Rhynchophylline Protects Cultured Rat Neurons against Methamphetamine Cytotoxicity

Rhynchophylline (Rhy) is an active component isolated from species of the genus Uncaria which has been used for the treatment of ailments to the central nervous system in traditional Chinese medicine. Besides acting as a calcium channel blocker, Rhy was also reported to be able to protect against glutamate-induced neuronal death. We thus hypothesize that Rhy may have neuroprotective activity against methamphetamine (MA). The primary neurons were cultured directly from the cerebral cortex of neonatal rats, acting as in vitro model in the present study. The neurotoxicity of MA and the protective effect of Rhy were evaluated by MTT assay. The effects of MA, Rhy or their combination on intracellular free calcium concentration ([Ca2+]i) were determined in individual neocortical neurons by the Fluo-3/AM tracing method. The MTT assay demonstrated that MA has a dose-dependent neurotoxicity in neuronal cultures. The addition of Rhy prior to the exposure to MA prevented neuronal death. Time course studies with the Fluo-3/AM probe showed that Rhy significantly decreased neuronal [Ca2+]i which was elevated by the exposure to MA. Our results suggested that Rhy can protect the neuronal cultures against MA exposure and promptly attenuate intracellular calcium overload triggered by MA challenge. This is the first report demonstrating an inhibitory effect of Rhy against MA impairment in cultured neurons in vitro.


Introduction
Rhynchophylline (Rhy) is a major tetracyclic oxindole alkaloid (the chemical structure of Rhy is shown in Figure 1), originally isolated from species of the genus Uncaria which is widely used in traditional Chinese medicine prescribed mainly to treat ailments to the central nervous and cardiovascular systems, such as lightheadedness, convulsions, numbness, and hypertension [1].
In the cardiovascular system, it is well documented that the vasodilative effect of Rhy is mainly due to the dysfunction of Ca 2+ transport, including influx of extracellular calcium and release of intracellular calcium by blocking the voltagedependent calcium channel and the receptor-regulation calcium channel [2]. There are records that an extraction with methanol from Uncaria rhynchophylla exhibits a significant neuroprotective effect in vitro, in which Rhy appears to be the active pharmacological component [3]. Further research on rat cerebellar granule cells showed Rhy to be able to protect against glutamate-induced neuronal death [4]. Moreover, Rhy is thought to have calcium channel blocking functions, which can further explain the neuroprotective functions. Calcium channel blockage can potentially have a protective effect because of the positive relation between increased calcium influx and cell death [1]. For this reason, we expect Rhy to have neuroprotective functions for the central nervous system. However, the mechanistic study for Rhy is limited, especially for the neural impairment induced by drug abuse. Methamphetamine (MA) is a commonly abused psychostimulant in the world, which leads to severely neurodegenerative changes in the human brain [5]. It  The dose-response curve of neuronal cultures exposed to methamphetamine (MA) determined by MTT assay, which was generated by Prism software. The cortical neurons were exposed to various concentrations of MA (0, 25, 50, 100, 200, and 400 µM) for 48 hours.
primarily acts on dopamine transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2), which when dysfunctioning result in high extracellular dopamine concentrations and subsequent neurotoxicity [6]. Repeated administration of MA has neurotoxic properties, which are caused by an intracellular calcium overload [7]. Several in vitro and in vivo studies suggest that MA might cause cell death via a process that resembles apoptosis in vitro and in vivo [8]. Calcium signaling plays an important role in apoptosis, which mediates several important steps in the apoptotic downstream pathway. The inhibition of Ca 2+ influx in a rat cerebellum neural cell line (R2) was reported recently to be able to attenuate the neurotoxicity of MA [9]. We thus hypothesize that Rhy may have neuroprotective activity against MA-induced intracellular calcium overload. In the present study, the neurotoxicity of MA was examined and then saved by the potential calcium antagonist Rhy in the primary rat neuronal cells in culture. Wister rats (postnatal day 1) were used for the primary neuronal cell culture. Rats were sacrificed by decapitation and brain regions of the cortex were dissected on ice. The tissues were put into an eppendorf with 1 mL minimal essential medium (MEM; Gibco, USA) with 1% penicillin/streptomycin (PSN; Gibco) in ice. Then the brain tissues were chopped using a razor blade in MEM. The chopped tissues were then treated with prewarmed trypsin/EDTA solution with 1% PSN for 20 minutes at 37 • C in a 5% CO 2 incubator and agitated with a dropper every 5 minutes. MEM solution with 10% fetal bovine serum (Gibco) and 1% PSN was added to stop the trypsinization. Then the solution was centrifuged at 1,300 rpm for 10 minutes. The supernatant was removed and the cells at the bottom were suspended in MEM solution and centrifuged again. Finally, the supernatant was removed and the cells inside were resuspended in the complete culture medium, composed of Neurobasal-A-Medium, 10% fetal bovine serum, 1% PSN, and 0.1% 50 × B27 supplement (Gibco). The neurons were then seeded on poly-L-lysine (Sigma) precoated 96well microplates for MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay, or seeded on glass bottom culture dishes (MatTek Corporation, USA) for intracellular calcium recording under confocal microscope. The cultures were maintained at 37 • C in a 5% CO 2 humidified incubator. We used serum-free medium (Neurobasal-A-Medium, 1% PSN, 0.1% 50 × B27 supplement; Gibco), complemented with 1 µM cytosine arabinoside (Sigma), to gradually replace complete medium. The addition of a selective DNA synthesis inhibitor (cytosine arabinoside) in culture eliminated proliferative glial cells allowed adequate numbers of neurons to selectively survive.

Cell Viability Assay.
Cell viability with MA treatment was determined by measuring the mitochondrial dehydrogenase activity using MTT assay in triplicate. The cortical neurons, with a seeding number of 10 4 cells per well in a 96well microplate, were cultured for 6 days before use. The cells were incubated with MA at serial concentrations (0, 25, 50, 100, 200, and 400 µM) for 48 hours at 37 • C. After 44 hours, 10 µL MTT solution (1 g/L; Sigma) was added into each well, and the microplate was incubated for 4 hours. Formazan crystals, produced by mitochondrial dehydrogenase activity in viable neurons, were dissolved by addition of 150 µL DMSO on a shaker at room temperature. Absorbances were read at 570 nm using a microplate reader. Vehicle controls were set up in parallel to offset the background during process. Each test was performed in triplicate in the 96-well microplates. The experiment was repeated for 3 times. Cytotoxic effect was analyzed by  generating dose-response curves as graphs of viable cells (yaxis) against the concentration of MA (x-axis) with assistance of Prism software (Graph Pad Software Inc, USA). The viable cells were measured in percentage with respect to the following formula:

Microscopy
Observation. The morphological changes in neurons were monitored under an inverted phase-contrast microscope before and after MA treatment.  The following experiments were conducted according to Tao-Cheng et al.'s protocols [10]. Briefly, cultures were observed on a laser scanning confocal microscope with 488 nm excitation. Time lapse images were collected in 10second intervals. Solution changes were prepared by adding fresh solution. We analyzed ten neuronal cells at 0, 430, and 860 seconds. Fluorescent intensity of each neuronal cell body at each time point was obtained with Image Pro Plus. Data were expressed as mean fluorescence intensity values ± SEM. The differences of [Ca 2+ ] i fluorescence between 860 and 430 seconds, 430 and 0 seconds were determined by paired-samples Mann-Whitney U test in each group, using SPSS software. Comparison of Fluo-3/AM-indicated [Ca 2+ ] i variances between 430 and 860 seconds of different groups, were performed with one-way ANOVA followed by post hoc test. The value of P < 0.05 was considered statistically significant.  Figure 3 shows the representative phase-contrast photomicrographs of control, MA and combination cultures after incubation for 48 hours.  Figure 4). However, MTT assay demonstrated that the number of viable neurons was dramatically decreased after exposure to 200 µM. Thus the intracellular Fluo-3/AM poured out from more dying cells, which still displayed relatively normal morphology but explicating a slightly decreased fluorescent intensity.

Rhy Inhibited Calcium Influx in Neurons after
Exposure to MA. One-way ANOVA showed significant difference in the comparison among groups (P < 0.001; Figure 5

Discussion
The majority of researches have been limited to MA neurotoxicity to dopaminergic neurons, in which the dopamine reuptake is inhibited by MA resulting in intracellular highconcentration dopamine [11]. Actually, MA can induce neural impairment in other brain areas too. For example, neurodegeneration has been found in the piriform and parietal cortex, thalamus and hippocampus, areas which receive only a sparse dopaminergic input, and in nondopamine striatal elements [7]. The main objective of the present study is to determine whether the neurotoxic effects of MA as a psychostimulatory drug, which causes neural death on rodent cortical cells in vitro, can be attenuated by the pretreatment with Rhy in cell cultures. The neurotoxicity is thought to be linked to intracellular calcium levels, which appear to correlate with MA incubation in a dose-dependent manner. The significant neurotoxicity caused by several types of amphetamines in rat neocortical neurons was previously reported to be accompanied by internucleosomal DNA cleavage and nuclear breakdown, as well as differential expression of the anti-and proapoptotic bcl-xL/S splice variants, indicating an involvement of the apoptotic pathways in amphetamine neurotoxicity [12]. It has been confirmed that MA can be neurotoxic to cortical cells directly, with the increase in calcium load. The [Ca 2+ ] i is therefore suggested to play a pivotal role in MA-induced cell death. MA is a cationic lipophilic molecule that can diffuse into the mitochondria and affects the mitochondrial calcium ATPase, which is responsible for pumping Ca 2+ into the inner mitochondrial space for storage purposes. The release of these stores, through the mitochondrial membrane that is made permeable by formation of transition pores, triggers the activation of the cell death pathway. Cell death requires the involvement of cysteine proteases and caspases, which are normally associated with the mitochondrial envelope in their inactive state [7]. Consequently, Ca 2+ influx is followed by mitochondrial damage and reactive species formation. It is well documented that MA induced neurological damage caused by the above described mechanisms, because it is responsible for Ca 2+ influx due to its effect on ionotropic glutamate receptors [11].
Rhy has been proven to be a potent calcium channel antagonist in blood vessels and it is assumed to act as one of the noncompetitive antagonists for glutamate receptors [2]. Therefore Rhy is thought to contribute to the neuroprotective and anticonvulsant activity of plant extracts of Uncaria species [13]. Moreover, another in vivo behavioral study indicates that Rhy has protective features against harmful effects of MA [14]. To conclude, our study demonstrates that Rhy decreases the high level of intracellular calcium induced by MA in cultured neurons, through the direct blockade of calcium channels and/or the inhibition of ionotropic glutamate receptors. This is the first report demonstrating an inhibitory effect of Rhy against MA impairment in cultured neurons in vitro. Our results suggested that Rhy can protect the neuronal cultures against MA exposure and promptly attenuate intracellular calcium overload triggered by the MA toxin.