Int J Clin Exp Med 2018;11(7):6547-6559 www.ijcem.com /ISSN:1940-5901/IJCEM0065327 Original Article Neurotoxicity of Veratrum nigrum L. and the molecular mechanism of veratridine toxicity Xianxie Zhang1,3*, Yanli Wang2,3*, Shuai Shao3, Yan Wu1, Yuguang Wang3, Zengchun Ma3, Hailong Yuan1, Yue Gao3 1Air Force General Hospital of People’s Liberation Army, Beijing, China; 2China Hospital Knowledge Depot, China National Knowledge Infrastructure, Beijing, China; 3Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing, China. *Equal contributors and co-first authors. Received April 6, 2017; Accepted February 14, 2018; Epub July 15, 2018; Published July 30, 2018 Abstract: Veratrum nigrum L. has been used as an herbal preparation in worldwide for treatment of blood-stroke, excessive phlegm, epilepsy, etc. Veratridine is both active and toxic constituents of Veratrum nigrum L., which has been reported to affect excitable membranes, peripheral nerves, and skeletal and cardiac muscle. Previous work and literature research are reported that veratridine can act on the central nervous system. Therefore, it is very meaningful to study the neurotoxicity of veratridine. Cell viability, LDH release, ROS generation, MMP expression, Ca2+ concentration and apoptosis were measured to evaluate the neurotoxic mechanism of veratridine in SH-SY5Y cells. A positive injury model was established with glutamic acid in SH-SY5Y cells and Western blot was used to cor- relate toxicity with the MAPK signaling pathway. The results show that veratridine causes, MMP expression changes, Ca2+ accumulation in the cytoplasm, increased ROS and LDH releasing and SH-SY5Y cell membrane damage. SH- SY5Y cell viability diminished due to apoptosis. The MAPK signaling pathway is activated by veratridine, and driving forward apoptosis. The study provided evidence of the neurotoxic caused by veratridine and revealed the molecular mechanism behind its neurotoxic effects. Keywords: Veratrum nigrum L., veratridine, SH-SY5Y cells, neurotoxicity, toxicity mechanism, MAPK Introduction administration accompanied by convulsions [7, 8]. Traditional Chinese medicines are gaining in- creasing popularity worldwide for disease treat- Veratridine (Ver), which accounts for 33% of the ment. Though the herb Veratrum nigrum L. has total plant alkaloid content, is considered to be been demonstrated to have toxic side effects, toxic and has been reported to affect excitable it has been used as an herbal preparation in membranes, peripheral nerves, and skeletal China, Japan, Southeast Asia, and by native and cardiac muscle [9-12]. Ver increases intra- Americans for the treatment of hemorrhagic cellular Na+, which subsequently increases in- stroke, excessive phlegm, epilepsy, etc [1, 2]. tracellular Ca2+ via Na+-Ca2+ exchange, Low do- Veratrum has been documented to be toxic to ses of Ver alkaloids have hypotensive effects, the digestive tract mucosa, the dorsal nucleus while high doses can be neurotoxic [13, 14]. It of the vagus nerve, and the central nervous has also been reported that Ver can act on the system [3, 4]. Modern phytochemical and phar- central nervous system and modify opioid and macological studies have shown that both the muscarinic binding sites in brain slices. The active and toxic constituents are alkaloids, and brain can be excited after these inhibitory some studies suggest that it is mutagenic and effects, leading to disturbances of conscious- teratogenic [5, 6]. We previously reported that ness such as convulsions, drowsiness, and the LD50 of aqueous extracts of Veratrum nig- coma [15, 16]. The human SH-SY5Y neuroblas- rum L. is 2.566 g/kg after intragastric adminis- toma cell line has similarities to nerve cell mor- tration, and that mice die 5-10 min after drug phology, physiology, and biochemical function, Neurotoxicity mechanism of veratridine Figure 1. Graphical abstract, The inner structure of the paper. and is used to model neurodegenerative dis- stimuli such as growth factors, cytokines, extra- eases [17]. We documented differentially ex- cellular mitogens, and stress [21, 22]. MAPKs pressed genes in SH-SY5Y cells treated with are involved in cell differentiation, proliferation, Ver using a cDNA microarray. These differen- and programmed cell death. Four pathways of tially expressed genes are involved with metab- MAPK signaling transduction have been identi- olism, mitochondrial function, the cell cycle, fied, ERK1/2, JNK, P38, and ERK5, among cell differentiation, cell signaling, and apopto- which p38/JNK/MAPK signaling transduction is sis, and their disturbance results in subacute or important in cell differentiation [23-25]. At chronic injury to the nervous system [18]. present, the activation of ERK signaling is Therefore, it is very meaningful to study the reported to promote cell proliferation, while neurotoxicity of Ver. JNK signaling is tied to neurodegenerative or neurological diseases [26, 27]. Both p38 and Apoptosis is involved in acute neurotoxicity and JNK signaling pathways are associated with chronic neurodegenerative disorders, so drugs apoptosis. Thus, we assessed mitochondrial that block this type of cell death are of interest. dysfunction-induced neurotoxicity in SH-SY5Y Apoptosis is initiated by Ver-mediated oxidative cells and observed the mechanisms underlying stress, which damages the membrane and neurotoxicity after treatment with the toxin Ver. causes lactate dehydrogenase (LDH) release Abstract mining the inner structure of the and decreased mitochondrial membrane po- graph, as shown in Figure 1. tential (MMP). Mitochondrial dysfunction is a significant source of reactive oxygen species Materials & methods (ROS) [19, 20]. Reagents Mitogen-activated protein kinase (MAPK) sig- naling pathways are composed of SerThr pro- Veratridine (Ver) and Glutamic acid (Glu) (purity tein kinases that activate as a cascade. MAPKs > 99%) was obtained from the National Institute respond to diverse cellular and extracellular for the control of Pharmaceutical and Biological 6548 Int J Clin Exp Med 2018;11(7):6547-6559 Neurotoxicity mechanism of veratridine Product (Beijing, China). RPMI-1640 medium LDH release and fetal bovine serum were purchased from Gibcol (Carlsbad, CA). Dichlorofluorescein di- LDH release was measured using a commerci- acetate (DCFH-DA), lactate dehydrogenase al kit. SH-SY5Y cells were plated at 1.5×105 (LDH) and a Cell Counting Kit-8 (CCK-8) were cells/ml in 96-well plates, with indicated con- purchased from Sigma (St. Louis, MO). An centrations of Glu for 16 h and Ver for 48 h. We annexinV-fluorescein isothiocyanate (FITC)- ran the reaction in the dark for 10 min prior to propidium iodide (PI) double-stained apoptosis measurement, and the absorbance was read at detection kit was obtained from Biosea bio- 450 nm using a microplate reader. Results are technology (Beijing, China). Fluo-3-acetoxy- expressed as percent relative to controls. methylester (Fluo-3-AM) and Hochest 3325 were purchased from Invitrogen Biotechnolo- MMP measurement gy (Carlsbad, CA). The ROS fluorescent probe- MMP was measured using flow cytometry and dihydroergotamine (DHE) was purchased from the mitochondrial-specific cationic dye JC-1. Vigorous Biotechnology Beijing (Beijing, China). SH-SY5Y cell (1.5×105 cells/ml) suspensions Polyvinylidene difuride membrane (PVDF) and were plated in 12-well plates with Glu for 16 h an ECL kit were purchased from Amersham or Ver for 48 h. Cells were harvested, washed (Arlington Heights, IL). Primary antibodies to twice, and incubated with 0.5 mL JC-1 (25 μM) β-actin (1:500), ERK1/2 (1:500), p-ERK1/2 for 20 min at 37°C. MMP was assayed and (1:500), c-Jun NH2-terminal kinase (JNK) green (JC-1 monomer) and red (JC-1 aggregate) (1:500), p-JNK (1:200), P38 (p38 MAPK) fluorescence were monitored at an emission (1:400), p-P38 (1:200), and secondary anti- wavelength of 525 nm and 595 nm. Changes in body horseradish peroxidase (HRP) conjugated ratios between measurements indicated MMP immunoglobulin G (IgG) (1:2,000) were pur- changes. chased from Santa Cruz biotechnology (Santa Cruz, CA). Other analytically pure reagents were Intracellular ROS and Ca2+ measurements from Promega. SH-SY5Y cell (1.5×105 cells/ml) suspensions Cell culture and treatment were plated in 12-well plates with the indicated concentrations of Glu for 16 h and Ver for 48 h. SH-SY5Y human neuroblastoma cells were Intracellular ROS and cytosolic Ca2+ were mea- grown in RPMI-1640 supplemented with 15% sured using the fluorescent probe DCFH-DA fetal calf serum. Cells were maintained at 37°C in an incubator with a saturated humid atmo- and Fluo-3-AM, respectively, and a fluorescent- activated cell sorter. DCFH-DA is converted to a sphere containing 95% air and 5% CO2. Cells were passaged once every three days. All fluorescent compound in the presence of ROS. experiments were conducted on cells between Fluo-3/AM was added to treated cells to mea- 2+ passages 10-20 after cells were purchased sure Ca . After treatment with the indicated from Peking Union Medical College Cell Bank drugs, cells were incubated with DCFH-DA (10 (Beijing, China). μM) for 20 min at 37°C in the dark. (ROS assay) and Fluo-3/AM (5 μmol/L) for 30 min at 37°C Cell viability analysis (Ca2+ assay), and cells were harvested and sus- pended in 500 μL HBSS. Intracellular ROS and Cytotoxicity was assayed in SH-SY5Y cells plat- Ca2+ were measured using a flow cytometer ed in 96-well plates (1.5×105 cells/ml) over- (excitation wavelength 488 nm; emission wave- night and replaced with serum-free medium length 535 nm). supplemented with Glu for 16 h or Ver for 48 h. 10 μL of CCK-8 was added to each well and Cell apoptosis incubated for 1-4 h. Absorbance was read at 450 nm with a PekinElmer Victor X Microplate Hoechst 33342 was used to assess apoptosis Reader (PekinElmer, Foster City, CA). Reductions in SH-SY5Y cell (1.5×105 cells/ml). Suspensions in optical density (OD) due to drug treatment were plated in 6-well plates with Glu for 16 h or were used to assess cell viability and normal- Ver for 48 h.