J Nat Med DOI 10.1007/s11418-015-0928-2
ORIGINAL PAPER
Protective effects of aloperine on neonatal rat primary cultured hippocampal neurons injured by oxygen–glucose deprivation and reperfusion Ning-Tian Ma1 • Ru Zhou1 • Ren-Yuan Chang1 • Yin-Ju Hao1 • Lin Ma1 Shao-Ju Jin1 • Juan Du1 • Jie Zheng1 • Cheng-Jun Zhao2 • Yang Niu3 • Tao Sun4 • Wei Li5 • Kazuo Koike5 • Jian-Qiang Yu1 • Yu-Xiang Li6
•
Received: 30 April 2015 / Accepted: 25 June 2015 Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract Aloperine (ALO), one of the alkaloids isolated from Sophora alopecuroides L., is traditionally used for various diseases including neuronal disorders. This study investigated the protective effects of ALO on neonatal rat primary-cultured hippocampal neurons injured by oxygen– glucose deprivation and reperfusion (OGD/RP). Treatment with ALO (25, 50, and 100 mg/l) attenuated neuronal damage (p \ 0.01), with evidence of increased cell viability (p \ 0.01) and decreased cell morphologic impairment. Furthermore, ALO increased mitochondrial membrane potential (p \ 0.01), but inhibited intracellular-free calcium [Ca2?]i (p \ 0.01) elevation in a dose-dependent manner at OGD/RP. ALO also reduced the intracellular reactive oxygen species and malondialdehyde production and enhanced the antioxidant enzymatic activities of catalase, N.-T. Ma, R. Zhou, R.-Y. Chang contributed equally to this work. & Wei Li [email protected] & Jian-Qiang Yu [email protected] 1
Department of Pharmacology, School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China
2
Key Lab of Fertility Preservation and Maintenance, Ministry of Education, Ningxia Medical University, Yinchuan 750004, China
3
School of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China
4
Ningxia Key Lab for Research on Craniocerebral Diseases of Ningxia Hui Autonomous Region, Yinchuan 750004, China
5
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
6
School of Nursing, Ningxia Medical University, Yinchuan 750004, China
superoxide dismutase, glutathione peroxidase and the total antioxidant capacity. The results suggested that ALO has significant neuroprotective effects that can be attributed to anti-oxidative stress. Keywords Aloperine Hippocampal neurons Oxygen– glucose deprivation and reperfusion Antioxidant capacity
Introduction Cerebrovascular disease has become the third leading cause of death among adults in China [1]. Strokes have become one of the most devastating and complicated diseases, causing mortality and high morbidity and disability rates in the aged population [2]. They cause an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration [3]. In the process of a stroke, a cascade of pathological mechanisms such as excessive release of excitatory amino acids, energy failure, increased oxidative stress and apoptosis will be activated, eventually resulting in acute cerebral ischemic injury [4]. These mechanisms eventually cause irreversible damage of the brain tissue. Recent studies have provided direct and indirect experimental evidence showing that oxygen-free radicals are released during ischemia and reperfusion (I/R) because of the failure of metabolic reactions [5]. Therefore, antioxidant defenses including free radical scavengers and antioxidant enzymes are considered a promising approach to limit the extent of damage of I/R injury [6]. Aloperine (ALO) is a quinolizidine alkaloid, which has been isolated from the roots of Sophora alopecuroides L., a traditional Chinese medicine used for the treatment of cancer [7]. ALO has been reported to have a large range of
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biological effects, including anti-inflammatory, anti-allergenic, anti-tumor, and antiviral reactions [7]. Recently, we reported ALO to have antinociceptive effects on neuropathic pain induced by chronic constriction injury, in which antioxidants are involved as one of the mechanisms [8]. Taking the importance of antioxidant capacity for ALO, we further examined the neuropreotective effect of ALO [9] in this study. The antioxidant effect was also investigated.
Materials and methods Animals and materials All animals were provided by the Animal Center of Ningxia Medical University. Experiments were approved by the Animal Research Ethics Committee, School of Pharmacy, Ningxia Medical University. ALO (purity [98.0 %) was obtained from a drug manufactory (Ningxia Zijinghua Pharmacy, Ningxia, China). Its chemical structure is given in Fig. 1. Nimodipine (Nim) injection (5 mg/ ml) was obtained from the German Bayer Company. The reagents used in this experiment were Dulbecco’s modified Eagles medium (DMEM, Gibco), neurobasal-A medium (NAM, Gibco), fetal bovine serum (FBS, Gibco), B-27 supplement (Gibco), 0.25 % trypsin (Gibco), poly-L-lysine (PLL, Gibco), HEPES buffered salt solution (Gibco), Earle’s balanced salt solution (EBSS) (mg/l: 6,800 NaCl, 400 KCl, 264 CaCl2 9 H2O, 200 MgCl2 9 H2O, 2200 NaHCO3, 140 NaH2PO4 9 H2O, and 1,000 glucose, pH 7.2), phosphate-buffered saline (PBS), 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Solarbio), and fura-3 acetoxymethyl ester (fura-3AM, Invitrogen). Other commercial kits for the detection of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), the total antioxidant capacity (TAOC) and reactive oxygen species (ROS) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents were from commercial sources and of standard biochemical quality. The commercial kit for the detection of mitochondrial membrane potential (MMP), calcium fluorescence probe (fluo-3 AM) and Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Nanjing, China). Fig. 1 Chemical structure of aloperine (ALO)
HN HH N
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H H
Cell cultures and oxygen–glucose deprivation and reperfusion (OGD/RP) Primary hippocampal neuronal cells were prepared from newborn Sprague–Dawley rats. After trituration and trypsinization, viability of hippocampal neurons was [95 % as calculated using conventional trypan blue staining under an optical microscope. The single-cell suspension was plated at a density of 1 9 106/ml, seeded in PLL-coated 6-well or 96-well plates and cultured in DMEM supplemented with 10 % FBS and 2 % HEPES buffered salt solution. Cells were maintained in a humidified incubator (37 °C with 5 % CO2) for 1.5 h and the well-adherent cell DMEM culture medium was discarded and replaced with 2 % B-27 of NAM culture medium, after which the medium was changed every 2 days. In preliminary experiments in our laboratory, hippocampal neurons fluorescense staining with rabbit anti-rat neuron specific enolase was performed to identify the neurons [10]. Briefly, on the seventh day, the medium of the vehicle group (n = 6) was replaced with pre-warmed EBSS without glucose. The cultures were then held in an incubator containing 95 % N2 and 5 % CO2 at 37 °C for 2 h as OGD. After 2 h, the cultures were placed back into the normoxic incubator with a normal culture medium for an additional 24 h as OGD/RP. ALO [final concentrations 25 mg/l (low dose), 50 mg/l (middle dose), 100 mg/l (high dose), n = 6] and Nim (final concentration 5 mg/l, n = 6) were added to the culture 24 h before reperfusion. The cultures of the control group (n = 6) were not exposed to OGD/RP.
MTT and LDH assay Cell viability was assessed by morphological observation with an inverted-phase contrast microscope and measurement of the reduction of MTT. Following the above cell treatment protocol, the morphology of the cells was observed and recorded as a photograph under an Olympus Microscope (Tokyo, Japan). After removal of the original culture medium, MTT was added to 96-well plates at a final concentration of 0.5 mg/ml, and the incubation continued for 4 h. The medium was then removed, and cells were dissolved in DMSO. The blue MTT-formazan was measured by spectrophotometry at 490 nm using an ELISA reader (ELx800uv; Bio Tek Instruments, USA). Neuronal damage in the cells was also quantitatively assessed by measuring the activity of LDH released from damaged or dead cells. A previous study has shown that the activity of LDH occurring from either necrotic or apoptotic cells is proportional to the number of neurons damaged or destroyed [11]. LDH activity in the medium and total activity was measured by an LDH assay kit at 440 nm. The
J Nat Med
percentage of LDH leakage was expressed as (LDH activity in medium/total activity) 9 100 %. Measurement of cellular SOD, GSH-PX, MDA, TAOC, CAT levels The cells were harvested and resuspended in 0.1 mol/l icecold PBS. The cell suspensions were sonicated for 25 s on ice and centrifuged at 1,0009g at 4 °C for 10 min, and supernatants were saved. SOD was assayed by a modification of the xanthine/xanthine oxidase method [12]. The GSH-Px activity was measured by the oxidizing speed of the GSH, which can be expressed by GSH reduction in a certain time. MDA, a compound produced during lipid peroxidation, was determined by the thiobarbituric acid method [13]. Catalase is an enzyme which catalyses decomposition of hydrogen peroxide into oxygen and water and is a peroxidase present in the cell in vivo. The catalase enzyme is a sign of peroxisomes, accounting for 40 % of the total peroxidase enzyme. Protein of cell homogenates was measured by the method of Coomassie brilliant blue using bovine serum albumin as the standard. Cellular SOD, GSH-Px, MDA, CAT, TAOC levels were determined by spectrophotometry. Hoechst 33342 staining Hoechst 33342 staining was used to determine cell apoptosis. The hippocampal neurons were stained by 10 lM Hoechst 33342 for 30 min and rinsed with PBS, and then checked under a DMR fluorescence microscope (Leica Microsystems, Germany).
times with Hank’s solution to remove the extracellular fluo-3/AM. The fluorescence intensity of [Ca2?]i in the hippocampal neurons was determined by laser scanning confocal microscopy. Total images were scanned and the data were placed in disks for analyzing [16]. Measurement of ROS Formation of ROS was determined using the fluorescent probe DCFH-DA. Cell-permeant nonfluorescent DCFHDA has been shown to be oxidized to the highly fluorescent 2,7-dichlorofluorescin in the presence of ROS. Neurons were washed twice with PBS, and then incubated with DCFH-DA (10 mM) for 30 min at 37 °C in the dark. The cells were harvested and suspended in PBS. The fluorescence intensity was measured by a fluorospectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 525 nm [17]. Statistical analysis All quantitative data were expressed as mean ± SD. The statistical significance of differences was analyzed using a one-way analysis of variance (ANOVA) (LSD, S-N-K, Dunnett) for between-group comparison and with Student’s t test for comparison between the two groups. The software package SPSS 18.0 was used for calculations. p values of \0.05 were considered statistically significant.
Results ALO ameliorated OGD/RP-induced cell viability
Effects of ALO on MMP of hippocampal neurons JC-1 was used to evaluate the loss of MMP in hippocampal cultures exposed to OGD/RP. We measured it by fluorescence microscope. JC-1 is a convenient voltage sensitive probe to monitor MMP [14]. Cells containing forming J-aggregates have high 4Wm, and showed fluorescence. Cells with low 4Wm are those in which JC-1 maintains (or reacquires) monomeric form, and show green fluorescence [15]. The relative proportions of red and green fluorescence were used to measure the ratio of mitochondrial depolarization. Measurement of [Ca21]i of hippocampal neurons Neurons were washed with Hank’s solution two or three times and were then loaded with 10 lM fluo-3/AM in the dark for 30 min at 37 °C. After this, cells were rinsed three
The cell viability was determined using MTT and LDH assay (n = 6, for each group). In order to investigate the protective effects of ALO on cell death caused by OGD/ RP, MTT assay, assessed by OD value, was used to assess cellular viability in neurons. As shown in Fig. 2a, OD value decreased in cells to 0.217 ± 0.08 at 24 h after OGD/RP, but reverted to 0.457 ± 0.03 (p \ 0.05 vs OGD/ RP group) and 0.513 ± 0.018 (p \ 0.01 vs OGD/RP group), respectively, by treatment with ALO at concentrations of 50 and 100 mg/l. Although 25 mg/l ALO did not show a remarkable reduction in OD value in Fig. 2a, there was a difference compared to the OGD/RP group 0.388 ± 0.019 (p \ 0.05). As illustrated in Fig. 2b, LDH leakage increased to 40.8 ± 5.0 % after OGD/RP. ALO significantly attenuated OGD/RP-induced cell death by reducing LDH leakage from 40.8 ± 5.0 % (vehicle group) to 28.3 ± 5.6–19.1 ± 1.72 % (Fig. 2b).
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increased by reperfusion, indicating an enhanced [Ca2?]i overload by OGD and reperfusion. Pretreatment with ALO obviously inhibited [Ca2?]i overload at both ischemic and reperfusion phases. The [Ca2?]i in ALO treated after [25 mg/l (L), 50 mg/l (M) and 100 mg/l (H)] OGD/RP injured neurons was lower (0.14 ± 0.005, 0.12 ± 0.005 and 0.1 ± 0.03) than that in OGD/RP-injured cells (0.17 ± 0.02) (Fig. 4), respectively, indicating that ALO significantly preserved the [Ca2?]i overload after ischemic neuronal injury. ALO attenuated OGD/RP-induced dissipation of MMP JC-1 was used to assess mitochondrial depolarization hippocampal cultures exposed to OGD/RP (n = 6, for each group). After OGD, the fluorescence intensity of JC-1 in hippocampal neurons was reduced, representing a dissipation of the MMP. However, treatment with ALO (25–100 mg/l) significantly stabilized the MMP (Fig. 5), which was 0.3 ± 0.09, 0.63 ± 0.05, 0.83 ± 0.05, respectively. The ALO-treated group (100 mg/l) was better than the other groups. ALO ameliorated OGD/RP-induced oxidative stress
Fig. 2 Effects of ALO on cell viability in primary hippocampal neuronal cells under oxygen–glucose deprivation for 2 h and reperfusion for 24 h, determined by MTT assay (a), and the extent of LDH release (b). Histograms represent mean ± SD. ##p \ 0.01 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
ALO attenuated OGD/RP-induced cell apoptosis Exposure to OGD also resulted in apoptotic death of hippocampal neurons (n = 6, for each group), as revealed by the appearance of condensed nuclei in Hoechst-stained cultures (Fig. 3(1)). Through cell counting, 50.8 ± 4.2 % of the cultured neurons in the vehicle group were determined to be apoptotic cells 24 h post-OGD compared to 3 % of apoptotic neurons in the total count of the control group. However, in the ALO-treated groups, the OGD/RPinduced apoptotic cell death rate was significantly decreased to 32.57 ± 6.22, 18.3 ± 1.85, and 7 ± 1.32 %, respectively (Fig. 3(2)). ALO attenuated OGD/RP-induced [Ca21]i Green fluorescence intensity was used to assess the expression of [Ca2?]i (n = 6, for each group). OGD/RP triggered a rapid rise in [Ca2?]i, which was further
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We next tested the effects of ALO on the OGD/RP-induced ROS generation in hippocampal neurons (n = 6, for each group). 24 h after OGD, a 2.1-fold increase in the intracellular ROS was found using a DCFH-DA assay. However, ALO (25–100 mg/l) decreased ROS production (Fig. 6). The effects of ALO on cellular MDA, GSH-Px, SOD, TAOC and CAT levels are presented in Fig. 6 (n = 6, for each group). OGD exposure markedly decreased GSH-Px content, TAOC and antioxidant enzymatic activities (CAT, SOD), while it increased the level of the lipid peroxidation product (MDA) in the neuronal cultures. However, concurrent treatment with ALO significantly decreased the MDA content. ALO also resulted in a noticeable increase in the activities of CAT, SOD and GSH-Px in comparison with the vehicle control.
Discussion The present study demonstrated the neuroprotective effect of ALO on neonatal rat primary cultured hippocampal neurons injured by OGD/RP. There were two important results—(1) pre-treatment with ALO significantly reduced the injury in primary cultured hippocampal neurons; (2) this neuroprotection was associated with attenuation of intracellular oxidation properties and increase of antioxidants during OGD/RP.
J Nat Med Fig. 3 (1) Effects of ALO on morphological changes of OGD/RP-injured primary hippocampal neurons (9400). Nuclei were labeled with Hoechst 33342 (A–F). A Control, B exposure to OGD/ RP, C treated with nimodipine (final concentration 5 mg/l), D treated with low concentration of ALO (final concentration 25 mg/l), E treated with middle concentration of ALO (final concentration 50 mg/l), F treated with high concentration ALO (final concentration 100 mg/l). (2) Effects of ALO on apoptosis rate in primary hippocampal neuronal cells under OGD 2 h/ RP 24 h. Histograms represent mean ± SD. ##p \ 0.01 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
We evaluated the degree of injury in hippocampal neuronal cell exposed to OGD/RP by MTT and LDH release. Treatment with ALO (25–100 mg/l) decreased the cell damage and inhibited LDH release, which indicated that ALO may have a protective effect on hippocampal neuronal cell exposed to OGD/RP. Oxygen-free radicals are known to be generated during periods of ischemia followed by reperfusion. Studies on the antioxidant changes and the significance during brain failure have provided a new insight into acute cerebral
ischemia. The biochemical profile of the neurons, like depletion of SOD, CAT and GSH-Px, provide strong evidence for oxidative stress occurring during ischemia– reperfusion. In this study, increased MDA level and decreased antioxidant activities of enzymes (SOD, CAT, GSH-Px and TAOC) were detected in OGD/RP neurons. This indicated that oxidative injury was closely associated with brain ischemia–reperfusion. Interestingly, in the current study, neurons in the hippocampal from the ALO-group showed a significantly decreased MDA and increased
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J Nat Med Fig. 4 (1) Effects of ALO on [Ca2?]i in primary hippocampal neuronal cells under OGD 2 h/ RP 24 as indicated by fluo-3/ AM. A Control, B exposure to OGD/RP, C treated with nimodipine (final concentration 5 mg/l), D treated with low concentration of ALO (final concentration 25 mg/l), E treated with middle concentration of ALO (final concentration 50 mg/l), F treated with high concentration ALO (final concentration 100 mg/l). (2) Effects of ALO on apoptosis rate in primary hippocampal neuronal cells under OGD 2 h/ RP 24 h. Histograms represent mean ± SD. ##p \ 0.01 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
endogenous antioxidants (GSH-Px, SOD, TAOC and CAT). This indicated that ALO can reduce OGD/RP-induced injury by increasing antioxidant enzymes like SOD, CAT, GSH-Px and TAOC activities. Excessive ROS production in the brain is believed to contribute to neurodegenerative processes [18]. Statistical analysis between the control and OGD/RP groups with the administration of ALO revealed that ALO significantly inhibited ROS production during OGD/RP. Taken together, the observations indicated that the protective mechanisms of ALO on cultured rat hippocampal neurons injured by OGD/RP were related to antioxidative stress.
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The protective effect of ALO was also supported by a higher MMP. MMP reflects the performance of the electron transport chain and indicates a pathological disorder of this system. High average MMP is related to higher viability [19]. After OGD/RP, the participation of free radicals increases the lipid peroxidation of the cell membrane, breakage of the DNA, degradation of protein, etc. Finally, these lead to cell dysfunction. The present study indicated that OGD led to mitochondrial membrane depolarization and ALO (100 mg/l) significantly prevented the loss of the MMP, suggesting that the electron transport chain was still maintained. Mitochondria are susceptible targets of free
J Nat Med Fig. 5 (1) Effects of ALO on MMP changes in primary hippocampal neuronal cells under OGD 2 h/RP 24 h as indicated by JC-1 (9400). Red fluorescence indicates polarized mitochondrial membranes whereas green fluorescence indicates depolarized mitochondrial membranes. A Control, B exposure to OGD/ RP, C treated with nimodipine (final concentration 5 mg/l), D treated with low concentration of ALO (final concentration 25 mg/l), E treated with middle concentration of ALO (final concentration 50 mg/l), F treated with high concentration ALO (final concentration 100 mg/l). (2) Effects of ALO on MMP changes in primary hippocampal neuronal cells under OGD 2 h/RP 24 h. Histograms represent mean ± SD. ##p \ 0.01 vs control group, *p \ 0.05, **p \ 0.01vs vehicle group
radical-mediated damage [20]. The antioxidant properties of ALO on cultured rat hippocampal neurons injured by OGD/RP were involved in anti-early apoptosis and the stabilization of MMP. Ischemia or hypoxia can cause cell death by necrosis and apoptosis [21]. Numerous studies have shown that OGD/RP induced hippocampal neuron death, mainly through apoptosis [22]. In our study, the OGD/RP insult increased the
apoptosis rate of neurons from 3.00 ± 1.732 to 50.8 ± 4.2 %, and ALO could inhibit it effectively. This was also consistent with morphological changes examined with a scanning electron microscope. To further explore the anti-apoptotic mechanism of ALO, we performed an additional study of mitochondria function and [Ca2?]i overload. [Ca2?]i overload during, and especially following cerebral ischemia has been considered to be one of the key
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Fig. 6 Effects of ALO on SOD (a) and GSH-Px (b) activities and the level of MDA (c) and CAT (d) activities and TAOC (e) and ROS (f) in primary hippocampal neuronal cells under oxygen–glucose
deprivation for 2 h and reperfusion for 24 h. Histograms represent mean ± SD. ##p \ 0.01, #p \ 0.05 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
pathogenic events in the post-ischemic cellular death pathway [23]. [Ca2?]i overload results in a loss of mitochondrial membrane integrity, secondary to an opening of the mitochondrial permeability transition pore [24]. Our findings showed that ALO (100 mg/l) decreased the elevation of calcium, which further confirmed the hypothesis that the protective effect of ALO was related to inhibition of calcium overload.
Although many compounds with neuroprotective action are in various stages of the pharmaceutical pipeline, ALO possesses unique advantages. Sophora alopecuroides L. and its related species have already had thousands of years of human exposure with little reported toxicity. Additionally, ALO can easily diffuse across biological membranes and the blood–brain barrier in an energy deficient environment.
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Conclusively, our results clearly demonstrated that ALO has a neuroprotective effect on OGD/RP neuronal injury in vitro. The effects have bearing on anti-oxidative stress, stabilization of MMP and suppression of the intracellular Ca2? elevation. These results should provide good evidence to explore the further neuroprotective mechanism of ALO. Acknowledgments This study was supported by Ningxia Hui Autonomous Region, colleges and universities of science and technology research projects (NGY2013073). We are indebted to the staff in the animal center and the Science and Technology Center who provided assistance in the study. Conflict of interest
The authors declare no conflict of interest.
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ORIGINAL PAPER
Protective effects of aloperine on neonatal rat primary cultured hippocampal neurons injured by oxygen–glucose deprivation and reperfusion Ning-Tian Ma1 • Ru Zhou1 • Ren-Yuan Chang1 • Yin-Ju Hao1 • Lin Ma1 Shao-Ju Jin1 • Juan Du1 • Jie Zheng1 • Cheng-Jun Zhao2 • Yang Niu3 • Tao Sun4 • Wei Li5 • Kazuo Koike5 • Jian-Qiang Yu1 • Yu-Xiang Li6
•
Received: 30 April 2015 / Accepted: 25 June 2015 Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract Aloperine (ALO), one of the alkaloids isolated from Sophora alopecuroides L., is traditionally used for various diseases including neuronal disorders. This study investigated the protective effects of ALO on neonatal rat primary-cultured hippocampal neurons injured by oxygen– glucose deprivation and reperfusion (OGD/RP). Treatment with ALO (25, 50, and 100 mg/l) attenuated neuronal damage (p \ 0.01), with evidence of increased cell viability (p \ 0.01) and decreased cell morphologic impairment. Furthermore, ALO increased mitochondrial membrane potential (p \ 0.01), but inhibited intracellular-free calcium [Ca2?]i (p \ 0.01) elevation in a dose-dependent manner at OGD/RP. ALO also reduced the intracellular reactive oxygen species and malondialdehyde production and enhanced the antioxidant enzymatic activities of catalase, N.-T. Ma, R. Zhou, R.-Y. Chang contributed equally to this work. & Wei Li [email protected] & Jian-Qiang Yu [email protected] 1
Department of Pharmacology, School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China
2
Key Lab of Fertility Preservation and Maintenance, Ministry of Education, Ningxia Medical University, Yinchuan 750004, China
3
School of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China
4
Ningxia Key Lab for Research on Craniocerebral Diseases of Ningxia Hui Autonomous Region, Yinchuan 750004, China
5
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
6
School of Nursing, Ningxia Medical University, Yinchuan 750004, China
superoxide dismutase, glutathione peroxidase and the total antioxidant capacity. The results suggested that ALO has significant neuroprotective effects that can be attributed to anti-oxidative stress. Keywords Aloperine Hippocampal neurons Oxygen– glucose deprivation and reperfusion Antioxidant capacity
Introduction Cerebrovascular disease has become the third leading cause of death among adults in China [1]. Strokes have become one of the most devastating and complicated diseases, causing mortality and high morbidity and disability rates in the aged population [2]. They cause an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration [3]. In the process of a stroke, a cascade of pathological mechanisms such as excessive release of excitatory amino acids, energy failure, increased oxidative stress and apoptosis will be activated, eventually resulting in acute cerebral ischemic injury [4]. These mechanisms eventually cause irreversible damage of the brain tissue. Recent studies have provided direct and indirect experimental evidence showing that oxygen-free radicals are released during ischemia and reperfusion (I/R) because of the failure of metabolic reactions [5]. Therefore, antioxidant defenses including free radical scavengers and antioxidant enzymes are considered a promising approach to limit the extent of damage of I/R injury [6]. Aloperine (ALO) is a quinolizidine alkaloid, which has been isolated from the roots of Sophora alopecuroides L., a traditional Chinese medicine used for the treatment of cancer [7]. ALO has been reported to have a large range of
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biological effects, including anti-inflammatory, anti-allergenic, anti-tumor, and antiviral reactions [7]. Recently, we reported ALO to have antinociceptive effects on neuropathic pain induced by chronic constriction injury, in which antioxidants are involved as one of the mechanisms [8]. Taking the importance of antioxidant capacity for ALO, we further examined the neuropreotective effect of ALO [9] in this study. The antioxidant effect was also investigated.
Materials and methods Animals and materials All animals were provided by the Animal Center of Ningxia Medical University. Experiments were approved by the Animal Research Ethics Committee, School of Pharmacy, Ningxia Medical University. ALO (purity [98.0 %) was obtained from a drug manufactory (Ningxia Zijinghua Pharmacy, Ningxia, China). Its chemical structure is given in Fig. 1. Nimodipine (Nim) injection (5 mg/ ml) was obtained from the German Bayer Company. The reagents used in this experiment were Dulbecco’s modified Eagles medium (DMEM, Gibco), neurobasal-A medium (NAM, Gibco), fetal bovine serum (FBS, Gibco), B-27 supplement (Gibco), 0.25 % trypsin (Gibco), poly-L-lysine (PLL, Gibco), HEPES buffered salt solution (Gibco), Earle’s balanced salt solution (EBSS) (mg/l: 6,800 NaCl, 400 KCl, 264 CaCl2 9 H2O, 200 MgCl2 9 H2O, 2200 NaHCO3, 140 NaH2PO4 9 H2O, and 1,000 glucose, pH 7.2), phosphate-buffered saline (PBS), 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Solarbio), and fura-3 acetoxymethyl ester (fura-3AM, Invitrogen). Other commercial kits for the detection of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), the total antioxidant capacity (TAOC) and reactive oxygen species (ROS) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents were from commercial sources and of standard biochemical quality. The commercial kit for the detection of mitochondrial membrane potential (MMP), calcium fluorescence probe (fluo-3 AM) and Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Nanjing, China). Fig. 1 Chemical structure of aloperine (ALO)
HN HH N
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H H
Cell cultures and oxygen–glucose deprivation and reperfusion (OGD/RP) Primary hippocampal neuronal cells were prepared from newborn Sprague–Dawley rats. After trituration and trypsinization, viability of hippocampal neurons was [95 % as calculated using conventional trypan blue staining under an optical microscope. The single-cell suspension was plated at a density of 1 9 106/ml, seeded in PLL-coated 6-well or 96-well plates and cultured in DMEM supplemented with 10 % FBS and 2 % HEPES buffered salt solution. Cells were maintained in a humidified incubator (37 °C with 5 % CO2) for 1.5 h and the well-adherent cell DMEM culture medium was discarded and replaced with 2 % B-27 of NAM culture medium, after which the medium was changed every 2 days. In preliminary experiments in our laboratory, hippocampal neurons fluorescense staining with rabbit anti-rat neuron specific enolase was performed to identify the neurons [10]. Briefly, on the seventh day, the medium of the vehicle group (n = 6) was replaced with pre-warmed EBSS without glucose. The cultures were then held in an incubator containing 95 % N2 and 5 % CO2 at 37 °C for 2 h as OGD. After 2 h, the cultures were placed back into the normoxic incubator with a normal culture medium for an additional 24 h as OGD/RP. ALO [final concentrations 25 mg/l (low dose), 50 mg/l (middle dose), 100 mg/l (high dose), n = 6] and Nim (final concentration 5 mg/l, n = 6) were added to the culture 24 h before reperfusion. The cultures of the control group (n = 6) were not exposed to OGD/RP.
MTT and LDH assay Cell viability was assessed by morphological observation with an inverted-phase contrast microscope and measurement of the reduction of MTT. Following the above cell treatment protocol, the morphology of the cells was observed and recorded as a photograph under an Olympus Microscope (Tokyo, Japan). After removal of the original culture medium, MTT was added to 96-well plates at a final concentration of 0.5 mg/ml, and the incubation continued for 4 h. The medium was then removed, and cells were dissolved in DMSO. The blue MTT-formazan was measured by spectrophotometry at 490 nm using an ELISA reader (ELx800uv; Bio Tek Instruments, USA). Neuronal damage in the cells was also quantitatively assessed by measuring the activity of LDH released from damaged or dead cells. A previous study has shown that the activity of LDH occurring from either necrotic or apoptotic cells is proportional to the number of neurons damaged or destroyed [11]. LDH activity in the medium and total activity was measured by an LDH assay kit at 440 nm. The
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percentage of LDH leakage was expressed as (LDH activity in medium/total activity) 9 100 %. Measurement of cellular SOD, GSH-PX, MDA, TAOC, CAT levels The cells were harvested and resuspended in 0.1 mol/l icecold PBS. The cell suspensions were sonicated for 25 s on ice and centrifuged at 1,0009g at 4 °C for 10 min, and supernatants were saved. SOD was assayed by a modification of the xanthine/xanthine oxidase method [12]. The GSH-Px activity was measured by the oxidizing speed of the GSH, which can be expressed by GSH reduction in a certain time. MDA, a compound produced during lipid peroxidation, was determined by the thiobarbituric acid method [13]. Catalase is an enzyme which catalyses decomposition of hydrogen peroxide into oxygen and water and is a peroxidase present in the cell in vivo. The catalase enzyme is a sign of peroxisomes, accounting for 40 % of the total peroxidase enzyme. Protein of cell homogenates was measured by the method of Coomassie brilliant blue using bovine serum albumin as the standard. Cellular SOD, GSH-Px, MDA, CAT, TAOC levels were determined by spectrophotometry. Hoechst 33342 staining Hoechst 33342 staining was used to determine cell apoptosis. The hippocampal neurons were stained by 10 lM Hoechst 33342 for 30 min and rinsed with PBS, and then checked under a DMR fluorescence microscope (Leica Microsystems, Germany).
times with Hank’s solution to remove the extracellular fluo-3/AM. The fluorescence intensity of [Ca2?]i in the hippocampal neurons was determined by laser scanning confocal microscopy. Total images were scanned and the data were placed in disks for analyzing [16]. Measurement of ROS Formation of ROS was determined using the fluorescent probe DCFH-DA. Cell-permeant nonfluorescent DCFHDA has been shown to be oxidized to the highly fluorescent 2,7-dichlorofluorescin in the presence of ROS. Neurons were washed twice with PBS, and then incubated with DCFH-DA (10 mM) for 30 min at 37 °C in the dark. The cells were harvested and suspended in PBS. The fluorescence intensity was measured by a fluorospectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 525 nm [17]. Statistical analysis All quantitative data were expressed as mean ± SD. The statistical significance of differences was analyzed using a one-way analysis of variance (ANOVA) (LSD, S-N-K, Dunnett) for between-group comparison and with Student’s t test for comparison between the two groups. The software package SPSS 18.0 was used for calculations. p values of \0.05 were considered statistically significant.
Results ALO ameliorated OGD/RP-induced cell viability
Effects of ALO on MMP of hippocampal neurons JC-1 was used to evaluate the loss of MMP in hippocampal cultures exposed to OGD/RP. We measured it by fluorescence microscope. JC-1 is a convenient voltage sensitive probe to monitor MMP [14]. Cells containing forming J-aggregates have high 4Wm, and showed fluorescence. Cells with low 4Wm are those in which JC-1 maintains (or reacquires) monomeric form, and show green fluorescence [15]. The relative proportions of red and green fluorescence were used to measure the ratio of mitochondrial depolarization. Measurement of [Ca21]i of hippocampal neurons Neurons were washed with Hank’s solution two or three times and were then loaded with 10 lM fluo-3/AM in the dark for 30 min at 37 °C. After this, cells were rinsed three
The cell viability was determined using MTT and LDH assay (n = 6, for each group). In order to investigate the protective effects of ALO on cell death caused by OGD/ RP, MTT assay, assessed by OD value, was used to assess cellular viability in neurons. As shown in Fig. 2a, OD value decreased in cells to 0.217 ± 0.08 at 24 h after OGD/RP, but reverted to 0.457 ± 0.03 (p \ 0.05 vs OGD/ RP group) and 0.513 ± 0.018 (p \ 0.01 vs OGD/RP group), respectively, by treatment with ALO at concentrations of 50 and 100 mg/l. Although 25 mg/l ALO did not show a remarkable reduction in OD value in Fig. 2a, there was a difference compared to the OGD/RP group 0.388 ± 0.019 (p \ 0.05). As illustrated in Fig. 2b, LDH leakage increased to 40.8 ± 5.0 % after OGD/RP. ALO significantly attenuated OGD/RP-induced cell death by reducing LDH leakage from 40.8 ± 5.0 % (vehicle group) to 28.3 ± 5.6–19.1 ± 1.72 % (Fig. 2b).
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increased by reperfusion, indicating an enhanced [Ca2?]i overload by OGD and reperfusion. Pretreatment with ALO obviously inhibited [Ca2?]i overload at both ischemic and reperfusion phases. The [Ca2?]i in ALO treated after [25 mg/l (L), 50 mg/l (M) and 100 mg/l (H)] OGD/RP injured neurons was lower (0.14 ± 0.005, 0.12 ± 0.005 and 0.1 ± 0.03) than that in OGD/RP-injured cells (0.17 ± 0.02) (Fig. 4), respectively, indicating that ALO significantly preserved the [Ca2?]i overload after ischemic neuronal injury. ALO attenuated OGD/RP-induced dissipation of MMP JC-1 was used to assess mitochondrial depolarization hippocampal cultures exposed to OGD/RP (n = 6, for each group). After OGD, the fluorescence intensity of JC-1 in hippocampal neurons was reduced, representing a dissipation of the MMP. However, treatment with ALO (25–100 mg/l) significantly stabilized the MMP (Fig. 5), which was 0.3 ± 0.09, 0.63 ± 0.05, 0.83 ± 0.05, respectively. The ALO-treated group (100 mg/l) was better than the other groups. ALO ameliorated OGD/RP-induced oxidative stress
Fig. 2 Effects of ALO on cell viability in primary hippocampal neuronal cells under oxygen–glucose deprivation for 2 h and reperfusion for 24 h, determined by MTT assay (a), and the extent of LDH release (b). Histograms represent mean ± SD. ##p \ 0.01 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
ALO attenuated OGD/RP-induced cell apoptosis Exposure to OGD also resulted in apoptotic death of hippocampal neurons (n = 6, for each group), as revealed by the appearance of condensed nuclei in Hoechst-stained cultures (Fig. 3(1)). Through cell counting, 50.8 ± 4.2 % of the cultured neurons in the vehicle group were determined to be apoptotic cells 24 h post-OGD compared to 3 % of apoptotic neurons in the total count of the control group. However, in the ALO-treated groups, the OGD/RPinduced apoptotic cell death rate was significantly decreased to 32.57 ± 6.22, 18.3 ± 1.85, and 7 ± 1.32 %, respectively (Fig. 3(2)). ALO attenuated OGD/RP-induced [Ca21]i Green fluorescence intensity was used to assess the expression of [Ca2?]i (n = 6, for each group). OGD/RP triggered a rapid rise in [Ca2?]i, which was further
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We next tested the effects of ALO on the OGD/RP-induced ROS generation in hippocampal neurons (n = 6, for each group). 24 h after OGD, a 2.1-fold increase in the intracellular ROS was found using a DCFH-DA assay. However, ALO (25–100 mg/l) decreased ROS production (Fig. 6). The effects of ALO on cellular MDA, GSH-Px, SOD, TAOC and CAT levels are presented in Fig. 6 (n = 6, for each group). OGD exposure markedly decreased GSH-Px content, TAOC and antioxidant enzymatic activities (CAT, SOD), while it increased the level of the lipid peroxidation product (MDA) in the neuronal cultures. However, concurrent treatment with ALO significantly decreased the MDA content. ALO also resulted in a noticeable increase in the activities of CAT, SOD and GSH-Px in comparison with the vehicle control.
Discussion The present study demonstrated the neuroprotective effect of ALO on neonatal rat primary cultured hippocampal neurons injured by OGD/RP. There were two important results—(1) pre-treatment with ALO significantly reduced the injury in primary cultured hippocampal neurons; (2) this neuroprotection was associated with attenuation of intracellular oxidation properties and increase of antioxidants during OGD/RP.
J Nat Med Fig. 3 (1) Effects of ALO on morphological changes of OGD/RP-injured primary hippocampal neurons (9400). Nuclei were labeled with Hoechst 33342 (A–F). A Control, B exposure to OGD/ RP, C treated with nimodipine (final concentration 5 mg/l), D treated with low concentration of ALO (final concentration 25 mg/l), E treated with middle concentration of ALO (final concentration 50 mg/l), F treated with high concentration ALO (final concentration 100 mg/l). (2) Effects of ALO on apoptosis rate in primary hippocampal neuronal cells under OGD 2 h/ RP 24 h. Histograms represent mean ± SD. ##p \ 0.01 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
We evaluated the degree of injury in hippocampal neuronal cell exposed to OGD/RP by MTT and LDH release. Treatment with ALO (25–100 mg/l) decreased the cell damage and inhibited LDH release, which indicated that ALO may have a protective effect on hippocampal neuronal cell exposed to OGD/RP. Oxygen-free radicals are known to be generated during periods of ischemia followed by reperfusion. Studies on the antioxidant changes and the significance during brain failure have provided a new insight into acute cerebral
ischemia. The biochemical profile of the neurons, like depletion of SOD, CAT and GSH-Px, provide strong evidence for oxidative stress occurring during ischemia– reperfusion. In this study, increased MDA level and decreased antioxidant activities of enzymes (SOD, CAT, GSH-Px and TAOC) were detected in OGD/RP neurons. This indicated that oxidative injury was closely associated with brain ischemia–reperfusion. Interestingly, in the current study, neurons in the hippocampal from the ALO-group showed a significantly decreased MDA and increased
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J Nat Med Fig. 4 (1) Effects of ALO on [Ca2?]i in primary hippocampal neuronal cells under OGD 2 h/ RP 24 as indicated by fluo-3/ AM. A Control, B exposure to OGD/RP, C treated with nimodipine (final concentration 5 mg/l), D treated with low concentration of ALO (final concentration 25 mg/l), E treated with middle concentration of ALO (final concentration 50 mg/l), F treated with high concentration ALO (final concentration 100 mg/l). (2) Effects of ALO on apoptosis rate in primary hippocampal neuronal cells under OGD 2 h/ RP 24 h. Histograms represent mean ± SD. ##p \ 0.01 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
endogenous antioxidants (GSH-Px, SOD, TAOC and CAT). This indicated that ALO can reduce OGD/RP-induced injury by increasing antioxidant enzymes like SOD, CAT, GSH-Px and TAOC activities. Excessive ROS production in the brain is believed to contribute to neurodegenerative processes [18]. Statistical analysis between the control and OGD/RP groups with the administration of ALO revealed that ALO significantly inhibited ROS production during OGD/RP. Taken together, the observations indicated that the protective mechanisms of ALO on cultured rat hippocampal neurons injured by OGD/RP were related to antioxidative stress.
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The protective effect of ALO was also supported by a higher MMP. MMP reflects the performance of the electron transport chain and indicates a pathological disorder of this system. High average MMP is related to higher viability [19]. After OGD/RP, the participation of free radicals increases the lipid peroxidation of the cell membrane, breakage of the DNA, degradation of protein, etc. Finally, these lead to cell dysfunction. The present study indicated that OGD led to mitochondrial membrane depolarization and ALO (100 mg/l) significantly prevented the loss of the MMP, suggesting that the electron transport chain was still maintained. Mitochondria are susceptible targets of free
J Nat Med Fig. 5 (1) Effects of ALO on MMP changes in primary hippocampal neuronal cells under OGD 2 h/RP 24 h as indicated by JC-1 (9400). Red fluorescence indicates polarized mitochondrial membranes whereas green fluorescence indicates depolarized mitochondrial membranes. A Control, B exposure to OGD/ RP, C treated with nimodipine (final concentration 5 mg/l), D treated with low concentration of ALO (final concentration 25 mg/l), E treated with middle concentration of ALO (final concentration 50 mg/l), F treated with high concentration ALO (final concentration 100 mg/l). (2) Effects of ALO on MMP changes in primary hippocampal neuronal cells under OGD 2 h/RP 24 h. Histograms represent mean ± SD. ##p \ 0.01 vs control group, *p \ 0.05, **p \ 0.01vs vehicle group
radical-mediated damage [20]. The antioxidant properties of ALO on cultured rat hippocampal neurons injured by OGD/RP were involved in anti-early apoptosis and the stabilization of MMP. Ischemia or hypoxia can cause cell death by necrosis and apoptosis [21]. Numerous studies have shown that OGD/RP induced hippocampal neuron death, mainly through apoptosis [22]. In our study, the OGD/RP insult increased the
apoptosis rate of neurons from 3.00 ± 1.732 to 50.8 ± 4.2 %, and ALO could inhibit it effectively. This was also consistent with morphological changes examined with a scanning electron microscope. To further explore the anti-apoptotic mechanism of ALO, we performed an additional study of mitochondria function and [Ca2?]i overload. [Ca2?]i overload during, and especially following cerebral ischemia has been considered to be one of the key
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Fig. 6 Effects of ALO on SOD (a) and GSH-Px (b) activities and the level of MDA (c) and CAT (d) activities and TAOC (e) and ROS (f) in primary hippocampal neuronal cells under oxygen–glucose
deprivation for 2 h and reperfusion for 24 h. Histograms represent mean ± SD. ##p \ 0.01, #p \ 0.05 vs control group. *p \ 0.05, **p \ 0.01 vs vehicle group
pathogenic events in the post-ischemic cellular death pathway [23]. [Ca2?]i overload results in a loss of mitochondrial membrane integrity, secondary to an opening of the mitochondrial permeability transition pore [24]. Our findings showed that ALO (100 mg/l) decreased the elevation of calcium, which further confirmed the hypothesis that the protective effect of ALO was related to inhibition of calcium overload.
Although many compounds with neuroprotective action are in various stages of the pharmaceutical pipeline, ALO possesses unique advantages. Sophora alopecuroides L. and its related species have already had thousands of years of human exposure with little reported toxicity. Additionally, ALO can easily diffuse across biological membranes and the blood–brain barrier in an energy deficient environment.
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Conclusively, our results clearly demonstrated that ALO has a neuroprotective effect on OGD/RP neuronal injury in vitro. The effects have bearing on anti-oxidative stress, stabilization of MMP and suppression of the intracellular Ca2? elevation. These results should provide good evidence to explore the further neuroprotective mechanism of ALO. Acknowledgments This study was supported by Ningxia Hui Autonomous Region, colleges and universities of science and technology research projects (NGY2013073). We are indebted to the staff in the animal center and the Science and Technology Center who provided assistance in the study. Conflict of interest
The authors declare no conflict of interest.
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