Introduction

Oxidative stress plays an important role in the loss of neurons in many neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and cerebral ischemia (Kim et al. 2015). Many reactive oxygen species (ROS), including hydrogen peroxide (H2O2), nitric oxide (NO), and highly reactive hydroxyl and monoxide radicals can be released immediately after injury of neurons during oxidative stress (Bhat et al. 2015). This excessive ROS further induces neuronal apoptosis via their interactions with macromolecules as well as their ability to regulate both pro-survival and pro-apoptotic signaling pathways (Yoon et al. 2002). H2O2, an uncharged and freely diffusible molecule, is widely used as a neurotoxin to establish in vitro models of oxidative stress-induced neuronal apoptosis (Lee et al. 2007). Neuronal apoptosis can be regulated by the inhibition of pro-survival signaling pathways such as the phosphoinositide 3-kinase (PI3-K)/Akt cascade and the activation of pro-apoptotic signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway. Inhibition of PI3-K/Akt led to the activation of glycogen synthase kinase 3β (GSK3β), and was reported to be involved in H2O2-induced neuronal apoptosis (Gao et al. 2012; Lin et al. 2016). Moreover, inhibition of extracellular signal-regulated kinase (ERK), a key intermediate of the MAPK signaling pathway, has been proposed as a pro-apoptotic mechanism underlying H2O2-induced neuronal apoptosis (Yang et al. 2005; Lin et al. 2016).

Indirubin-3-oxime is a derivative of indirubin, an active constituent of the traditional Chinese medicine recipe Danggui Longhui Wan used to treat chronic myelocytic leukemia (Smith et al. 2006). Interestingly, a recent pharmacokinetics study has shown that indirubin-3-oxime could easily cross the blood–brain barrier, suggesting that this chemical might be used to treat brain disorders (Selenica et al. 2007). Previous studies have demonstrated that indirubin-3-oxime inhibits neuronal apoptosis induced by β-amyloid, 6-hydroxydopamine, and potassium deprivation in vitro (Zhang et al. 2009; Hu et al. 2015). Moreover, indirubin-3-oxime prevents behavioral abnormities induced by many neurotoxins in rodents, possibly by inhibiting oxidative stress-induced neuronal loss (Wang et al. 2007; Ding et al. 2010). However, the underlying molecular mechanisms by which indirubin-3-oxime protects against oxidative stress-induced neuronal apoptosis are largely unknown.

SH-SY5Y cells are rat human neuroblastoma which has been reported to be sensitive to oxidants (Li et al. 1996). Therefore, SH-SY5Y cells could be used for studying the molecular mechanisms of drugs against oxidative stress-induced neuronal apoptosis (Nirmaladevi et al. 2014; Tian et al. 2015). In addition, homogenous cerebellar granule neurons (CGNs) are easy to be acquired because more than 90 % of neurons in cerebellum are CGNs (Gonzalez-Polo et al. 2004). Therefore, CGNs could be used to investigate the effects of neuroprotective drugs on primary neurons.

In this study, we have shown that indirubin-3-oxime effectively prevents H2O2-induced neuronal apoptosis in both SH-SY5Y cells and primary CGNs. Moreover, our results have demonstrated that indirubin-3-oxime protects against H2O2-induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway.

Materials and Methods

Chemicals and Reagents

H2O2 was obtained from Calbiochem (San Diego, CA, USA). Indirubin-3-oxime and SB415286 were obtained from Sigma Chemicals (St Louis, MO, USA). PD98059, U0126, Wortmannin, and LY294002 were purchased from LC Laboratories (Woburn, MA, USA). Antibodies against pSer473-Akt, Akt, pSer9 glycogen synthase kinase 3β (GSK3β), GSK3β, phospho-Thr202/Tyr204-p44/42 (pERK), and ERK were obtained from Cell Signaling Technology (Beverly, MA, USA). Unless otherwise noted, all media and supplements used for cell cultures were purchased from Invitrogen (Carlsbad, CA, USA).

SH-SY5Y Cells Culture

SH-SY5Y cells were purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences), and cultured in high glucose modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 μg/ml). The cells were cultured in an incubator at 37 °C and 5 % CO2. The medium was replaced every other day. For the experiments with H2O2, SH-SY5Y cells (1 × 105 cells/ml) in DMEM with low serum content (1 % FBS) were seeded in 6-well or 96-well plates. All experiments were carried out 24 h after the cells were seeded.

Primary CGNs Culture

CGNs were prepared from 8-day-old Sprague–Dawley rats as described in our previous publication (Luo et al. 2010). Briefly, neurons were seeded at a density of 2.7 × 105 cells/cm2 in basal modified Eagle’s medium containing 10 % FBS, 25 mM KCl, 2 mM glutamine, and penicillin (100 U/ml)/streptomycin (100 μg/ml). Cytosine arabinoside (10 μM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. Granule cells were identified by a combination of several criteria including their size, shape and relative proportion of the total cell population as determined by phase contrast microscopy.

Measurement of Cell Viability

Cell viability was determined by the activity of mitochondrial dehydrogenases via the 3(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay as previously described (Cui et al. 2013, 2014). Briefly, after treatment, 10 μl of 5 mg/ml MTT solution was added to each well. Plates were incubated at 37 °C for 4 h in a humidified incubator. 100 μl of the solvating solution (0.01 N HCl in 10 % SDS solution) was then added to each well for 16–20 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm as a reference wavelength. Unless otherwise indicated, the extent of MTT conversion in cells without treatment is expressed as a percentage of the control.

Measurements of Lactate Dehydrogenase (LDH) Release

Cytotoxicity was evaluated by measuring the release of LDH after drug treatment for 24 h. Cells were incubated with 2 % (v/v) Triton X-100 in culture medium for 30 min at 37 °C to obtain a maximal LDH release as the positive control with 100 % toxicity. Extracellular LDH release was further determined in the conditioned media collected from the culture dishes using the LDH assay kit (Roche Diagnostics, Indianapolis, IN, USA) according to manufacturer’s instructions. Briefly, 50 μl culture supernatants were collected from each well with the addition of 50 μl reaction buffer. Thirty minutes after mixing at room temperature, the release of LDH was measured at a wavelength of 490 nm with 655 nm as a reference wavelength.

Fluorescein Diacetate/Propidium Iodide Double Staining Assay

Viable cells were visualized by the fluorescein formed from fluorescein diacetate (FDA) by esterase activity in viable cells. Non-viable cells were stained by propidium iodide (PI) intercalation into DNA, which only penetrates the cell membranes of dead cells. Briefly, after incubation with 10 μg/ml of FDA and 5 μg/ml of PI for 15 min, the cells were examined and images were acquired using UV light microscopy for comparison with photos taken under phase contrast microscopy. To quantitative evaluate cell viability, photos of each well were taken from five random fields and the number of PI-positive cells and FDA-positive cells was counted. % of cell viability = [number of FDA-positive cells/(number of PI-positive cells + number of FDA-positive cells)] × 100 % was then averaged (Jones and Senft 1985).

Hoechst Staining

Chromatin condensation was detected by staining the cell nuclei with Hoechst-33342 as described previously (Cui et al. 2012; Hu et al. 2014). After treatment, cells grown in six-well plates were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4 % formaldehyde in PBS for 15 min, membrane permeabilized in 0.1 % Triton X-100 for 15 min, and blocked in 1 % bovine serum albumin (BSA) for 15 min. Cells were then stained with Hoechst-33342 (5 μg/ml) at 4 °C for 5 min. Images were acquired using a fluorescence microscope (Nikon Instruments Inc. Melville, NY, USA) at 100× magnification. Ultraviolet excitation and emission wavelengths were used to obtain images of nuclei labeled with Hoechst-33342. To quantify the percentage of apoptotic nuclei in each group, photos of each well were taken from five random fields and the number of pyknotic nuclei and total nuclei counted. The percentage of pyknotic nuclei was then averaged.

Western Blot Analysis

Western blotting was performed as previously described (Hu et al. 2013). Protein lysates were separated on SDS-polyacrylamide gels and transferred onto polyvinyldifluoride membranes. After membrane blocking, proteins were detected using primary antibodies. After incubation at 4 °C overnight, signals were obtained after binding to HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were developed using the enhanced chemiluminescence plus kit (Amersham Bioscience, Aylesbury, UK) and exposed to autoradiographic film.

Statistical Analysis

Results are expressed as mean ± SEM. Analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s test was used for statistical comparisons. Levels of p < 0.05 were considered to be of statistical significance.

Results

Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis in SH-SY5Y Cells

It was previously reported that treatment with 150 μM H2O2 for about 24 h could induce typical apoptosis in SH-SY5Y cells (Tian et al. 2015). Therefore, we used this model to investigate the neuroprotective effects of indirubin-3-oxime. SH-SY5Y cells were pre-treated with a dose curve of indirubin-3-oxime (0.1–3 μM) for 2 h, followed by treatment with 150 μM H2O2 for 24 h. Cell viability was then measured using the MTT assay. It was found that indirubin-3-oxime significantly prevented H2O2-induced neuronal death in a dose-dependent manner (Fig. 1a). We also assayed LDH release to evaluate alterations in cell membrane permeability. As shown in Fig. 1b, indirubin-3-oxime (0.3–3 μM) significantly prevented the H2O2-induced increase of LDH release in a concentration-dependent manner, providing further support that indirubin-3-oxime can prevent H2O2-induced neuronal death. Treatment with 3 μM indirubin-3-oxime alone for 26 h was not cytotoxic and did not alter cell proliferation.

Fig. 1
figure 1

Indirubin-3-oxime prevents H2O2-induced neuronal death in SH-SY5Y cells in a dose-dependent manner. SH-SY5Y cells were pre-treated with indirubin-3-oxime at the indicated concentrations for 2 h, and then exposed to 150 μM H2O2. Cell viability was measured by a the MTT assay or b the LDH assay at 24 h after H2O2 challenge. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus the H2O2-treated group (ANOVA and Dunnett’s test)

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To further characterize the protective effect of indirubin-3-oxime against neurotoxicity induced by H2O2, treated and control SH-SY5Y cells were examined by FDA/PI double staining. It was found that indirubin-3-oxime significantly blocked the loss of neurons induced by H2O2 (Fig. 2). Moreover, according to the number of pyknotic bodies stained by Hoechst-33342, indirubin-3-oxime significantly prevented apoptosis induced by H2O2 in SH-SY5Y cells (Fig. 3).

Fig. 2
figure 2

Indirubin-3-oxime blocks H2O2-induced cell death in SH-SY5Y cells. SH-SY5Y cells were pre-treated with 3 μM indirubin-3-oxime (I3O) or vehicle control for 2 h, and then exposed to 150 μM H2O2. After 24 h of H2O2 challenge, SH-SY5Y cells were assayed with FDA/PI double staining. Cell viability was analyzed from representative photomicrographs. Data were the mean ± SEM of three separate experiments; **p < 0.01 versus the H2O2-treated group (ANOVA and Dunnett’s test)

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Fig. 3
figure 3

Indirubin-3-oxime significantly prevents H2O2-induced apoptosis in SH-SY5Y cells. SH-SY5Y cells were pre-treated with 3 μM indirubin-3-oxime or vehicle control for 2 h, and then exposed to 150 μM H2O2. After 24 h of H2O2 challenge, SH-SY5Y cells were assayed with Hoechst staining. The number of pyknotic nuclei with condensed chromatin were counted from representative Hoechst staining photomicrographs and represented as a percentage of the total number of nuclei counted. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus the H2O2-treated group (ANOVA and Dunnett’s test)

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Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis in CGNs

We have reported that the treatment of 30 μM H2O2 for 6 h could lead to neuronal death in primary CGNs (Cui et al. 2011). In this study, we further investigated whether indirubin-3-oxime could produce neuroprotective effects in primary neurons. CGNs were pre-treated with indirubin-3-oxime for 2 h, and then treated with 30 μM H2O2 for another 6 h. CGNs were examined by MTT assay or FDA/PI double staining. It was found that indirubin-3-oxime significantly blocked neuronal death induced by H2O2 in CGNs (Fig. 4).

Fig. 4
figure 4

Indirubin-3-oxime blocks H2O2-induced neuronal death in primary CGNs. a CGNs were pre-treated with indirubin-3-oxime (I3O) at the indicated concentrations for 2 h, and then exposed to 30 μM H2O2. Cell viability was measured by the MTT assay at 6 h after H2O2 challenge. b CGNs were pre-treated with 3 μM indirubin-3-oxime for 2 h, and then exposed to 30 μM H2O2. After 6 h of H2O2 challenge, CGNs were assayed with FDA/PI double staining. Cell viability was analyzed from representative photomicrographs. Data were the mean ± SEM of three separate experiments; **p < 0.01 versus the H2O2-treated group (ANOVA and Dunnett’s test)

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Activation of GSK3β and the ERK Signaling are Involved in the Neurotoxicity Induced by H2O2 in SH-SY5Y Cells

It has been reported that inhibition of the activation of GSK3β and the ERK signaling are involved in the neurotoxicity induced by oxidative stress (Gao et al. 2012; Lin et al. 2016). To determine whether the alteration of these molecules is involved in our model, Western blotting analysis was used. As shown in Fig. 5, H2O2 decreased the levels of pSer473-Akt and pSer9-GSK3β in SH-SY5Y cells in a time-dependent manner. Moreover, the level of phospho-ERK increased significantly during the first hour post-treatment, although it returned to near basal level 2 h after H2O2 challenge (Fig. 6). Furthermore, SB415286, a specific inhibitor of GSK3β, and PD98059 and U0126, two specific inhibitors of MEK, significantly attenuated H2O2-induced neuronal death in SH-SY5Y cells (Fig. 7). These results suggest that the activation of GSK3β and MEK are involved in the neurotoxicity induced by H2O2 in SH-SY5Y cells. Interestingly, co-application of SB415286 and PD98059 significantly produced the neuroprotective effects, which are similar as those of indirubin-3-oxime.

Fig. 5
figure 5

H2O2 decreases the levels of pSer473-Akt and pSer9-GSK3β in a time-dependent manner in SH-SY5Y cells. SH-SY5Y cells were incubated with 150 μM H2O2 for various durations as indicated. Western blotting analysis was performed to detect protein expression of a pSer473-Akt and b pSer9-GSK3β. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *p < 0.05 and **p < 0.01 versus the control group (ANOVA and Dunnett’s test)

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Fig. 6
figure 6

H2O2 increases the levels of phospho-ERK in SH-SY5Y cells. SH-SY5Y cells were incubated with 150 μM H2O2 for various durations as indicated. Western blotting analysis was used to detect protein expression of phospho-ERK. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus the control group (ANOVA and Dunnett’s test)

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Fig. 7
figure 7

Inhibition of GSK3β and MEK prevent H2O2-induced neuronal death. SH-SY5Y cells were pre-treated with SB415286, PD98059, U0126, or SB415286/PD98059 at the indicated concentrations for 2 h, and then exposed to 150 μM H2O2. Cell viability was measured by the MTT assay 24 h after H2O2 challenge. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *p < 0.05 and **p < 0.01 versus H2O2-treated group, # p < 0.05 versus H2O2 + 30 μM SB415286 group (ANOVA and Dunnett’s test)

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Indirubin-3-Oxime Prevents the Activation of GSK3β by H2O2

The levels of pSer473-Akt and pSer9-GSK3β were determined in indirubin-3-oxime-treated cells by Western blotting. Pre-treatment with 3 μM indirubin-3-oxime prevented the decrease of both pSer473-Akt and pSer9-GSK3β that were caused by H2O2 (Fig. 8). Additionally, two PI3-K specific inhibitors, LY294002 and wortmannin, were used to investigate the mechanism of neuroprotection by indirubin-3-oxime. The inhibition of PI3-K by either 10 μM LY294002 or 0.05 μM wortmannin significantly blocked the neuroprotective effects of indirubin-3-oxime against H2O2-induced neuronal death in our model (Fig. 9).

Fig. 8
figure 8

Indirubin-3-oxime prevents the H2O2-induced decrease of pSer473-Akt and pSer9-GSK3β in SH-SY5Y cells. SH-SY5Y cells were pre-treated with indirubin-3-oxime (I3O) at the indicated concentrations for 2 h, and then exposed to 150 μM H2O2. Western blotting analysis was used at 2 h after H2O2 challenge to detect levels of a pSer473-Akt and b pSer9-GSK3β. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus H2O2-treated group (ANOVA and Dunnett’s test)

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Fig. 9
figure 9

PI3-K specific inhibitors abolish the neuroprotective effects of indirubin-3-oxime on H2O2-induced cell death in SH-SY5Y cells. SH-SY5Y cells were pre-treated with LY294002 (LY) or wortmannin (Wort) at the indicated concentrations for 0.5 h, and then supplemented with 3 μM indirubin-3-oxime (I3O) for 2 h before the exposure to 150 μM H2O2. Cell viability was then measured by the MTT assay 24 h after H2O2 challenge. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus H2O2-treated group, ## p < 0.01 versus H2O2 plus I3O group (ANOVA and Tukey’s test)

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Indirubin-3-Oxime Inhibits the Activation of ERK Signaling Induced by H2O2

To further examine whether indirubin-3-oxime protected SH-SY5Y cells against neuronal death via inhibition of the ERK pathway, the level of phospho-ERK was also determined in indirubin-3-oxime-treated cells by Western blotting. As shown in Fig. 10, pre-treatment with indirubin-3-oxime at 3 μM for 2 h significantly prevented the increase of phospho-ERK that was induced by H2O2 at 0.5 h, suggesting that indirubin-3-oxime also prevents H2O2-induced neuronal death by preventing activation of the ERK pathway.

Fig. 10
figure 10

Indirubin-3-oxime prevents the H2O2-induced increase of phospho-ERK in SH-SY5Y cells. SH-SY5Y cells were pre-treated with indirubin-3-oxime (I3O) at the indicated concentrations for 2 h, and then exposed to 150 μM H2O2. Western blotting analysis was used at 30 min after H2O2 challenge to detect levels of phospho-ERK. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus H2O2-treated group (ANOVA and Dunnett’s test)

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Discussion

In this study we have shown that indirubin-3-oxime effectively prevents H2O2-induced neuronal apoptosis. Moreover, our results suggest that the neuroprotective effects of indirubin-3-oxime were mediated through simultaneous inhibition of GSK3β and the ERK signaling.

H2O2-induced oxidative stress in neurons can lead to both apoptotic and necrotic death depending on the concentration of H2O2 used (Valencia and Moran 2004; Fatokun et al. 2007). Compounds with anti-apoptotic properties may have therapeutic utility in neurodegenerative diseases characterized by neuronal apoptosis (Bachis et al. 2001). In our study, we have shown that H2O2 could significantly increase the number of pyknotic bodies, suggesting that H2O2 mainly induces neuronal apoptosis rather than necrosis in SH-SY5Y cells, which is consistent with other reports that exposure of 150 μM H2O2 for 24 h produces neuronal apoptosis in SH-SY5Y cells (Tian et al. 2015).

Indirubin-3-oxime has previously been reported to have neuroprotective effects (Sharma and Taliyan 2014; 2015). It was reported that systemic administration of indirubin-3-oxime (30 mg/kg i.v. or 20 mg/kg i.p.) could easily cross the blood–brain barrier, and retain in the brain at 4 h after administration, suggesting that indirubin-3-oxime might be used in the treatment of neurological diseases (Selenica et al. 2007). Interestingly, previous studies have also shown that indirubin-3-oxime can suppress excessive ROS production in both lipopolysaccharide-induced primary microglia cultures and 6OHDA-induced PC12 cells, suggesting that its neuroprotective activity may be mediated by anti-oxidative properties (Jung et al. 2010; Hu et al. 2015). In our study, we demonstrated that indirubin-3-oxime prevented H2O2-induced neuronal death not only in SH-SY5Y cells, but also in primary CGNs, providing a strong support that indirubin-3-oxime could effectively protected against ROS-induced neuronal loss and may be used in the treatment of neurological diseases.

How does indirubin-3-oxime produce neuroprotective effects in our model? We have evaluated the effects of indirubin-3-oxime on ROS scavenge using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and found that indirubin-3-oxime (0.1–10 μM) could not decrease DPPH radical, suggesting that the neuroprotective effects of indirubin-3-oxime might not due to ROS scavenge (Data not shown). We speculated that indirubin-3-oxime might regulate pro-survival and pro-apoptotic signaling pathways that are involved in H2O2-induced apoptosis. Indirubin-3-oxime is a potent and direct inhibitor of GSK3β with an IC50 of 22 nM (Eisenbrand et al. 2004). In our study, we found that indirubin-3-oxime prevents the H2O2-induced decrease of pSer9-GSK3β. We also showed that SB415286, another small molecule of GSK3β inhibitor, prevented H2O2-induced neuronal apoptosis, supporting the role of GSK3β inhibition in the neuroprotective effects of indirubin-3-oxime. However, indirubin-3-oxime (3 μM) could almost fully prevent H2O2-induced cell death. SB415286 could not fully reversed H2O2-induced cell death at high concentrations, suggesting that indirubin-3-oxime might act on other neuroprotective target(s) besides GSK3β.

Activation of the ERK signaling has been regarded as one of the key pathways mediating neuronal apoptosis (Jiang et al. 2005). We found that indirubin-3-oxime prevents the H2O2-induced increase of pERK. Furthermore, we show that treatment with MEK inhibitors can also prevent H2O2-induced neuronal apoptosis, suggesting that inhibition of the ERK signaling may also be involved in the neuroprotective effects of indirubin-3-oxime. How could indirubin-3-oxime act on the ERK pathway? Indirubin-3-oxime could compete with ATP for binding to the catalytic subunit of kinase (Zhang et al. 2009). Due to the similarities of ATP binding site among different kinases, indirubin-3-oxime inhibits many kinases, including GSK3β, cyclin dependent kinase (CDK) 1, CDK2, and CDK5 (Rivest et al. 2011). We speculated that indirubin-3-oxime might directly act on the ATP binding site of the upstream kinases of ERK, e.g., MEK, and inhibit the phosphorylation of ERK. To further determine which kinase(s) are directly inhibited by indirubin-3-oxime, additional experiments are being undertaken in our lab.

How could PI3-K inhibitors abolish the neuroprotective effects of indirubin-3-oxime when GSK3β was inhibited by indirubin-3-oxime? Previous studies have shown that there is a crosstalk between the PI3-K and the ERK pathways (Frebel and Wiese 2006; Yang et al. 2005; Heras-Sandoval et al. 2014). Inactivation of PI3-K triggered the activation of the ERK pathway. We speculated that PI3-K inhibitors might abolish indirubin-3-oxime-induced neuroprotective effects independent of GSK3β activation, possibly via the activation of the ERK pathway.

In summary, we have found that indirubin-3-oxime prevents H2O2-induced neuronal apoptosis via concurrent inhibition of GSK3β and the ERK signaling. Our results also provide support for the use of indirubin-3-oxime or similar compounds in the treatment neurodegenerative disorders caused or exacerbated by oxidative stress.