Article pubs.acs.org/JAFC
Effect of Swainsonine in Oxytropis kansuensis on Golgi α‑Mannosidase II Expression in the Brain Tissues of Sprague−Dawley Rats Hao Lu, Shan-shan Wang, Wen-long Wang, Liang Zhang, and Bao-yu Zhao* College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China ABSTRACT: The purpose of this study was to observe the effects of swainsonine in Oxytropis kansuensis on the expression of Golgi α-mannosidase II (MAN2A1) in the brain tissues of Sprague−Dawley (SD) rats. Twenty-four SD rats were randomly divided into four groups (experimental groups I, II, and III and a control group) of six animals each. The rats were penned as groups and fed feeds containing either 15% (swainsonine content = 0.003%), 30% (swainsonine content = 0.006%), or 45% (swainsonine content = 0.009%) O. kansuensis for experimental groups I−III, respectively, or complete feed for the control group. One hundred and nineteen days after poisoning, all rats showed neurological disorders at different degrees, which were considered to be successful establishment of a chronic poisoning model of O. kansuensis. Rats were sacrificed, and MAN2A1 expression of brain tissues was detected by immunohistochemistry and RT-PCR. The results showed that MAN2A1 was either not expressed or lowly expressed in the molecular layer of the cerebral cortex and hippocampal layers, but was found to be highly expressed in other areas of the brain. MAN2A1 expression decreased in the cerebrum and cerebellum in experimental groups when compared to the control group, whereas the expression of MAN2A1 mRNA was inhibited in cerebral and cerebellar tissues by O. kansuensis. These results indicated that O. kansuensis treatment could reduce the expression of MAN2A1 in brain tissues of SD rats. KEYWORDS: locoweeds, Oxytropis kansuensis, swainsonine, MAN2A1
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INTRODUCTION Oxytropis kansuensis is a perennial plant belonging to the genus Oxytropis and is mainly distributed the western provinces of China, including Qinghai, Gansu, and Sichuan.1 It can cause cell vacuolization in many tissues, which leads to the death of livestock and affects their breeding; for these reasons, it is regarded as one of the major poisonous plants in western pastures.2 Previous studies have demonstrated that the main toxic component of O. kansuensis is the indolizidine alkaloid swainsonine (Figure 1).3 The toxicity of swainsonine is due to
N-glycosylation, decreasing the synthesis of complex type Nlinked oligosaccharides while increasing the synthesis of hybrid type N-glycosylation.8 N-linked glycoproteins are widely distributed and have an essential physiological function in the central nervous system (CNS). They also play an important role in mediating cellular communication, signal transduction, and the regulation of cell growth and migration.4 However, the distribution of Golgi α-mannosidase II in the CNS of Sprague− Dawley (SD) rats remains unclear. Aside from inhibition of its activity by swainsonine, the effects of swainsonine on the distribution, transcription, translation, and modification of Golgi α-mannosidase II in the CNS have not been elucidated. In this study we used O. kansuensis as a source of the toxin swainsonine to investigate its effects on the distribution and expression of MAN2A1 in the brain tissues of SD rats utilizing immunohistochemistry and real-time quantitative PCR. These results provide an experimental reference for further research into the toxic mechanisms of O. kansuensis and other kinds of locoweeds.
Figure 1. Structure of the indolizidine alkaloid swainsonine.
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its ability to inhibit acid or lysosomal α-mannosidase and Golgi α-mannosidase II.4 It has been confirmed that Ipomoea carnea induces an intralysosomal accumulation of mannose-containing oligosaccharides in guinea pigs.5 Research shows that the inhibitory ability of swainsonine on Golgi apparatus α-mannosidase II is 10 times greater than its inhibition of lysosomal α-mannosidase.6 Golgi α-mannosidase II (MAN2A1) is a key enzyme in the maturation process of Nglycans, mainly contributing to the clipping and processing of different kinds of mannoses on glycoprotein sugar chains during N-glycosylation.7 Swainsonine-mediated inhibition of Golgi α-mannosidase II can lead to abnormal modification of © 2014 American Chemical Society
MATERIALS AND METHODS
Plants Material. The aerial portion of O. kansuensis was collected in Huangzhong County (36°29.286′ N; 101°41.499′ E), Qinghai province, in August 2008 (Figure 2). The plants were preserved in a Special Issue: Poisonous Plant Symposium, Inner Mongolia Received: Revised: Accepted: Published: 7407
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Figure 2. O. kansuensis distributed in Qinghai. shady place after air-drying and grinding. Swainsonine was analyzed by TLC and GC-MS methods.9 Swainsonine content was determined by gas chromatography as 0.021% by weight of the total plant. The plant was identified by the Institute of Botany (College of Life Science, Northwest A&F University). Animals and Animal Feed. The animals and protocols used in this study were approved by the Animal Care Committee of Xi’an Jiaotong University. Sprague−Dawley rats were purchased from the Experimental Animal Center of College of Medicine, Xi’an Jiaotong University (Xi’an, China). The aerial portion of O. kansuensis was ground completely and sifted through a 200 mesh screen. The resulting O. kansuensis meal was mixed with whole feed (25% flour, 28% cornmeal, 20% soybean cake, 10% wheat bran, 10% fish meal, 1% vegetable oil, 1% yeast powder, 2% bone meal, 1% cooking salt, 1% cod-liver oil, 0.9% mineral additive, 0.1% vitamin additive)10 to reach 15, 30, and 45% plant content (0.003, 0.006, and 0.009% swainsonine, respectively).11 Water was then added to this mixture and stirred into 1−3 cm3 diced mixed rations, dried for preservation. Chemical Reagents and Apparatuses. NRNAiso Plus Total RNA Extraction kit, RT-PCR kit, and Real-Time PCR Kit were obtained from TaKaRa Bio Inc. (Dalian, China). DNA marker (Topsun, Jiangmen, China), PCR-mix (TransGen, Beijing, China), PCR primers (GenScript, Nanjing, China), EB (RUNDE, Sichuan, China), APES (Boster, Wuhan, China), DAB (Boster, Wuhan, China), IHC Antibody Diluent (Beyotime, Shanghai, China), S-P UltraSensitive SAP kit (Maixin, Fuzhou, China),and primary antibody (MAN2A1, Abcam, London, UK) were obtained as noted. Establishment of a Chronic Poisoning Model of O. kansuensis in SD Rats. Experiments were performed on male and female SD rats (200−220 g total body weight). After a week-long adaptation period in a room with controlled temperature (21 ± 1 °C) and lighting (12 h light/12 h dark), 24 SD rats were assigned to either a control group (complete feed) or experimental groups I (15% O. kansuensis containing 0.003% swainsonine), II (30% O. kansuensis containing 0.006% swainsonine), or III (45% O. kansuensis containing 0.009% swainsonine), and the 6 SD rats of each group were fed in three cages separately. All treated rats were fed for 119 days and showed neurological disorders to different degrees, which was considered to be successful establishment of a chronic poisoning model of O. kansuensis. All SD rats were anesthetized with ether vapor and sacrificed by decapitation. The cerebrum and cerebellum were collected and divided into two parts: one was preserved in liquid nitrogen, whereas the other was fixed in 4% paraformaldehyde. Immunohistochemistry. Immunohistochemistry was performed according to instructions of the S-P UltraSensitive SAP kit (Maixin, Fuzhou, China). Paraformaldehyde fixed brain tissues were paraffinembedded and sliced at 6 μm thickness onto slides. Tissue slices were deparaffinized, rehydrated, treated using microwave heat-induced epitope retrieval, and incubated in 3% hydrogen peroxide to block endogenous peroxidases with healthy nonimmune goat serum to block endogenous biotin. Subsequently, tissues were incubated in a 1:100 dilution of primary antibody for 12 h at 4 °C. A negative control slice
was incubated with antibody diluent alone. Tissues were then incubated with a biotin-labeled secondary antibody for 10 min at room temperature, followed by treatment with streptavidin− peroxidase and staining by DAB. The stained tissues were observed through a light microscope, and the resulting images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics). Real-Time Quantitative PCR. PCR primers were designed by Primer 5.0. The primer sequences for MAN2A1 were as follows: forward primer, 5′-TCAGCTACCCTTCCCTCCTC-3′; reverse primer, 5′-TGCCCACCTTTGACTGTATTG-3′. The length of the PCR amplicon was 189 bp. β-Actin was used as an internal reference with primer sequences as follows: forward primer, 5′-GACAGGATGCAGAAGGAGATTACT-3′; reverse primer, 5′-ATAGAGCCACCAATCCACACAG-3′. The length of the PCR amplicon was 104 bp. All primers were synthesized by GenScript Co. (Nanjing, China). The tissues preserved in liquid nitrogen were prepared as tissue homogenate for total RNA extraction according to the manufacturer’s instructions for the NRNAiso Plus kit. RT-PCR was performed following the manufacturer’s instructions for the PrimeScript RT Reagent Kit with gDNA Eraser; the resulting cDNA was then used as input for real-time PCR performed as described by the manufacturer’s protocol for SYBR Premix Ex TaqII. The reaction mixture was 1 μL of cDNA, 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 7.5 μL of SYBR, premix Ex TaqII (2×), and ddH2O up to 15 μL. The reaction conditions were as follows: initial denaturation for 3 min at 95 °C, followed by 40 cycles of denaturation at 94 °C for 3 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. After cycling, a final step was performed at 72 °C for 10 min. Statistical Analysis. Results are expressed as the mean ± standard deviation. All data were analyzed in SPASS 18.0 using one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons. P < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION
Effects of O. kansuensis on the Distribution and Expression of MAN2A1 in Brain Tissues of SD Rats. MAN2A1 was expressed in all areas of the brain except the molecular layer of the cerebral cortex and certain hippocampal layers (the stratum radiatum, the stratum oriens, and the molecular layer of the gyrus dentatus). The strongest expression levels were observed in the pyramidal cell layer of the cerebral cortex, the mesencephalon, the mesocephalon, the cerebellar nuclei or nerve cells of the medulla oblongata, and the Purkinje cells. The expression level of MAN2A1 in neurons is higher when compared to other cell types. Among neurons, MAN2A1 expression was strongest in cells located in the nucleus of the facial nerve and the pyramidal cell layer of the cerebral cortex, followed by Purkinje cells. The lowest expression was in the hippocampus cells. The expression of MAN2A1 decreased proportionally with increasing doses of 7408
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Figure 3. MAN2A1 expression in different areas of the cerebrum and cerebellum: (A) cerebral cortex in the control group (IHC × 100); (B) hippocampal formation in the control group (IHC × 100); (C) external granular cells in the control group (IHC × 400); (D) external granular cells in experimental group III (IHC × 400); (E) hippocampus of the control group (IHC × 400); (F) hippocampus of experimental group III (the arrow indicates pyramidal cells) (IHC × 400); (G) corpus callosum of the control group (IHC × 400); (H) corpus callosum of experimental group III (IHC × 400); (I) cerebellar cortex of the control group (the arrow indicates Purkinje’s layer) (IHC × 400); (J) cerebellar cortex of experimental group III (some of the Purkinje cells turned pale) (IHC × 400).
Figure 4. MAN2A1 expression in different areas of the cerebrum. MAN2A1 expression was detected in the cerebrum of SD rats by immunohistochemistry. The stained tissues were observed through a light microscope, and the resulting images were analyzed using Image-Pro Plus 6.0. Results are the mean ± SD. Identical letters denote nonsignificant deviation (P > 0.05); adjacents denote significant deviation (P < 0.05), and alternates denote extreme significance (P < 0.01) versus the control group.
swainsonine, and it significantly decreased in the external granular layer of the cerebral cortex, nerve cells in the hippocampus, cerebellar medulla, and molecular layer. Expression of MAN2A1 in Different Areas of the Cerebrum. We found MAN2A1 was widely distributed in the cerebral cortex, with expression in all regions except the molecular layer (Figure 3A). It was most strongly expressed in
the granular layer and the external pyramidal layer, whereas its expression was relatively weaker in the polymorphic layer. In the hippocampus, MAN2A1 was expressed most strongly in the stratum pyramidale, followed by the molecular layer; however, there was almost no expression in the hippocampal medulla, and the expression level was even lower in the dentate gyrus (Figure 3B). When the poisoning dose was increased, the 7409
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Figure 5. MAN2A1 expression in different areas of the cerebellum. MAN2A1 expression was detected in the cerebellum of SD rats by immunohistochemistry. The stained tissues were observed through a light microscope, and the resulting images were analyzed using Image-Pro Plus 6.0. Results are the mean ± SD. Identical letters denote nonsignificant deviation (P > 0.05); adjacents denote significant deviation (P < 0.05), and alternates denote extreme significance (P < 0.01) versus the control group.
expression of MAN2A1 decreased in the cerebrum; the decrease in expression was more obvious in the external granular layer of the cerebral cortex than in either the external pyramidal layer, ganglion cell layer, or polymorphic layer. Furthermore, the difference in expression in the polymorphic layer was more significant than that in the ganglion cell layer (Figure 3C,D). MAN2A1 was expressed at much lower levels in the hippocampus of experimental groups when compared to the control group (Figure 3E,F). MAN2A1 was more strongly and widely expressed in the corpus callosum (Figure 3G), and its expression in this region was much weaker in experimental groups when compared to the control group (Figure 3G,H). The expression of MAN2A1 in different groups is shown in Figure 4. When compared with the control group, the expression of MAN2A1 showed a tendency to decrease in the external granular layer of the cerebral cortex in the experimental groups. The expression levels of experimental groups II and III decreased by 30.8 and 31.0%, respectively, and this difference was found to be extremely significant (P < 0.01). When compared with the control group, the expression of MAN2A1 in the external pyramidal layer of the cerebral cortex was 18.7% lower in experimental group II and 5.9 and 13.6% higher in experimental groups I and III, respectively. However, these differences were not significant (P > 0.05). The expression of MAN2A1 fell by 13.1, 42.4, and 44.8% in the hippocampus and by 11.7, 61.1, and 52.9% in the gyrus dentatus in the experimental groups, respectively. The differences between the control group and experimental groups II and III were extremely significant (P < 0.01), as were those between experimental group I and experimental groups II and III (P < 0.01). In the corpus callosum, the expression of MAN2A1 decreased by 20.1, 33.0, and 38.2% in experimental groups, respectively, and these differences were either significant or extremely significant when compared with the control group (P < 0.05 or P < 0.01) (Figure 3G,H). Expression of MAN2A1 in Different Areas of the Cerebellum. The Purkinje cell layer had the highest expression, whereas the molecular layer and the granular layer had the lowest (Figure 3I). With increasing locoweed doses, the expression of MAN2A1 in the cerebellum decreased clearly and uniformly in the cerebellar medulla and the molecular layer, whereas in the Purkinje cell layer, parts of Purkinje cells showed decreased or complete loss of expression, but in some cells the expression level tended to increase (Figure 3I,J).
The expression of MAN2A1 of the different groups is shown in Figure 5. Upon increasing the locoweed dose, MAN2A1 expression tended to decrease in the molecular layer of the cerebellum, the Purkinje cell layer, the granular layer, and the medulla. When compared with the control group, the expression in groups I, II, and III, respectively, decreased by 30.7, 40.3, and 29.3% in the molecular layer; by 29.4, 47.2, and 42.0% in the Purkinje cell layer; by 38.9, 67.8, and 64.9% in the granular layer; and by 33.7, 31.5, and 39.0% in the medulla, and all differences were extremely significant with respect to the control group (P < 0.01); however, differences between experimental groups were either significant or nonsignificant (P < 0.05 or P > 0.05). The expression in the medial cerebellar nucleus of experimental groups I, II, and III dropped by 20.8, 29.9, and 10.8%, respectively, when compared with the control group; these differences were either significant or extremely significant (P < 0.05 or P < 0.01). Effects of O. kansuensis on the Expression of MAN2A1 mRNA in Brain Tissues of SD Rats. Expression of MAN2A1 mRNA in the Cerebrum. The MAN2A1 mRNA expression showed a decreasing trend in the cerebrum of SD rats as the poisoning dose increased in experimental groups (Figure 6). When compared with the control group, MAN2A1 mRNA expression was reduced by 8.1, 11.8, and 21.3% in the cerebrum of experimental groups I, II, and III, respectively. However, these differences were not significant (P > 0.05).
Figure 6. MAN2A1 mRNA expression in the cerebrum and cerebellum. MAN2A1 mRNA expression was measured using realtime quantitative PCR. Swainsonine treatment decreased the expression of MAN2A1 at mRNA levels. Results are the mean ± SD. Identical letters denote nonsignificant deviation (P > 0.05); adjacents denote significant deviation (P < 0.05), and alternates denote extreme significance (P < 0.01) versus the control group. 7410
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Expression of MAN2A1 mRNA in the Cerebellum. Expressions of MAN2A1 mRNA also had a decreasing trend in the cerebellum of SD rats as the poisoning dose increased in experimental groups (Figure 6). When compared with the control group, MAN2A1 mRNA expression was reduced by 14.2, 27.7, and 67.1% in the cerebellum of experimental groups, respectively. The difference was not significant for experimental group I (P > 0.05), whereas differences were either significant or extremely significant in experimental groups II and III (P < 0.05 or P < 0.01). MAN2A1 contains an amino acid sequence from family 38 of the glycoside hydrolases that distributes it to the Golgi apparatus, but its location within the Golgi apparatus can vary in different cell types.12 It plays a key catalytic role during the N-glycosylation process of proteins, specifically cleaving mannose α-1,3 and α-1,6 glycosidic bonds.13,14 Immunohistochemical staining showed that MAN2A1 is widely distributed throughout brain of SD rats, consistent with reports that Nlinked glycoprotein is also widely distributed throughout the brain.8 The expression level of MAN2A1 is variable in different kinds of cells: it is high in cells involved in information transmission and low in cells that do not participate in information transmission. These differences may due to the important role of N-linked glycoprotein in mediating signal transduction and intracellular communication.4 Swainsonine can specifically inhibit MAN2A1;14 therefore, it can interfere with the glycosylation pathway, leading to abnormal N-glycosylation of proteins and effects on the processing, modification, and transport of proteins, which subsequently affect cell functions.7,15 The results from this study clearly showed that swainsonine can inhibit the expression of MAN2A1 in external granular layer cells, stratum pyramidale cells, granular cells of the dentate gyrus, Purkinje cells in the cerebellum, and cerebellar granular cells. The hippocampus plays an important role in the process of memory formation and, when damaged, may cause dysmnesia. The Purkinje cell, originating from the cerebellar cortex, is the only neuron that can transmit the efferent impulses from the cerebellum. Its axon goes through the granular layer and white substance, reaching the cerebellar central nuclei, and plays a significant role in the motor coordination of animals.16 Abnormal N-glycosylation may affect signal transmission and, as a result, produce clinical symptoms such as ataxia in animals. MAN2A1 mRNA levels decreased in the cerebrum and cerebellum of SD rats from experimental groups, but the absence of significant differences when compared to the control group shows that swainsonine can weakly inhibit the expression of MAN2A1 at the transcriptional level. Swainsonine inhibited the expression of MAN2A1 in many areas of the cerebrum and cerebellum of rats, potentially causing abnormal N-glycosylation that would decrease the synthesis of complex N-linked oligosaccharides while increasing the synthesis of hybrid N-linked oligosaccharides.17 Proteins that undergo N-glycosylation include receptors, secreted proteins, hormones, and intracellular proteins, and abnormal N-glycosylation can affect the bioactivity, folding, and location of these proteins. The complex N-linked oligosaccharide chain of glycoproteins participates in the posttranslational modification of intracellular and secreted proteins, and their inhibition can cause cellular dysfunction.17−19 Tomoya et al.20 found that rats that are deficient in Golgi α-mannosidase II genes die quickly during the postnatal period due to dyspnea, proving that complex N-linked oligosaccharide chains play an important
role in the respiratory system of newborn animals. The effects of MAN2A1 on the CNS of animals have not been reported. Researchers have also shown that the development and metastasis of cancer cells are related to abnormal Nglycosylation of glycoprotein on the surface of these cells. Finding an inhibitor of the key enzyme in this process has clinical significance, and MAN2A1 is one of the key enzymes in catalyzing N-glycosylation.15 It has been reported that giving terminal cancer patients a specific inhibitor of MAN2A1, swainsonine, could inhibit tumor growth and metastasis with relatively minor side effects.21−24 At the same time, ongoing research highlighted the search for a MAN2A1inhibitor with lower toxicity.25,26 mRNA is translated into polypeptide chains in the ribosome; these polypeptide chains are then folded and modified by processes such as acylation and hydroxylation in the endoplasmic reticulum. Subsequently, these chains undergo further processing, such as glycosylation and are packaged into the Golgi apparatus, before mature proteins are finally transported to the lysosome and cytomembrane or secreted as extracellular proteins.27 If a protein is folded incorrectly, it will be degraded through the ubiquitin pathway. When compared with the control group, differences in MAN2A1 expression of the experimental group at mRNA level were not as significant as differences at the protein level. This may be due to the abnormal metabolism and processing of glycoproteins after swainsonine poisoning, which can affect the translation and posttranslational modification of MAN2A1, ultimately leading to MAN2A1 being degraded because it cannot mature or is incorrectly folded. Furthermore, the differences could also be due to how much swainsonine each cell type is exposed to. In this study, the influence of swainsonine on the expression of MAN2A1 in the cerebrum is not as significant as in the cerebellum. These data are consistent with the fact that clinical signs in poisoned rats are characterized by dyskinesia. Our results further demonstrate the inhibition of MAN2A1 by swainsonine in SD rats and provide experimental evidence for the underlying neurotoxicological mechanism of swainsonine.
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AUTHOR INFORMATION
Corresponding Author
*(B.Z.) Phone: +86 29 87092429. Fax: +86 29 87091032. Email:
[email protected]. Funding
This work was cofinanced by grants from the National Natural Science Foundation (Grants 31072175, 31201958), the Ph.D. Programs Foundation of Ministry of Education of China (Grant 20100204120018), and the Special Scientific Research Fund of Agriculture Public Welfare industry (Grant 201203062). Notes
The authors declare no competing financial interest.
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REFERENCES
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