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Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta25–35 via the Autophagy Pathway

Evidence shows that an abnormal deposition of amyloid beta-peptide25–35 (Aβ25–35) was the primary cause of the pathogenesis of Alzheimer’s disease (AD). And the elimination of Aβ25–35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta25–35 (Aβ25–35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ25–35–induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ25–35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ25–35-treated differentiated PC12 cells. Meanwhile, Aβ25–35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ25–35-induced cytotoxicity and attenuate Aβ25–35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ25–35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ25–35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the neurological damage in AD caused by deposition of Aβ25–35 via the autophagy pathway, all of which may provide an exciting view of the potential application of triptolide or TWHF as a future research for AD.

Based on these, the purpose of this study was to assess whether triptolide could protect against Aβ induced cytotoxicity in differentiated PC12 cells. In our experiments, we use MTT assay and flow cytometry to investigate the protective effects of triptolide. Western blot and acridine orange staining were chosen to detect the mechanism of triptolide on differentiated PC12 cells treated with Aβ [25][26][27][28][29][30][31][32][33][34][35] . All of these may provide an interesting view of the potential application of triptolide or TWHF in future research for AD.
Triptolide (PG490, molecular formula: C 20 H 24 O 6 , molecular weight: 360.4) was purchased from Sigma. The material was composed of white to off-white crystals, had a melting point of 235-237°C, and conformed to standard triptolide preparation by proton nuclear magnetic resonance. The material was 98% pure by reverse phase high pressure liquid chromatography evaluation. Before using, triptolide was soluble in dimethylsulfoxide (DMSO). After reconsititution, triptolide was stored at -20°C at a concentration of 1 mg/mL. When using, it was diluted to different concentrations with RPMI 1640 medium.

Cell culture
The rat pheochromocytoma cell line (PC12, derived from the American Type Culture Collection) was purchased from the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. It has been described in our previously work [23,36]. The cell line was derived from a rat adrenal medulla pheochromocytoma. In the presence of nerve growth factor (NGF), the undifferentiated PC12 cells could differentiate into sympathetic-like neurons, which were widely used as the model of neurons in vitro [37].
The undifferentiated PC12 cells were cultured in an incubator aerated with 95% humidified air with 5% CO 2 at 37°C, supplemented with 10% FBS, 5% horse serum, and 1% antibiotics (penicillin and streptomycin). Then the medium was replaced with serum-free RPMI1640 supplemented with 50 ng/mL NGF for 7 days to obtain neuronal differentiated PC12 cells. Then differentiated PC12 cells were cultured in RPMI 1640 medium (pH = 7.4) supplemented with 5% FBS and 1% antibiotics (penicillin and streptomycin). Cells were grown at 37°C in 95% humidified air with 5% CO 2 . All subsequent experiments in the present study were undertaken with these differentiated PC12 cells.

Cytotoxicity induced by Aβ 25-35 on differentiated PC12 cells
In vitro cytotoxicity induced by Aβ 25-35 on differentiated PC12 cells was assessed by the MTT assay, which was widely used to evaluate the cytotoxic activity. Differentiated PC12 cells were cultured on 96-well plates with RPMI 1640 medium for stabilization. 24 hours later, cells were incubated with different concentrations of Aβ [25][26][27][28][29][30][31][32][33][34][35] (5,10,20 μmol/L) for 24 hours. Subsequently, MTT was added and incubated for 4 hours at 37°C. After that, formazan crystals were dissolved by DMSO and measured at a wavelength of 570 nm. The cell viability was expressed as a percentage of viability of the control culture. Each condition and experiment was repeated three times.

The viability of differentiated PC12 cells treated with different concentrations of triptolide
After differentiated PC12 cells were cultured on 96-well plates with RPMI 1640 medium for stabilization, differentiated PC12 cells were incubated with different concentrations of triptolide (10 −11 , 10 −10 , 10 −9 mol/L) for 24 hours. The concentrations in this study were chosen according to the published data [26,35]. Then cell viability was determined by the MTT assay. Each condition and experiment was repeated three times.

Detection of apoptotic cells
Annexin V-FITC and PI staining analyzed by flow cytometry (Beckman-Coulter, USA) was used to detect the apoptotic index. The cells were plated in 6-well plates and exposed to 10 μmol/L Aβ [25][26][27][28][29][30][31][32][33][34][35] and /or 10 −10 mol/L triptolide or cell culture medium without treatment (control) for 24 hours. Then cells were harvested and rinsed with PBS. After that, cells were resuspended in 400 μL 1×binding buffer containing 10 μL PI and 5 μL V-FITC, and incubated for 15 min at room temperature in the dark. The cell suspension was determined by flow cytometry to analyze the apoptotic rate. The apoptosis ratio was calculated as follows: apoptosis ratio (%) = (the percentage of early apoptotic cells) + (the percentage of late apoptotic cells). The percentage of the cells is presented in the area of respective quadrant profiles. All experiments were performed a minimum of three times.

Measurement of ROS generation
The level of ROS induced by different conditions was measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Cells were exposed to cell culture medium, 10 μmol/L Aβ 25-35, and 10 μmol/L Aβ 25-35 +10 −10 mol/L triptolide for 24 hours. Then differentiated PC12 cells were incubated in the staining solution containing DCFH-DA for 20 min at 37°C and washed with PBS. The methodology followed the procedures as described in the ROS assay kit. The intracellular accumulation of ROS was measured by flow cytometry. The level of ROS generation was calculated as follows: the level of ROS (%) = the percentage of DCF-positive cells in M1 region. All experiments were performed a minimum of three times.

Acridine orange staining
Cellular acidic compartments were examined by acridine orange staining. Acridine orange (molecular formula: C 17 H 19 N 3 ÁHClÁZnCl 2 , molecular weight: 438.1, purity:!98%) was diluted to 1μmol/L with PBS. Differentiated PC12 cells were seeded in 6-well plates and treated with cell culture medium, 10 μmol/L Aβ 25-35 and /or 10 −10 mol/L triptolide for 24 hours. After treatment, the cells were stained with acridine orange at 37°C for 15 min. After washing three times with PBS, the cells were immediately visualized by a Leica TCS SP5 laser-scanning confocal microscope for the detection of acidic vesicular organelles. For the quantitation analysis, fluorescent intensity was quantified using Image-Pro Plus 6.0 (IPP 6.0) [38]. Data were analyzed from several cells of one sample, and there were seven samples for each group. The TIFF images were not processed before measurement of signal intensities.

Immunofluorescence
The cells were treated with cell culture medium, 10 μmol/L Aβ 25-35 , 10 μmol/L Aβ 25-35 +10 −10 mol/L triptolide and 10 −10 mol/L triptolide for 24 hours. Following a 30 min-fixation in 4% paraformaldehyde at 4°C, cells were washed with PBS, and then were permeabilized with 0.5% Triton X-100 and blocked with 10% NGS for 2 hours at room temperature. After that, cells were incubated with mouse anti-LC3 antibody as the primary antibody overnight at 4°C. After washing with PBS, cells were incubated with Alexa 594-conjugated anti-mouse IgG for 3 hours at room temperature. Thereafter, the cell nuclei were stained by DAPI. The fluorescent signals were examined using an Olympus FV1000 laser-scanning confocal microscope, and fluorescent intensity was quantified using IPP 6.0. Data were analyzed from several cells of one sample, and there were seven samples for each group. The TIFF images were not processed before measurement of signal intensities. All experiments were performed a minimum of three times.

Western blot assay
Differentiated PC12 cells were incubated with cell culture medium, 10 −10 mol/L triptolide, 10 μmol/L Aβ 25-35 and /or 10 −10 mol/L triptolide for 24 hours. Then cells were lysed for 30 min on ice. Protein samples were centrifuged for 15 min at 4°C. Samples were subjected to electrophoresis on SDS-polyacrylamide gel and the separated proteins were electrotransferred to polyvinylidenedifluoride (nitrocellulose) membranes. Membranes were blocked in 5% non-fat milk for 2 hours at room temperature, and then incubated with primary antibody (anti-LC3) overnight at 4°C. Subsequently, membranes were washed and incubated with a 1:2500 dilution of secondary antibodies for 1 hour at room temperature. The bands were visualized by a chemiluminescent HRP substrate detection kit. For analysis, quantization was performed by scanning and determination of the intensity of the hybridization signals. We used the LC3 II/LC3 I ratio to comprehensive assess autophagy flux. Protein loading and transferring of LC3 II and LC3 I were standardized by preliminary experiments in which β-actin expression on the same Western blot sample was quantitated by use of the Imge J software [39]. All experiments were performed a minimum of three times.

Statistical analysis
All of the results were expressed as mean ±S.E.M and analyzed by Origin 8.1 and SPSS 17.0. The results of the groups treated with the triptolide were compared to those of the no-triptolide -treated group and represented as the percentage of the control value. The test of Kolmogorov-Smirnov with the correction of Lilliefors was used to evaluate the normal distribution and the test of Levene to evaluate the homogeneity of variance. Statistical analysis was performed using one-way ANOVA followed by a Turkey's multiple range test and P<0.05 was considered significant.

Cytotoxicity induced by Aβ 25-35 on differentiated PC12 cells
We first tested the cytotoxicity of Aβ 25-35 on differentiated PC12 cells by MTT assay. Kolmogorov-Smirnov with the correction of Lilliefors and Levene test showed that all of our data satisfied the normal distribution (P>0.05) and the homogeneity of variance (P>0.05). Therefore, it was omitted in next experiments.

Effect of triptolide on Aβ 25-35 -induced apoptosis in differentiated PC12 cells
The effect of triptolide on Aβ 25-35 -induced apoptosis in differentiated PC12 cells was tested by flow cytometry, as shown in Fig 4. After incubation with 10 μmol/L Aβ 25-35 for 24 hours, the apoptotic rates of cells were increased significantly compared to those of the control group without treatment (Fig 4d). When treated with 10 −10 mol/L triptolide and 10 μmol/L Aβ 25-35 for 24 hours, the apoptotic rate of differentiated PC12 cells was significantly decreased. The results indicate that triptolide has neuroprotective effects on differentiated PC12 cells treated with Aβ 25-35 .

Morphological change observations using acridine orange staining
Acridine orange is a nucleic acid dye that also enters acidic compartments, such as acidic vesicular organelles, where it becomes protonated and sequestered. Acridine orange staining is often used to detect the occurrence of autophagy. As shown in Fig 6a, green fluorescence with minimal orange fluorescence was displayed in the control group; in the Aβ 25-35 -treated cells the acidic compartments displayed orange fluorescence and green fluorescence with minimal orange fluorescence being displayed in the triptolide-treated cells. Moreover, acidic compartments of Aβ 25-35 -treated differentiated PC12 cells (the level of red fluorescence) were markedly more than triptolide -treated cells (P<0.01) in Fig 6b. Our data (the raw data was showed in S1 Table) provide evidence that the number of acidic vesicular organelles increased when the cells were treated with Aβ [25][26][27][28][29][30][31][32][33][34][35] , which means that autophagy processes were activated, and triptolide could inhibit autophagy.

Morphological evaluation of autophagy
Staining with anti-LC3 is a recognized marker for autophagosomes. The fluorescence intensity and the number of bright fluorescent particles are related to the extent of lysosome acidity and  Table).

The level of LC3 in differentiated PC12 cells
To identify the level of autophagy, the expression of LC3 was examined by Western blot analysis. Conjugated LC3, called LC3 II, is the canonical marker of autophagosomes. During autophagy, LC3 I is cleaved to LC3 II, while the LC3 II/LC3 I ratio increases. As a result, we found that the LC3 II/LC3 I ratio of Aβ 25-35 -treated cells increased compared to the control groups (the raw figure was showed in S1 Fig). When incubated with triptolide, the LC3 II/LC3 I ratio in differentiated PC12 cells was significantly decreased (Fig 8). Our experiments suggest that autophagy was greatly and continually activated when differentiated PC12 cells were treated with Aβ 25-35 and triptolide could inhibit this autophagy process.
Conventional wisdom suggests that ROS was one of the indicators of oxidative stress and an important factor in the apoptotic process [36,44]. Accumulating evidence suggests that ROS act as signaling molecules involving a variety of intracellular processes. Furthermore, the intracellular excessive accumulation of ROS might mediate autophagy [45,46]. For example, the addition of H 2 O 2 could induce autophagy [47]. In some diseases, endogenous ROS levels were The Protective Effect of Triptolide on Cytotoxicity Induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] raised which then activated mitochondrial autophagy [47,48]. In our research, the data of flow cytometry showed that the ROS level significantly increased when the differentiated PC12 cells were incubated with Aβ 25-35, and treatment with triptolide could decrease the intracellular ROS level in cells (Fig 5). We speculated that Aβ 25-35 might induce autophagy through elevating the ROS level in differentiated PC12 cells and triptolide protected the cell by reducing the generation of ROS to weaken autophagy induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] . However, ROS could induce autophagy or play an important role in the process, and various circumstances could cause an increase of intracellular ROS. Meanwhile, we also found that ROS generation of triptolide group was intermediate of the Aβ 25-35 group and higher than control group, which meant that the pathway activated by ROS was just one of mediators in autophagy. The Protective Effect of Triptolide on Cytotoxicity Induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] Acridine orange is a fluorescent dye which stains acidic compartments (such as lysosomes and autolysosomes) orange/red, while it stains cytoplasm and nuclei bright green. Acidic vacuoles, which are formed by autophagosomes fusing with lysosomes, can usually bind acridine orange in the process of cells' autophagy. Therefore, acridine orange staining is often used to The Protective Effect of Triptolide on Cytotoxicity Induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] detect the occurrence of autophagy [49,50]. Furthermore, in Fig 6, Aβ 25-35 -treated cells showed an obvious increase in orange fluorescence compared with that of the control group (P<0.01), which was indicated by the level of red fluorescence. And triptolide could decrease the level of acidic vesicular organelle during the autophagy process (P<0.01).
3-MA is an inhibitor of the class I phosphatidylinositol 3-kinase (PI3K) [56]. Further in vitro enzymatic assays show that 3-methyladenine also inhibits class III PI3K activity [57]. Recent studies indicate that PI3K, especially class III PI3K, is essential in the regulation of autophagy. 3-MA inhibits autophagy by inhibition of these enzymes. Rapamycin is commonly used as a specific activator of autophagic sequestration. The literature observes that the increase is accompanied by a significant increase in autophagy due to the mammalian target of the rapamycin (mTOR) pathway inhibition [58]. As a result, cell viability was increased by 3-MA, and there was a significant difference between the Aβ 25-35 +3-MA-treated group and Aβ-treated group (P<0.01). 10 ng/mL of rapamycin could markedly reduce the viability compared with the triptolide-treated group (P<0.01) (Fig 9). These results further confirm that autophagy plays an important role in the protection of triptolide on differentiated PC12 cells.
In summary, we discovered that Aβ 25-35 could induce cell death and cell apoptosis via the activation of autophagy in differentiated PC12 cells. Furthermore, triptolide has a protective effect against Aβ 25-35 -induced cytotoxicity in differentiated PC12 cells by inhibiting the autophagy pathway. In our further experiments, we will detect the molecular signaling mechanism of oxidative stress (because of the generation of ROS in triptolide group) and investigate whether triptolide inhibits autophagy in an mTOR-dependent manner (because of the function of rapamycin), and we will also check the effect of triptolide in primary cells and in in vivo models to compare the difference of the molecular signaling mechanism of autophagy in pathogenesis of AD between in vivo and in vitro. These results may suggest that triptolide will be a promising tool for AD research. It will be helpful to provide an interesting view of the potential application of triptolide or TWHF in the future study of AD.  Table. Raw data of acridine orange staining. For the quantitation analysis, red fluorescent intensity and green fluorescent intensity were quantified using IPP 6.0. (DOCX) S2 Table. Raw data of the expression of LC3 with immunofluorescence staining. For the quantitation analysis, fluorescent intensity was quantified using IPP 6.0. (DOCX)