Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells.
Background: Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned.
Purpose: In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PCI 2 cells.
Study design: PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway.
Methods: PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting.
Results: Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells.
Conclusion: Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression.
Protein kinase B
Pyrrolizidine alkaloids (PAs) are common toxic agents found in about 3% of the world's flowering plants. Until now, more than 600 PAs have been found in nature, especially those from the families of Asteraceae, Boraginaceae and Fabaceae: these plants are commonly used as herbal medicine in the treatment of traumatic injury, pain, inflammation and other ailments (Roeder, 1995 and 2000). Unfortunately, PAs possess serious toxicity: because they contain an unsaturated 1, 2-double bond in the necine base, one of the most toxic compounds of plant origin. More importantly, PAs have been detected in many food products, such as honey and milk. The toxic substances, therefore, may be transferred to humans through the food chain causing intoxication (Goeger et al., 1982; Medeiros et al., 1999; Dubecke et al., 2011). As the result, special attention has been paid on the safety of PA worldwide (World Health Organization, 1989; German Federal Health Bureau, 1992; Pharmacopoeia of the People's Republic of China, 2010). Despite PAs are primarily considered as hepatotoxic, an extra-hepatoxic effect, such as pneumotoxic and neurotoxic, was also related to its intoxication in animals (Lee et al., 2005; Cooper et al., 1999).
Clivorine is an otonecine PA found in the plants from various Ligularia species (Asteraceae), including Ligularia hodgsonii and L. wilsoniana. Both species are commonly called Chuanziwan, which are commonly used as erroneously substitute of an herbal medicine Asteris Radix et Rhizoma (commonly called Ziwan, the dried root and rhizome of Aster tataricus) for antitussive function. The recommended dosage of Ziwan intake should be 5-10 g per day (Pharmacopoeia of the People's Republic of China, 2010). Considering about 0.8 mg/g clivorine found in Chuanziwan (Cheng et al., 2011), over 4 mg of clivorine should be taken by usage of the herb; this dose is 4000-fold higher than the safety limit of PA according to German Federal Health Bureau, i.e. 1 [micro]g/day (German Federal Health Bureau, 1992). Therefore, it should be a great threaten to human health. Multiple administrations of Chuanziwan water extracts could cause significant liver injury with elevated serum ALT and AST activities in rats (Cheng et al., 2011). In divorine-induced hepatotoxicity, the activations of growth-related kinase and caspase signaling pathways, as well as the oxidation system, were revealed (Ji et al., 2002, 2005, and 2010). However, very limited study has been done on the neurotoxicity of clivorine. Owing to its unique structure, clivorine exists in either a lipophilic non-ionized form or a hydrophilic ionized form (Lin et al., 2000): the unique dual solubilities may influence its toxicity by enhancing penetration via blood-brain barrier. On the other hand, clivorine showed toxicity on non-hepatic cells without P450 enzymes, such as human embryonic kidney 293 (HEK293) cells (Ji et al., 2002 and 2008). Thus, clivorine should be an excellent candidate for investigating the extra-hepatic toxicity on non-hepatic cell models, e.g. neuron.
PC12 cell is a rat pheochromocytoma cell line. Although they are not considered adult neurons, they undergo changes in phenotype exhibited by normal neurons when treated with nerve growth factor (NGF), including outgrowth and extension of neurites and maintenance of the structural basis of neural communication. Thus, PC12 cell model has been accepted as a widely used model for studying of neuronal properties and neurotoxicity in vitro (Greene et al., 1987). The challenge of clivorine in PC12 cultures showed robust decrease of markers for neuronal differentiation, including (i) NGF-induced neurite outgrowth; (ii) mRNAs and proteins encoding for neurofilaments; and (iii) downstream signaling of NGF, i.e. phosphorylation of TrkA, protein kinase B (Akt) and cAMP response element-binding protein (CREB).
Material and methods
Chemicals and reagents
Clivorine was purchased from Shanghai R&D Centre for Standardization of Traditional Chinese Medicine (>98% purity; Shanghai, China; molecular weight of 405 Da). The 13CNMR spectrum of clivorine was run on a Bruker AVANCE-III instrument operating at 600 MHz with tetra-methylsilane (TMS) as internal standard and compared with those reported (Tan et al., 2001) (Suppl. Table 1). Clivorine was dissolved in dimethyl sulfoxide (DMSO) forming 100 mM stock solution and stored at -20 [degrees]C, and the final concentration of DMSO in all the culture medium was kept at 0.1%. The roots of L. hodgsonii were collected from Hehuachi Market (Sichuan, China) and authorized by the authors. The water extract of herb was prepared by boiling with water according to our reported method (Xiong et al., 2011 and 2014), and the content of clivorine was determined to be about 2.10 mg/g using an UPLCMS system (Supplementary Text 1). Proper amount of extract was dissolved in water by ultra-sonication to make a stock solution of 250mg/ml, which equaled to 1.27 mM of clivorine, and stored at -20 [degrees]C.
Rat pheochromatocytoma PC12 cell was obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 6% fetal bovine serum, 6% horse serum, 100 U/mL penicillin, and 100 [micro]g/mL streptomycin in a humidified at 37 [degrees]C with 7.5% C[O.sub.2]. Fresh medium was supplied every other day. All culture reagents were purchased from Invitrogen Technologies (Carlsbad, CA). Experiments were performed on cells that had undergone fewer than fifteen passages.
After being treated with clivorine, the cell viability was measured by the colorimetric 3-(4,5-dimethylthioazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. In brief, cells were cultured in 96-well plate and treated with series concentrations of clivorine and water extracts of L. hodgsonii root. After the drug treatment for 48 h, MTT solution (0.5 mg/ml) was added into the cultures and then incubated for additional 3 h. After the medium was removed, DMSO was added into each well before measuring the absorbance at 570 nm. The cell viability was expressed as the percentage of absorbance value of control (DMSO treatment), where the absorbance value was set as 100%. Besides, the cell viability was also measured by trypan blue staining according to the reported method (Ji et al., 2008). Cells were incubated with clivorine for 48 h before trypsinization; then the cells were mixed with 0.4% trypan blue-PBS for 2 min, and the dead cells were stained blue by trypan blue. The number of stained and unstained cells was counted using a hemocytometer.
PC12 cells were grown on glass cover-slip. After being treated with NGF and series concentrations of clivorine for 48 h, the cells were then fixed with 4% paraformaldehyde (PFA) in PBS for 15 min, followed by 50 mM ammonium chloride (N[H.sub.4]Cl) treatment for 25 min. Cultures were permeablized by 0.1% Triton X-100 in PBS for 10 min and blocked by 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Anti-NF68 primary antibody (1:500) was then applied on the cells for 16 h at 4 [degrees]C. Then, cells were stained with Alexa 555-conjugated secondary antibody (1:1000) and diamidino-phenyl-indole (DAPI; 5 [micro]g/ml; Sigma-Aldrich, St Louis, MO) for 1 h at room temperature. After being washed with PBS for 4 times in 1 h, the cells were dehydrated serially with 50%, 75%, 95% and 100% ethanol and mounted with fluorescence mounting medium. Samples were then examined by ZEISS LSM710 Laser Scanning Confocal Microscope (Zeiss Inc., Jena, Germany). Images were captured with Ex 405 / Em 400-515 nm for DAPI (blue color) while Ex 560 / Em 560-695 nm for NF68 (red color).
Determination of neurite outgrowth
The determination of neurite outgrowth in PC12 cell cultures was performed according to a previous paper (Xu et al., 2012). PC12 cells (5 x [10.sup.4] cells/well) were seeded onto 6-well plates and treated with NGF, with or without series concentrations of divorine for 48 h. A light microscope (Diagnostic Instruments, Sterling Heights, MI) equipped with a phase-contrast condenser, 10X objective len, a digital camera and SPOT imaging software were used to capture the images with the manual setting. For analyzing the neurite presence and neurite length, approximately 50 cells were counted from 20 randomly chosen visual fields for each culture, i.e. 1000 cells were counted in total for each culture. First, the cells were scored as differentiated if one or more neurites had a length equal to or longer than the cell body diameter, approximate 30 [micro]m. Second, cells were also classified according to their neurite length (length of the longest neurite) in < 30, 30-60, 60-90 and >90 [micro]m. In this study, NGF (20ng/ml) was used as a positive control.
Quantitative real time PCR
To determine the effect of divorine on mRNA expression, PC12 cells (2 x 105 cells/well) were seeded onto 6-well plates and treated with NGF and/or series concentrations of divorine and water extracts of L. hodgsonii root for 48 h. Expression levels of genes encoded with NF68, NF160, NF200, and TrkA were determined. Total RNA from cultured cells was isolated with RNAzol reagent according to the manufacturer's protocol (Molecular Research Center, Cincinnati, OH). In addition, cDNA was generated from 3 [micro]g of RNA using High Capacity cDNA Reverse Transcription Kit of Applied Biosystems (Foster City, CA). Real-time quantitative PCR for the target genes was performed on equal amounts of cDNA by using Roche SYBR Green Master, according to the manufacturer's instructions (Roche Applied Science, Pleasanton, CA). Each sample was run in triplicate. Relative expression levels of genes were calculated using 18S rRNA as the internal control. The primer pairs designed using qPrimerDepot are listed in Suppl. Table 2. The amplification protocol was as follows: initial denaturation for 30 s at 95 [degrees]C, followed by 40 cycles of 95 X for 40 s and 60 [degrees]C for 31 s.
Western blot analysis
PC12 cells (2 x [10.sup.5] cells/well) were seeded onto 6-well plates and subjected to the same treatment as the above groups. Cultures were collected in the low salt lysis buffer (10 mM HEPES, pH 7.5, 100 [micro]M NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 5 mM benzamidine HC1, 10 [micro]M aprotinin, and 10 [micro]M leupeptin). Then, the lysates were centrifuged at 4 [degrees]C for 10 min at 16,100 g. After quantifying the protein concentration, the lysates were denatured at 100 [degrees]C for 10 min with lysis buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 2% 2-mercaptoethanol). The cell lysate was then subjected to 8% and 12% (cleaved caspase-3 was separated on 12% gel) SDS-PAGE and western blotting. The specific antibodies for NF68 (-68 kDa; Catalog No. 2835), NF160 (-160 kDa; Catalog No. 2838), TrkA (-140 kDa; Catalog No. 2505), cleaved caspase3 (-17/19 kDa; Catalog No. 9661) and cleaved poly ADP ribose polymerase (PARP, -89 kDa; Catalog No. 9545) were employed together with horseradish peroxidase (HRP)-conjugated anti-mouse (for NF68 and NF160) and anti-rabbit (for TrkA, cleaved caspase3 and cleaved PARP) secondary antibody. All antibodies were from Cell Signaling (Danvers, MA). The immune complexes were visualized using the enhanced chemiluminescence (ECL) method (GE Healthcare, Piscataway, NJ). The band intensities in the control and different samples, run on the same gel and under strictly standardized ECL conditions, were compared on an image analyzer. The expression level of proteins was calculated using glyceraldehyde 3phosphate dehydrogenase (GAPDH, -37 kDa; anti-mouse primary and secondary antibodies from Cell Signaling) as the internal control.
Protein phosphorylation on PC12 cells
PC12 cells (1 x [10.sup.5] cells/well) were seeded onto 12-well plates and treated with DMSO or 25 [micro]M divorine for 48 h. Then, the culture medium was changed to DMEM medium without serum over 3 h. The cells were treated with DMEM or NGF (20 ng/ml) at different time points (0 min, 5 min and 10 min). Then, the cells were harvested and digested with lysis buffer. The degrees of kinase and receptor phosphorylation were then evaluated by the immunoblotting with specific anti-phospho-kinase and anti-phospho-receptor and total kinase/receptor antibodies. All primary antibodies were purchased from Cell Signaling, including p-TrkA Y490 (~140 kDa; Catalog No. 9141), total TrkA (~140 kDa; Catalog No. 2505), p-Akt S473 (~60 kDa; Catalog No. 4060), total Akt (~60 kDa; Catalog No. 9272), p-CREB S133 (~43 kDa; Catalog No. 9196), total CREB (~43 kDa; Catalog No. 9197), p-Erkl/2 T202/Y204 (~42/44 kDa; Catalog No. 9101) and total Erkl/2 (~42/44 kDa; Catalog No. 9102).
Protein concentration was measured routinely by Bradford's method (Hercules, Bio-Rad, CA). Statistical tests were done by using one-way analysis of variance. Data were expressed as mean [+ or -] SEM, where n = 4-8 and statistically analyzed using two-tailed unpaired Student's t-test. Data were considered significant where p < 0.05 (* or #), and highly significant where p < 0.01 (** or ##) and p < 0.001 (*** or ###).
Clivorine reduces cell viability
Figs. 1A and IB show a common chemical structure of PAs and clivorine. Clivorine is found in Ligularia plants, e.g. L. hodgsonii. L. hodgsonii roots (Fig. 2A) however are commonly found as substitute of a herbal medicine Asteris Radix et Rhizoma (Fig. 2B) for antitussive function. Using MTT assay in cultured PC12 cells, the cell viability was decreased as the concentration of clivorine increased after 48 h of treatment (Suppl. Fig. 1 A). Beyond 50 [micro]M of clivorine, the cell viability was significantly reduced, as further confirmed by cell counting (Suppl. Fig. IB). In addition, the treatment of clivorine at 50 [micro]M over 4h showed significant apoptotic signals, i.e. activated cleavage of caspase-3 and PARP (Suppl. Fig. 1C). After 48 h exposure, a significant (p <0.05) reduction of cell viability was evidenced at higher concentrations of clivorine (50 - 100 [micro]M) tested, which might be an indicative role of apoptosis in divorine-induced cytotoxicity.
Besides, the cytotoxicity of L. hodgsonii root extract was also determined. The cell viability was decreased as the concentration of herbal extract increased (Suppl. Fig. 2A). After 48 h of exposure, the cell viability was significantly reduced by -50% (p < 0.001) in cells incubated with 5 mg/g herbal extract, which was determined to contain 25.4 [micro]M clivorine (Supplementary Text 1). No obvious inhibition on cell viability was found in those cells incubated with lower concentrations of herbal extracts, i.e. 0.1-2 mg/ml (Suppl. Fig. 2A).
Clivorine attenuates NGF-induced neuronal differentiation
Low concentration of clivorine, i.e. 5 to 25 [micro]M, showed no significant reduction in cell viability. Thus, the effects on cell morphology were further investigated. As shown in Fig. 3A, the cell morphology did not change under clivorine treatments. On the other hand, cultured PC12 cells responded to NGF having a dramatic growth in differentiated cell number and neurite length, as well as showing reticular connections between the cells. NGF treatment (20 ng/ml) for 48 h triggered the changes of PC12 cells into neuron-like cells, and which resulted in larger cell bodies and elaboration of an extensive network of neuritis (Fig. 3B). To investigate the effect of divorine on NGF-induced neurite outgrowth, the number of cells exhibiting neurites and neurite length were measured under this co-treatment condition (Figs. 3B and 3C). Compared with those cells treated with NGF only, a gradual decrease in the number of cells having longer neurite was revealed when that was exposure to divorine. The decrease was robust in cells of neurite over 60 [micro]m in length (Fig. 3C), and which showed a dose-dependent manner. On the other hand, nuclear chromatin staining by DAPI showed no change of chromatins in clivorine-treated PCI 2 cells (5 to 25 [micro]M); while neurofilament, staining by NF68, showed significantly decrease of neurite length in PC12 cells exposed to 25 [micro]M divorine (Fig. 4).
Clivorine reduces expression level of neuronal differentiation markers
The molecular events of clivorine and water extracts of L. hodgsonii root in attenuating NGF-induced neurite outgrowth were determined. In cultured PC12 cells, clivorine application at 25 [micro]M reduced the mRNAs encoding NF68, NF160 and NF200 by ~40% (Fig. 5A). Those proteins are structural components responsible for neurite outgrowth of PC12 cells. In parallel, the differentiation markers for neuron were reduced under the influence of 25 [micro]M clivorine. By quantitative PCR analysis, the receptor for NGF, TrkA, was reduced by -30% (Fig. 5B). The application of clivorine here suppressed the basal expression of neural markers for differentiation. The suppressive role of clivorine was further tested in NGF-treated PC12 cells. Here, a minimal amount of NGF at 20 ng/ml was used here as to have a higher sensitive response to clivorine. NGF at 20 ng/ml application caused the up regulation of neurofilaments, e.g. NF68, NF160 and NF200 (Fig. 6A). The NGF-induced gene expressions were suppressed by treatment of clivorine and the herbal extract in a dose-dependent manner (Fig. 6A and Suppl. Fig. 2B). Similarly, clivorine and the herbal extract caused a reduction of NGF-induced TrkA mRNA expression (Fig. 6B and Suppl. Fig. 2B).
Besides the mRNA, the protein level was determined here for those regulated genes. The application of clivorine reduced the protein levels of NF68 (~68kDa), NF160 (-160 kDa) and TrkA (-140 l<Da) in dose-dependent manners (Fig. 7A). The control protein GAPDH was not altered. The protein suppression by clivorine was also revealed in the scenario of NGF-treated PC12 cells. Again, the treatment of clivorine at low concentration markedly reduced the NGF-induced protein expressions (Fig. 7B).
Clivorine inhibits the downstream signaling of NGF
The downstream signaling of NGF in PC12 cells was elucidated. NGF induced the phosphorylations of TrkA (-140 kDa), Akt (-60 kDa), CREB (-43 kDa) and Erkl/2 (-42/44 kDa) in a transit manner (Figs. 8A and 8B). The pre-treatment of clivorine reduced the responsiveness of PC12 cells to NGF challenge, including the phosphorylations of TrkA, Akt and CREB: the reduction was from 30% to 50% (Fig. 8B). The NGF-induced phosphorylation of Erkl/2 however was not altered under the treatment of clivorine, indicating the suppression could be specific for certain signaling pathway.
Discussion and conclusions
PA contamination has become one of the major issues of food safety. Most of PAs, especially the retronecine type, e.g. monocrotaline, need to be metabolized by cytochrome P450 enzymes as to generate unstable dehydro-PAs (DHPs) triggering the toxicity by binding to DNA or proteins (Fu et al., 2004). Therefore, abundant studies have been performed to investigate their toxicity in liver, where contains metabolic P450 enzymes. Although extrahepatotoxic effects were also related to the intoxication in animals (Lee et al., 2005; Cooper et al., 1999), the study on extra-hepatic toxicity of PA is very limited. This is partially because of inefficiency of P450 enzymes in cell cultures for metabolic activation (Ji et al., 2004). On the other hands, the otonecine type PA, e.g. clivorine, is believed to possess toxic effects without metabolic activation, which have been evidenced by showing significant toxicity on non-hepatic cells without P450 enzymes (Ji et al., 2002 and 2008). In addition, the unique chemical structure of clivorine could enhance the abilities to penetrate the blood-brain barrier, suggesting that clivorine might cause neuronal damage in the central nerve system (CNS) (Lin et al., 2000 and 2002). Clivorine exists at large amount in L. hodgsonii, which is commonly used as erroneously substitute of an herbal medicine Asteris Radix et Rhizoma for antitussive function. Therefore, the evaluation on the neurotoxicity of clivorine as well as L. hodgsonii is urgently needed.
The cytotoxicity of clivorine varied among cells, which might be due to contribution of metabolic activation. As reported, clivorine showed stronger toxicity on mouse hepatocytes than on L-02 cells, as well as other non-hepatic cells, where metabolic activation might not work as efficiently as in hepatocytes (Ji et al., 2005). Here, the cytotoxicity of clivorine on PC12 cells suggested that clivorine itself had direct toxicity on PC12 cells without the requirement of P450 enzymes. More importantly, a low concentration of clivorine might inhibit the NGF-induced differentiation in PCI 2 cells. Besides, the extract of L. hodgsonii root showed more server cytotoxicity than clivorine in cultured cells. Quercetin showed protection effect against clivorine-induced acute liver injury (Ji et al., 2014); however, the concentration of quercetin in L. hodgsonii was about 0.01 mg/g (Wang et al., 2009). Therefore, the antagonistic effect of quercetin in our case should be minimum. Thus, clivorine, as well as L. hodgsonii, showed their neurotoxicities partially by impairing the neuronal differentiation, which could result in the failed formation of neural network structures.
Viable connections between cytoskeletal networks are essential for the survival of neurons. In vitro studies have revealed that significant decrease in expression of neuronal cytoskeleton proteins, including glial fibrillary acidic protein (GFAP) and [beta]-tubulin III protein after exposure to monocrotaline (Barreto et al., 2008; SilvaNeto et al., 2010; Pitanga et al., 2011). Neurofilaments, composing three subunits NF68, NF160 and NF200, are synthesized within cell body and travelled along axon to reach their final destination therefore working together to provide mechanical strength. Changes in neurofilament expression have been found during development in response to neurotoxins both in vitro and in vivo. Here, clivorine and the extract of L. hodgsonii root reduced the expression levels of neurofilaments on PC12 cells. As neurofilament is one of the most important neuronal cytoskeleton elements and the key components during extension of neurite, the expression level could serve as a marker for neuronal differentiation. This is in line to the morphological changes, and this evidence supports the notion of neuronal loss is triggered partially by impairing neuronal differentiation.
Depression was reported to be the dominant neurological abnormality in horses poisoned by PA-containing herbs (McLean, 1970; Stegelmeier et al., 1996). The poisoned animals were depressed, and it compulsively walked in a straight line until they came to an object and then stood with their heads pressed against an object, indicating specific lesions in the CNS. Although hepatic edema and/or ammonia production in hepatic failure might contribute to the CNS effects, in vitro studies, as described here, have provided direct evidence for the cytotoxicity of PA and its active metabolite in neurons. Monocrotaline, a retronecine PA, and dehydro-monocrotaline showed cytotoxicity on neurons mainly by triggering apoptosis and damaging cytoskeletal stability (Barreto et al., 2008; Silva-Neto et al., 2010; Pitanga et al., 2011). All these observations provided useful information for neurotoxicity of PAs; however, the mechanism is still not fully elucidated.
The underlying mechanism by PAs in impairing neuronal differentiation remains unclear. NGF is one of the important neurotrophic factors that play a crucial role of neuronal differentiation and survival by binding TrkA receptor on the surface of neuronal cells. Our results have shown that clivorine inhibited NGF-induced neuronal differentiation via TrkA/Akt signaling pathway. The pre-treatment of clivorine inhibited the NGF-triggered phosphorylations of TrkA and Akt, thus to attenuate the activation of CREB. The phosphorylation of Erk1/2, however, showed no significantly change. Besides the blockage of NGF signaling, the treatment of high dose of clivorine reduced the mRNA level of TrkA. The reduced level of TrkA therefore could account partly the suppression on NGF signaling by clivorine; however, the mechanism triggering this gene transcription has not been determined. On the other hand, the expression of TrkB receptor was shown to be regulated by G-protein coupled receptor via cAMP signaling (Aloyz et al., 1999; Samarajeewa et al., 2014). In line to this notion, the treatment of clivorine in cultured PC12 cells reduced synthesis of dopamine and mRNA expression of dopamine receptor D2 (unpublished result). Dopamine D2 receptor is known to be coupled with G-protein in inhibiting the formation of cAMP (Neves et al., 2002). Thus, clivorine might reduce TrkA receptor expression via impairing the activity of G-protein coupled receptor, and which subsequently enhanced the neurotoxicity of clivorine.
Taken together, we firstly revealed that clivorine inhibited NGF-induced TrkA phosphorylation, which in turn reduced the phosphorylations of the downstream molecule Akt and CREB. These protein phosphorylations are important to maintain the self-renewal and survival in a number of different cell types, including neurons. Thus, our study might suggest that PAs and its preparations not only damage the liver, but also possess neurotoxicities, resulting in brain disorders, such as depression. And great attention should be paid on taking medical herbs and preparation containing PAs, such as Ligularia species.
Received 16 July 2015
Revised 2 June 2016
Accepted 8 June 2016
Conflict of interest
The authors declare that there are no conflict of interest. Acknowledgments
This work was supported by Hong Kong Scholars Program (XJ2012031), China Postdoctoral Science Foundation (2012T50451), Shanghai Nature Science Foundation (12ZR145300 and 16ZR1434200), Foundation for University Key Teachers of Shanghai Municipal Science and Technology (12CG50) to AX and ZW, and Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), GRF (661110, 662911, 660411, 663012, 662713), M-HKUST604/13, TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club Charities Trust, Foundation of The Awareness of Nature (TAON12SC01) to KT.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.06.006.
Aloyz, R., Fawcett, J.P., Kaplan, D.R., Murphy, R.A., Miller, F.D., 1999. Activity-dependent activation of TrkB neurotrophin receptors in the adult CNS. Learn Mem 6 (3), 216-231.
Barreto, R.A., Sousa, C.S., Silva, V.D., Silva, A.R., Veloso, E.S., Cunha, S.D., Costa, S.L., 2008. Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures. Toxicol. Vitro 22 (5), 1191-1197.
Cheng, M., Tang, J., Gao, Q.F., Lin, G., 2011. Analysis on clivorine from alkaloid in aqueous extract of Ligularia hodgsonii and its hepatotoxicity in rats. Chin. Tra. Herbal Drugs 42 (12), 2507-2511.
Cooper, R.A., Huxtable, R.J., 1999. The relationship between reactivity of metabolites of pyrrolizidine alkaloids and extra-hepatic toxicity. Proc. West Pharmacol. Soc. 42, 13-16.
Dubecke, A., Beckh, G., Luilmann, C., 2011. Pyrrolizidine alkaloids in honey and bee pollen. Food Addit. Contam. Part A Chem. Anal. Control Expo Risk Assess. 28 (3), 348-358.
Fu, P.P, Xia, Q.S., Lin, G., Chou, M.W., 2004. Pyrrolizidine alkaloids- genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metabo. Rev. 36, 1-55.
Goeger, D.E., Cheeke, P.R., Schmitz, JA, Buhler, D.R., 1982. Effect of feeding milk from goats fed tansy ragwort (Senerio jacobaea) to rats and calves. Am. J Vet. Res. 43 (9), 1631-1633.
German Federal Health Bureau, Bundesanzeiger, June 17, 4805. Dtsch. Apoth. Ztg., 1992, 132, 1406.
Greene, LA., Aletta, J.M., Rukenstein, A., Green, S.H., 1987. PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation. Meth. Enzymol 147, 207-216.
Ji, L.L., Zhao, X.G., Chen, L, Zhang, M., Wang, Z.T., 2002. Pyrrolizidine alkaloid clivorine inhibits human normal liver L-02 cells growth and activates p38 mitogen-activated protein kinase in L-02 cells. Toxicon 40 (12), 1685-1690.
Ji, L.L., Tan, A.M., Tang, J., Zhang, M., Wang, Z.T., 2004. Toxicity of several pyrrolizidine alkaloids on hepatocytes. Chin. J Nat. Med. 2, 239-241.
Ji, L.L., Zhang, M., Sheng, Y.C., Wang, Z.T., 2005. Pyrrolizidine alkaloid clivorine induces apoptosis in human normal liver L-02 cells and reduces the expression of p53 protein. Toxicol, in Vitro 19 (1), 41-46.
Ji, L.L., Chen, Y., Wang, Z.T., 2008. The toxic effect of pyrrolizidine alkaloid clivorine on the human embryonic kidney 293 cells and its primary mechanism. Exp. Toxicol. Pathol. 60 (1), 87-93.
Ji, L.L, Liu, T.Y., Wang, Z.T., 2010. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes. Hum. Exp. Toxicol. 29 (4), 303-309.
Ji, L.L., Ma, Y.B., Wang, Z.Y., Cai, Z., Pang, C., Wang, Z.T., 2014. Quercetin prevents pyrrolizidine alkaloid clivorine-induced liver injury in mice by elevating body defense capacity. PLoS One 9 (6), e98970.
Lee, Y.S., Byun, J., Kim, J.A., Lee, J.S., Kim, K.L., Suh, Y.L., Kim, D.K., 2005. Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung. Exp Mol Med 37 (1), 27-35.
Lin, G., Rose, R, Chatson, K.B., Hawes, E.M., Zhao, X.G., Wang, Z.T., 2000. Characterization of two structural forms of otonecine-type pyrrolizidine alkaloids from Ligularia hodgsonii by NMR spectroscopy. J Nat. Prod. 63 (6), 857-860.
Lin, G., Cui, Y.Y., Liu, X.Q., Wang, Z.T., 2002. Species differences in the in vitro metabolic activation of the hepatotoxic pyrrolizidine alkaloid clivorine. Chem. Res. Toxicol. 15, 1421-1428.
Medeiros. R.M.T., Gorniak, S.L., Guerra, J.L., 1999. Effects of milk from goat fed Crotalaria spectabilis seeds on growing rats. Braz. J Vet. Res. Anim. Sci. 36, 97-100.
McLean, E.K., 1970. The toxic actions of pyrrolizidine (senecio) alkaloids. Pharmacol. Rev. 22 (4), 429-483.
Neves, S.R., Ram, P.T., Iyengar, R., 2002. G-protein pathways. Science 296 ( 5573), 1636-1639.
Pharmacopoeia of the People's Republic of China, 2010. State pharmacopoeia commission of the P R. China. China Medicinal Science and Technology Press, Beijing 2010.
Pitanga, B.P., Silva, V.D., Souza, C.S., Junqueira, HA., Fragomeni, B.O., Nascimento, R.P. Costa, S.L., 2011. Assessment of neurotoxicity of monocrotaline, an alkaloid extracted from Crotalaria retusa in astrocyte/neuron co-culture system, Neurotoxicol 32 (6), 776-784.
Roeder, E., 1995. Medicinal plants in Europe containing pyrrolizidine alkaloids. Pharmazie 50, 83-98.
Roeder, E., 2000. Medicinal plants in China containing pyrrolizidine alkaloids. Pharmazie 55, 711-726.
Samarajeewa, A., Goldemann, L, Vasefi, M.S., Ahmed, N., Gondora, N., Khanderia, C., Beazely, MA., 2014. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation. Front Behav Neurosci 8, 391.
Stegelmeier, B.L., Gardner, D.R., James, L.F., Molyneux, R.J., 1996. Pyrrole detection and the pathologic progression of Cynoglossum officinale (houndstongue) poisoning in horses. J Vet. Diagn. Invest 8 (1), 81-90.
Silva-Neto, J.P., Barreto, R.A., Pitanga. B.P.S., Souza, C.S., Silva, V.D., Silva, A.R., Costa, S.L., 2010. Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells. Toxicon 55, 105-117.
Tan, A.M, Huang, J.F, Zhang, M., Wang, Z.T., Shen, Y.M., 2001. Studies on the alkaloids of Ligularia japonica. J China Pharmaceutical Uni 32, 250-252.
Wang, W.S., Lu, P, Zhao, K., 2009. Studies on the chemical constituents of Ligularia hodgsonii. J Liaoning Uni. TCM 11, 176-177.
World Health Organization, 1989. International Programme on Chemical Safety. Pyrrolizidine alkaloids health and safety guide 26 WHO, Geneva.
Xu, S.L., Choi, R.C., Zhu, K.Y., Leung, K.W., Guo, A.J., Bi, D., Tsim, K.W., 2012. Isorhamnetin, a flavonol aglycone from Ginkgo biloba L., induces neuronal differentiation of cultured PC12 cells: potentiating the effect of nerve growth factor. Evid. Based Complement Alternat. Med, 278273.
Xiong, A.Z., Yang, L, Yang, X.J., Ji, L.L., Wang, Z.Y., Chen, Y....Wang, Z.T., 2011. Metabolomics 8, 614-623.
Xiong, A.Z., Fang, L.X., Yang, X., Yang, F., Qi, M., Kang, H., Wang, Z.T., 2014. An application of target profiling analyses in the hepatotoxicity assessment of herbal medicines: comparative characteristic fingerprint and bile acid profiling of Senecio vulgaris. L. and Senecio scandens Buch.-Ham. Anal Bioanal Chem 406, 7715-7727.
Abbreviations: Akt, protein kinase B; CREB, cAMP response element-binding protein; DHP, dehydro-PA; Erk, extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NGF, nerve growth factor; NF, neurofilaments; PA, pyrrolizidine alkaloid; PARP, poly ADP ribose polymerase; TrkA, tyrosine kinase receptor A.
Aizhen Xiong (a,b), Artemis Lu Yan (b), Cathy W.C. Bi (b), Kelly Y.C. Lam (b), Gallant K.L. Chan (b), Kitty K.M. Lau (b), Tina T.X. Dong (b), Huangquan Lin (b), Li Yang (a), Zhengtao Wang (a), Karl W.K. Tsim (b), *
(a) The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China
(b) Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China
* Corresponding author. Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China. Tel.: (852) 2358 7332.
E-mail address: email@example.com (K.W.K. Tsim).
Please note: Some tables or figures were omitted from this article.
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Original Article; nerve growth factor|
|Author:||Xiong, Aizhen; Yan, Artemis Lu; Bi, Cathy W.C.; Lam, Kelly Y.C.; Chan, Gallant K.L.; Lau, Kitty K.M.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Aug 15, 2016|
|Previous Article:||Reversal of diabetes-induced behavioral and neurochemical deficits by cinnamaldehyde.|
|Next Article:||Psoralidin induced reactive oxygen species (ROS)-dependent DNA damage and protective autophagy mediated by NOX4 in breast cancer cells.|