- Email: [email protected]
β-catenin signaling pathway accounts for the neurorestorative effects of morroniside against cerebral ischemia injury
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Q1 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside against cerebral ischemia injury Fang-Ling Sun a, Wen Wang a,n, Wei Zuo a, Jin-Long Xue a, Jing-dong Xu b, Hou-Xi Ai a, Li Zhang a, Xiao-Min Wang c,nn, Xun-Ming Ji d,nnn a
Department of Pharmacology, Xuanwu Hospital of Capital Medical University, 45 Chang-chun Street, Beijing 100053, China Department of Physiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China Department of Neurobiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men Beijing 100069, China d Department of Neurosurgery, Xuanwu Hospital of Capital Medical University, 45 Chang-chun Street, Beijing 100053, China b c
art ic l e i nf o
a b s t r a c t
Article history: Received 25 October 2013 Received in revised form 4 May 2014 Accepted 10 May 2014
Ischemic stroke is a leading cause of mortality and permanent disability in adults worldwide. Neurogenesis triggered by ischemia in the adult mammalian brain may provide insights into stroke treatment. Morroniside is an active component of sarcocarp of C. officinalis that have shown neuroprotective effects. The aim of the present study is to test whether morroniside promotes neurogenesis via Wnt/β-catenin signaling pathway for brain recovery in a rat model of focal cerebral ischemia. Morroniside was administered intragastrically once daily at the concentrations of 30, 90 and 270 mg/kg for 7 days postischemia. Neurological functions were detected by Ludmila Belayev score tests. Endogenous neural stem cells responses were investigated with immunofluorescence staining of Ki-67 and Nestin to identify the neurogenesis in the subventricular zone (SVZ). The expression of proteins involved in and related to Wnt/β-catenin signaling pathway was detected by western blotting analysis. Morroniside significantly promoted neurogenesis for brain recovery 7 days post-ischemia. Increased expression of Wnt 3a, β-catenin and T-cell transcription factor-4 (Tcf-4), along with activation of downstream transcription factors Pax6 and neurogenin2 (Ngn2), indicated that the neurorestorative effects of morroniside may be associated with Wnt/β-catenin signaling pathway. These data provide support for understanding the mechanisms of morroniside in neurorestorative effects and suggest a potential new strategy for ischemic stroke treatment. & 2014 Published by Elsevier B.V.
Keywords: Ischemic stroke Morroniside Neurogenesis Wnt/β-catenin signaling
1. Introduction Stroke is the third leading cause of death and the most common cause of permanent disability in adults worldwide (Navarro-Sobrino et al., 2011). Despite substantial progress in understanding the mechanisms of ischemic stroke over the past few decades, there is still no effective therapy for neurorestorative treatment (Yemisci et al., 2009). Evidence is accumulating that cerebral ischemia can stimulate endogenous neurogenesis in the subventricular zone (SVZ) of adult mammals (Marti-Fabregas et al., 2010; Ohira et al., 2010). Although the increase of stem cells in the SVZ of the adult
n
Corresponding author. Tel.: þ 86 10 83198881; fax: þ 86 10 83154745. Corresponding author. Tel./fax: þ86 10 83911829. Corresponding author. Tel.: þ 86 10 83198952; fax: þ 86 10 83911496. E-mail addresses: [email protected] (W. Wang), [email protected] (X.-M. Wang), [email protected] (X.-M. Ji). nn
nnn
brain may lead to morphological and functional improvements following ischemic injuries, this internal response is unable to functionally compensate for the ischemic damage. Thus, there is an urgent need to search for therapeutic approaches that can augment endogenous neurogenesis. Wnt/β-catenin signaling pathway is well known for its important role in nervous system development. In the presence of Wnt signals, β-catenin is stabilized and translocates to the nucleus, where it interacts with T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to induce changes in gene expression (Baarsma et al., 2013; Clevers, 2006). This signaling pathway has been shown to regulate neurogenesis in the adult SVZ, by promoting proliferation of progenitor cells (Adachi et al., 2007). A recent study in focal cerebral ischemic injury model revealed that treatment with lentivirus expressing Wnt3a-HA into SVZ enhanced functional recovery from the 2nd day after injury and increased the number of immature neurons in the striatum
http://dx.doi.org/10.1016/j.ejphar.2014.05.019 0014-2999/& 2014 Published by Elsevier B.V.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
and SVZ (Shruster et al., 2013). The findings indicate that Wnt signaling could induce an appropriate neurogenic environment for neuronal differentiation and survival post-ischemic injury. Morroniside is an iridoid glycoside extracted from the sarcocarp of Cornus officinalis. It has been shown to possess antioxidative and anti-apoptosis activities in vitro and in vivo (Wang et al., 2008, 2009); moreover, our previous studies have also demonstrated that morroniside reduces the infarct volume and promotes neurological functional recovery 3 days after focal ischemic stroke (Wang et al., 2010). However, the long-term effects and mechanisms of morroniside against ischemic stroke are not clear. We hypothesized that morroniside could promote neurogenesis for neurorepair from ischemic injury. In the present study, we used a rat model of middle cerebral artery occlusion (MCAO) to identify the effects of morroniside on neurogenesis 7 days postischemia, and to examine whether the Wnt signaling pathway involved in the neurorestorative mechanism of morroniside.
2. Materials and methods 2.1. Morroniside extraction and identification C. officinalis was purchased from Tong Ren Tang Company, Beijing, China, and authenticated by Professor Wen Wang. Morroniside was extracted from the sarcocarp of C. officinalis and purified as previously described (Wang et al., 2010). The final purity was determined to be 98.5% by high performance liquid chromatography (HPLC) analysis.
2.4. Immunofluorescence analysis and immunohistochemistry analysis. Immunofluorescence staining was used to visualize Nestin in the SVZ. After the rats were perfused with 4% paraformaldehyde in PBS, the brains were extracted and post-fixed overnight. Ice-cold sections (40 μm) were prepared using standard protocols and freefloating sections were incubated with 3% H2O2 in 0.01 M PBS. After incubation with a blocking solution, the sections were incubated with primary antibody anti-Ki67 (Cell Signaling Technology) or anti-Nestin (Sigma-Aldrich) for 48 h at 4 1C and then incubated with CY2 or CY3-conjugated secondary antibody (Invitrogen) for 0.5 h. The fluorescence signals were visualized using a confocal laser scanning microscope system (MRC1024, Bio-Rad, Hercules, CA, USA). For immunohistochemistry analysis, the sections prepared as above were incubated with primary antibody anti-βcatenin (Santa Cruz Biotechnology) for 24 h at 4 1C. The secondary antibody was biotinylated goat anti-rabbit IgG (Santa Cruz Biotechnology). The sections were stained using the avidin-biotin peroxidase complex method with 3,30 -diaminobenzidine as a chromogen. The stained sections were then dehydrated in a graded alcohol series, cleared in xylene, coverslipped with neutral balsam and examined under a light microscope equipped with a computerized image analysis system (Olympus BX51, Japan). Image Pros Plus 5.0 software (Media Cybernetics, Inc., Bethesda, MD, USA) was used to determine the average number of positive cells in area of interest (AOI) in the dorsal lateral corner of SVZ per section. 2.5. Protein expression analysis.
2.2. Animals and the middle cerebral artery occlusion (MCAO) model Male Sprague-Dawley (SD) rats weighing 260–280 g were purchased from Beijing Vitalriver Experimental Animal Co. (Beijing, China), and were housed under a 12/12 h dark/light cycle and specific-pathogen-free (SPF) conditions. Before the MCAO model was performed, the rats were fasted without water deprivation for 12 h. Anesthesia was induced and maintained with 3.5% enflurane in a mixture of 70% nitrous oxide and a balance of oxygen (Bickford veterinary anesthesia equipment model no. 61010, AM Bickford Inc., Wales Center, NY, USA). The rats were orally intubated, immobilized with intra-arterial pancuronium bromide (0.6 mg/kg) and mechanically ventilated (Rodent Ventilator Model 683, Harvard Apparatus Inc., Holliston, MA, USA). A piece of nylon monofilament was inserted into the left internal carotid artery via an arteriotomy and lodged in the narrow proximal anterior cerebral artery, which blocked the MCA at its origin. After 30 min of ischemia, reperfusion was established by the withdrawal of the filament. When both cerebral and rectal temperatures returned to normal levels, the animals were allowed to regain consciousness and were placed under warm conditions for an additional 3 h. Morroniside was dissolved in normal saline and administered intragastrically once a day at the dose of 30, 90, 270 mg/kg for 7 days, starting at 3 h after MCAO. The vehicle control groups of the ischemic rats received an equal volume of normal saline. Sham-operated rats were treated with an equal volume of normal saline or 270 mg/kg morroniside. Animal protocols for these studies were approved by the Animal Care and Use Committee of Xuanwu Hospital in Capital Medical University. 2.3. Neurological scoring Seven days post-MCAO, neurological deficits were evaluated by Ludmila Belayev score test as previously described (Belayev et al., 1996).
The rats were euthanized under 10% chloral hydrate (0.4 mL/kg) anesthesia. The infarct side of the cortex was harvested and homogenized in ice-cold buffer (tris-(hydroxymethyl)-aminomethane 50 mM, pH 7.4, NaCl 150 mM, 0.5% Triton X-100, edetic acid 1 mM, phenylmethylsulfonyl fluoride 1 M and aprotinin 5 mg/L). Following the homogenate centrifuged at 14,000 g at 4 1C for 30 min, the supernatants were collected as the total protein. The proteins were then electrophoresed through a 10–15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and electrically transferred onto a nitrocellulose membrane. This membrane was detected using primary antibodies, including anti-Wnt 3a (Millipore), anti-β catenin (Santa Cruz Biotechnology), anti-Tcf-4 (Santa Cruz Biotechnology), anti-Pax 6 (Abcam), anti-Tbr 2 (Abcam), anti-neurogenin2 (Ngn2) (Abcam). The amount of protein was normalized with β-actin values in the same lane. 2.6. Statistical analysis. All data are presented as the mean 7SEM. Analysis of variance (ANOVA) followed by Scheffe (when number of sample is not equal) or Tukey's multiple range post-hoc test was carried out by using Statistical Package for the Social Sciences software (SPSS 17.0 Statistical Software). A p value of less than 0.05 was considered significant.
3. Results 3.1. Morroniside improved neurological deficits 7 days following MCAO In Ludmila Belayev score test, the score of the vehicle-treated rats was significantly increased to 5.36 70.61 from 0 compared with the sham-operated group 7 days after MCAO (P o0.001, n¼ 11), indicating that neurological deficits of the rats caused by cerebral ischemia-reperfusion remained 7 days after MCAO.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
However, treatment with morroniside at the concentrations of 90 and 270 mg/kg/day for 7 days after MCAO respectively reduced the Ludmila Belayev score from 5.36 7 0.61 to 2.697 0.56 (P o0.05, n ¼13) and 1.36 7 0.49 (P o0.001, n ¼ 14) (Fig. 1). These results suggested the effects of morroniside on the recovery of neurological function 7 days post-ischemic injury.
3.2. Morroniside promotes neurogenesis after cerebral ischemia To study the effects of morroniside on neurogenesis, we first examined cell proliferation in the SVZ 7 days after MCAO by using immunostaining for Ki-67. In the vehicle-treated ischemic rats, cerebral ischemia induced Ki-67-positive cells were clearly
3
increased in the ischemic ipsilateral dorsolateral corner of SVZ 7 days after MCAO (Fig. 2C). Treatment with morroniside at dose of 90 and 270 mg/kg respectively increased the number of Ki-67positive cells in SVZ from 123.177 3.66 to 310.5 73.47 (P o0.001) and 423.83 75.76 (P o0.001) (Fig. 2E–G). Next, we detected Nestin-positive cells in the SVZ by using immunofluorescence. In the vehicle-treated ischemic rats, Nestinpositive cells were also increased in the ischemic ipsilateral dorsolateral corner of the SVZ 7 days after MCAO (Fig. 3C). Quantitation confirmed that the number of Nestin-positive cells was significantly increased compared with the sham-operated group (Po0.001) (Fig. 3H). Treatment with morroniside at dose of 30, 90 and 270 mg/kg respectively increased the density of Nestin-positive cells in SVZ from 254.6876.03% to 363.3675.89% (Po0.001), 442.167 7.25% (Po0.001) and 475.5974.97% (Po0.001) (Fig. 3H). Moreover, Nestin-positive cells in morroniside-treated rats were found not only in the SVZ area but also appeared to penetrate into peri-infarct cortex (Fig. 3G). In addition, the results of Ki-67- and Nestin-positive cells indicated that there were no effects on the cell proliferation of the sham-operated rats after morroniside treatment (Figs. 2B and 3B). Taken together, these data indicate that administration of morroniside promotes neural stem cell proliferation after focal cerebral ischemia. 3.3. Morroniside activated Wnt /β-catenin signaling pathway after ischemic stroke
Fig. 1. Effects of morroniside on neurological function 7 days post-ischemia. Ludmila Belayev scores are expressed as absolute values and Scheffe post-hoc test is used for data analysis. The data of each group were obtained from at least 11 rats. Data are presented as mean 7 S.E.M. ###Po 0.001 as compared to the shamoperated control group. nPo 0.05 and nnnPo 0.001 as compared to the vehicletreated ischemic group.
In order to study the potential mechanisms of morroniside in neurogenesis, we examined the role of Wnt/β-catenin signaling pathway in this neurorestorative process. The results showed β-catenin expression in the SVZ (Fig. 4A and B), indicating the potential relationship between the Wnt signaling pathway and neurogenesis. As Fig. 4C–F shows, the expression of Wnt3a, β-catenin and Tcf-4 was increased at 7 days after ischemia in the morroniside-treated rats, compared with the vehicle-treated ischemic rats. These results indicate that the Wnt signaling pathway is involved in the effects of morroniside on ischemic recovery. It was also observed that there were no significant differences in the above-mentioned protein expression between
Fig. 2. Effects of morroniside on Ki-67-labeled cells proliferation in the ischemic ipsilateral SVZ. (A–F) Ki-67-immunoreactive cells in the ischemic ipsilateral SVZ in shamoperated rats (A), a sham-operated group treated with morroniside of 270 mg/kg/day (B), a vehicle-treated ischemic group (C) and groups of ischemic rats treated with morroniside at dose of 30 mg/kg/day (D), 90 mg/kg/day (E), and 270 mg/kg/day (F). (G) Quantitative analysis of the density of Ki-67-positive cells. n¼6 for each group. Data are presented as mean 7 S.E.M. ###Po 0.001 as compared to sham-operated control group; nnnP o 0.001 as compared to vehicle-treated ischemic group. Scale bar: 100 μm (A–F).
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 3. Effects of morroniside on Nestin-labeled neural stem cells proliferation in the ischemic ipsilateral SVZ. (A–F) Nestin-immunoreactive cells in the ischemic ipsilateral SVZ in sham-operated rats (A), a sham-operated group treated with morroniside of 270 mg/kg/day (B), a vehicle-treated ischemic group (C) and groups of ischemic rats treated with morroniside at dose of 30 mg/kg/day (D), 90 mg/kg/day (E), and 270 mg/kg/day (F). (G) Low magnified image of Nestin-immunoreactive cells from a 90 mg/kg/ day morroniside-treated ischemic rat. Nestin-positive cells appeared to penetrated into peri-infarct cortex. (G’) A high magnified image of Nestin-immunoreactive cells in the peri-infarct cortex of (G). (H) Quantitative analysis of the density of Nestin-positive cells. n ¼6 for each group. Data are presented as mean 7S.E.M. ###Po 0.001 as compared to sham-operated control group; nnnPo 0.001 as compared to vehicle-treated ischemic group. Scale bar: 100 μm (A–F); 500 μm (G); 25 μm (G’).
the vehicle-treated ischemia rats and the sham-operated group (Fig. 4C–F). These results suggest that long term activation of Wnt signaling is sustained by morroniside per se, and not non-specifically caused by lingering effects of ischemia. Consistent with a role for this mechanism in brain recovery, the expression profiles of Wnt3a continued to evolve at 1 day and 3 days after ischemia. Compared with the corresponding shamoperated group, the expression of Wnt3a was increased about 57.1% at 1 day (Fig. 5A; Wnt 3a/actin at 1 day, MCAO, 1.05 70.03 versus sham, 0.7 70.05, P o0.05; n¼ 3), but no change occurred at 3 day (Fig. 5B; Wnt 3a/actin at 3 day, MCAO, 0.89 70.15 versus sham, 0.89 70.14, P4 0.05; n ¼3). This result indicates that activation of Wnt signaling triggered by ischemia may last up to 24 h; however, treatment with morroniside may extend this active period to 7 days. To further confirm the effects of morroniside, we treated non-ischemic sham-operated rats with morroniside at dose of 270 mg/kg/day for 1 day and 3 days, and then examined the Wnt3a expression on the first and third day after morroniside administration. The results showed that there was no difference between morroniside-treated animals and untreated control groups (Fig. 5A and B), which indicate that morroniside may not
be a direct agonist of Wnt signaling per se, but may only activate Wnt signaling in the context of ischemia-injured brain. 3.4. Morroniside regulated the expression of Wnt signaling-related transcription factors after ischemic stroke To further study the effects of morroniside on Wnt signaling pathway, the expression of transcription factors downstreams of Wnt signaling pathway was examined by immunoblot assay. The results showed that the expression of Ngn2 was significantly increased by 1.76-fold 7 days after MCAO, compared with the sham-operated group (P o0.05, n ¼3). Administration of morroniside at dose of 90 and 270 mg/kg enhanced this increase in Ngn 2 expression by 1.59-fold (P o0.01, n ¼4) and 1.76-fold (P o0.01, n¼ 4) respectively, compared with the vehicle-treated group (Fig. 6A and B). The Pax6 level is also up-regulated by morroniside of 90 and 270 mg/kg 7 days after MCAO, although no change occurred in the vehicle-treated group compared with the shamoperated group (Fig. 6A and C). As to the expression of Tbr2, post-hoc analysis showed that there were no significant differences between the morroniside-treated, the vehicle-treated ischemia rats and the sham-operated group.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
5
Fig. 4. Effects of morroniside on the Wnt/β-catenin signaling pathway after stroke. (A and B) Immunohistochemistry staining of brain sections from a vehicle-treated ischemic rat reveals β-catenin expression in the SVZ, indicating the potential relationship of Wnt signaling pathway with neurogenesis. (C) Representative image of immunoblots for Wnt3a (40 kDa), β-catenin (92 kDa) and Tcf-4 (71 kDa) in the cortex of rat 7 days after MCAO. (D–F) Quantitative analysis of Wnt3a (D), β-catenin (E) and Tcf-4 (F) expressed as a fraction of the respective levels of β-actin. n¼ 3 (the data of each group were obtained from three rats in three experiments). Data are presented as mean 7 S.E.M. nPo 0.05, nnP o 0.01 and nnnPo 0.001 as compared to vehicle-treated ischemic group. Scale bar: 500 μm.
Fig. 5. Immunoblot analysis of Wnt3a expression in the cortex of rats 1 and 3 day after MCAO. Quantitative analysis of Wnt3a 1 day (A) and 3 day (B) after MCAO, expressed as a fraction of the respective level of β-actin, suggests that the activation of Wnt signaling induced by ischemia may be limited to 24 h. n¼ 3 (the data of each group were obtained from three rats in three experiments). Data are presented as mean 7 S.E.M. #Po 0.05 as compared to sham-operated control group, and nPo 0.05 as compared to vehicle-treated ischemic group.
4. Discussion In the present study, we identified the neuroprotective effects of morroniside of 7 days-administration in a rat model of MCAO, characterized by improving neurological function. We proposed that promoting neurogenesis after cerebral ischemia plays an
important role for morroniside intervention. As the results of immunohistochemistry and immunoblotting shown, Wnt/β-catenin signaling pathway might be involved in the neurorestorative mechanisms of morroniside on neurogenesis after cerebral ischemia. Neurogensis is a primary neurovascular response during stroke recovery phase. Cerebral ischemia stimulates neurogenesis in the
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Q2 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 6. Effects of morroniside on the transcription factors downstream related to Wnt/β-catenin signaling pathway. (A) Representative image of immunoblots for Ngn2 (28 kDa), Pax6 (50.6 kDa) and Tbr2 (72 kDa). (B–D) Quantitative analysis of Ngn2 (B), Pax6 (C) and Tbr2 (D) expressed as a fraction of the respective levels of β-actin. The data of each group were obtained from at least three rats of three independent experiments. Data are presented as mean 7 S.E.M. #Po 0.05 as compared to sham-operated control group, nPo 0.05 and nnPo 0.01 as compared to vehicle-treated ischemic group.
SVZ and subgranular zone (SGZ) of adult brain (Delavaran et al., 2013; Marti-Fabregas et al., 2010; Ohira et al., 2010). Ki-67 is a nuclear nonhistone protein expressed among proliferating cells but absent in quiescent cells, therefore being used as a proliferation marker (Yerushalmi et al., 2010; Varela-Nallar et al., 2014), while Nestin is a class VI intermediate filament protein that serves as a predominate marker for neural stem and progenitor cells (Messam et al., 2000; Wu et al., 2013). Hence, the increased levels of Ki-67 or Nestin-immunoreactive cells in the SVZ of the ischemia-treated rats indicated endogenous neural stem cells response 7 days following MCAO. After morroniside administration, this increase in the number of Ki-67 or Nestin-postive cells was enhanced. Morever, the distribution of Nestin-immunoreactive cells of ischemia-treated rats was observed not only in the lateral ventricle wall and the dorsolateral corner of the SVZ, but also in the peri-infarct cortex. The results indicate that morroniside promote neurogenesis post-ischemia, and the upregulation of these neural progenitor cells is thought to contribute to functional recovery. Several studies have addressed the role of Wnt/β-catenin signaling in adult neurogenesis, mediating cell survival, proliferation of neural progenitor cells and differentiation into neuron (Inestrosa and Arenas, 2010; Kuwabara et al., 2009). It has been also previously reported that components of the canonical Wnt/βcatenin pathway are expressed within the adult SVZ and SGZ (Adachi et al., 2007), and Wnt/β-catenin signals activate downstream target genes to promote adult neurogenesis (Kuwabara et al., 2009; Lie et al., 2005). Growing evidence from recent studies suggests that this pathway is upregulated in the SVZ following
stroke and may exert an important role in ischemia-induced neurogenesis (Lei et al., 2008; Mastroiacovo et al., 2009). The results in the present research show that the Wnt signaling pathway is activated in the early phase of ischemia and is limited to 24 h; however, its activation extends to 7 days after treatment with morroniside. The absence of any effects on the shamoperated rats suggests that morroniside may not directly act on Wnt signaling, while it may only exert its activation role on the ischemic-injured brain. Thus, morroniside may function as a stem cell proliferation enhancer rather than an initiator. In order to confirm the effects and possible mechanisms of morroniside on neurogenesis, we further studied the changes of the expression of downstreams factors in the Wnt/β-catenin signaling pathway 7 days post-ischemia. Transcription factors Pax6, Tbr2 and Ngn2 have been demonstrated to function as downstream mediator of Wnt-induced neurogenesis from adult hippocampal neural progenitors (Cho and Dressler, 1998; Faigle and Song, 2013). Kohwi et al. (2005) found that transcription factor Pax6 was expressed in most proliferating SVZ progenitors but only in a subpopulation of migrating neuroblasts, promoting neuronal differentiation. The studies on Pax6 mutant Small Eye mice have shown that Pax6 plays an important role in the regulation of forebrain development by Wnt–Tcf signaling pathway (Cho and Dressler, 1998), and regulates the differentiation of specific subtypes of interneurons in the olfactory bulb (OB) (Ninkovic et al., 2010). Tbr2, a pro-neurogenic T-box transcription factor expressed by intermediate neuronal progenitors (INPs), has been shown to be critically required for neurogenesis in the adult subventricular zone (SVZ) and in the dentate gyrus of adult mice
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Q3 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
(Brill et al., 2009). Functional inactivation of Tbr2 consistently results in increased proliferation of NSCs, but in the long-time absence of Tbr2, neurogenesis is halted as the result of failed neuronal differentiation (Hodge et al., 2012). There is a crosstalk between Pax6 and Tbr2 that down-regulation of Pax6 upregulates Tbr2 expressions in olfactory bulb mitral cells in developing neocortex while overexpression of Pax6 leads the postmitotic cells differentiate into interneurons (Bayatti et al., 2008; Imamura and Greer, 2013). Ngn2 is a member of the basic helix-loop-helix (bHLH) transcription factors, and it is well known that Ngn2 is associated with neural differentiation and inhibits astrocytic differentiation. The previous studies have shown that β-catenin signaling could upregulate Ngn2 expression although the mechanism is still not clarified (Hong and Bain, 2012; Matsumoto et al., 2013). The results of this present study demonstrated that morroniside-treatment upregulated the expressions of Pax6 and Ngn2. It is indicated that the regulation of these factors might be involved in the augment of neural stem cells proliferation and next neuronal differention by morroniside through Wnt/β-catenin pathway. As to the negative-effects of morroniside on Tbr2, we considered it be associated with the complex expression patterns of Pax6 and Tbr2 (Bayatti et al., 2008; Muzio et al., 2002). In summary, morroniside improves the endogenous mechanisms of neurogenesis for brain recovery following ischemic stroke. Morroniside could sustain the activation of Wnt/β-catenin signaling pathway to enhance the proliferation of neural stem cells 7 days post-ischemia, and could regulate the expression of downstream transcription factors Pax6 and Ngn2 to promote neurogenesis. Though the exact correlation between the Wnt/β-catenin signaling pathway and neurogenesis promoted by morroniside still needs to be fully defined, these results suggest that morroniside offers a prospective medicine for stroke recovery. One caveat remains, i.e. the temporal relationship between acute neuroprotection and delayed neurorepair. In this initial study, treatments with morroniside were started at 3 h after MCAO. Thus, overlap between its known acute protective effects and our present findings of promoting neurogenesis cannot be fully separated, and ultimately, the correlation between delayed neurorepair effect and whole-animal neurological function still needs to be fully defined. Acknowledgments This work was supported by the National Science and Technology Major Project (2012ZX09102201-106); National Natural Science Foundation of China (81173575, 81373994, 30973893 and 81274173); Beijing Natural Science Foundation (7102077 and 7211017); Beijing Municipal Commission of Education & Capital Medical University Base-Technological Innovation Platform Fund (111219); Beijing Health System Experts of High-level grant (20113-097); China Postdoctoral Science Foundation funded project and Beijing Postdoctoral Science Foundation funded project to FL Sun. We also express our appreciation to Professor Deyu Guo and Yumin Luo with their colleagues for their kind help on the MCAO model-required technical assistance. References Adachi, K., Mirzadeh, Z., Sakaguchi, M., Yamashita, T., Nikolcheva, T., Gotoh, Y., Peltz, G., Gong, L., Kawase, T., Alvarez-Buylla, A., Okano, H., Sawamoto, K., 2007. Betacatenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone. Stem Cells25, 2827–2836. Baarsma, H.A., Konigshoff, M., Gosens, R., 2013. The WNT signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets. Pharmacol. Ther. 138, 66–83. Bayatti, N., Sarma, S., Shaw, C., Eyre, J.A., Vouyiouklis, D.A., Lindsay, S., Clowry, G.J., 2008. Progressive loss of PAX6, TBR2, NEUROD and TBR1 mRNA gradients
7
correlates with translocation of EMX2 to the cortical plate during human cortical development. Eur. J. Neurosci. 28, 1449–1456. Belayev, L., Alonso, O.F., Busto, R., Zhao, W., Ginsberg, M.D., 1996. Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. Stroke 27, 1616–1622. Brill, M.S., Ninkovic, J., Winpenny, E., Hodge, R.D., Ozen, I., Yang, R., Lepier, A., Gascon, S., Erdelyi, F., Szabo, G., Parras, C., Guillemot, F., Frotscher, M., Berninger, B., Hevner, R.F., Raineteau, O., Gotz, M., 2009. Adult generation of glutamatergic olfactory bulb interneurons. Nat. Neurosci. 12, 1524–1533. Cho, E.A., Dressler, G.R., 1998. TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs. Mech. Dev. 77, 9–18. Clevers, H., 2006. Wnt/beta-catenin signaling in development and disease. Cell 127, 469–480. Delavaran, H., Sjunnesson, H., Arvidsson, A., Lindvall, O., Norrving, B., van Westen, D., Kokaia, Z., Lindgren, A., 2013. Proximity of brain infarcts to regions of endogenous neurogenesis and involvement of striatum in ischaemic stroke. Eur. J. Neurol. 20, 473–479. Faigle, R., Song, H., 2013. Signaling mechanisms regulating adult neural stem cells and neurogenesis. Biochim. Biophys. Acta 1830, 2435–2448. Hodge, R.D., Nelson, B.R., Kahoud, R.J., Yang, R., Mussar, K.E., Reiner, S.L., Hevner, R. F., 2012. Tbr2 is essential for hippocampal lineage progression from neural stem cells to intermediate progenitors and neurons. J. Neurosci. 32, 6275–6287. Hong, G.M., Bain, L.J., 2012. Arsenic exposure inhibits myogenesis and neurogenesis in P19 stem cells through repression of the beta-catenin signaling pathway. Toxicol. Sci. 129, 146–156. Imamura, F., Greer, C.A., 2013. Pax6 regulates Tbr1 and Tbr2 expressions in olfactory bulb mitral cells. Mol. Cell. Neurosci. 54, 58–70. Inestrosa, N.C., Arenas, E., 2010. Emerging roles of Wnts in the adult nervous system. Nat. Rev. Neurosci. 11, 77–86. Kohwi, M., Osumi, N., Rubenstein, J.L., Alvarez-Buylla, A., 2005. Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb. J. Neurosci. 25, 6997–7003. Kuwabara, T., Hsieh, J., Muotri, A., Yeo, G., Warashina, M., Lie, D.C., Moore, L., Nakashima, K., Asashima, M., Gage, F.H., 2009. Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nat. Neurosci. 12, 1097–1105. Lei, Z.N., Zhang, L.M., Sun, F.Y., 2008. Beta-catenin siRNA inhibits ischemia-induced striatal neurogenesis in adult rat brain following a transient middle cerebral artery occlusion. Neurosci. Lett. 435, 108–112. Lie, D.C., Colamarino, S.A., Song, H.J., Desire, L., Mira, H., Consiglio, A., Lein, E.S., Jessberger, S., Lansford, H., Dearie, A.R., Gage, F.H., 2005. Wnt signalling regulates adult hippocampal neurogenesis. Nature 437, 1370–1375. Marti-Fabregas, J., Romaguera-Ros, M., Gomez-Pinedo, U., Martinez-Ramirez, S., Jimenez-Xarrie, E., Marin, R., Marti-Vilalta, J.L., Garcia-Verdugo, J.M., 2010. Proliferation in the human ipsilateral subventricular zone after ischemic stroke. Neurology 74, 357–365. Mastroiacovo, F., Busceti, C.L., Biagioni, F., Moyanova, S.G., Meisler, M.H., Battaglia, G., Caricasole, A., Bruno, V., Nicoletti, F., 2009. Induction of the Wnt antagonist, Dickkopf-1, contributes to the development of neuronal death in models of brain focal ischemia. J. Cereb. Blood Flow Metab. 29, 264–276. Matsumoto, M., Imura, T., Fukazawa, T., Sun, Y., Takeda, M., Kajiume, T., Kawahara, Y., Yuge, L., 2013. Electrical stimulation enhances neurogenin2 expression through beta-catenin signaling pathway of mouse bone marrow stromal cells and intensifies the effect of cell transplantation on brain injury. Neurosci. Lett. 533, 71–76. Messam, C.A., Hou, J., Major, E.O., 2000. Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody. Exp. Neurol. 161, 585–596. Muzio, L., Di Benedetto, B., Stoykova, A., Boncinelli, E., Gruss, P., Mallamaci, A., 2002. Conversion of cerebral cortex into basal ganglia in Emx2(-/-) Pax6(Sey/Sey) double-mutant mice. Nat. Neurosci. 5, 737–745. Navarro-Sobrino, M., Rosell, A., Hernandez-Guillamon, M., Penalba, A., Boada, C., Domingues-Montanari, S., Ribo, M., Alvarez-Sabin, J., Montaner, J., 2011. A large screening of angiogenesis biomarkers and their association with neurological outcome after ischemic stroke. Atherosclerosis 216, 205–211. Ninkovic, J., Pinto, L., Petricca, S., Lepier, A., Sun, J., Rieger, M.A., Schroeder, T., Cvekl, A., Favor, J., Gotz, M., 2010. The transcription factor Pax6 regulates survival of dopaminergic olfactory bulb neurons via crystallin alpha. Neuron 68, 682–694. Ohira, K., Furuta, T., Hioki, H., Nakamura, K.C., Kuramoto, E., Tanaka, Y., Funatsu, N., Shimizu, K., Oishi, T., Hayashi, M., Miyakawa, T., Kaneko, T., Nakamura, S., 2010. Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells. Nat. Neurosci. 13, 173–179. Shruster, A., Ben-Zur, T., Melamed, E., Offen, D., 2013. Wnt signaling enhances neurogenesis and improves neurological function after focal ischemic injury. PloS One 7, e40843. Varela-Nallar, L., Rojas-Abalos, M., Abbott, A.C., Moya, E.A., Iturriaga, R., Inestrosa, N. C., 2014. Chronic hypoxia induces the activation of the Wnt/β-catenin signaling Q4 pathway and stimulates hippocampal neurogenesis in wild-type and APPswePS1ΔE9 transgenic mice in vivo. Front Cell Neurosci., 8, http://dx.doi.org/ 10.3389/fncel.2014.00017. Wang, W., Huang, W., Li, L., Ai, H., Sun, F., Liu, C., An, Y., 2008. Morroniside prevents peroxide-induced apoptosis by induction of endogenous glutathione in human neuroblastoma cells. Cell. Mol. Neurobiol. 28, 293–305. Wang, W., Sun, F., An, Y., Ai, H., Zhang, L., Huang, W., Li, L., 2009. Morroniside protects human neuroblastoma SH-SY5Y cells against hydrogen peroxideinduced cytotoxicity. Eur. J. Pharmacol. 613, 19–23.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
8
1 2 3 4 5 6 7
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Wang, W., Xu, J., Li, L., Wang, P., Ji, X., Ai, H., Zhang, L., Li, L., 2010. Neuroprotective effect of morroniside on focal cerebral ischemia in rats. Brain Res. Bull. 83, 196–201. Wu, M.D., Montgomery, S.L., Rivera-Escalera, F., Olschowka, J.A., O’Banion, M.K., 2013. Sustained IL-1beta expression impairs adult hippocampal neurogenesis independent of IL-1 signaling in nestin þ neural precursor cells. Brain Behav. Immun. 32, 9–18.
Yemisci, M., Gursoy-Ozdemir, Y., Vural, A., Can, A., Topalkara, K., Dalkara, T., 2009. Pericyte contraction induced by oxidative–nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat. Med. 15, 1031–1037. Yerushalmi, R., Woods, R., Ravdin, P.M., Hayes, M.M., Gelmon, K.A., 2010. Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 11, 174–183.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Q1 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside against cerebral ischemia injury Fang-Ling Sun a, Wen Wang a,n, Wei Zuo a, Jin-Long Xue a, Jing-dong Xu b, Hou-Xi Ai a, Li Zhang a, Xiao-Min Wang c,nn, Xun-Ming Ji d,nnn a
Department of Pharmacology, Xuanwu Hospital of Capital Medical University, 45 Chang-chun Street, Beijing 100053, China Department of Physiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China Department of Neurobiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men Beijing 100069, China d Department of Neurosurgery, Xuanwu Hospital of Capital Medical University, 45 Chang-chun Street, Beijing 100053, China b c
art ic l e i nf o
a b s t r a c t
Article history: Received 25 October 2013 Received in revised form 4 May 2014 Accepted 10 May 2014
Ischemic stroke is a leading cause of mortality and permanent disability in adults worldwide. Neurogenesis triggered by ischemia in the adult mammalian brain may provide insights into stroke treatment. Morroniside is an active component of sarcocarp of C. officinalis that have shown neuroprotective effects. The aim of the present study is to test whether morroniside promotes neurogenesis via Wnt/β-catenin signaling pathway for brain recovery in a rat model of focal cerebral ischemia. Morroniside was administered intragastrically once daily at the concentrations of 30, 90 and 270 mg/kg for 7 days postischemia. Neurological functions were detected by Ludmila Belayev score tests. Endogenous neural stem cells responses were investigated with immunofluorescence staining of Ki-67 and Nestin to identify the neurogenesis in the subventricular zone (SVZ). The expression of proteins involved in and related to Wnt/β-catenin signaling pathway was detected by western blotting analysis. Morroniside significantly promoted neurogenesis for brain recovery 7 days post-ischemia. Increased expression of Wnt 3a, β-catenin and T-cell transcription factor-4 (Tcf-4), along with activation of downstream transcription factors Pax6 and neurogenin2 (Ngn2), indicated that the neurorestorative effects of morroniside may be associated with Wnt/β-catenin signaling pathway. These data provide support for understanding the mechanisms of morroniside in neurorestorative effects and suggest a potential new strategy for ischemic stroke treatment. & 2014 Published by Elsevier B.V.
Keywords: Ischemic stroke Morroniside Neurogenesis Wnt/β-catenin signaling
1. Introduction Stroke is the third leading cause of death and the most common cause of permanent disability in adults worldwide (Navarro-Sobrino et al., 2011). Despite substantial progress in understanding the mechanisms of ischemic stroke over the past few decades, there is still no effective therapy for neurorestorative treatment (Yemisci et al., 2009). Evidence is accumulating that cerebral ischemia can stimulate endogenous neurogenesis in the subventricular zone (SVZ) of adult mammals (Marti-Fabregas et al., 2010; Ohira et al., 2010). Although the increase of stem cells in the SVZ of the adult
n
Corresponding author. Tel.: þ 86 10 83198881; fax: þ 86 10 83154745. Corresponding author. Tel./fax: þ86 10 83911829. Corresponding author. Tel.: þ 86 10 83198952; fax: þ 86 10 83911496. E-mail addresses: [email protected] (W. Wang), [email protected] (X.-M. Wang), [email protected] (X.-M. Ji). nn
nnn
brain may lead to morphological and functional improvements following ischemic injuries, this internal response is unable to functionally compensate for the ischemic damage. Thus, there is an urgent need to search for therapeutic approaches that can augment endogenous neurogenesis. Wnt/β-catenin signaling pathway is well known for its important role in nervous system development. In the presence of Wnt signals, β-catenin is stabilized and translocates to the nucleus, where it interacts with T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to induce changes in gene expression (Baarsma et al., 2013; Clevers, 2006). This signaling pathway has been shown to regulate neurogenesis in the adult SVZ, by promoting proliferation of progenitor cells (Adachi et al., 2007). A recent study in focal cerebral ischemic injury model revealed that treatment with lentivirus expressing Wnt3a-HA into SVZ enhanced functional recovery from the 2nd day after injury and increased the number of immature neurons in the striatum
http://dx.doi.org/10.1016/j.ejphar.2014.05.019 0014-2999/& 2014 Published by Elsevier B.V.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
and SVZ (Shruster et al., 2013). The findings indicate that Wnt signaling could induce an appropriate neurogenic environment for neuronal differentiation and survival post-ischemic injury. Morroniside is an iridoid glycoside extracted from the sarcocarp of Cornus officinalis. It has been shown to possess antioxidative and anti-apoptosis activities in vitro and in vivo (Wang et al., 2008, 2009); moreover, our previous studies have also demonstrated that morroniside reduces the infarct volume and promotes neurological functional recovery 3 days after focal ischemic stroke (Wang et al., 2010). However, the long-term effects and mechanisms of morroniside against ischemic stroke are not clear. We hypothesized that morroniside could promote neurogenesis for neurorepair from ischemic injury. In the present study, we used a rat model of middle cerebral artery occlusion (MCAO) to identify the effects of morroniside on neurogenesis 7 days postischemia, and to examine whether the Wnt signaling pathway involved in the neurorestorative mechanism of morroniside.
2. Materials and methods 2.1. Morroniside extraction and identification C. officinalis was purchased from Tong Ren Tang Company, Beijing, China, and authenticated by Professor Wen Wang. Morroniside was extracted from the sarcocarp of C. officinalis and purified as previously described (Wang et al., 2010). The final purity was determined to be 98.5% by high performance liquid chromatography (HPLC) analysis.
2.4. Immunofluorescence analysis and immunohistochemistry analysis. Immunofluorescence staining was used to visualize Nestin in the SVZ. After the rats were perfused with 4% paraformaldehyde in PBS, the brains were extracted and post-fixed overnight. Ice-cold sections (40 μm) were prepared using standard protocols and freefloating sections were incubated with 3% H2O2 in 0.01 M PBS. After incubation with a blocking solution, the sections were incubated with primary antibody anti-Ki67 (Cell Signaling Technology) or anti-Nestin (Sigma-Aldrich) for 48 h at 4 1C and then incubated with CY2 or CY3-conjugated secondary antibody (Invitrogen) for 0.5 h. The fluorescence signals were visualized using a confocal laser scanning microscope system (MRC1024, Bio-Rad, Hercules, CA, USA). For immunohistochemistry analysis, the sections prepared as above were incubated with primary antibody anti-βcatenin (Santa Cruz Biotechnology) for 24 h at 4 1C. The secondary antibody was biotinylated goat anti-rabbit IgG (Santa Cruz Biotechnology). The sections were stained using the avidin-biotin peroxidase complex method with 3,30 -diaminobenzidine as a chromogen. The stained sections were then dehydrated in a graded alcohol series, cleared in xylene, coverslipped with neutral balsam and examined under a light microscope equipped with a computerized image analysis system (Olympus BX51, Japan). Image Pros Plus 5.0 software (Media Cybernetics, Inc., Bethesda, MD, USA) was used to determine the average number of positive cells in area of interest (AOI) in the dorsal lateral corner of SVZ per section. 2.5. Protein expression analysis.
2.2. Animals and the middle cerebral artery occlusion (MCAO) model Male Sprague-Dawley (SD) rats weighing 260–280 g were purchased from Beijing Vitalriver Experimental Animal Co. (Beijing, China), and were housed under a 12/12 h dark/light cycle and specific-pathogen-free (SPF) conditions. Before the MCAO model was performed, the rats were fasted without water deprivation for 12 h. Anesthesia was induced and maintained with 3.5% enflurane in a mixture of 70% nitrous oxide and a balance of oxygen (Bickford veterinary anesthesia equipment model no. 61010, AM Bickford Inc., Wales Center, NY, USA). The rats were orally intubated, immobilized with intra-arterial pancuronium bromide (0.6 mg/kg) and mechanically ventilated (Rodent Ventilator Model 683, Harvard Apparatus Inc., Holliston, MA, USA). A piece of nylon monofilament was inserted into the left internal carotid artery via an arteriotomy and lodged in the narrow proximal anterior cerebral artery, which blocked the MCA at its origin. After 30 min of ischemia, reperfusion was established by the withdrawal of the filament. When both cerebral and rectal temperatures returned to normal levels, the animals were allowed to regain consciousness and were placed under warm conditions for an additional 3 h. Morroniside was dissolved in normal saline and administered intragastrically once a day at the dose of 30, 90, 270 mg/kg for 7 days, starting at 3 h after MCAO. The vehicle control groups of the ischemic rats received an equal volume of normal saline. Sham-operated rats were treated with an equal volume of normal saline or 270 mg/kg morroniside. Animal protocols for these studies were approved by the Animal Care and Use Committee of Xuanwu Hospital in Capital Medical University. 2.3. Neurological scoring Seven days post-MCAO, neurological deficits were evaluated by Ludmila Belayev score test as previously described (Belayev et al., 1996).
The rats were euthanized under 10% chloral hydrate (0.4 mL/kg) anesthesia. The infarct side of the cortex was harvested and homogenized in ice-cold buffer (tris-(hydroxymethyl)-aminomethane 50 mM, pH 7.4, NaCl 150 mM, 0.5% Triton X-100, edetic acid 1 mM, phenylmethylsulfonyl fluoride 1 M and aprotinin 5 mg/L). Following the homogenate centrifuged at 14,000 g at 4 1C for 30 min, the supernatants were collected as the total protein. The proteins were then electrophoresed through a 10–15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and electrically transferred onto a nitrocellulose membrane. This membrane was detected using primary antibodies, including anti-Wnt 3a (Millipore), anti-β catenin (Santa Cruz Biotechnology), anti-Tcf-4 (Santa Cruz Biotechnology), anti-Pax 6 (Abcam), anti-Tbr 2 (Abcam), anti-neurogenin2 (Ngn2) (Abcam). The amount of protein was normalized with β-actin values in the same lane. 2.6. Statistical analysis. All data are presented as the mean 7SEM. Analysis of variance (ANOVA) followed by Scheffe (when number of sample is not equal) or Tukey's multiple range post-hoc test was carried out by using Statistical Package for the Social Sciences software (SPSS 17.0 Statistical Software). A p value of less than 0.05 was considered significant.
3. Results 3.1. Morroniside improved neurological deficits 7 days following MCAO In Ludmila Belayev score test, the score of the vehicle-treated rats was significantly increased to 5.36 70.61 from 0 compared with the sham-operated group 7 days after MCAO (P o0.001, n¼ 11), indicating that neurological deficits of the rats caused by cerebral ischemia-reperfusion remained 7 days after MCAO.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
However, treatment with morroniside at the concentrations of 90 and 270 mg/kg/day for 7 days after MCAO respectively reduced the Ludmila Belayev score from 5.36 7 0.61 to 2.697 0.56 (P o0.05, n ¼13) and 1.36 7 0.49 (P o0.001, n ¼ 14) (Fig. 1). These results suggested the effects of morroniside on the recovery of neurological function 7 days post-ischemic injury.
3.2. Morroniside promotes neurogenesis after cerebral ischemia To study the effects of morroniside on neurogenesis, we first examined cell proliferation in the SVZ 7 days after MCAO by using immunostaining for Ki-67. In the vehicle-treated ischemic rats, cerebral ischemia induced Ki-67-positive cells were clearly
3
increased in the ischemic ipsilateral dorsolateral corner of SVZ 7 days after MCAO (Fig. 2C). Treatment with morroniside at dose of 90 and 270 mg/kg respectively increased the number of Ki-67positive cells in SVZ from 123.177 3.66 to 310.5 73.47 (P o0.001) and 423.83 75.76 (P o0.001) (Fig. 2E–G). Next, we detected Nestin-positive cells in the SVZ by using immunofluorescence. In the vehicle-treated ischemic rats, Nestinpositive cells were also increased in the ischemic ipsilateral dorsolateral corner of the SVZ 7 days after MCAO (Fig. 3C). Quantitation confirmed that the number of Nestin-positive cells was significantly increased compared with the sham-operated group (Po0.001) (Fig. 3H). Treatment with morroniside at dose of 30, 90 and 270 mg/kg respectively increased the density of Nestin-positive cells in SVZ from 254.6876.03% to 363.3675.89% (Po0.001), 442.167 7.25% (Po0.001) and 475.5974.97% (Po0.001) (Fig. 3H). Moreover, Nestin-positive cells in morroniside-treated rats were found not only in the SVZ area but also appeared to penetrate into peri-infarct cortex (Fig. 3G). In addition, the results of Ki-67- and Nestin-positive cells indicated that there were no effects on the cell proliferation of the sham-operated rats after morroniside treatment (Figs. 2B and 3B). Taken together, these data indicate that administration of morroniside promotes neural stem cell proliferation after focal cerebral ischemia. 3.3. Morroniside activated Wnt /β-catenin signaling pathway after ischemic stroke
Fig. 1. Effects of morroniside on neurological function 7 days post-ischemia. Ludmila Belayev scores are expressed as absolute values and Scheffe post-hoc test is used for data analysis. The data of each group were obtained from at least 11 rats. Data are presented as mean 7 S.E.M. ###Po 0.001 as compared to the shamoperated control group. nPo 0.05 and nnnPo 0.001 as compared to the vehicletreated ischemic group.
In order to study the potential mechanisms of morroniside in neurogenesis, we examined the role of Wnt/β-catenin signaling pathway in this neurorestorative process. The results showed β-catenin expression in the SVZ (Fig. 4A and B), indicating the potential relationship between the Wnt signaling pathway and neurogenesis. As Fig. 4C–F shows, the expression of Wnt3a, β-catenin and Tcf-4 was increased at 7 days after ischemia in the morroniside-treated rats, compared with the vehicle-treated ischemic rats. These results indicate that the Wnt signaling pathway is involved in the effects of morroniside on ischemic recovery. It was also observed that there were no significant differences in the above-mentioned protein expression between
Fig. 2. Effects of morroniside on Ki-67-labeled cells proliferation in the ischemic ipsilateral SVZ. (A–F) Ki-67-immunoreactive cells in the ischemic ipsilateral SVZ in shamoperated rats (A), a sham-operated group treated with morroniside of 270 mg/kg/day (B), a vehicle-treated ischemic group (C) and groups of ischemic rats treated with morroniside at dose of 30 mg/kg/day (D), 90 mg/kg/day (E), and 270 mg/kg/day (F). (G) Quantitative analysis of the density of Ki-67-positive cells. n¼6 for each group. Data are presented as mean 7 S.E.M. ###Po 0.001 as compared to sham-operated control group; nnnP o 0.001 as compared to vehicle-treated ischemic group. Scale bar: 100 μm (A–F).
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 3. Effects of morroniside on Nestin-labeled neural stem cells proliferation in the ischemic ipsilateral SVZ. (A–F) Nestin-immunoreactive cells in the ischemic ipsilateral SVZ in sham-operated rats (A), a sham-operated group treated with morroniside of 270 mg/kg/day (B), a vehicle-treated ischemic group (C) and groups of ischemic rats treated with morroniside at dose of 30 mg/kg/day (D), 90 mg/kg/day (E), and 270 mg/kg/day (F). (G) Low magnified image of Nestin-immunoreactive cells from a 90 mg/kg/ day morroniside-treated ischemic rat. Nestin-positive cells appeared to penetrated into peri-infarct cortex. (G’) A high magnified image of Nestin-immunoreactive cells in the peri-infarct cortex of (G). (H) Quantitative analysis of the density of Nestin-positive cells. n ¼6 for each group. Data are presented as mean 7S.E.M. ###Po 0.001 as compared to sham-operated control group; nnnPo 0.001 as compared to vehicle-treated ischemic group. Scale bar: 100 μm (A–F); 500 μm (G); 25 μm (G’).
the vehicle-treated ischemia rats and the sham-operated group (Fig. 4C–F). These results suggest that long term activation of Wnt signaling is sustained by morroniside per se, and not non-specifically caused by lingering effects of ischemia. Consistent with a role for this mechanism in brain recovery, the expression profiles of Wnt3a continued to evolve at 1 day and 3 days after ischemia. Compared with the corresponding shamoperated group, the expression of Wnt3a was increased about 57.1% at 1 day (Fig. 5A; Wnt 3a/actin at 1 day, MCAO, 1.05 70.03 versus sham, 0.7 70.05, P o0.05; n¼ 3), but no change occurred at 3 day (Fig. 5B; Wnt 3a/actin at 3 day, MCAO, 0.89 70.15 versus sham, 0.89 70.14, P4 0.05; n ¼3). This result indicates that activation of Wnt signaling triggered by ischemia may last up to 24 h; however, treatment with morroniside may extend this active period to 7 days. To further confirm the effects of morroniside, we treated non-ischemic sham-operated rats with morroniside at dose of 270 mg/kg/day for 1 day and 3 days, and then examined the Wnt3a expression on the first and third day after morroniside administration. The results showed that there was no difference between morroniside-treated animals and untreated control groups (Fig. 5A and B), which indicate that morroniside may not
be a direct agonist of Wnt signaling per se, but may only activate Wnt signaling in the context of ischemia-injured brain. 3.4. Morroniside regulated the expression of Wnt signaling-related transcription factors after ischemic stroke To further study the effects of morroniside on Wnt signaling pathway, the expression of transcription factors downstreams of Wnt signaling pathway was examined by immunoblot assay. The results showed that the expression of Ngn2 was significantly increased by 1.76-fold 7 days after MCAO, compared with the sham-operated group (P o0.05, n ¼3). Administration of morroniside at dose of 90 and 270 mg/kg enhanced this increase in Ngn 2 expression by 1.59-fold (P o0.01, n ¼4) and 1.76-fold (P o0.01, n¼ 4) respectively, compared with the vehicle-treated group (Fig. 6A and B). The Pax6 level is also up-regulated by morroniside of 90 and 270 mg/kg 7 days after MCAO, although no change occurred in the vehicle-treated group compared with the shamoperated group (Fig. 6A and C). As to the expression of Tbr2, post-hoc analysis showed that there were no significant differences between the morroniside-treated, the vehicle-treated ischemia rats and the sham-operated group.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
5
Fig. 4. Effects of morroniside on the Wnt/β-catenin signaling pathway after stroke. (A and B) Immunohistochemistry staining of brain sections from a vehicle-treated ischemic rat reveals β-catenin expression in the SVZ, indicating the potential relationship of Wnt signaling pathway with neurogenesis. (C) Representative image of immunoblots for Wnt3a (40 kDa), β-catenin (92 kDa) and Tcf-4 (71 kDa) in the cortex of rat 7 days after MCAO. (D–F) Quantitative analysis of Wnt3a (D), β-catenin (E) and Tcf-4 (F) expressed as a fraction of the respective levels of β-actin. n¼ 3 (the data of each group were obtained from three rats in three experiments). Data are presented as mean 7 S.E.M. nPo 0.05, nnP o 0.01 and nnnPo 0.001 as compared to vehicle-treated ischemic group. Scale bar: 500 μm.
Fig. 5. Immunoblot analysis of Wnt3a expression in the cortex of rats 1 and 3 day after MCAO. Quantitative analysis of Wnt3a 1 day (A) and 3 day (B) after MCAO, expressed as a fraction of the respective level of β-actin, suggests that the activation of Wnt signaling induced by ischemia may be limited to 24 h. n¼ 3 (the data of each group were obtained from three rats in three experiments). Data are presented as mean 7 S.E.M. #Po 0.05 as compared to sham-operated control group, and nPo 0.05 as compared to vehicle-treated ischemic group.
4. Discussion In the present study, we identified the neuroprotective effects of morroniside of 7 days-administration in a rat model of MCAO, characterized by improving neurological function. We proposed that promoting neurogenesis after cerebral ischemia plays an
important role for morroniside intervention. As the results of immunohistochemistry and immunoblotting shown, Wnt/β-catenin signaling pathway might be involved in the neurorestorative mechanisms of morroniside on neurogenesis after cerebral ischemia. Neurogensis is a primary neurovascular response during stroke recovery phase. Cerebral ischemia stimulates neurogenesis in the
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Q2 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 6. Effects of morroniside on the transcription factors downstream related to Wnt/β-catenin signaling pathway. (A) Representative image of immunoblots for Ngn2 (28 kDa), Pax6 (50.6 kDa) and Tbr2 (72 kDa). (B–D) Quantitative analysis of Ngn2 (B), Pax6 (C) and Tbr2 (D) expressed as a fraction of the respective levels of β-actin. The data of each group were obtained from at least three rats of three independent experiments. Data are presented as mean 7 S.E.M. #Po 0.05 as compared to sham-operated control group, nPo 0.05 and nnPo 0.01 as compared to vehicle-treated ischemic group.
SVZ and subgranular zone (SGZ) of adult brain (Delavaran et al., 2013; Marti-Fabregas et al., 2010; Ohira et al., 2010). Ki-67 is a nuclear nonhistone protein expressed among proliferating cells but absent in quiescent cells, therefore being used as a proliferation marker (Yerushalmi et al., 2010; Varela-Nallar et al., 2014), while Nestin is a class VI intermediate filament protein that serves as a predominate marker for neural stem and progenitor cells (Messam et al., 2000; Wu et al., 2013). Hence, the increased levels of Ki-67 or Nestin-immunoreactive cells in the SVZ of the ischemia-treated rats indicated endogenous neural stem cells response 7 days following MCAO. After morroniside administration, this increase in the number of Ki-67 or Nestin-postive cells was enhanced. Morever, the distribution of Nestin-immunoreactive cells of ischemia-treated rats was observed not only in the lateral ventricle wall and the dorsolateral corner of the SVZ, but also in the peri-infarct cortex. The results indicate that morroniside promote neurogenesis post-ischemia, and the upregulation of these neural progenitor cells is thought to contribute to functional recovery. Several studies have addressed the role of Wnt/β-catenin signaling in adult neurogenesis, mediating cell survival, proliferation of neural progenitor cells and differentiation into neuron (Inestrosa and Arenas, 2010; Kuwabara et al., 2009). It has been also previously reported that components of the canonical Wnt/βcatenin pathway are expressed within the adult SVZ and SGZ (Adachi et al., 2007), and Wnt/β-catenin signals activate downstream target genes to promote adult neurogenesis (Kuwabara et al., 2009; Lie et al., 2005). Growing evidence from recent studies suggests that this pathway is upregulated in the SVZ following
stroke and may exert an important role in ischemia-induced neurogenesis (Lei et al., 2008; Mastroiacovo et al., 2009). The results in the present research show that the Wnt signaling pathway is activated in the early phase of ischemia and is limited to 24 h; however, its activation extends to 7 days after treatment with morroniside. The absence of any effects on the shamoperated rats suggests that morroniside may not directly act on Wnt signaling, while it may only exert its activation role on the ischemic-injured brain. Thus, morroniside may function as a stem cell proliferation enhancer rather than an initiator. In order to confirm the effects and possible mechanisms of morroniside on neurogenesis, we further studied the changes of the expression of downstreams factors in the Wnt/β-catenin signaling pathway 7 days post-ischemia. Transcription factors Pax6, Tbr2 and Ngn2 have been demonstrated to function as downstream mediator of Wnt-induced neurogenesis from adult hippocampal neural progenitors (Cho and Dressler, 1998; Faigle and Song, 2013). Kohwi et al. (2005) found that transcription factor Pax6 was expressed in most proliferating SVZ progenitors but only in a subpopulation of migrating neuroblasts, promoting neuronal differentiation. The studies on Pax6 mutant Small Eye mice have shown that Pax6 plays an important role in the regulation of forebrain development by Wnt–Tcf signaling pathway (Cho and Dressler, 1998), and regulates the differentiation of specific subtypes of interneurons in the olfactory bulb (OB) (Ninkovic et al., 2010). Tbr2, a pro-neurogenic T-box transcription factor expressed by intermediate neuronal progenitors (INPs), has been shown to be critically required for neurogenesis in the adult subventricular zone (SVZ) and in the dentate gyrus of adult mice
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Q3 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
(Brill et al., 2009). Functional inactivation of Tbr2 consistently results in increased proliferation of NSCs, but in the long-time absence of Tbr2, neurogenesis is halted as the result of failed neuronal differentiation (Hodge et al., 2012). There is a crosstalk between Pax6 and Tbr2 that down-regulation of Pax6 upregulates Tbr2 expressions in olfactory bulb mitral cells in developing neocortex while overexpression of Pax6 leads the postmitotic cells differentiate into interneurons (Bayatti et al., 2008; Imamura and Greer, 2013). Ngn2 is a member of the basic helix-loop-helix (bHLH) transcription factors, and it is well known that Ngn2 is associated with neural differentiation and inhibits astrocytic differentiation. The previous studies have shown that β-catenin signaling could upregulate Ngn2 expression although the mechanism is still not clarified (Hong and Bain, 2012; Matsumoto et al., 2013). The results of this present study demonstrated that morroniside-treatment upregulated the expressions of Pax6 and Ngn2. It is indicated that the regulation of these factors might be involved in the augment of neural stem cells proliferation and next neuronal differention by morroniside through Wnt/β-catenin pathway. As to the negative-effects of morroniside on Tbr2, we considered it be associated with the complex expression patterns of Pax6 and Tbr2 (Bayatti et al., 2008; Muzio et al., 2002). In summary, morroniside improves the endogenous mechanisms of neurogenesis for brain recovery following ischemic stroke. Morroniside could sustain the activation of Wnt/β-catenin signaling pathway to enhance the proliferation of neural stem cells 7 days post-ischemia, and could regulate the expression of downstream transcription factors Pax6 and Ngn2 to promote neurogenesis. Though the exact correlation between the Wnt/β-catenin signaling pathway and neurogenesis promoted by morroniside still needs to be fully defined, these results suggest that morroniside offers a prospective medicine for stroke recovery. One caveat remains, i.e. the temporal relationship between acute neuroprotection and delayed neurorepair. In this initial study, treatments with morroniside were started at 3 h after MCAO. Thus, overlap between its known acute protective effects and our present findings of promoting neurogenesis cannot be fully separated, and ultimately, the correlation between delayed neurorepair effect and whole-animal neurological function still needs to be fully defined. Acknowledgments This work was supported by the National Science and Technology Major Project (2012ZX09102201-106); National Natural Science Foundation of China (81173575, 81373994, 30973893 and 81274173); Beijing Natural Science Foundation (7102077 and 7211017); Beijing Municipal Commission of Education & Capital Medical University Base-Technological Innovation Platform Fund (111219); Beijing Health System Experts of High-level grant (20113-097); China Postdoctoral Science Foundation funded project and Beijing Postdoctoral Science Foundation funded project to FL Sun. We also express our appreciation to Professor Deyu Guo and Yumin Luo with their colleagues for their kind help on the MCAO model-required technical assistance. References Adachi, K., Mirzadeh, Z., Sakaguchi, M., Yamashita, T., Nikolcheva, T., Gotoh, Y., Peltz, G., Gong, L., Kawase, T., Alvarez-Buylla, A., Okano, H., Sawamoto, K., 2007. Betacatenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone. Stem Cells25, 2827–2836. Baarsma, H.A., Konigshoff, M., Gosens, R., 2013. The WNT signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets. Pharmacol. Ther. 138, 66–83. Bayatti, N., Sarma, S., Shaw, C., Eyre, J.A., Vouyiouklis, D.A., Lindsay, S., Clowry, G.J., 2008. Progressive loss of PAX6, TBR2, NEUROD and TBR1 mRNA gradients
7
correlates with translocation of EMX2 to the cortical plate during human cortical development. Eur. J. Neurosci. 28, 1449–1456. Belayev, L., Alonso, O.F., Busto, R., Zhao, W., Ginsberg, M.D., 1996. Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. Stroke 27, 1616–1622. Brill, M.S., Ninkovic, J., Winpenny, E., Hodge, R.D., Ozen, I., Yang, R., Lepier, A., Gascon, S., Erdelyi, F., Szabo, G., Parras, C., Guillemot, F., Frotscher, M., Berninger, B., Hevner, R.F., Raineteau, O., Gotz, M., 2009. Adult generation of glutamatergic olfactory bulb interneurons. Nat. Neurosci. 12, 1524–1533. Cho, E.A., Dressler, G.R., 1998. TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs. Mech. Dev. 77, 9–18. Clevers, H., 2006. Wnt/beta-catenin signaling in development and disease. Cell 127, 469–480. Delavaran, H., Sjunnesson, H., Arvidsson, A., Lindvall, O., Norrving, B., van Westen, D., Kokaia, Z., Lindgren, A., 2013. Proximity of brain infarcts to regions of endogenous neurogenesis and involvement of striatum in ischaemic stroke. Eur. J. Neurol. 20, 473–479. Faigle, R., Song, H., 2013. Signaling mechanisms regulating adult neural stem cells and neurogenesis. Biochim. Biophys. Acta 1830, 2435–2448. Hodge, R.D., Nelson, B.R., Kahoud, R.J., Yang, R., Mussar, K.E., Reiner, S.L., Hevner, R. F., 2012. Tbr2 is essential for hippocampal lineage progression from neural stem cells to intermediate progenitors and neurons. J. Neurosci. 32, 6275–6287. Hong, G.M., Bain, L.J., 2012. Arsenic exposure inhibits myogenesis and neurogenesis in P19 stem cells through repression of the beta-catenin signaling pathway. Toxicol. Sci. 129, 146–156. Imamura, F., Greer, C.A., 2013. Pax6 regulates Tbr1 and Tbr2 expressions in olfactory bulb mitral cells. Mol. Cell. Neurosci. 54, 58–70. Inestrosa, N.C., Arenas, E., 2010. Emerging roles of Wnts in the adult nervous system. Nat. Rev. Neurosci. 11, 77–86. Kohwi, M., Osumi, N., Rubenstein, J.L., Alvarez-Buylla, A., 2005. Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb. J. Neurosci. 25, 6997–7003. Kuwabara, T., Hsieh, J., Muotri, A., Yeo, G., Warashina, M., Lie, D.C., Moore, L., Nakashima, K., Asashima, M., Gage, F.H., 2009. Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nat. Neurosci. 12, 1097–1105. Lei, Z.N., Zhang, L.M., Sun, F.Y., 2008. Beta-catenin siRNA inhibits ischemia-induced striatal neurogenesis in adult rat brain following a transient middle cerebral artery occlusion. Neurosci. Lett. 435, 108–112. Lie, D.C., Colamarino, S.A., Song, H.J., Desire, L., Mira, H., Consiglio, A., Lein, E.S., Jessberger, S., Lansford, H., Dearie, A.R., Gage, F.H., 2005. Wnt signalling regulates adult hippocampal neurogenesis. Nature 437, 1370–1375. Marti-Fabregas, J., Romaguera-Ros, M., Gomez-Pinedo, U., Martinez-Ramirez, S., Jimenez-Xarrie, E., Marin, R., Marti-Vilalta, J.L., Garcia-Verdugo, J.M., 2010. Proliferation in the human ipsilateral subventricular zone after ischemic stroke. Neurology 74, 357–365. Mastroiacovo, F., Busceti, C.L., Biagioni, F., Moyanova, S.G., Meisler, M.H., Battaglia, G., Caricasole, A., Bruno, V., Nicoletti, F., 2009. Induction of the Wnt antagonist, Dickkopf-1, contributes to the development of neuronal death in models of brain focal ischemia. J. Cereb. Blood Flow Metab. 29, 264–276. Matsumoto, M., Imura, T., Fukazawa, T., Sun, Y., Takeda, M., Kajiume, T., Kawahara, Y., Yuge, L., 2013. Electrical stimulation enhances neurogenin2 expression through beta-catenin signaling pathway of mouse bone marrow stromal cells and intensifies the effect of cell transplantation on brain injury. Neurosci. Lett. 533, 71–76. Messam, C.A., Hou, J., Major, E.O., 2000. Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody. Exp. Neurol. 161, 585–596. Muzio, L., Di Benedetto, B., Stoykova, A., Boncinelli, E., Gruss, P., Mallamaci, A., 2002. Conversion of cerebral cortex into basal ganglia in Emx2(-/-) Pax6(Sey/Sey) double-mutant mice. Nat. Neurosci. 5, 737–745. Navarro-Sobrino, M., Rosell, A., Hernandez-Guillamon, M., Penalba, A., Boada, C., Domingues-Montanari, S., Ribo, M., Alvarez-Sabin, J., Montaner, J., 2011. A large screening of angiogenesis biomarkers and their association with neurological outcome after ischemic stroke. Atherosclerosis 216, 205–211. Ninkovic, J., Pinto, L., Petricca, S., Lepier, A., Sun, J., Rieger, M.A., Schroeder, T., Cvekl, A., Favor, J., Gotz, M., 2010. The transcription factor Pax6 regulates survival of dopaminergic olfactory bulb neurons via crystallin alpha. Neuron 68, 682–694. Ohira, K., Furuta, T., Hioki, H., Nakamura, K.C., Kuramoto, E., Tanaka, Y., Funatsu, N., Shimizu, K., Oishi, T., Hayashi, M., Miyakawa, T., Kaneko, T., Nakamura, S., 2010. Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells. Nat. Neurosci. 13, 173–179. Shruster, A., Ben-Zur, T., Melamed, E., Offen, D., 2013. Wnt signaling enhances neurogenesis and improves neurological function after focal ischemic injury. PloS One 7, e40843. Varela-Nallar, L., Rojas-Abalos, M., Abbott, A.C., Moya, E.A., Iturriaga, R., Inestrosa, N. C., 2014. Chronic hypoxia induces the activation of the Wnt/β-catenin signaling Q4 pathway and stimulates hippocampal neurogenesis in wild-type and APPswePS1ΔE9 transgenic mice in vivo. Front Cell Neurosci., 8, http://dx.doi.org/ 10.3389/fncel.2014.00017. Wang, W., Huang, W., Li, L., Ai, H., Sun, F., Liu, C., An, Y., 2008. Morroniside prevents peroxide-induced apoptosis by induction of endogenous glutathione in human neuroblastoma cells. Cell. Mol. Neurobiol. 28, 293–305. Wang, W., Sun, F., An, Y., Ai, H., Zhang, L., Huang, W., Li, L., 2009. Morroniside protects human neuroblastoma SH-SY5Y cells against hydrogen peroxideinduced cytotoxicity. Eur. J. Pharmacol. 613, 19–23.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i
8
1 2 3 4 5 6 7
F.-L. Sun et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Wang, W., Xu, J., Li, L., Wang, P., Ji, X., Ai, H., Zhang, L., Li, L., 2010. Neuroprotective effect of morroniside on focal cerebral ischemia in rats. Brain Res. Bull. 83, 196–201. Wu, M.D., Montgomery, S.L., Rivera-Escalera, F., Olschowka, J.A., O’Banion, M.K., 2013. Sustained IL-1beta expression impairs adult hippocampal neurogenesis independent of IL-1 signaling in nestin þ neural precursor cells. Brain Behav. Immun. 32, 9–18.
Yemisci, M., Gursoy-Ozdemir, Y., Vural, A., Can, A., Topalkara, K., Dalkara, T., 2009. Pericyte contraction induced by oxidative–nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat. Med. 15, 1031–1037. Yerushalmi, R., Woods, R., Ravdin, P.M., Hayes, M.M., Gelmon, K.A., 2010. Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 11, 174–183.
Please cite this article as: Sun, F.-L., et al., Promoting neurogenesis via Wnt/β-catenin signaling pathway accounts for the neurorestorative effects of morroniside.... Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.019i