of AD patients, treatment and prevention of AD have stimulated the search for novel agents that can confer protection against learning and memory impairment. Notoginsenoside R1 (NR1) (Fig. 1) is the main compound in Panax notoginseng, an herbal medicine widely used in Asia for the treatment of cardiovascular disease and cerebral vascular diseases [8]. Several recent studies have indicated that NR1 can attenuate the risk of human cardiovascular and cerebrovascular diseases through antioxidant, antiinflammatory, anti-angiogenic, anti-apoptosis, and other biological activities [9–11]. Furthermore, More and more researches have shown that NR1 has remarkable neuroprotective effect, it can improve ischemia–reperfusion injury in vivo and prevent various damages (oxidative, glutamate, LPS, Aβ, etc.) of nerve cells (primary neurons, neurogliocytes, PC12 cells, etc.) in vitro [12–16]. NR1 has also been demonstrated that it could be absorbed into the bloodstream by pharmacokinetic studies [17,18], so it is becoming a new potential drug
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Fig. 1. Molecular structure of notoginsenoside R1.
candidate. Here, we mainly investigated the protective effects of NR1 and their possible mechanisms in PC12 cells exposed to Aβ25–35. 2. Materials and methods 2.1. Materials Aβ25–35, Aβ1–42, Fluorescent dyes Hoechst 33342, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and rhodamine 123 were purchased from Sigma (St. Louis, Missouri, USA). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), Annexin V/fluorescein isothiocyanate/propidium iodide (FITC/PI) apoptosis kit, and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) kit were purchased from Invitrogen (Carlsbad, California, USA). Dulbecco's modified Eagle medium (DMEM), Neurobasal™ medium, B-27 serum-free supplement, fetal bovine serum (FBS), and penicillin–streptomycin were purchased from Gibco BRL (Life Technologies, Paisley, Scotland). Anti-ERK1/2, antip-ERK1/2, anti-p38, anti-p-p38, anti-JNK, anti-p-JNK, anti-caspase 3, anti-BAX, anti-BCL2, and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (CA, USA). Notoginsenoside R1 (purity ≥ 98%) was obtained from the Shanghai Winherb Medical S & T Development Co. Ltd. (Shanghai, China). It was dissolved in 0.1% acetic acid at a concentration of 1 mM as stock solutions, the stock was diluted to the desired final concentrations in treatment medium prior to use. 2.2. Preparation of aggregated Aβ Aβ25–35 and Aβ1–42 was dissolved in deionized distilled water at a concentration of 1 mM and incubated at 37 °C for 4 days to induce aggregation separately [19]. After aggregation, the solution was stored at −20 °C until use. 2.3. Cell culture Rat PC12 pheochromocytoma cells were obtained from the Institute of Basic Medical Sciences at the Chinese Academy of Medical Sciences.
Cells were cultured in DMEM supplemented with 5% horse serum, 10% FBS, and 100 U/mL of penicillin–streptomycin at 37 °C with a 5% CO2 atmosphere in a humidified incubator. PC12 cells were subcultured about twice a week and split 1:4 when the culture was 80–90% confluent. Primary cultured cortical neurons were obtained from embryonic (E18d) Sprague–Dawley rat fetuses. Briefly, the brains were removed and the cortices were dissected out. The tissues were cut into fragments, and incubated in 0.2% trypsin for 20 min in 37 °C. Then, DMEM medium containing 10% FBS was added for trypsin inactivation, and the tissues were dissociated by mild mechanical trituration. About 1 × 105 cells/mL were plated onto poly-D-lysine-coated 96-well plates for further culture. After 4 h, the DMEM medium was changed into Neurobasal™ medium supplemented with 2% B-27 serum-free supplement and 0.5 mM L-glutamine in a humidified incubator containing 95% air and 5% CO2 at 37 °C. Primary neuronal cultures were maintained for 6–7 days before experiments.
2.4. MTT assay The MTT assay measures cell proliferation rate and cell viability by chromogenic changes of MTT dye, which is reduced to a blue-violet formazan by mitochondrial succinate dehydrogenase in viable cells. Approximately 10,000 cells/well were seeded in a flat 96-well microplate in triplicate. After 36 h, cells were then incubated with freshly prepared Aβ25–35/Aβ1–42 (2.5–40 μM) for 24 h, cell viability and half maximal inhibitory concentrations (IC50) were determined by MTT. To determine the protective effects of NR1 on Aβ-induced neurotoxicity, both PC12 cells and primary neurons were pretreated with different concentrations of NR1 (1–100 μM) for 24 h. Cells were then incubated with Aβ25–35 (20 μM) or Aβ1–42 (10 μM) for an additional 24 h. MTT solution (20 μL of 5 mg/mL stock solution in PBS per well) was added for 3 h at 37 °C, medium was aspirated, and 150 μL DMSO was added. The absorbance at 570 nm was determined using a microplate reader (SpectraFluor; Tecan, Sunrise, Austria). Experiments were repeated in triplicate and data were expressed as percentages of control.
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2.5. Hoechst 33342 and PI double staining In this study, cells were double-stained with Hoechst 33342 and propidium iodide (PI) for qualitative analysis of apoptosis. Hoechst 33342 is a vital nucleic acid stain readily taken up by all cells, and PI binds to DNA but is impermeable to cells with intact plasma membranes. Before 20 μM Aβ25–35 treatment in the presence or absence of NR1 for 24 h, briefly, the PC12 cells were incubated with 10 μL Hoechst 33342 (10 μg/mL, Ex/Em = 350 nm/461 nm) for 15 min, then 5 μL PI (100 μg/mL, Ex/Em = 535 nm/617 nm) for 5 min, visualized by fluorescence microscopy (Q9, Leica, Wetzlar, Germany) [20]. The percentage of
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total surviving cells was also assessed under 200 × magnification. All experiments were carried out in triplicate. 2.6. Apoptosis assays Two different assays quantified apoptosis. First, apoptotic cells were determined by the TUNEL kit (Invitrogen). PC12 cells were cultured on cover slips, following 20 μM Aβ25–35 treatment for 24 h, the cells were fixed by incubation in 10% neutral buffered formalin solution for 30 min at room temperature. Then cells were incubated with a methanol solution containing 0.3% H2O2 for 30 min to inactivate endogenous
Fig. 2. MTT assay measures the protective effects of notoginsenoside R1 (NR1) on Aβ-induced cytotoxicity in PC12 cells and primary neurons. (A) PC12 cells were treated with various concentrations of Aβ25–35 or Aβ1–42 (2.5–40 μM) for 24 h and cell viability was determined by MTT assay. *p b 0.05, **p b 0.01 compared to control group. (B) PC12 cells were pretreated with different concentrations of NR1 (1–100 μM) for 24 h, incubated with or without Aβ25–35 (20 μM)/Aβ1–42 (10 μM) for an additional 24 h, and cell viability was determined by MTT assay. *p b 0.05, **p b 0.01 compared to Aβ-treated group. (C) Primary neurons were pretreated with different concentrations of NR1 (1–100 μM) for 24 h, incubated with or without Aβ25–35 (20 μM)/Aβ1–42 (10 μM) for an additional 24 h, and cell viability was determined by MTT assay. *p b 0.05, **p b 0.01 compared to Aβ-treated group. All values represent cell viability as a percent of control and represent mean ± SEM from at least three independent experiments.
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peroxidase activity. PC12 cells were treated with a permeabilizing solution (0.1% sodium citrate and 0.1% Triton X-100) for 2 min at 4 °C. Subsequently, PC12 cells were incubated in the TUNEL reaction mixture for 60 min at 37 °C and visualized by fluorescence microscopy [20]. The apoptotic cells were counted in at least 100 cells from four randomly selected fields in each sample and counts were expressed as a percentage of the total number of cells. Apoptosis was also assessed by flow cytometry analysis. Apoptotic cells were detected with an Annexin V-FITC/PI detection kit (Invitrogen). PC12 cells (1 × 104) were plated in 6-well plates and exposed to 20 μM Aβ25–35 for 24 h in the presence or absence of 10 μM NR1. After incubation, cells were harvested and centrifuged at 1000 rpm for 10 min, rinsed with cold PBS twice, and incubated with 5 μL FITC-Annexin V and 1 μL PI working solution (100 μg/mL) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry analysis with a flow cytometer (FACS Calibur™, BD Biosciences, CA, USA) [20]. 2.7. Intracellular reactive oxygen species (ROS) Intracellular ROS were detected by a fluorescence assay in which the non-fluorescent 2′,7′-dichlorofluorescein diacetate (DCF-DA) passively diffuses into cells and is then converted into 2′,7′-dichlorofluorescein
(DCFH) by intracellular esterase. Intracellular ROS further oxidizes the non-fluorescent DCFH into fluorescent 2′,7′-dichlorofluorescein (DCF). Exponentially growing PC12 cells were seeded into a 96-well plate. After 12 h, cells were incubated with various concentrations of NR1 (10 μM) for 24 h, then incubated with 20 μM Aβ25–35 for an additional 24 h. After the medium was removed, cells were incubated with 25 μM carboxy-H2DCFDA in the dark at 37 °C for 30 min. Cells were washed with PBS twice and resuspended in PBS. The fluorescence intensity (relative fluorescence units) was detected with a microplate reader (SpectraFluor, Tecan, Sunrise, Austria) at the excitation wavelength of 495 nm and emission wavelength of 529 nm. The levels of intracellular ROS were expressed as a percentage of control. 2.8. Mitochondrial membrane potential To determine the mitochondrial membrane potential (Ψm), exponentially growing PC12 cells were seeded into a 96-well plate. After 12 h, cells were incubated with 10 μM NR1 for 24 h and then incubated with 20 μM Aβ25–35 for an additional 24 h. Fluorescent dye rhodamine 123 was added to the culture medium at a concentration of 1 μM for 15 min. Cells were washed five times with PBS and examined under a Nikon TE300 confocal fluorescence microscope equipped with an argon laser. The digital images were analyzed with Image-Pro Plus
Fig. 3. Hoechst 33342/propidium iodide (PI) staining measures the effects of notoginsenoside R1 (NR1) on Aβ25–35-induced cell death in PC12 neuronal cells. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and stained with Hoechst 33342/ PI. Panels (top) depict representative fields and graph and (below) mean ± SEM of cell survival as a percentage of controls counted from five individual fields/treatment. **p b 0.01 compared to control; ##p b 0.01 compared to Aβ25–35-treated group.
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software (v5.0). The average fluorescent density of intracellular areas was measured to index Ψm.
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with post hoc Tukey t-tests were used to determine statistical significance. p b 0.05 was considered statistically significant.
2.9. Western blotting 3. Results For immunoblotting, cells were lysed with RIPA buffer containing 50 mM Tris–HCl pH 7.2, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.02% sodium azide, 100 μg/mL phenylmethylsulfonyl fluoride, and 1 μg/mL each of protease inhibitors leupeptin, aprotinin, and pepstatin A. The lysate was centrifuged for 15 min at 12,000 ×g and 4 °C to remove insoluble materials. Protein concentrations were determined by Bradford assay and equalized lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). Membranes were blocked for 2 h in 5% nonfat milk in Tris-buffered saline (TBS)/Tween 20 and probed with the following antibodies: anti-BAX (1:200), anti-BCL-2 (1:200), anti-p-p38 (1:500), anti-p-38 (1:500), anti-p-ERK (1:200), anti-ERK (1:200), anti-cleaved caspase-3 (1:500), anti-p-JNK (1:200), anti-JNK (1:200), and anti-βactin (1:1000). The membranes were than washed 3 times with TBST and probed with horseradish peroxidase-conjugated secondary antibodies (1:2000) at room temperature for 1 h. Following a 30 min wash, the membranes were visualized by enhanced chemiluminescence. Band intensity was measured and quantified. 2.10. Statistical analysis Data represent percentage of values in untreated controls and each value represents mean ± SEM. One-way analysis of variance (ANOVA)
3.1. NR1 rescues cell viability The cytotoxicity of Aβ25–35/Aβ1–42 at different concentrations (2.5, 5, 10, 20, 40 μM) for 24 h, was evaluated by MTT assay. Aβ25–35 significantly reduced the viability of PC12 cells in a dose- and time-dependent manner (Fig. 2A). IC50 value of Aβ1–42 at 24 h was 10 μM, we also can speculate that IC50 value of Aβ25–35 at 24 h must be higher than 40 μM. Treatment with 20 μM Aβ25–35 induced approximately 35% cell death at 24 h, so this concentration was used for further studies. To investigate the possible neuroprotective effects of NR1, we evaluated PC12 and primary neurons cell viability in the presence of Aβ25–35/Aβ1–42 with or without different concentrations of NR1. About 69.1% (52.0%) of PC12 cells survived after treatment with 20 μM Aβ25–35 (10 μM Aβ1–42) for 24 h, and PC12 cell viability increased to 75.0% (55.0%), 77.0% (58.4%), 88.0% (74.8%), 94.7% (81.1%) and 89.2% (72.7%) when pretreated with 1, 5, 10, 50 and 100 μM NR1 for 24 h, respectively (Fig. 2B). Moreover, about 62.0% (48.3%) of primary neurons survived after treatment with 20 μM Aβ25–35 (10 μM Aβ1–42) for 24 h, and primary neurons viability increased to 65.1% (49.2%), 69.7% (50.1%), 73.4% (55.6%), 85.8% (65.7%) and 71.8% (58.6%) when pretreated with 1, 5, 10, 50 and 100 μM NR1 for 24 h, respectively (Fig. 2C). Therefore, the results showed that NR1 could protect PC12 cells and primary neurons from Aβ-induced cell death and apoptosis in a dose-dependent manner. In addition, 100 μM NR1 slightly increased
Fig. 4. TUNEL assay and annexin V-FITC/propidium iodide (PI) staining measure the effects of notoginsenoside R1 (NR1) on Aβ25–35-induced apoptosis in PC12 neuronal cells. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and apoptotic cells were evaluated by TUNEL assay. Panels (top) depict representative fields and graph and (below) mean ± SEM of apoptotic cells as a percentage of controls counted from five individual fields/treatment. (B) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and apoptotic cells were evaluated by flow cytometry of annexin V-FITC/PI double staining. Percentages of positive cells within a quadrant are indicated. The lower right quadrant represents apoptotic cells (PI-negative, annexin V-FITC-positive). Data represent mean ± SEM from at least three independent experiments. **p b 0.01 compared to control; ##p b 0.01 compared to Aβ25–35-treated group.
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cell viability compared to PBS-treated controls. To avoid potential cytotoxic effects, we used 10 μM NR1 for the remaining investigations. 3.2. NR1 prevents cell death Cell death was further analyzed by fluorescence microscopy using Hoechst/PI staining. Viable Hoechst-stained nuclei were found in control and NR1-treated cells, while necrotic cells stained with PI and apoptotic cells with Hoechst-stained nuclear fragments were observed after Aβ25–35-treatment [20]. Cells with blue nuclei were considered normal, while cells with bright blue or red/pink nuclei were considered apoptotic (Fig. 3). Pretreatment with 10 μM NR1 significantly decreased cell necrosis and apoptosis induced by Aβ25–35, shifting the percentage of surviving Aβ25–35-treated cells from 65% to 86% after NR1 pretreatment (p b 0.01). 3.3. NR1 inhibits apoptosis Similar to the quantification of Hoechst/PI staining, the TUNEL assay also assessed cell death and DNA fragmentation in NR1- and/or Aβ25–35treated cells. More TUNEL-positive staining was observed in PC12 cells treated with Aβ25–35, whereas positive staining was rarely detectable in control or NR1-treated cells (Fig. 4A). NR1 pretreatment (10 μM) significantly reduced apoptotic cells induced by Aβ25–35 (p b 0.01). To further study the protective effects of NR1, annexin-V/PI double staining was performed to evaluate the occurrence of apoptosis. Flow cytometry detected 14.27% apoptotic cells after treatment with 20 μM Aβ25–35 (Fig. 4B). NR1 and PBS (control) induced only 4.59% and 4.18% apoptosis, respectively. After pretreatment with 10 μM NR1, however, apoptosis was reduced to 9.61% (p b 0.01). Active caspase-3 and the BCL-2/BAX ratio are additional markers of apoptosis. Western blot analysis revealed that 24 h of 20 μM Aβ25–35 treatment decreased BCL-2 and increased BAX expression (Fig. 5A), altering the BCL-2/BAX ratio (Fig. 5B). Aβ25–35 treatment also induced a significant increase in total and cleaved caspase-3 expression (Fig. 5C). NR1 pretreatment before Aβ25–35 treatment partially reversed these apoptotic changes, although NR1 treatment alone had no effect. 3.4. NR1 attenuates Aβ25–35-induced ROS production and mitochondrial damage To evaluate the effect of NR1 on Aβ-induced ROS and mitochondrial damage, PC12 cells were treated with Aβ25–35 for 24 h in the presence or absence of NR1. Single-cell changes in ROS were assessed by the redoxsensitive dye carboxy-H2DCFDA; mitochondrial membrane potential was assessed by the membrane potential-dependent fluorescent dye rhodamine 123. Aβ25–35 treatment (20 μM) for 24 h induced a 1.26-fold average increase in carboxy-H2DCFDA staining intensity (Fig. 6A). Aβ25–35 treatment also significantly depolarized membrane potentials (p b 0.01) (Fig. 6B). NR1 pretreatment (10 μM) before Aβ25–35 treatment both prevented increased ROS and maintained membrane potentials at levels indistinguishable from controls; NR1 treatment alone had no significant effect. 3.5. NR1 inhibits Aβ25–35-induced MAPK activation ROS induce the activation of mitogen-activated protein kinases (MAPK), including JNK, p38 and ERK [21]. Hence, we evaluated MAPK expression in Aβ25–35-treated PC12 cells. Although total protein levels of ERK, JNK, and p38 were unaffected, phosphorylation of ERK, JNK, and p38 (p-ERK, p-JNK and p-p38) were increased after treatment with 20 μM Aβ25–35 for 24 h, p-ERK, p-JNK increased markedly (Fig. 7). Further, p-ERK, p-JNK, and p-p38 levels were inhibited by 10 μM NR1 pretreatment before Aβ25–35 treatment, although NR1 treatment alone had no effect.
Fig. 5. Western blots of apoptotic markers. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and protein lysates were blotted for BCL-2 and BAX expression. Blot for β-actin represents a loading control. (B) BCL-2/BAX ratios from Western blots (A) were determined by densitometry and values were normalized to β-actin. (C) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and protein lysates were blotted for caspase-3 and cleaved caspase-3. Blot for β-actin represents a loading control. *p b 0.01 compared to control; #p b 0.01 compared to Aβ25–35-treated group.
4. Discussion AD pathology shows significant oxidative damage associated with a marked accumulation of Aβ, the main constituent of senile brain plaques, and deposition of neurofibrillary tangles and neuropil threads [22,23]. Consequently, the loss of neurons and synapses leads to cognitive impairment and the development of dementia [24]. Therefore, the search for neuroprotective drugs is an important step toward the development of effective treatment strategies for neurodegenerative disorders. The PC12 cell line, derived from a rat pheochromocytoma, exhibits unique sensitivity to changes in oxygen availability. PC12 cells stop dividing and terminally differentiate when treated with NGF, making them a useful model for neuronal differentiation to examine drug effects on cellular toxicity [25]. The dried root of P. notoginseng of the Araliaceae family is a Chinese herbal medicine widely used in the treatment of microcirculatory disturbance-related diseases, such as cardiovascular disease and cerebral vascular diseases [26]. P. notoginseng contains more than 30 different types of saponin, including ginsenoside Rg1, ginsenoside Rb1, and NR1; NR1 is the main compound in P. notoginseng [27]. NR1 shows remarkable pharmacological activities, including anti-oxidation, antitumor, and anti-inflammation [28]. We investigated the protective effects of NR1 on Aβ-induced neuronal damage in PC12 neurons and found that NR1 can rescue cell viability after Aβ25–35 treatment.
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Fig. 6. Effects of notoginsenoside R1 (NR1) on Aβ25–35-induced reactive oxygen species (ROS) production and mitochondrial damage in PC12 neuronal cells. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and ROS levels were measured by intracellular conversion to fluorescent 2′,7′-dichlorofluorescein (DCF). Panels (left) depict representative fields and graph and (right) mean ± SEM of intracellular ROS levels expressed as a percentage of control. (B) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and mitochondrial membrane potential was determined by flow cytometry after staining with rhodamine 123. Values represent mean ± SEM fluorescence as a percentage of control from at least three independent experiments. **p b 0.01 compared to control; ##p b 0.01 compared to Aβ25–35-treated group.
Aβ25–35 induces neuronal degeneration through apoptosis in vitro and in vivo [29,30] by inducing mitochondrial dysfunction, which subsequently can trigger sequential activation of apoptosis-inducing enzymes such as caspase-3. BCL-2 family proteins play a pivotal role in regulating apoptotic cell death; some members, such as BCL-2 and BCL-xL, inhibit apoptosis, while others, such as BAX, induce apoptosis [31,32]. The relative ratio of pro-apoptotic and anti-apoptotic proteins is important to determine cell survival or death. Our study revealed that NR1 significantly increased cell viability against Aβ-induced (Aβ25–35 and Aβ1–42) neurotoxicity in PC12 cells and primary neurons. Hoechst/PI and Annexin V-FITC/PI double staining showed that Aβ25–35 treatment induced both apoptosis and necrosis, while pretreatment with 10 μM NR1 decreased the apoptotic rate from 14.27% to 9.16% but did not affect necrotic rate (5.31% vs. 4.91%). These results imply that NR1 is capable of alleviating apoptosis but not necrosis. During apoptosis, increasing active caspase signals activates the specific nuclease caspase-activated DNase, leading to chromatin condensing and cleavage of DNA into massive oligonucleosomal fragments. The effects of NR1 in attenuating Aβ25–35-induced apoptosis were confirmed by the TUNEL assay. Aβ25–35-induced apoptosis was associated with upregulation of the pro-apoptotic molecule BAX and downregulation of the anti-apoptotic molecule BCL-2. Further, upregulation of cleaved caspase-3, an enzyme critically involved in the execution of mammalian apoptosis, following Aβ25–35 treatment further confirmed cytotoxicity. NR1 pretreatment downregulated caspase-3 and decreased
the BAX/BCL-2 ratio. Collectively, these results suggest that the neuroprotective effects of NR1 are due to apoptosis suppression. The generation of ROS and oxidative damage are believed to be involved in the pathogenesis of neurodegenerative disorders [7]. Recent evidence indicates that oxidative stress occurs early in the progression of AD, before the development of senile plaques [33–35]. The interaction of abnormal mitochondria, redox transition metals, and oxidative stress response elements contributes to the generation of ROS in diseased neurons [36,37]. Several agents, including antioxidants and free radical scavengers, are neuroprotective both in vitro and in vivo against Aβ25–35-induced cytotoxicity [37]. These results may be due to mitochondrial function, as oxidative damage increases the permeability of the mitochondrial membrane [38]. Increased permeability causes membrane depolarization and uncoupling of oxidative phosphorylation reactions in the mitochondrial lumen. Membrane potential reduction is an early and sensitive indicator of cellular damage that precedes adenosine 5′-triphosphate depletion and increased membrane permeability. We observed a decreased membrane potential in Aβ25–35-treated cells, indicating the presence of mitochondrial damage; these cells also accumulated ROS. NR1 pretreatment significantly prevented membrane potential decreases and ROS accumulation caused by Aβ25–35 treatment, suggesting NR1 can prevent mitochondrial damage that may lead to the induction of apoptosis. MAPK family members, including ERK, JNK, and p38, are important signaling components linking extracellular stimuli to cellular responses
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Fig. 7. Western blots of MAPK (ERK, p38 and JNK) activation. PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and protein lysates were blotted for p-p38, p38, p-ERK, ERK, p-JNK, and JNK. Blot for β-actin represents a loading control.
[39,40]. The ERK pathway plays a major role in regulating cell growth and differentiation, and JNK and p38 are highly activated in response to a variety of stress signals, including tumor necrosis factor, ultraviolet irradiation, and hyperosmotic stress [41]. MAPK activation is most frequently associated with apoptosis induction [42]. Recent evidence suggests that Aβ could stimulate p38, JNK, and ERK activation and that this activation might be involved in AD pathogenesis [43]. Here, we found that total ERK, JNK, and p38 levels were unaffected but that phosphorylated ERK, JNK, and p38 were transiently and markedly increased after Aβ25–35 treatment. Further, NR1 significantly inhibited ERK, JNK, and p38 phosphorylation. We demonstrated that NR1 can protect PC12 neuronal cells from Aβ25–35-induced neurotoxicity by inhibiting oxidative stress, apoptosis, and stress-activated MAPK signaling pathways. Therefore, we hypothesize that NR1 has potential for the treatment of neurodegenerative diseases. However, because our experiments were limited to an in vitro model of AD, further in vivo studies are needed to confirm the neuroprotective effects of NR1 against Aβ25–35-induced neurotoxicity. Acknowledgments The present work was supported by China Postdoctoral Science Foundation (2012M510360) and the Major Scientific and Technological Special Project for ‘Significant New Drugs Formulation’ (Nos. 2012ZX09501001; 2012ZX09301002-001). References [1] Hardy J. Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci 1997;20:154–9. [2] Mattson Mark P. Pathways towards and away from Alzheimer's disease. Nature 2004;430:631–9. [3] Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, Shaffer LM, et al. Production of the Alzheimer amyloid β protein by normal proteolytic processing. Science 1992; 258:126–9. [4] Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 2001; 81:741–66. [5] Casley CS, Land JM, Sharpe MA, Clark JB, Duchen MR, Canevari L. β-amyloid fragment 25–35 causes mitochondrial dysfunction in primary cortical neurons. Neurobiol Dis 2002;10:258–67.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Notoginsenoside R1 attenuates amyloid-β-induced damage in neurons by inhibiting reactive oxygen species and modulating MAPK activation Bo Ma a, Xiangbao Meng a, Jing Wang a, Jing Sun a, Xiaoyu Ren a,b, Meng Qin a, Jie Sun a, Guibo Sun a,⁎, Xiaobo Sun a,⁎ a b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 151, Malianwa North Road, Haidian District, Beijing 100193, PR China Eastern Liaoning University, No. 325 Wenhua Street, Yuanbao District, Dandong Liaoning 118003, PR China
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 7 June 2014 Accepted 11 June 2014 Available online 24 June 2014 Keywords: Amyloid-β Alzheimer's disease PC12 Apoptosis MAPK Notoginsenoside R1
a b s t r a c t Progressive accumulation of amyloid-β (Aβ) is a pathological hallmark of Alzheimer's disease (AD). Aβ increases free radical production in neuronal cells, leading to oxidative stress and cell death. An intervention that would reduce Aβ-related neurotoxicity through free radical reduction could advance the treatment of AD. Notoginsenoside R1 (NR1), the major and most active ingredient in the herb Panax notoginseng, can reduce reactive oxygen species and confer some neuroprotective effects. Here, NR1 was applied in a cell-based model of Alzheimer's disease. Cell viability, cell death, reactive oxygen species generation, and mitochondrial membrane potential were assessed in cultured PC12 neuronal cells incubated with Aβ25–35. In this model, Aβ was neurotoxic and induced necrosis and apoptosis; however, NR1 significantly counteracted the effects of Aβ by increasing cell viability, reducing oxidative damage (including apoptosis), restoring mitochondrial membrane potential, and suppressing stress-activated MAPK signaling pathways. These results promise a great potential agent for Alzheimer's disease and other Aβ pathology-related neuronal degenerative disease. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer's disease (AD) is the most common human age-related neurodegenerative disorder. AD pathology is characterized by deposition of extracellular senile plaques and intracellular neurofibrillary tangles and selective loss of synapses and neurons [1]. A common feature of AD is the accumulation of amyloid-β (Aβ) [2], 39–43 amino acid peptides produced by cleavage of the amyloid precursor protein by β- and γ-secretases [3]. Aβ peptides aggregate to form fibrillar deposits that are the principal component of senile plaques. Numerous studies have demonstrated that fibrillar Aβ aggregates, but not soluble Aβ, are neurotoxic [4]. Therefore, Aβ aggregates are thought to be the central molecules in the pathological development of AD. Aβ-induced toxicity involves oxidative stress, inflammation, and perturbation of calcium homeostasis [5]. In addition, both necrotic and apoptotic processes occur in primary neurons and neuronal cell lines after exposure to micromolar
Abbreviations: AD, Alzheimer's disease; ANOVA, analysis of variance; Aβ, amyloid-β; DA, diacetate; DCF, 2′,7′-dichlorofluorescein; DMSO, dimethyl sulfoxide; ERK 1/2, extracellular signal-regulated kinases 1/2; H2DCF, reduced DCF; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinase; NR1, notoginsenoside R1; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species. ⁎ Corresponding authors. Tel./fax: +86 10 62898496. E-mail addresses: [email protected] (G. Sun), [email protected] (X. Sun).
http://dx.doi.org/10.1016/j.intimp.2014.06.018 1567-5769/© 2014 Elsevier B.V. All rights reserved.
concentrations of Aβ [6,7]. However, the underlying mechanisms of toxicity and the neuronal cellular signaling cascades activated by Aβ are not fully understood. Between its two predominant forms, Aβ1–42 is dominant in senile plaques and has stronger aggregation propensity and increased toxicity than Aβ1–40. Aβ25–35 exerts neurotoxic effects similar to those produced by parent Aβ1–40/42 peptides, such as learning and memory impairment, neuronal apoptosis, and oxidative stress; therefore, Aβ25–35 is used to establish AD models for studying the neurotoxic properties of Aβ and for drug screening [5]. Because memory loss and cognitive dysfunction are the main clinical symptoms of AD patients, treatment and prevention of AD have stimulated the search for novel agents that can confer protection against learning and memory impairment. Notoginsenoside R1 (NR1) (Fig. 1) is the main compound in Panax notoginseng, an herbal medicine widely used in Asia for the treatment of cardiovascular disease and cerebral vascular diseases [8]. Several recent studies have indicated that NR1 can attenuate the risk of human cardiovascular and cerebrovascular diseases through antioxidant, antiinflammatory, anti-angiogenic, anti-apoptosis, and other biological activities [9–11]. Furthermore, More and more researches have shown that NR1 has remarkable neuroprotective effect, it can improve ischemia–reperfusion injury in vivo and prevent various damages (oxidative, glutamate, LPS, Aβ, etc.) of nerve cells (primary neurons, neurogliocytes, PC12 cells, etc.) in vitro [12–16]. NR1 has also been demonstrated that it could be absorbed into the bloodstream by pharmacokinetic studies [17,18], so it is becoming a new potential drug
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Fig. 1. Molecular structure of notoginsenoside R1.
candidate. Here, we mainly investigated the protective effects of NR1 and their possible mechanisms in PC12 cells exposed to Aβ25–35. 2. Materials and methods 2.1. Materials Aβ25–35, Aβ1–42, Fluorescent dyes Hoechst 33342, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and rhodamine 123 were purchased from Sigma (St. Louis, Missouri, USA). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), Annexin V/fluorescein isothiocyanate/propidium iodide (FITC/PI) apoptosis kit, and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) kit were purchased from Invitrogen (Carlsbad, California, USA). Dulbecco's modified Eagle medium (DMEM), Neurobasal™ medium, B-27 serum-free supplement, fetal bovine serum (FBS), and penicillin–streptomycin were purchased from Gibco BRL (Life Technologies, Paisley, Scotland). Anti-ERK1/2, antip-ERK1/2, anti-p38, anti-p-p38, anti-JNK, anti-p-JNK, anti-caspase 3, anti-BAX, anti-BCL2, and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (CA, USA). Notoginsenoside R1 (purity ≥ 98%) was obtained from the Shanghai Winherb Medical S & T Development Co. Ltd. (Shanghai, China). It was dissolved in 0.1% acetic acid at a concentration of 1 mM as stock solutions, the stock was diluted to the desired final concentrations in treatment medium prior to use. 2.2. Preparation of aggregated Aβ Aβ25–35 and Aβ1–42 was dissolved in deionized distilled water at a concentration of 1 mM and incubated at 37 °C for 4 days to induce aggregation separately [19]. After aggregation, the solution was stored at −20 °C until use. 2.3. Cell culture Rat PC12 pheochromocytoma cells were obtained from the Institute of Basic Medical Sciences at the Chinese Academy of Medical Sciences.
Cells were cultured in DMEM supplemented with 5% horse serum, 10% FBS, and 100 U/mL of penicillin–streptomycin at 37 °C with a 5% CO2 atmosphere in a humidified incubator. PC12 cells were subcultured about twice a week and split 1:4 when the culture was 80–90% confluent. Primary cultured cortical neurons were obtained from embryonic (E18d) Sprague–Dawley rat fetuses. Briefly, the brains were removed and the cortices were dissected out. The tissues were cut into fragments, and incubated in 0.2% trypsin for 20 min in 37 °C. Then, DMEM medium containing 10% FBS was added for trypsin inactivation, and the tissues were dissociated by mild mechanical trituration. About 1 × 105 cells/mL were plated onto poly-D-lysine-coated 96-well plates for further culture. After 4 h, the DMEM medium was changed into Neurobasal™ medium supplemented with 2% B-27 serum-free supplement and 0.5 mM L-glutamine in a humidified incubator containing 95% air and 5% CO2 at 37 °C. Primary neuronal cultures were maintained for 6–7 days before experiments.
2.4. MTT assay The MTT assay measures cell proliferation rate and cell viability by chromogenic changes of MTT dye, which is reduced to a blue-violet formazan by mitochondrial succinate dehydrogenase in viable cells. Approximately 10,000 cells/well were seeded in a flat 96-well microplate in triplicate. After 36 h, cells were then incubated with freshly prepared Aβ25–35/Aβ1–42 (2.5–40 μM) for 24 h, cell viability and half maximal inhibitory concentrations (IC50) were determined by MTT. To determine the protective effects of NR1 on Aβ-induced neurotoxicity, both PC12 cells and primary neurons were pretreated with different concentrations of NR1 (1–100 μM) for 24 h. Cells were then incubated with Aβ25–35 (20 μM) or Aβ1–42 (10 μM) for an additional 24 h. MTT solution (20 μL of 5 mg/mL stock solution in PBS per well) was added for 3 h at 37 °C, medium was aspirated, and 150 μL DMSO was added. The absorbance at 570 nm was determined using a microplate reader (SpectraFluor; Tecan, Sunrise, Austria). Experiments were repeated in triplicate and data were expressed as percentages of control.
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2.5. Hoechst 33342 and PI double staining In this study, cells were double-stained with Hoechst 33342 and propidium iodide (PI) for qualitative analysis of apoptosis. Hoechst 33342 is a vital nucleic acid stain readily taken up by all cells, and PI binds to DNA but is impermeable to cells with intact plasma membranes. Before 20 μM Aβ25–35 treatment in the presence or absence of NR1 for 24 h, briefly, the PC12 cells were incubated with 10 μL Hoechst 33342 (10 μg/mL, Ex/Em = 350 nm/461 nm) for 15 min, then 5 μL PI (100 μg/mL, Ex/Em = 535 nm/617 nm) for 5 min, visualized by fluorescence microscopy (Q9, Leica, Wetzlar, Germany) [20]. The percentage of
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total surviving cells was also assessed under 200 × magnification. All experiments were carried out in triplicate. 2.6. Apoptosis assays Two different assays quantified apoptosis. First, apoptotic cells were determined by the TUNEL kit (Invitrogen). PC12 cells were cultured on cover slips, following 20 μM Aβ25–35 treatment for 24 h, the cells were fixed by incubation in 10% neutral buffered formalin solution for 30 min at room temperature. Then cells were incubated with a methanol solution containing 0.3% H2O2 for 30 min to inactivate endogenous
Fig. 2. MTT assay measures the protective effects of notoginsenoside R1 (NR1) on Aβ-induced cytotoxicity in PC12 cells and primary neurons. (A) PC12 cells were treated with various concentrations of Aβ25–35 or Aβ1–42 (2.5–40 μM) for 24 h and cell viability was determined by MTT assay. *p b 0.05, **p b 0.01 compared to control group. (B) PC12 cells were pretreated with different concentrations of NR1 (1–100 μM) for 24 h, incubated with or without Aβ25–35 (20 μM)/Aβ1–42 (10 μM) for an additional 24 h, and cell viability was determined by MTT assay. *p b 0.05, **p b 0.01 compared to Aβ-treated group. (C) Primary neurons were pretreated with different concentrations of NR1 (1–100 μM) for 24 h, incubated with or without Aβ25–35 (20 μM)/Aβ1–42 (10 μM) for an additional 24 h, and cell viability was determined by MTT assay. *p b 0.05, **p b 0.01 compared to Aβ-treated group. All values represent cell viability as a percent of control and represent mean ± SEM from at least three independent experiments.
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peroxidase activity. PC12 cells were treated with a permeabilizing solution (0.1% sodium citrate and 0.1% Triton X-100) for 2 min at 4 °C. Subsequently, PC12 cells were incubated in the TUNEL reaction mixture for 60 min at 37 °C and visualized by fluorescence microscopy [20]. The apoptotic cells were counted in at least 100 cells from four randomly selected fields in each sample and counts were expressed as a percentage of the total number of cells. Apoptosis was also assessed by flow cytometry analysis. Apoptotic cells were detected with an Annexin V-FITC/PI detection kit (Invitrogen). PC12 cells (1 × 104) were plated in 6-well plates and exposed to 20 μM Aβ25–35 for 24 h in the presence or absence of 10 μM NR1. After incubation, cells were harvested and centrifuged at 1000 rpm for 10 min, rinsed with cold PBS twice, and incubated with 5 μL FITC-Annexin V and 1 μL PI working solution (100 μg/mL) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry analysis with a flow cytometer (FACS Calibur™, BD Biosciences, CA, USA) [20]. 2.7. Intracellular reactive oxygen species (ROS) Intracellular ROS were detected by a fluorescence assay in which the non-fluorescent 2′,7′-dichlorofluorescein diacetate (DCF-DA) passively diffuses into cells and is then converted into 2′,7′-dichlorofluorescein
(DCFH) by intracellular esterase. Intracellular ROS further oxidizes the non-fluorescent DCFH into fluorescent 2′,7′-dichlorofluorescein (DCF). Exponentially growing PC12 cells were seeded into a 96-well plate. After 12 h, cells were incubated with various concentrations of NR1 (10 μM) for 24 h, then incubated with 20 μM Aβ25–35 for an additional 24 h. After the medium was removed, cells were incubated with 25 μM carboxy-H2DCFDA in the dark at 37 °C for 30 min. Cells were washed with PBS twice and resuspended in PBS. The fluorescence intensity (relative fluorescence units) was detected with a microplate reader (SpectraFluor, Tecan, Sunrise, Austria) at the excitation wavelength of 495 nm and emission wavelength of 529 nm. The levels of intracellular ROS were expressed as a percentage of control. 2.8. Mitochondrial membrane potential To determine the mitochondrial membrane potential (Ψm), exponentially growing PC12 cells were seeded into a 96-well plate. After 12 h, cells were incubated with 10 μM NR1 for 24 h and then incubated with 20 μM Aβ25–35 for an additional 24 h. Fluorescent dye rhodamine 123 was added to the culture medium at a concentration of 1 μM for 15 min. Cells were washed five times with PBS and examined under a Nikon TE300 confocal fluorescence microscope equipped with an argon laser. The digital images were analyzed with Image-Pro Plus
Fig. 3. Hoechst 33342/propidium iodide (PI) staining measures the effects of notoginsenoside R1 (NR1) on Aβ25–35-induced cell death in PC12 neuronal cells. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and stained with Hoechst 33342/ PI. Panels (top) depict representative fields and graph and (below) mean ± SEM of cell survival as a percentage of controls counted from five individual fields/treatment. **p b 0.01 compared to control; ##p b 0.01 compared to Aβ25–35-treated group.
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software (v5.0). The average fluorescent density of intracellular areas was measured to index Ψm.
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with post hoc Tukey t-tests were used to determine statistical significance. p b 0.05 was considered statistically significant.
2.9. Western blotting 3. Results For immunoblotting, cells were lysed with RIPA buffer containing 50 mM Tris–HCl pH 7.2, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.02% sodium azide, 100 μg/mL phenylmethylsulfonyl fluoride, and 1 μg/mL each of protease inhibitors leupeptin, aprotinin, and pepstatin A. The lysate was centrifuged for 15 min at 12,000 ×g and 4 °C to remove insoluble materials. Protein concentrations were determined by Bradford assay and equalized lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). Membranes were blocked for 2 h in 5% nonfat milk in Tris-buffered saline (TBS)/Tween 20 and probed with the following antibodies: anti-BAX (1:200), anti-BCL-2 (1:200), anti-p-p38 (1:500), anti-p-38 (1:500), anti-p-ERK (1:200), anti-ERK (1:200), anti-cleaved caspase-3 (1:500), anti-p-JNK (1:200), anti-JNK (1:200), and anti-βactin (1:1000). The membranes were than washed 3 times with TBST and probed with horseradish peroxidase-conjugated secondary antibodies (1:2000) at room temperature for 1 h. Following a 30 min wash, the membranes were visualized by enhanced chemiluminescence. Band intensity was measured and quantified. 2.10. Statistical analysis Data represent percentage of values in untreated controls and each value represents mean ± SEM. One-way analysis of variance (ANOVA)
3.1. NR1 rescues cell viability The cytotoxicity of Aβ25–35/Aβ1–42 at different concentrations (2.5, 5, 10, 20, 40 μM) for 24 h, was evaluated by MTT assay. Aβ25–35 significantly reduced the viability of PC12 cells in a dose- and time-dependent manner (Fig. 2A). IC50 value of Aβ1–42 at 24 h was 10 μM, we also can speculate that IC50 value of Aβ25–35 at 24 h must be higher than 40 μM. Treatment with 20 μM Aβ25–35 induced approximately 35% cell death at 24 h, so this concentration was used for further studies. To investigate the possible neuroprotective effects of NR1, we evaluated PC12 and primary neurons cell viability in the presence of Aβ25–35/Aβ1–42 with or without different concentrations of NR1. About 69.1% (52.0%) of PC12 cells survived after treatment with 20 μM Aβ25–35 (10 μM Aβ1–42) for 24 h, and PC12 cell viability increased to 75.0% (55.0%), 77.0% (58.4%), 88.0% (74.8%), 94.7% (81.1%) and 89.2% (72.7%) when pretreated with 1, 5, 10, 50 and 100 μM NR1 for 24 h, respectively (Fig. 2B). Moreover, about 62.0% (48.3%) of primary neurons survived after treatment with 20 μM Aβ25–35 (10 μM Aβ1–42) for 24 h, and primary neurons viability increased to 65.1% (49.2%), 69.7% (50.1%), 73.4% (55.6%), 85.8% (65.7%) and 71.8% (58.6%) when pretreated with 1, 5, 10, 50 and 100 μM NR1 for 24 h, respectively (Fig. 2C). Therefore, the results showed that NR1 could protect PC12 cells and primary neurons from Aβ-induced cell death and apoptosis in a dose-dependent manner. In addition, 100 μM NR1 slightly increased
Fig. 4. TUNEL assay and annexin V-FITC/propidium iodide (PI) staining measure the effects of notoginsenoside R1 (NR1) on Aβ25–35-induced apoptosis in PC12 neuronal cells. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and apoptotic cells were evaluated by TUNEL assay. Panels (top) depict representative fields and graph and (below) mean ± SEM of apoptotic cells as a percentage of controls counted from five individual fields/treatment. (B) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and apoptotic cells were evaluated by flow cytometry of annexin V-FITC/PI double staining. Percentages of positive cells within a quadrant are indicated. The lower right quadrant represents apoptotic cells (PI-negative, annexin V-FITC-positive). Data represent mean ± SEM from at least three independent experiments. **p b 0.01 compared to control; ##p b 0.01 compared to Aβ25–35-treated group.
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cell viability compared to PBS-treated controls. To avoid potential cytotoxic effects, we used 10 μM NR1 for the remaining investigations. 3.2. NR1 prevents cell death Cell death was further analyzed by fluorescence microscopy using Hoechst/PI staining. Viable Hoechst-stained nuclei were found in control and NR1-treated cells, while necrotic cells stained with PI and apoptotic cells with Hoechst-stained nuclear fragments were observed after Aβ25–35-treatment [20]. Cells with blue nuclei were considered normal, while cells with bright blue or red/pink nuclei were considered apoptotic (Fig. 3). Pretreatment with 10 μM NR1 significantly decreased cell necrosis and apoptosis induced by Aβ25–35, shifting the percentage of surviving Aβ25–35-treated cells from 65% to 86% after NR1 pretreatment (p b 0.01). 3.3. NR1 inhibits apoptosis Similar to the quantification of Hoechst/PI staining, the TUNEL assay also assessed cell death and DNA fragmentation in NR1- and/or Aβ25–35treated cells. More TUNEL-positive staining was observed in PC12 cells treated with Aβ25–35, whereas positive staining was rarely detectable in control or NR1-treated cells (Fig. 4A). NR1 pretreatment (10 μM) significantly reduced apoptotic cells induced by Aβ25–35 (p b 0.01). To further study the protective effects of NR1, annexin-V/PI double staining was performed to evaluate the occurrence of apoptosis. Flow cytometry detected 14.27% apoptotic cells after treatment with 20 μM Aβ25–35 (Fig. 4B). NR1 and PBS (control) induced only 4.59% and 4.18% apoptosis, respectively. After pretreatment with 10 μM NR1, however, apoptosis was reduced to 9.61% (p b 0.01). Active caspase-3 and the BCL-2/BAX ratio are additional markers of apoptosis. Western blot analysis revealed that 24 h of 20 μM Aβ25–35 treatment decreased BCL-2 and increased BAX expression (Fig. 5A), altering the BCL-2/BAX ratio (Fig. 5B). Aβ25–35 treatment also induced a significant increase in total and cleaved caspase-3 expression (Fig. 5C). NR1 pretreatment before Aβ25–35 treatment partially reversed these apoptotic changes, although NR1 treatment alone had no effect. 3.4. NR1 attenuates Aβ25–35-induced ROS production and mitochondrial damage To evaluate the effect of NR1 on Aβ-induced ROS and mitochondrial damage, PC12 cells were treated with Aβ25–35 for 24 h in the presence or absence of NR1. Single-cell changes in ROS were assessed by the redoxsensitive dye carboxy-H2DCFDA; mitochondrial membrane potential was assessed by the membrane potential-dependent fluorescent dye rhodamine 123. Aβ25–35 treatment (20 μM) for 24 h induced a 1.26-fold average increase in carboxy-H2DCFDA staining intensity (Fig. 6A). Aβ25–35 treatment also significantly depolarized membrane potentials (p b 0.01) (Fig. 6B). NR1 pretreatment (10 μM) before Aβ25–35 treatment both prevented increased ROS and maintained membrane potentials at levels indistinguishable from controls; NR1 treatment alone had no significant effect. 3.5. NR1 inhibits Aβ25–35-induced MAPK activation ROS induce the activation of mitogen-activated protein kinases (MAPK), including JNK, p38 and ERK [21]. Hence, we evaluated MAPK expression in Aβ25–35-treated PC12 cells. Although total protein levels of ERK, JNK, and p38 were unaffected, phosphorylation of ERK, JNK, and p38 (p-ERK, p-JNK and p-p38) were increased after treatment with 20 μM Aβ25–35 for 24 h, p-ERK, p-JNK increased markedly (Fig. 7). Further, p-ERK, p-JNK, and p-p38 levels were inhibited by 10 μM NR1 pretreatment before Aβ25–35 treatment, although NR1 treatment alone had no effect.
Fig. 5. Western blots of apoptotic markers. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and protein lysates were blotted for BCL-2 and BAX expression. Blot for β-actin represents a loading control. (B) BCL-2/BAX ratios from Western blots (A) were determined by densitometry and values were normalized to β-actin. (C) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and protein lysates were blotted for caspase-3 and cleaved caspase-3. Blot for β-actin represents a loading control. *p b 0.01 compared to control; #p b 0.01 compared to Aβ25–35-treated group.
4. Discussion AD pathology shows significant oxidative damage associated with a marked accumulation of Aβ, the main constituent of senile brain plaques, and deposition of neurofibrillary tangles and neuropil threads [22,23]. Consequently, the loss of neurons and synapses leads to cognitive impairment and the development of dementia [24]. Therefore, the search for neuroprotective drugs is an important step toward the development of effective treatment strategies for neurodegenerative disorders. The PC12 cell line, derived from a rat pheochromocytoma, exhibits unique sensitivity to changes in oxygen availability. PC12 cells stop dividing and terminally differentiate when treated with NGF, making them a useful model for neuronal differentiation to examine drug effects on cellular toxicity [25]. The dried root of P. notoginseng of the Araliaceae family is a Chinese herbal medicine widely used in the treatment of microcirculatory disturbance-related diseases, such as cardiovascular disease and cerebral vascular diseases [26]. P. notoginseng contains more than 30 different types of saponin, including ginsenoside Rg1, ginsenoside Rb1, and NR1; NR1 is the main compound in P. notoginseng [27]. NR1 shows remarkable pharmacological activities, including anti-oxidation, antitumor, and anti-inflammation [28]. We investigated the protective effects of NR1 on Aβ-induced neuronal damage in PC12 neurons and found that NR1 can rescue cell viability after Aβ25–35 treatment.
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Fig. 6. Effects of notoginsenoside R1 (NR1) on Aβ25–35-induced reactive oxygen species (ROS) production and mitochondrial damage in PC12 neuronal cells. (A) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and ROS levels were measured by intracellular conversion to fluorescent 2′,7′-dichlorofluorescein (DCF). Panels (left) depict representative fields and graph and (right) mean ± SEM of intracellular ROS levels expressed as a percentage of control. (B) PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and mitochondrial membrane potential was determined by flow cytometry after staining with rhodamine 123. Values represent mean ± SEM fluorescence as a percentage of control from at least three independent experiments. **p b 0.01 compared to control; ##p b 0.01 compared to Aβ25–35-treated group.
Aβ25–35 induces neuronal degeneration through apoptosis in vitro and in vivo [29,30] by inducing mitochondrial dysfunction, which subsequently can trigger sequential activation of apoptosis-inducing enzymes such as caspase-3. BCL-2 family proteins play a pivotal role in regulating apoptotic cell death; some members, such as BCL-2 and BCL-xL, inhibit apoptosis, while others, such as BAX, induce apoptosis [31,32]. The relative ratio of pro-apoptotic and anti-apoptotic proteins is important to determine cell survival or death. Our study revealed that NR1 significantly increased cell viability against Aβ-induced (Aβ25–35 and Aβ1–42) neurotoxicity in PC12 cells and primary neurons. Hoechst/PI and Annexin V-FITC/PI double staining showed that Aβ25–35 treatment induced both apoptosis and necrosis, while pretreatment with 10 μM NR1 decreased the apoptotic rate from 14.27% to 9.16% but did not affect necrotic rate (5.31% vs. 4.91%). These results imply that NR1 is capable of alleviating apoptosis but not necrosis. During apoptosis, increasing active caspase signals activates the specific nuclease caspase-activated DNase, leading to chromatin condensing and cleavage of DNA into massive oligonucleosomal fragments. The effects of NR1 in attenuating Aβ25–35-induced apoptosis were confirmed by the TUNEL assay. Aβ25–35-induced apoptosis was associated with upregulation of the pro-apoptotic molecule BAX and downregulation of the anti-apoptotic molecule BCL-2. Further, upregulation of cleaved caspase-3, an enzyme critically involved in the execution of mammalian apoptosis, following Aβ25–35 treatment further confirmed cytotoxicity. NR1 pretreatment downregulated caspase-3 and decreased
the BAX/BCL-2 ratio. Collectively, these results suggest that the neuroprotective effects of NR1 are due to apoptosis suppression. The generation of ROS and oxidative damage are believed to be involved in the pathogenesis of neurodegenerative disorders [7]. Recent evidence indicates that oxidative stress occurs early in the progression of AD, before the development of senile plaques [33–35]. The interaction of abnormal mitochondria, redox transition metals, and oxidative stress response elements contributes to the generation of ROS in diseased neurons [36,37]. Several agents, including antioxidants and free radical scavengers, are neuroprotective both in vitro and in vivo against Aβ25–35-induced cytotoxicity [37]. These results may be due to mitochondrial function, as oxidative damage increases the permeability of the mitochondrial membrane [38]. Increased permeability causes membrane depolarization and uncoupling of oxidative phosphorylation reactions in the mitochondrial lumen. Membrane potential reduction is an early and sensitive indicator of cellular damage that precedes adenosine 5′-triphosphate depletion and increased membrane permeability. We observed a decreased membrane potential in Aβ25–35-treated cells, indicating the presence of mitochondrial damage; these cells also accumulated ROS. NR1 pretreatment significantly prevented membrane potential decreases and ROS accumulation caused by Aβ25–35 treatment, suggesting NR1 can prevent mitochondrial damage that may lead to the induction of apoptosis. MAPK family members, including ERK, JNK, and p38, are important signaling components linking extracellular stimuli to cellular responses
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Fig. 7. Western blots of MAPK (ERK, p38 and JNK) activation. PC12 cells were untreated, treated with 10 μM NR1 only (NR1), 20 μM Aβ25–35 only (Aβ), or pretreated with 10 μM NR1 before 20 μM Aβ25–35 treatment (NR1 + Aβ) and protein lysates were blotted for p-p38, p38, p-ERK, ERK, p-JNK, and JNK. Blot for β-actin represents a loading control.
[39,40]. The ERK pathway plays a major role in regulating cell growth and differentiation, and JNK and p38 are highly activated in response to a variety of stress signals, including tumor necrosis factor, ultraviolet irradiation, and hyperosmotic stress [41]. MAPK activation is most frequently associated with apoptosis induction [42]. Recent evidence suggests that Aβ could stimulate p38, JNK, and ERK activation and that this activation might be involved in AD pathogenesis [43]. Here, we found that total ERK, JNK, and p38 levels were unaffected but that phosphorylated ERK, JNK, and p38 were transiently and markedly increased after Aβ25–35 treatment. Further, NR1 significantly inhibited ERK, JNK, and p38 phosphorylation. We demonstrated that NR1 can protect PC12 neuronal cells from Aβ25–35-induced neurotoxicity by inhibiting oxidative stress, apoptosis, and stress-activated MAPK signaling pathways. Therefore, we hypothesize that NR1 has potential for the treatment of neurodegenerative diseases. However, because our experiments were limited to an in vitro model of AD, further in vivo studies are needed to confirm the neuroprotective effects of NR1 against Aβ25–35-induced neurotoxicity. Acknowledgments The present work was supported by China Postdoctoral Science Foundation (2012M510360) and the Major Scientific and Technological Special Project for ‘Significant New Drugs Formulation’ (Nos. 2012ZX09501001; 2012ZX09301002-001). References [1] Hardy J. Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci 1997;20:154–9. [2] Mattson Mark P. Pathways towards and away from Alzheimer's disease. Nature 2004;430:631–9. [3] Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, Shaffer LM, et al. Production of the Alzheimer amyloid β protein by normal proteolytic processing. Science 1992; 258:126–9. [4] Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 2001; 81:741–66. [5] Casley CS, Land JM, Sharpe MA, Clark JB, Duchen MR, Canevari L. β-amyloid fragment 25–35 causes mitochondrial dysfunction in primary cortical neurons. Neurobiol Dis 2002;10:258–67.
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