Environmental Toxicology and Pharmacology 40 (2015) 230–240

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Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap

Neuroprotective effects of Kukoamine B against hydrogen peroxide-induced apoptosis and potential mechanisms in SH-SY5Y cells Xiao-Long Hu a,b , Yi-Xuan Niu b , Qiao Zhang b , Xing Tian b , Ling-Yue Gao b , Li-Ping Guo b , Wei-Hong Meng a , Qing-Chun Zhao a,∗ a b

Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China

a r t i c l e

i n f o

Article history: Received 22 April 2015 Received in revised form 9 June 2015 Accepted 12 June 2015 Available online 22 June 2015 Keywords: Neuroprotection Kukoamine B Oxidative stress Hydrogen peroxide SH-SY5Y cells

a b s t r a c t Oxidative stress mediates the cell damage in several neurodegenerative diseases, including multiple sclerosis, Alzheimer’s disease (AD) and Parkinson’s disease (PD). This study aimed at investigating the protective effects of Kukoamine B (KuB) against hydrogen peroxide (H2 O2 ) induced cell injury and potential mechanisms in SH-SY5Y cells. Our results revealed that treatment with KuB prior to H2 O2 exposure effectively increased the cell viability, and restored the mitochondria membrane potential (MMP). Furthermore, KuB enhanced the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and decreased the malondialdehyde (MDA) content. Moreover, KuB minimized the ROS formation and inhibited mitochondria-apoptotic pathway, MAPKs (p-p38, p-JNK, pERK) pathways, but activated PI3K-AKT pathway. In conclusion, we believed that KuB may potentially serve as an agent for prevention of several human neurodegenerative and other disorders caused by oxidative stress. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Brain is a vital organ in human body and more predisposed to oxidative stress because of their high metabolic rate and high content of polyunsaturated fatty acids. Therefore, oxidative stress results in Reactive Oxygen Species (ROS) overload is commonly in the etiology of human diseases, especially in brain injury diseases, and ROS associate with the neuron death results in various chronic neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (Barnham et al., 2004; Everse and Coates, 2009; Jenner, 2003; Simonian and Coyle, 1996; Yan et al., 2013). In addition, the DNA damage, oxidative of proteins and peroxidation of lipids caused by ROS will further result in neuronal apoptosis (Halliwell and Aruoma, 1991). Hydrogen peroxide (H2 O2 ), a major ROS contributor, could induce apoptosis in many cells especially in neuronal cells (Sherer et al., 2002) and is generated as a by-product of enzymatic action during the processes

∗ Corresponding author at: Department of Pharmacy, General Hospital of Shenyang Military Area Command, 83 Wenhua Road, Shenyang 110016, China. E-mail address: [email protected] (Q.-C. Zhao). http://dx.doi.org/10.1016/j.etap.2015.06.017 1382-6689/© 2015 Elsevier B.V. All rights reserved.

of dopamine oxidation, amyloid aggregation, and brain ischemia reperfusion (Nelson et al., 2005). In recent years, accumulated evidences have suggested that H2 O2 induces cytotoxicity in SH-SY5Y cells, which provides a suitable model system in studying neuronal cell death caused by oxidative stress, such as: (1) membrane and nuclear damage, (2) MMP loss and antioxidant enzyme activities decline, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and (3) increasing the expression of p-MAPKs and decreasing the p-AKT (Nirmaladevi et al., 2014; Xiao et al., 2013; Ruffels et al., 2004). Therefore, H2 O2 -induced SH-SY5Y cells apoptosis model was employed in our research. Natural products are rich in diverse bioactive with suited health benefits. Many plant species have been found to provide neuroprotective activity. Recently, increasing attentions have been given to natural products with the potential neuroprotection of protecting the cells from oxidative damage (Ju et al., 2012; Si et al., 2013). However, many antioxidant compounds could prevent the damage by scavenging ROS, but usually accompanied with some adverse effects (Slemmer et al., 2008). Consequently, compounds that have the ability of scavenging excessive ROS, the capacity of anti-oxidative stress, might be a great potential therapeutic agent of brain diseases.

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Fig. 1. Effects of KuB on H2 O2 -induced cytotoxicity in SH-SY5Y cells. (A) The chemical structure of Kukoamine B. (B) Different concentrations of KuB (5–40 ␮M) on cell viability. (C) Different concentrations of H2 O2 on cell viability. (D) The effect of KuB on SH-SY5Y cell viability. (E) The release of LDH. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

Cortex lycii radicis, a traditional Chinese herbal medicine, is generally used as a tonic and the activity of its exhibit hypotensive, hypoglycaemic, antipyretic and anti-stress ulcer were reported in animal experiments. However, there is almost no reports of Cortex lycii radicis and its bioactive compounds concerning the neuroprotective effects. In our previous study, we found Kukoamine A (KuA), the isomeride of Kukoamine B (KuB) (Fig. 1A), had the ability of anti-oxidative stress and NMDA receptors antagonism (Hu et al., 2015). However, we found (KuB) has stronger anti-oxidative stress ability than KuA in our subsequently research. But the neuroprotective effects of KuB against H2 O2 -induced neuronal cell death and its underlying mechanisms are still unclear. Therefore, we focused on the anti-oxidative stress ability of KuB and the potential mechanisms of its neuroprotection in our study.

2. Materials and methods 2.1. Reagents and antibodies KuB was purchased from the Chengdu Biopurify Phytochemicals Ltd. (China) with more than 98.22% purity. 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), 0.25% trypsin – EDTA and dimethyl sulfoxide (DMSO) were purchased from Amresco (Solon, OH, USA). The kit for lactate dehydrogenase (LDH) assay was obtained from JianCheng Ltd. (Nanjing, China). The 2,7 dichlorofluorescin diacetate (DCF-DA), SOD, MDA, CAT and GSH-Px assay kits were acquired from Beyotime (Nanjing, China). The AnnexinV-PI double staining assay kit was purchased from BD Pharmingen (CA, USA). Rhodamine123, Hoechst33342, and H2 O2 were obtained from Sigma-Chemical (St. Louis, MO, USA). We purchased fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium from Hyclone (Logan, UT, USA). Rabbit antibodies for caspase-3, caspase-9, Bcl-2 (C21), Bax (P19) and mouse antibodies for cytochrome c, ␤-actin and the secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit antibodies for p-ERK1/2, t-ERK, p-JNK, t-JNK, p-p38, t-p38, p-AKT, t-AKT were purchased from Cell Signalling

Technology (Inc. USA). All other reagents and chemicals are analytical grade. 2.2. Cell culture and drug treatments Cell culture: SH-SY5Y cells were cultured in DMEM-F12 (1:1) medium supplemented with 10% FBS. Cells were incubated at 37 ◦ C in a humidified atmosphere incubator of 5% CO2 . The culture medium was changed on alternative day. Drug treatments: KuB was dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was less than 0.1% (v/v). SH-SY5Y cells were seeded in 96-well plates (1 × 104 cells/well) or 6-well plates (2 × 105 cells/well) for 24 h and pretreated with different concentrations of KuB (5, 10, 20 ␮M) for 2 h. Then, supernatant was replaced with fresh medium containing H2 O2 (final concentration: 100 ␮M) for another 12 h. After these treatments, the supernatant or the cells were collected for the following experiments. All the data were obtained from three independent experiments. 2.3. Cell viability assay Cytoprotective activity of KuB on H2 O2 -induced cell injury was assessed by MTT assay. The cells were cultured in 96-well plates and the subsequent procedures were operated as described above. After these treatments, MTT (final concentration: 0.5 mg/mL) was added to the medium and cells were incubated for 4 h at 37 ◦ C in the dark. Then, the formed insoluble formazan crystals were dissolved with 150 ␮L DMSO and the absorbance at 490 nm was measured by a microplate reader (Elx 800 Bio-Tek, USA). 2.4. Lactate dehydrogenase (LDH) release assay The cytotoxicity of H2 O2 and the protective effects of KuB were further evaluated by LDH assay. The cells were seeded into 96-well plates and then treated as mentioned above. After these treatments, the supernatant was collected, which was used with the LDH assay

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Fig. 2. Effects of KuB on H2 O2 -induced nuclear condensation in SH-SY5Y cells. Cells were pretreated with KuB (5, 10, 20 ␮M) for 2 h and then exposed to H2 O2 for 12 h. Representative fluorescence images were obtained after Hoechst 33342 staining in SH-SY5Y cells (200×).

Kit (Beyotime, Nanjing, China) according to manufacturer’s instructions. The absorbance at 450 nm was measured by a microplate reader (Elx 800 Bio-Tek, USA).

2.5. Hoechst 33342 staining assay SH-SY5Y cells were seeded into 6-well plates and then treated as described above. After these treatments, the culture medium was removed and the cells were stained with Hoechst 33342 dye (final concentration: 10 ␮g/mL) for 15 min in the dark (Tan et al., 2013). Then the dye liquor was removed and the cells were washed three times with PBS. Finally, the cells were visualized under a fluorescence microscope (IX71, Olympus, Japan).

2.6. Annexin V-FITC and propidium iodide (PI) double staining assay SH-SY5Y cells were seeded into 6-well plates and treated as mentioned above. After these treatments, cells were collected and then washed twice with cold PBS. According to the manufacturer’s instructions of AV-PI kit (Becton Dickinson, NJ, USA), cells (1 × 106 cells/mL, 100 ␮L) were counted and then loaded with PI (5 ␮L) and Annexin V-FITC (5 ␮L) for 15 min in the dark at room temperature. After the incubation, 400 ␮L buffer was added in each sample and then detected by a flow cytometer (Becton Dickinson, NJ, USA).

2.7. Measurement of intracellular ROS levels assay The cells were seeded into 6-well plates and treated as mentioned above. After these treatments, the cells were washed twice with PBS and loaded with DCFH-DA (final concentration: 5 ␮M) in serum-free medium (Menazza et al., 2010). The supernatant was discarded after incubation for 30 min at 37 ◦ C in the dark, and then the cells were washed twice with PBS again. Finally, images were obtained with a fluorescence microscope at 525 nm (IX71, Olympus, Japan). Image J was used to quantify the fluorescent intensity.

2.8. Mitochondrial membrane potential (MMP) assay Rhodamine 123 (Rho 123), a fluorescent dye, was used for the quantitative expression of MMP (Luo et al., 2011). SH-SY5Y cells were seeded into 6-well plates and treated as described above. After these treatments, Rho 123 (final concentration: 10 ␮g/mL) was added and the cells were incubated for 20 min at 37 ◦ C in the dark. Then the cells were washed twice with PBS and the fluorescence images were captured by a fluorescence microscope at 529 nm (IX71, Olympus, Japan). 2.9. Measurement of GSH-Px, SOD, CAT activities and MDA level SH-SY5Y cells were seeded into 6-well plates and treated as described earlier. Then cells were incubated with lysis buffer on ice for 30 min. After that, the lysed cells were centrifuged at 10,000 × g for 10 min. After these treatments, the supernatant was used to determine the protein concentrations using the BCA protein assay kit (Beyotime, Nanjing, China). And the supernatant was also used in the assays of GSH-Px, SOD, CAT activities and MDA level according to the manufacturer’s instructions (Tian et al., 2014; Zhou et al., 2011). GSH-Px activities were determined at 340 nm by the ability to oxidize GSH, using cumene hydroperoxide (Cum-OOH) as a substrate, coupled to the reduction rate of triphosphopyridine nucleotide (NADPH) by glutathione reductase (GR). Activities of SOD were measured by the ability to inhibit the formation of formazan dye with maximum absorbance at 450 nm. The formazan dye was produced by monosodium salt reacting with O2 − from the xanthine oxidase system. The content of MDA was assessed at 532 nm by reacting with thiobarbituric acid to form a chromophoric product. Hydrogen peroxide can be oxidized by peroxidase and generates red product, which can be assessed at 520 nm, to indirectly show the CAT activity. The activities of GSH-Px, SOD, CAT were expressed as % of control, and the level of MDA were expressed ␮mol/mg of protein. 2.10. Western-blot analysis The sample processing: SH-SY5Y cells were seeded in 6-well plates. At the end of described treatments, the cells were harvested and washed once with cold PBS. Then cells were incubated with

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Fig. 3. The effects of KuB on apoptosis. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. (A) Representative pictures were the obtained from flow cytometry. (B) Quantitative analysis for apoptotic ratio. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

lysis buffer on ice for 30 min. After these treatments, the lysed cells were centrifuged at 10,000 × g for 10 min. The supernatant was used to determine the protein concentrations by using the BCA protein assay kit (Beyotime, Nanjing, China). Finally, the protein concentrations were quantified to 3 mg/mL for loading. Importantly, the incubation time of H2 O2 to the samples of p38, JNK, ERK and AKT was only 120 min. Western-blot processing: The samples were separated on a 12% SDS polyacrylamide gel. After transferring to PVDF membranes, the membranes were blocked with 5% non-fat milk or 5% BSA for 2 h at room temperature. After that, protein samples were incubated at 4 ◦ C overnight with primary antibodies Bax, Bcl-2, caspase-3, caspase-9, cytochrome c, p38, ERK, JNK and ␤-actin (all dilutions in 1:1000). Then the membranes were washed with PBS and reacted with horseradish peroxidase-conjugated secondary antibodies (1:12,000) for 1 h at room temperature. Finally, the membranes were washed with PBS again and the antibody-bound

proteins were detected by an enhanced chemiluminescence (ECL) method. ␤-actin served as an internal control in western blot analysis. Image J was used to quantify the intensity of the bands. 2.11. Statistical analysis Data were represented as the Mean ± S.E.M. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. 3. Results 3.1. Effects of KuB on H2 O2 -induced cytotoxicity in SH-SY5Y cells Different concentrations of KuB (5–40 ␮M) had no effects on the cell viability (Fig. 1B). Various concentrations of H2 O2 (25–200 ␮M)

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Fig. 4. Effects of KuB on MMP in SH-SY5Y cells. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. Fifteen random fields of immunostained cells were chosen by using a 40× objective. (A) The representative fluorescence images obtained after Rho123. (B) The quantitative analysis of the mean fluorescence intensity of Rho 123. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

were added to the cells for 12 h and cell viability was inhibited in a dose-dependent manner. 100 ␮M H2 O2 could decrease the cell viability to 47.00 ± 1.76% compared with the control group (Fig. 1C). Therefore, 100 ␮M H2 O2 was chosen for the subsequent experiments. As is shown in Fig. 1D, pretreatment with KuB (5, 10, 20 ␮M) could prevent cell death and improve cell viability to 65.26 ± 2.34%, 85.61 ± 1.20%, 91.18 ± 1.11%, respectively. These results suggested that KuB has the capacity of cytoprotection. 3.2. Protective effects of KuB against plasma membrane damage The protective effect of KuB was further evaluated by LDH assay. The LDH release in H2 O2 -stimulated group (486.67 ± 12.02 U/L) was much more than the control group (60.33 ± 2.60 U/L). In contrast, pretreatment with different concentrations of KuB (5, 10, 20 ␮M) lowered the LDH release to 281.70 ± 7.90, 127.00 ± 3.05 and 64.67 ± 3.18 U/L, respectively (Fig. 1E). These results revealed that KuB could prevent plasma membrane from damaging. 3.3. Effects of KuB on H2 O2 -induced morphological changes in SH-SY5Y cells Hoechst 33342 staining showed DNA condensation and nuclear fragmentation in cells treated with H2 O2 . However, pretreatment

with KuB (5, 10, 20 ␮M) inhibited these characteristics of apoptosis (Fig. 2). These results indicated that KuB has the ability of antiapoptotic. 3.4. KuB attenuated H2 O2 -induced apoptosis and MMP loss in SH-SY5Y cells Annexin V and PI dual staining was used to further distinguish the early and late apoptotic cells. As is shown in Fig. 3A and B, a typical representative dot plot analysis of control cells showed total apoptotic cells (15.18 ± 0.39% p < 0.01) and late apoptotic cells (7.90 ± 0.64% p < 0.01). H2 O2 significantly increased the number of total apoptotic cells (50.91 ± 1.47% p < 0.01) and late apoptotic cells (30.55 ± 2.10% p < 0.01) compared with the control group. Pretreatment with KuB (5, 10, 20 ␮M) decreased total apoptotic cells (39.45 ± 0.66% p < 0.01) late apoptotic cells (23.22 ± 0.86% p < 0.01); total apoptotic cells (32.31 ± 1.17% p < 0.01) late apoptotic cells (18.08 ± 0.41% p < 0.01); total apoptotic cells (20.15 ± 1.03% p < 0.01) late apoptotic cells (8.94 ± 0.43% p < 0.01), respectively. These results indicated KuB could effectively reduce the apoptotic rate. In addition, as shown in Fig. 4A and B, cells treated with 100 ␮M H2 O2 showed a significant decrease in the fluorescence intensity of Rho123 (43.23 ± 3.05%) of the control group. However,

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Fig. 5. Effects of KuB on ROS generation. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. Fifteen random fields of immunostained cells were chosen by using a fluorescence microscope with 40× objective. (A) The representative fluorescence images obtained after DHC dye staining. (B) Bar graphs showed the quantitative analysis of the mean fluorescence intensity of DHC. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

pretreatment with KuB (5, 10, 20 ␮M) could increase the fluorescence intensity of Rho123 to 61.33 ± 4.82%, 78.98 ± 1.59% and 89.19 ± 3.15%, respectively. These results suggested that KuB could improve the MMP loss to provide neuroprotection. 3.5. Effects of KuB on intracellular ROS production ROS act as a key role in cellular signalling transduction and generates oxidative-stress. H2 O2 could increase the ROS production to 242.00 ± 6.68% of the control group. However, pretreatment with KuB (5, 10, 20 ␮M) could significantly decrease the ROS production to 173.51 ± 10.85%, 137.45 ± 9.86%, and 121.54 ± 8.22%, respectively (Fig. 5A and B). This result showed that KuB has strong ability of scavenging ROS. 3.6. Effects of KuB on the activity of CAT, SOD, GSH-Px and the MDA level Scavenging of ROS is achieved with several antioxidative enzymes, including the three important endogenous enzymes: CAT, SOD, and GSH-Px. As shown in Fig. 6A–C, activities of SOD, CAT and GSH-Px decreased to 44.00 ± 1.52%, 47.45 ± 2.31%, and 55.33 ± 2.33%, respectively, as compared to control group

(relative value = 100%) after the exposure of SH-SY5Y cells to H2 O2 . However, pretreatment with KuB (5, 10, 20 ␮M) significantly increased the activity of CAT (52.00 ± 2.08%, 67.67 ± 1.76%, and 87.33 ± 1.45%, respectively), SOD (51.54 ± 1.96%, 74.56 ± 2.11%, and 89.78 ± 1.56%, respectively), and GSH-Px (65.00 ± 2.31%, 74.00 ± 2.08%, and 84.33 ± 1.53%, respectively). Moreover, the MDA level was significantly increased (4.98 ± 0.33 ␮mol/mg) in H2 O2 -treated cells compared with the control group (3.32 ± 0.01 ␮mol/mg), whereas KuB (5, 10, 20 ␮M) could reduce the MDA production to 3.56 ± 0.11 ␮mol/mg, 3.09 ± 0.05 ␮mol/mg, and 2.12 ± 0.06 ␮mol/mg, respectively (Fig. 6D). These results indicated that KuB has strong ability of anti-oxidative stress.

3.7. Effects of KuB on the expression of Bcl-2 family, including Bcl-2 and Bax To further investigate the potential mechanisms of KuB on cell injury, the expression of Bcl-2 family were detected. As is shown in Fig. 7A and B, H2 O2 could decrease the ratio of Bcl-2/Bax to 0.41 ± 0.06 compared with control cells (ratio = 1). However, different concentrations of KuB (5, 10, 20 ␮M) could rescue the decreasing ratio to 0.68 ± 0.11, 0.9 ± 0.17 and 1.54 ± 0.17,

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Fig. 6. Effects of KuB the activity of CAT, SOD, GSH-Px and the MDA level. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. (A) The CAT activity. (B) The SOD activity. (C) The GSH-Px activity. (D) The MDA level. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

respectively. These results suggested that KuB restored the mitochondria function via inhibiting the ratio of Bcl-2/Bax. 3.8. Effects of KuB on the release of cytochrome c The mitochondrial dysfunction would result in cytochrome c release. As is shown in Fig. 8A and B, the expression of cytochrome c (295.66 ± 5.89%) in H2 O2 -treated cells significantly increased compared with control group (relative value = 100%). However, pretreatment with KuB (5, 10, 20 ␮M) could decrease the cytochrome c expression to 242.66 ± 6.94%, 148.00 ± 8.14% and 97.85 ± 3.89%, respectively. These results suggested that KuB could restrain the cytochrome c release. 3.9. Effects of KuB on the expression of caspase-3 and caspase-9

Fig. 7. Effects of KuB on the expression of Bcl-2 family. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. (A) The original bands of Bcl-2, Bax. (B) The quantitative analysis of Bcl-2/Bax ratio. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

As shown in Fig. 9A and B, H2 O2 could increase the caspase-3 expression to 308.22 ± 6.14% compared with control group (relative value = 100%), however, pretreatment with KuB (5, 10, 20 ␮M) could decrease the expression of caspase-3 to 236.08 ± 2.21%, 154.44 ± 6.71% and 104.11 ± 2.56%, respectively. The same tendency as above, caspase-9 expression was increased to 308.33 ± 5.78% compared with control cells, while pretreatment with KuB (5, 10, 20 ␮M) could decrease the expression of caspase9 to 277.67 ± 6.06%, 153.00 ± 7.73%, 111.00 ± 5.77%, respectively (Fig. 9C and D). These results showed that KuB could anti-late apoptotic by modulating the caspase cascades.

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Fig. 8. Effects of KuB on the release of cytochrome c. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. (A) The original bands of cytochrome c. (B) The quantitative analysis of cytochrome c. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

3.10. Effects of KuB on the expression of p-MAPKs (p38, JNK and ERK) and p-AKT In order to study the possible action mechanisms of the anti-oxidative stress ability of KuB, the expression of pMAPKs and p-AKT were detected by western-blot assay. As is shown in (Fig. 10A–E), comparing with the control group (relative value = 100%), the expression of p-p38, pERK and p-JNK in H2 O2 -treated cells were significantly increased to 204.67 ± 4.35%, 354.00 ± 3.56%, 203.33 ± 3.75%, respectively. However, KuB (5, 10, 20 ␮M) could effectively decrease the expression of these proteins: p-p38 (96.70 ± 3.99%, and 84.41 ± 2.89%, 33.81 ± 1.75%, respectively); p-ERK (173.45 ± 3.56%, 160.45 ± 4.44%, 95.45 ± 6.40%, respectively); p-JNK (168.66 ± 1.85%, 105.68 ± 2.40%, 86.00 ± 2.08%, respectively). In addition, H2 O2 -treated group could decrease the expression of p-AKT to 34.63 ± 1.76% compared with control group. And pretreatment with indicated concentration of KuB could increase the p-AKT expression to 38.62 ± 0.66%, 54.55 ± 1.79% and 90.45 ± 0.58%, respectively. These results suggested that the antioxidative stress ability of KuB involved in MAPKs and PI3K-AKT pathways. 4. Discussion Neuronal apoptosis has been linked to promote levels of oxidative stress, which is believed to be one of the most sensitive biological markers, caused by the imbalance between ROS production and antioxidant system (Gutteridge, 1995; Kassie et al., 2000). The apoptotic cells are characterized by some morphological changes, such as cell shrinkage, DNA condensation that further result in the degradation of chromosomal DNA and cell death (Enari et al., 1998). In our study, we evaluated the cytotoxic effects of H2 O2 and the protective effects of KuB by MTT and LDH assays. The results of Hoechst 33342 and AV-PI dual staining assays were further

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supported by the morphological observation. Our data showed that KuB prevents cell death, decreases LDH release and reduces apoptosis. These data had been fully proved that KuB has a strong ability of anti-apoptosis induced by H2 O2 in SH-SY5Y cells. What are the potential mechanisms? Oxidative stress and free radical production have been believed to play vital role in regulating redox reactions and contributing ROS, which are the main murderer in neurodegeneration. Accumulated reports indicated that oxidative stress also plays a key role in regulating the biochemical changes that leads to neurodegenerative disorders (Uttara et al., 2009). The oxidative stress could be inhibited by antioxidant enzymes, which were declined in neurodegenerative disorders (Nikam et al., 2009). In our study, H2 O2 caused an imbalance of antioxidant defence system, which further lead to the reduction of the activity of SOD, CAT, and GSH-Px (Fig. 6A–C). This phenomenon was in line with the previous studies (Azmi et al., 2013; Guizani et al., 2011). However, KuB-treated cells dramatically increased the level of these antioxidant enzymes and decreased MDA level. These data suggested KuB could attenuate H2 O2 -induced oxidative damage via decreasing intracellular lipid peroxidation levels and increasing the antioxidant enzymes activity. In addition, mitochondria plays a central role in apoptosis and the overgeneration of ROS induced by oxidative stress could lead to the MMP loss, which makes a contribution to open the mitochondrial permeability transition pore (Beal, 2005; Venuprasad et al., 2013). However, even worse is that the destruction of the mitochondria integrity could cause the increasing consumption of ATP and the generation of ROS (Dumont and Beal, 2011). Therefore, the MMP is a very important indicator of apoptosis. Rho 123, a mitochondrial selective dye, was used to monitor the MMP. Mitochondrial energization induces quenching of Rho 123 fluorescence and the rate of fluorescence decay is proportion to the MMP (Baraccaa et al., 2003). In our study, we found that the fluorescence intensity of the Rho 123 in H2 O2 -treated cells was signally declined compared with control cells. However, pretreatment with KuB can reverse this tendency in a dose-dependent manner. The results indicated that KuB has the ability of preventing mitochondrial dysfunction. Because ROS can break mitochondrial function in turn, so we used the DCFH-DA, a ROS fluorescence dye, to evaluate the ROS generation. Our data revealed that KuB could effectively decrease the fluorescence intensity in a dose-dependent manner, that is to say KuB has the ability of scavenging excessive ROS production. Taken collectively, it was clear that KuB has the ability of protecting SH-SY5Y cells against H2 O2 -induced death by preventing mitochondrial dysfunction and scavenging ROS. In order to study the potential mechanism of KuB, western-blot assay was used to detect the expression of mitochondria-related apoptotic proteins. Previous study has shown that mitochondria-related apoptotic pathways are associated with H2 O2 -induced cytotoxicity in SH-SY5Y cells (Kwon et al., 2011). Permeabilization of mitochondrial membrane is regulated by proteins of the Bcl-2 family (Miquel and Serge, 2003). The anti-apoptotic factor Bcl-2, residing in the outer mitochondrial membrane, inhibits the release of cytochrome c (Borner, 2003). The pro-apoptotic factor Bax residing in the cytosol and translocates from the cytoplasm to the mitochondria following apoptotic signaling, where it promotes the permeabilization of the mitochondrial membrane. Increased mitochondrial permeability results in the release of cytochrome c from the mitochondria (Chinnaiyan et al., 1996). Released cytochrome c triggers activation of caspase-9 which in turn activates caspase-3, and activated caspase-3 induces cell death (Kitamura et al., 1999). In our study, we found KuB could increase the ratio of Bcl-2/Bax compared with H2 O2 -stimulated cells and decrease the release of cytochrome c, inhibit the expression of caspase-9 and caspase-3. Therefore, we concluded that KuB provides neuroprotection at least via mitochondria dependent apoptotic pathways.

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Fig. 9. Effects of KuB on the expression of caspase-3 and caspase-9. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 12 h. (A) The original bands of capase-3 and caspase-9. (B) The quantitative analysis of caspase-3/actin. (C) The quantitative analysis of capase-9/actin. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with p value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

Fig. 10. Effects of KuB on the expression of p-MAPKs and p-AKT. The cells were incubated with indicated concentrations of KuB for 2 h, then stimulated with or without H2 O2 (100 ␮M) for 120 min. (A) The original bands of p- and t-p38, AKT, ERK and JNK. (B) The quantitative analysis of p-AKT/t-AKT. (C) The quantitative analysis of p-p38/t-p38. (D) The quantitative analysis of p-ERK/t-ERK. (E) The quantitative analysis of p-JNK/t-JNK. The data were represented as mean ± S.E.M of three independent experiments. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey’s HSD-post hoc test. Differences with P value less than 0.05 were considered statistically significant. & p < 0.05, && p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with H2 O2 treatment cells.

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Acknowledgments This work was supported by the National Science and Technology Major Project, China. (Project number: 2014ZX09J14101-05C).

References

Fig. 11. The action mechanisms of KuB against H2 O2 -induced SH-SY5Y cells injury. The green line represents the anti-apoptotic pathway and the red line represents the apoptotic pathways.

Last but not least, ROS play a crucial role as a second messenger in signal transduction cascades and could result in the activation of MAP kinases (Eguchi et al., 2003). Previous studies have shown that H2 O2 could rapidly activate ERK, JNK and p38, which are closely related to cell death induced by ROS (Ruffels et al., 2004; Spicer and Millhorn, 2003; Suzaki et al., 2002; Pan et al., 2013). In line with these studies, our data showed that H2 O2 activates MAPKs. However, pretreatment with KuB could effectively block H2 O2 -induced phosphorylation of ERK, JNK and p38. In contrast to MAPKs, PI3K-AKT signaling pathway controls many cellular events, such as oxidative stress-induced cell death. The expression of p-AKT was down-regulated in H2 O2 -treated SH-SY5Y cells (Pan et al., 2013), but pretreatment with KuB could up-regulate the p-AKT expression. These results indicated that the neuroprotection of KuB may involved in p-MAPKs inactivation and PI3K/AKT activation. 5. Conclusions In summary, this study demonstrated that KuB inhibited H2 O2 induced oxidative stress in SH-SY5Y cells, as indicated by its ability to increase the cell viability and inhibit LDH release. The effectiveness of KuB against H2 O2 -induced oxidative stress may due to restore antioxidant defense system, thereby attenuate ROS production, improve MMP and prevent the development of apoptosis. Moreover, mechanisms of neuroprotective effects of KuB against H2 O2 -induced apoptosis were related with an increase in the Bcl-2/Bax ratio and decreased level of cytochrome c, caspase-3, caspase-9 as well as the phosphorylation of p-MAPKs (See Fig. 11). Taken together, our findings suggested that KuB can act as protective agent against oxidative stress induced neurodegenerative disorders. Further work is needed to testify its activity and elucidate its underlying mechanisms in vivo. Conflict of interest There is no conflict of interest to declare. Transparency document The Transparency document associated with this article can be found in the online version.

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