- Email: [email protected]
or tetramethylpyrazine in cerebral ischemic injury in vivo and in vitro
brain research 1488 (2012) 81–91
Available online at www.sciencedirect.com
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Research Report
Neuroprotective effects of tanshinone IIA and/or tetramethylpyrazine in cerebral ischemic injury in vivo and in vitro Qiqiang Tanga,1, Ruodong Hana,1, Han Xiaoa, Jilong Shenb, Qingli Luob, Jun Lic,n a
Department of Neurology Disease, Province Affiliated Hospital, Anhui Medical University, Hefei 230022, Anhui, China Institute of Clinical Pharmacology, Anhui Medical University, the Key Laboratories of Zoonoses and Pathogen Biology, Hefei 230022, Anhui, China c School of Pharmacy, Anhui Medical University, No. 69 Meishan Road, Hefei 230032, Anhui, China b
art i cle i nfo
ab st rac t
Article history:
The present study compared the potential neuroprotective effects of tanshinone (Tan) IIA
Accepted 21 September 2012
monotherapy, tetramethylpyrazine (TMP) monotherapy, and Tan IIAþTMP combination
Available online 11 October 2012
therapy in adult rat subjected to cerebral ischemic injury using the permanent middle
Keywords:
cerebral artery occlusion (MCAO) model and in primary cortical neuron culture exposed to
Tanshinone
oxygen-glucose deprivation (OGD) model. Male Sprague Dawley rats (n¼ 84) were randomly
Tetramethylpy
divided into sham-operated, MCAO, cmc-Na (sodium carboxymethyl cellulose), TMP, Tan
Middle cerebral artery
IIAþTMP, and Tan IIA groups. In agreement with the in vivo experiment, primary cortical
occlusion (MCAO)
neuron culture was prepared from one-day-old SD rats and grouped according to exposure:
Neuroprotection
normoxia control (NC), OGD, dimethyl sulfoxide, TMP, Tan IIAþTMP, and Tan IIA groups. The neurological deficits and infarct volume were evaluated at 24 h after the MCAO models. Oxidative stress (malondialdehyde, glutathione, and superoxide dismutase) and intracellular [Ca2þ]i concentration were measured through spectrophotometric analysis. Neurocyte apoptosis and viability were respectively evaluated through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay, respectively. Apoptosis factors (Bax, Bcl-2, caspase-3, and trmp-7) were analyzed using western blot and immunohistochemistry. The results suggest that Tan IIAþTMP combination therapy was more effective than TMP monotherapy but not Tan IIA monotherapy. Tan IIA monotherapy is more effective than TMP monotherapy in protecting the neuron against hypoxia/ischemia both in vitro and in vivo. Interestingly, Tan IIA significantly increased the phosphorylation of AKT in primary cortical neuronal culture exposed to OGD, which was abolished by PI3K inhibitor LY294002. The PI3K/AKT signaling pathway may be involved in the neuroprotective mechanism of Tan IIA on primary cortical neurons. & 2012 Elsevier B.V. All rights reserved.
n
Corresponding author. Fax: þ86 551 5161001. E-mail address: [email protected] (J. Li). 1 Co-first authors. (Qiqiang Tang); (Ruodong Han).
0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.09.034
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1.
brain research 1488 (2012) 81–91
Introduction
Stroke is one of the most serious diseases that threaten the human health. The number of patients who suffer from new stroke cases is estimated to reach more than 200 million every year and almost 150 deaths annually. Cerebral ischemia accounts for 87% of all stroke cases (Rosamond et al., 2008) and is one of the major causes of death and subsequent sequela (Rathore et al., 2002; Suethanapornkul et al., 2008; Urban et al., 2010). Various therapeutic strategies have been used to prevent the neuronal damage of ischemic brain injury. In traditional Oriental medicine, Tanshinone IIA (Tan IIA) and tetramethylpyrazine (TMP) are widely used in the neurovascular and cardiovascular diseases such as ischemic stroke by Chinese herbalists. Recent studies have shown that these two drugs have been proposed as potential therapeutic agents in heart diseases, tumor, disease of heart head blood-vessel, liver diseases (Dai et al., 2012; Dong et al., 2011; Gao et al., 2012; Peng et al., 1992; Xu et al., 2007). Tan IIA is a lipid-soluble active compound extracted from the rhizome of herb miltiorrhiza. Similarly, TMP is one of the most important active ingredients in traditional Chinese herbal medicine. Synergistically, the combined therapy using Tan IIA and TMP is commonly applied in the treatment of ischemic stroke in traditional Chinese herbal medicine. Tan IIA and TMP have been separately proven to protect the brain from ischemic injury (Dong et al., 2009; Liao et al., 2004), which may be associated with their potential anti-inflammatory, anti-oxidant, and anticell apoptotic properties (Chen et al., 2007; Hintz and Ren, 2003; Liu et al., 2005). However, whether the neuroprotective effect of combination therapy (Tan IIA plus TMP) on brain injury is superior to monotherapy (Tan IIA or TMP) is yet to be determined. The exact mechanism of its neuroprotective effect in cerebral ischemic diseases remains unclear. PI3K/AKT plays an important role in facilitating cellular processes by activating various anti-apoptotic pathways. The overexpression of AKT can markedly reduce infarct volume
(Ohba et al., 2004). Furthermore, the activation of AKT has been demonstrated to provide protection against ischemic injury (Pignataro et al., 2008). Numerous neuroprotectants, including vascular endothelial growth factor, glial cell line-derived neurotrophic factor, human neuronal glial, and erythropoietin (Xu et al., 2008), exert their protective effects through the PI3K/AKT pathway. Considering the neuroprotective role of the PI3K/AKT pathway in ischemic brain injury, we speculated that PI3K/AKT signaling is involved in the neuroprotective mechanisms of Tan IIA. In this study, the protein expressions of AKT and p-AKTwere explored in oxygen-glucose deprivation (OGD) models. Moreover, we explored the neuroprotective effect of TMP monotherapy, Tan IIA monotherapy, and Tan IIAþTMP combination therapy against brain injury induced by middle cerebral artery occlusion (MCAO) and OGD models.
2.
Results
2.1.
Neurological deficits
Neurological deficits were assessed at 24 h after sham operation. The sham-operated rats achieved a score of 0 in the four-score system. The neurological deficit score was significantly higher in the MCAO rats (2.9370.30) than in the shamoperated ones (Po0.01). The groups treated with 40 mg/kg TMP, 40 mg/kg TMPþ30 mg/kg Tan IIA, and 30 mg/kg Tan IIA exhibited remarkably reduced neurological deficit scores from 2.9370.30 to 2.4870.10, 2.0570.19, and 2.1970.37, respectively (Fig. 1A).
2.2.
Infarct volume
The effects of TMP (40 mg/kg/day) and/or Tan IIA (30 mg/kg/ day) on brain infarct volume are shown in Fig. 1B. No increase in infarct volume was observed in the sham-operated rats. A significantly higher infarct ratio was noted after 24 h of
Fig. 1 – In the MCAO model, treatment with Tan IIA and/or TMP (with a dose of 30 and 40 mg/kg/day respectively, i. p. 3 days prior to induction of operation and continued for next 1 day following the operation) significantly improve the neurological function (A. n ¼14 per group, Po0.05) and attenuated the infarct compared ratio (B. n¼ 4 per group, Po0.05). Values given are the mean 7S.E.M. np-value o0.05 as with sham-operation, #p-valueo0.05 as compared with MCAO, and p-value o0.05 as compared with TMP.
brain research 1488 (2012) 81–91
suffering from ischemia in the MCAO group (38%70.82%). The treatment with TMP, TMPþTan IIA, and Tan IIA significantly decreased the infarct volume to 34.75%70.95%, 27.25%70.96%, and 27.50%71.29%, respectively, compared with those in the MCAO groups (Po0.05).
2.3.
Oxidative stress level
We further investigated the effect of TMP (40 mg/kg/day) and/ or Tan IIA (30 mg/kg/day) on oxidative stress by evaluating malondialdehyde (MDA) content, glutathione (GSH) activity, and superoxide dismutase (SOD) activity in the brain (Fig. 2A–C). In contrast to the sham-operated rats, a significant increase in MDA contents was observed in the MCAO rats (Po0.05). The activity levels of GSH and SOD in the MCAO rats significantly decreased compared with the shamoperated rats (Po0.05). In the TMP- and/or Tan IIA-treated groups after MCAO for 24 h, the MDA level was significantly decreased, whereas the GSH and SOD activity was increased (Po0.05).
2.4.
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Intercellular free [Ca2þ]i concentration
Compared with sham-operated rats, the concentration of intercellular free [Ca2þ]i in MCAO increased from 391.87 74.64 to 774.07112.38 (Fig. 2D; Po0.05). The concentration of [Ca2þ]i in MCAO was then decreased to 573.6761.68, 446.2742.81, and 432.2749.91 in the groups treated with TMP (40 mg/kg/day), TMP (40 mg/kg/day)þTan IIA (30 mg/kg/ day), and Tan IIA (30 mg/kg/day), respectively (Po0.05 as compared with MCAO rats).
2.5. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The apoptotic cells caused by MCAO were determined using the TUNEL assay. As shown in Fig. 3E, the increased number in TUNEL positive cell death induced by MCAO (79.273.27) was significantly reduced after treatment with TMP (38.271.48), TMPþTan IIA (3272.92), and Tan IIA (3071.58).
Fig. 2 – In the MCAO model, effect of Tan IIA and/or TMP treatment (with a dose of 30 and 40 mg/kg/day respectively, i. p. 3 days prior to induction of operation and continued for next 1 days following the operation) on the levels of (A) MDA content, (B) SOD activity, (C) GSH activity and (D) Intercellular free [Ca2þ]i concentration. Values expressed as mean 7S.E.M, n¼ 5 per group. np-value o0.05 as compared with sham-operation, #p-valueo0.05 as compared with MCAO, and p-value o0.05 as compared with TMP.
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Fig. 3 – In the MCAO model, effect of Tan IIA and/or TMP treatment (with a dose of 30 and 40 mg/kg/day respectively, i. p. 3 days prior to induction of operation and continued for next 1 days following the operation) on the expression levels of Bax, Bcl-2, Caspase-3, TRPM-7 proteins by western blot analysis (Fig. 3A–D) and IHC (Fig. 3F and G). In Fig. 3-F and G, the picture shows hippocampal region of brain tissue cells in MCAO model, of which the positive cell was presented brown in membrane or cytoplasm. Datas were expressed as mean7S.E.M, Significance of differences was evaluated by one-way analysis of variance. n¼ 5 per group. np-value o0.05 as compared with sham-operation, #p-valueo0.05 as compared with MCAO, and p-valueo0.05 as compared with TMP.
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Quantitative analyses of cell death in different culture models were performed using the MTT assay (Fig. 4E). The neurons exposed to OGD indicated only 42.2% viability (% of NC group). TMP and/or Tan IIA treatment protected the neurons against cell death induced by OGD from 42.2%77.94% to 79.8%73.03%, 89.80%73.11%, and 91.4%7 3.84% in the groups treated with 10 mmol TMP, TMP (10 mmol)þTan IIA (8 mmol), and 8 mmol Tan IIA, respectively (Po0.05 as compared with the OGD group).
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2.6. Western blot analysis for Bax, Bcl-2, Caspase-3, and TRPM-7 The western blot results of MCAO-induced cerebral tissues are shown in Fig. 3A–D. Tan IIA can function in various cancer types as one of the underlying mechanisms in the antiapoptotic effect by inhibiting Bax and/or increasing the levels of anti-apoptotic Bcl-2 (Sukumari-Ramesh et al., 2011). The anti-apoptotic role of Tan IIA was also observed in this study.
Fig. 4 – In primary cortical neurons culture models, expression of (A)Bax, (B)Bcl-2, (C)Caspase-3 and (D)TRPM-7 proteins were determined by western blotting, with results normalized relative to b-actin. Cell viability were evaluated by MTT assay. Datas were expressed as % NC. n¼ 5 per group. np-valueo0.05as compared with sham-operation, #p-valueo0.05 as compared with MCAO, and p-value o0.05 as compared with TMP.
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That is, the MCAO-induced ischemic brain injury was accompanied by a decrease in the expression of Bcl-2 and an increase in the Bax expression (Po0.05 compared with the sham-operated rats). Treatment with TMP and/or Tan IIA inhibited the decrease in Bcl-2 expression but suppressed the increase in Bax expression. We also measured the protein expression of TRPM-7 and caspase-3. Furthermore, MCAO significantly increased the expression of active caspase-3 (17 kDa) and TRPM-7 (216 kDa) compared with the levels obtained from the sham-operated group (Po0.05). TMP and/or Tan IIA treatment can attenuate the increased caspase-3 and TRPM-7 induced by MCAO.
2.7.
Immunohistochemistry
The results from the immunohistochemistry experiments revealed a significant increase in the immunoreactivity of Bax, caspase-3, and TRPM-7 protein, whereas a decrease in Bcl-2 was observed in the ischemic penumbral region of the MCAO rats (Po0.05; compared with the sham-operated rats). Treatment with TMP and/or Tan IIA significantly reduced the immunoreactivity of Bax, caspase-3, and TRPM-7. However, an increased immunoreactivity in Bcl-2 was observed (Fig. 3G and F). Similar to the results of western blot, Tan IIA monotherapy indicated the remarkable effect compared with the other treatments. TRPM-7 was found residing on the body and associated processes in cortical neurons, whereas Bax and Bcl-2 were found in the neuronal cytolemma rather than the cytoplasm.
2.8. Western blot analysis for Bax, Bcl-2, caspase-3, and TRPM-7 The expression levels of Bax, Bcl-2, caspase-3, and trmp-7 protein were examined through western blot of the following groups: NC, OGD, TMP, TMPþTan IIA, and Tan IIA treatment
groups. Relative to the NC group, the expression of pro-apoptotic markers (Bax and caspase-3) in the OGD group was significantly increased, whereas that of the antiapoptotic marker (Bcl-2) was reduced. Neuronal cultures were incubated with TMP and/or Tan IIA 30 min before and 2 and 4 h after exposure to OGD. This procedure was performed to address the possible role of TMP and/or Tan IIA in the regulation of Bax, Bcl-2, and caspase-3. As shown in Fig. 4A–D, Bax, Bcl-2, caspase-3, and TRPM-7 proteins could be significantly affected by the TMP and/or Tan IIA treatment. Recently, TRPM-7 has been reported capable of mediating the dominant damage mechanism of antiexcitotoxic therapy in unmasked anoxic neurons (Aarts et al., 2003). Increased TRPM-7 was also observed in the OGD group (Po0.05; compared with the NC group). Interestingly, the increased TRPM-7 induced by OGD could be reduced with TMP and/or Tan IIA treatment. Tan IIA monotherapy elicited a better effect than the other treatments (Fig. 4A–D).
2.9.
Recently, TRPM-7 has been recognized as a nonselective cation channel and has been reported to play a dominant role in anoxic neuronal damage. PI3K/AKT pathways have been reported to participate in regulating the gene expression of apoptotic and anti-apoptotic molecules against ischemia (Saito et al., 2004; Tabakman et al., 2005). We demonstrated that Tan IIA monotherapy is more effective than TMP monotherapy (Po0.05) and that no significant difference was found between Tan IIA monotherapy and TMPþTan IIA combination therapy (P40.05). We also explored whether the PI3K/AKT signaling pathway contributed to the protective effect of Tan IIA in cortical neuronal cultures after OGD. The inhibitor LY294002 (10 mmol) was separately co-incubated and solo incubated with Tan IIA in the OGD group for 30 min. The expressions of AKT and p-AKT proteins were evaluated using western blot in cultures (Fig. 5A and B). The protein expression of p-AKT in the OGD group was markedly decreased compared with that in the NC group (Po0.05). Tan IIA with a concentration of 8 mmol significantly increased p-AKT protein expression compared with the OGD group (Po0.05). However, the effect of Tan IIA on p-AKT protein could be reversed with the treatment of PI3K inhibitor LY294002. The p-AKT protein level in the OGDþLY294002 group was also reduced by the PI3K inhibitor LY294002 (Po0.05; compared with the OGD group). Otherwise, the p-AKT protein expression in the OGDþTan IIAþLY group was higher than that in the OGDþLY group (Po0.05; Fig. 5A and B).
3.
Fig. 5 – In primary cortical neurons cultures, the effects of LY294002 (8lM) inhibitor on AKT and p-AKT proteins by western blotting and densitometric analysis gave the related ratio of b-actin. Datas expressed as mean 7S.E.M. n p-value o0.05 as compared with NC group, # p-value o0.05 as compared with OGD groups. and p-valueo0.05 as compared with TSA (8 lM) groups.
PI3K/AKT in neuronal culture
Discussion
The present study systematically evaluated the therapeutic neuroprotective effect of Tan IIA monotherapy, TMP monotherapy, and Tan IIAþTMP combination therapy in rats with ischemic brain injury induced by MCAO (with 30 mg/kg/day of Tan IIA and 40 mg/kg/day of TMP) and neuronal cultures exposed to OGD (8 mmol of Tan IIA and 10 mmol of TMP). Two new findings were revealed from this study. First, Tan IIAþTMP combination therapy was more effective than TMP
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monotherapy but not Tan IIA monotherapy. Tan IIA monotherapy is superior to TMP monotherapy in protecting the neuron against hypoxia/ischemia both in vitro and in vivo. Second, the neuroprotective role of Tan IIA monotherapy may be mediated by regulating the PI3k/AKT signaling pathway because of an increase in p-AKT phosphorylation levels. Measurement of infarct volume is one of the tools in evaluating the functional outcome after suffering from stroke. In rats, administering both Tan IIA and TMP can decrease the infarct volume in a dose-dependent manner in MCAO models (Liao et al., 2004). In addition, neurological scores can also improve as ischemia progresses (Dong et al., 2009). In the present study, the increased infarct volume and improved neurological scores induced 24 h after MCAO was significantly reversed with TMP and/or Tan IIA treatment (Po0.05; compared with the MCAO group). Furthermore, TMPþTan IIA combination therapy is superior to TMP monotherapy but not Tan IIA monotherapy. Tan IIA monotherapy resulted in a more significant decrease in infarct volume and neurological scores (Po0.05; compared with TMP monotherapy; Fig. 1A and B). The potential mechanism of ischemic brain injury remains unclear. However, neuronal apoptosis is one of the major pathogenic mechanisms that govern ischemic brain injury. The increased activation of oxidative stress, sustained increase in [Ca2þ]i levels, and a series of apoptosis-related proteins contributed to the pathogenic processes of neuronal death following ischemia, which ultimately lead to cell death (Mattson, 2000). Various drugs have been used in the treatment of ischemic brain injury in MCAO rats or in neurons exposed to OGD to elicit effects on oxidative stress, [Ca2þ]i, apoptotic factors, and neuronal apoptosis. In the present study, the increased MDA content as well as the decreased GSH and SOD activities induced in MCAO were significantly reversed with the treatment of Tan IIA monotherapy, TMP monotherapy, and Tan IIAþTMP combination therapy (Po0.05; compared with the MCAO group). This finding indicates that Tan IIA and TMP may exhibit a direct effect on oxidative stress. The increased [Ca2þ]i and the number of neuronal apoptosis induced by ischemic brain injury were markedly decreased with TMP monotherapy, Tan IIA monotherapy, and TMPþTan IIA combination therapy in vivo (Po0.05; compared with MCAO). Caspase-3, as one of the caspase family members, plays an important role in medicating cell apoptosis (Watanabe et al., 2004). However, Bcl-2 decreases cell apoptosis by inhibiting caspase-3 activity. Bcl-2 is enzymolied to shorter fragments (22 kDa), and its role as an anti-apoptotic agent is reversed to apoptotic while caspase-3 becomes activated (Fu and Fan, 2002). Obviously, an association exists between Bcl-2 and caspase-3 family in modulating apoptosis. In the present study, TMP and/or Tan IIA treatment can also markedly interfere the increased pro-apoptosis factors Bax and caspase-3, the decreased anti-apoptosis factor Bcl-2, and the increased TRPM-7 protein induced by ischemic brain injury in vivo and in vitro (Po0.05; compared with MCAO/OGD). Similar to previous findings, the results of the present study demonstrate the potential character of TMP and Tan IIA to induce anti-apoptotic effects (Dong et al., 2009; Liao et al., 2004). TRPM-7 is a ubiquitous divalent-selective channel with its own kinase domain, and the knockdown of TRPM-7 in cultured neurons can lead to anoxic cell death in cortical neurons (Aarts et al., 2003; Kraft and Harteneck, 2005). Wei et al. demonstrated the
87
expression of TRPM-7 channels in neurons and identified them as key mediators of OGD-induced neuronal death (Sun et al., 2009). The finding may lead to new insights for further development of neuroprotective therapies. The results further suggest that TRPM-7 may be involved in the neuroprotective effects of Tan IIA and TMP. The Bcl-2 protein family members are potent regulators of apoptosis. The anti-apoptotic Bcl-2 and pro-apoptotic Bax also belong to the Bcl-2 protein family. Bcl-2/ Bax ratio is often used to represent the extent of apoptosis (Gallogly et al., 2010). Tan IIAþTMP combination therapy produced no significant effect on increasing Bcl-2 compared with TMP monotherapy. A more significant effect on increasing the level of Bax/Bcl-2 ratio was demonstrated in Tan IIAþTMP combination therapy (Po0.05; compared with the TMP monotherapy). In traditional Chinese herbal medicine, a recommended strategy in the management of ischemic stroke is the combination therapy rather than monotherapy. However, our results demonstrated a different concept. In the present study, a series of bioindices was evaluated, including oxidative stress (MDA content, SOD activity, and GSH activity), apoptotic factors (Bax, Bcl-2, Bax/Bcl-2, and caspase-3), [Ca2þ]i content, neuronal apoptosis, and viability in both MCAO and OGD models. Tan IIAþTMP combination therapy was more effective than TMP monotherapy but not Tan IIA monotherapy. PI3K/AKT pathways play important roles in the regulation of cell survival and death (Arai and Lo, 2010; Langenbach and Rando, 2002; Zhang et al., 2010). They also participate in the regulation of apoptotic and anti-apoptotic molecules against ischemic injuries (Jiang et al., 2008). The results of the present study demonstrate that the neuroprotective effect of Tan IIA relies on the increased levels of phosphorylated AKT. The PI3K inhibitor LY294002 decreased the Tan II-mediated protection. This result indicates that the PI3K/Akt pathway plays a role in the neuroprotective effect of Tan II. Several issues could be addressed in this study. First, we speculated that extensive insights may be obtained from the ischemic reperfusion MCAO model with prolonged therapy because the neuroprotective effects of TMP and/or Tan IIA treatment were evaluated 24 h after permanent MCAO. Second, the therapeutic effect observed in stroke patients in clinical settings may directly provide evidence. Last, the characteristics of the metabolic pathway, half-life period, and medication may also contribute to the results of the present study. These issues should be addressed in future studies. The present study is the first to reveal that Tan IIAþTMP combination therapy is more effective than TMP monotherapy but not Tan IIA monotherapy. In addition, Tan IIA monotherapy exhibits a more effective neuroprotection than TMP monotherapy against hypoxia/ischemia both in vitro and in vivo. The present study also proves that PI3K/AKT signaling pathway is involved in the protective mechanism of Tan IIA on primary cortical neurons.
4.
Experimental procedures
4.1.
Experimental materials
Adult male rats (Sprague Dawley) weighing 200–220 g (Vital River Laboratory Animal Technology Co., Ltd. China) were
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used in this study. The experiments were conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, China. All experimental protocols were approved by the University Animal Care and Use Committee.
4.2.
MCAO model
Permanent cerebral ischemia was performed through the intraluminal suture occlusion based on previously reported procedures (Foley et al., 2010). In brief, the animals were anesthetized with 10% chloral hydrate. The temperature was maintained at 37.0 1C and controlled using a thermostatic blanket. A 6-0 nylon monofilament with a round tip (0.24–0.26 mm) was inserted into the right internal carotid artery via an external carotid artery stump, whereas the common carotid artery was occluded. Successful occlusion was verified using a laser Doppler flowmetry. The local cerebral blood flow in the right MCA territory declined to approximately 10–15% of the baseline. The sham-operated rats were treated similarly, except that the middle cerebral arteries were not occluded after the neck incision.
4.3.
4.5.
Oxygen-glucose deprivation model
OGD was induced as previously described (Aarts et al., 2003; Chen et al., 2011) with slight modification. The cells were fed with glucose-free DMEM and infused with 5% CO2 and 95% N2 at 37 1C for 1 h. The maintenance medium was used to terminate OGD. Primary NC neurons were maintained in DMEM containing 5% CO2 and 95% air. In all experiments, the pH of the medium was maintained at 7.2 under OGD conditions.
4.6. Treatments and study design in neuronal culture models
Study design in animal models
In this study, 84 rats were randomly grouped into six groups (n ¼14, each group): group 1, sham-operated (control); group 2, MCAO rats; group 3, cmc-Na treatment (MCAO rats treated with cmc-Na); group 4, TMP (MCAO rats treated with 40 mg/ kg/day TMP); group 5, Tan IIAþTMP (MCAO rats treated with 30 mg/kg/day Tan IIAþ40 mg/kg/day TMP); group 6, Tan IIA (MCAO rats treated with 30 mg/kg/day Tan IIA). In all animals, the treatments were given via intragastric administration. The drugs were dissolved in 0.5% cmc-Na and administered once a day for 5 d [i.e., 3 d before operation and 2 twice after operation (include treated 4 h and 24 h after surgery]. MCAO surgery was performed on day 3, 1 h after TMP and/or Tan IIA administration. The MCAO group received an equal volume of cmc-Na.
4.4.
arabinoside and maintained for 48 h. The medium was replaced with fresh neurobasal medium after 24 h, and half of the culture medium was changed every 2 d. Arabinosylcytosine (5 mg/mL) was added on the third day of incubation to prevent non-neuronal cell growth. The cultures were used for 8 d to 10 d in vitro. All drugs were diluted in DMSO and administered 30 min before and 2 and 4 h after exposure to OGD in neuronal culture. The OGD group received an equal volume of DMSO.
Cell culture and treatment
Primary culture of cortical neurons was isolated and cultured as previously described (Jiang et al., 2008). Briefly, cerebral cortical neurons were prepared from the cerebrum of oneday-old Sprague Dawley rats (Qi et al., 2010) and dissected in ice cold dissection buffer. After the meninges of the cortex were peeled off and separated from the olfactory bulb and hippocampus, the dissected cortical tissues were treated in supplemented Hank’s balanced salt solution for 25 min at 37 1C, followed by mechanical dissociation. The cells were digested with trypsin. Subsequently, the cells were centrifuged at 1000g for 10 min. The cell pellets were resuspended and cultivated in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) suplemented with 15% fetal bovine serum, 5% horse serum (Sigma, St. Louis, MO), and 100 U/mL penicillin/ streptomycin. Cortical neurons at a density of 1 105/mL were seeded onto plates pre-coated with poly-l-lysine (25 mg/mL) and placed in a humidified incubator (37 1C with 5% CO2). After 3 d, the cells were cultured with 1 mM cytosine
Primary cortical neuron cultures were grouped into six (n ¼4, each group): group 1, NC (cells exposed to normoxia control); group 2, OGD (cultures exposed to OGD); group 3, DMSO (cultures exposed to OGD and incubated with DMSO); group 4, TMP (cultures exposed to OGD and treated with 8 mmol TMP); group 5, Tan IIAþTMP (cultures exposed to OGD and treated with 8 mmol Tan IIAþ10 mmol TMP); group 6, Tan IIA (cultures exposed to OGD and treated with 8 mmol Tan IIA). In culture models, the drugs were diluted in DMSO of the same volume and administered 30 min before and 2 and 4 h after exposure to OGD in the neuronal culture. The OGD group received cortical neurons with an equal volume to DMSO. The regulatory effect of the PI3K/AKT pathway on Tan IIA neuronal protection in cortical neuronal cultures after OGD was assessed. The expressions of AKT and p-AKT proteins were evaluated in the culture before conducting the following experiments (five groups): group 1, NC (cells were exposed to normoxia control); group 2, OGD (cultures exposed to OGD); group 3, LY294002 (cultures exposed to OGD and incubated with 10 mmol LY294002 for 30 min); group 4, Tan IIA (cultures exposed to OGD A and incubated with 8 mmol Tan IIA for 30 min); group 5, Tan IIAþLY294002 (cultures exposed to OGD and incubated with 8 mmol Tan IIAþ10 mmol LY294002 for 30 min). These levels were normalized to NC (incubated in DMEM high-glucose medium and maintained at 5% CO2 and 95% N2 at 37 1C). Whole proteins were then isolated from cortical neuronal cultures.
4.7.
Neurological deficit examination
Neurological deficits were carried out 24 h after MCAO or sham-operation as previously reported (Longa et al., 1989) with slight modifications. A 0–4 point scale was used to evaluate the neurological deficits observed in all groups (rats subjected to sham operation or MCAO procedure): 0, no neurological symptoms; 1, unable to stretch the upper
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extremities completely; 2, rotation to the left; 3, unable to walk without help; and 4, no spontaneous motor activity.
4.8.
Evaluation of Infarct volume
2,3,5-Tripenyltetrazolium chloride (TTC) staining was carried out to evaluate the infarct volume. The rat’s brains were quickly removed and sectioned into 2 mm thick slices. Subsequently, cornel slices were immersed in 0.25% TTC at 37 1C for 30 min and then fixed in 10% phosphate-buffered formalin for 45 min (Liao et al., 2004). Areas of the infarcted regions were captured using a digital camera (AV-P960C, JVC, Japan) and analyzed with an image C morphology analysis system (Chansan Instrument Co. Shanghai, China). The results were represented as the percentage of the total volume.
4.9.
TUNEL staining
TUNEL methods were used to detect and quantify apoptotic cell death on paraffin-embedded sections using an In Situ Cell Death Detection Kit (Roche, Germany). According to the manufacturers’ instructions, the rats were sacrificed 24 h after MCAO. Subsequently, brain sections (10 mm) were prepared using the same methods as in immunohistochemistry. The cells displaying brown stain within the nucleus were counted as TUNEL-positive cells. The number of TUNELpositive cells was determined in three randomly selected microscopic eyeshots and expressed as percentage under high-power magnification. The apoptotic index¼ TUNELpositive cell number/the number of nucleated cells 100%.
4.10.
[Ca2þ]i measurement
Intercellular free [Ca2þ]i was determined using the fluorescence of fura-2 in a spectrofluorometer as described in a previous study (Vinje et al., 2002). Briefly, the cortical neurons were dissected and washed thrice with Hank’s solution. The tissues were homogenized and then centrifuged for 3 min at 3000g and the supernatant for 30 min at 15000g. The formed pellet was two-layered and diluted in calcium-free hepes-buffered medium (HBM) for the released experiments (suspension for the evaluation of [Ca2þ]i and the supernatant for MDA content, GSH activity, and SOD activity). Fura-2/AM was added to the suspension of tissue cell (whereas the neural cells were incubated in HBM containing fura-2/AM) to a final concentration of 5 mmol for 30 min at 37 1C (neural cells were incubated in CO2 at 37 1C for 30 min). Then, the cells were centrifuged, and the supernatant was washed twice with Hank’s solution containing 0.2% bovine serum medium and resuspended into 1 106/mL cell concentration. The fluorescence light (F) was monitored using a Hitachi Fluorescence Spectrophotometer as previously reported (Vinje et al., 2002). Maximum (F max) and minimum (F min) fluorescence were obtained after 0.5% sodium dodecyl sulfate and 7.5 mM EGTA were added, respectively. The level of [Ca2þ]i was calculated according to the following equation: [Ca2þ]i ¼ [(FF min)/(F maxF)] kd (224 nM) is the dissociation constant of fura-2 with calcium.
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4.11. Evaluation of oxidative stress (MDA, GSH-Px, and SOD) The suspension was used to evaluate [Ca2þ]i, and the supernatant was used to assess oxidative stress (MDA, GSH-Px, and SOD) with a spectrofluorometer according to the manufacturer’s instructions (Bora and Sharma, 2010). (1) The MDA content was evaluated by measuring the content of thiobarbituric acid (TBA) reactant substances according to the TBA test method with slight modifications. The optical density of the supernatant was determined using a spectrophotometer at l¼532 nm. The MDA content was expressed as nmol/mg tissue. (2) The activity of GSH was measured using the method described by Jollow et al. (1974). Equal quantity of brain homogenate was mixed with 10% trichloroacetic acid and centrifuged at 1200g for 15 min. Phosphate buffer (0.1 M) and 5-50 -dithiobis[2-nitrobenzoic acid] (100 mM in 0.1 M phosphate buffer) were added to the mixture. The mixture was vortexed, and the absorbance was determined at l ¼412 nm. The concentration of GSH was expressed as nmol/g of tissue. (3) The activity of SOD in the cerebral tissues of all the rats was estimated according to a previous study (Kakkar et al., 1984) based on the inhibition of the formation of nicotinamide adenine dinucleotide, phenazine methosulphate (PMS), and nitroblue tetrazolium (NBT) formazan. In brief, 10 mL of the homogenate was mixed with 90 mL of 30 mM sodium tetrapyrophosphate buffer (pH 8.3), 30 mL of 0.3 mM NBT, 10 mL of 0.96 mM PMS, and 40 mL of doubledistilled water. Absorbance was obtained at l¼ 560 nm. A single unit of enzyme was expressed as 50% inhibition of NBT reduction/min/mg protein. The results were expressed as a unit of SOD/mg protein.
4.12.
MTT assay
MTT solution was added to 96-well plates at a final concentration of 500 mg/mL and then incubated at 37 1C for 4 h. After the supernatant was removed, DMSO was added into each well, which was shaken for 10 min. The absorbance was obtained at 570 nm, and the survival rate (%) was determined and compared with the control group.
4.13.
Bax, Bcl-2, Caspase-3, and TRPM-7 western blot
The total proteins were separately extracted from the cortical neuronal tissues or cultures according to previous methods (Jiang et al., 2008; Mattsson et al., 2003). The protein concentration was measured using a BCA protein assay kit (Cwbio Ltd., China). Equal amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. After the membranes were blocked with 5% nonfat dry milk, they were incubated overnight at 4 1C with rabbit anti-b action (1:1000, cell signaling), rabbit anti-Bcl-2 (1:1000, cell signaling), rabbit anti-Bax (1:1000, cell signaling), rabbit anti-caspase-3 (1:1000, cell signaling), and mouse anti-trmp-7 (1:1000, Abcam, Hong Kong). HRP conjugate goat anti-rabbit IgG and HRP conjugate goat anti-mouse IgG were used as secondary antibodies (1:2500, Promega, USA). The reaction was developed using a chemiluminescent substrate (Thermo Scientific), and signal
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density was measured using Alpha Image. Equal protein loading was confirmed by measuring b-actin. Densitometric analysis was performed using NIH Image J analysis software (USA). The values were normalized with b-actin.
4.14. Bax, Bcl-2, Caspase-3, and TRPM-7 immunohistochemistry Immunohistochemistry analysis for the in situ expression of Bax, Bcl-2, caspase-3, and TRPM-7 proteins were performed as previously described (Kaundal and Sharma, 2011). The sections were dewaxed with xylene, rehydrated through an alcohol gradient, and heated with 10 mm/L Na citrate (pH 6) in a microwave for 15 min to retrieve the antigenic determinants. The resulting product was treated with 1% H2O2 to inactivate endogenous peroxidase activity and was blocked with 5% goat serum in phosphate at 4 1C with the primary antibody in phosphate-buffered saline/2% goat serum. The following primary antibodies were used: anti-b-actin (1:1000, cell signaling), anti-Bcl-2 (1:200, Boster, China), anti-Bax (1:200, Boster, China), anti-caspase-3 (1:500, cell signaling), anti-TRPM-7 (1:200, ABCam, Hong Kong). The sections were treated with Vecta stain ABC kit (Vector Labs, Burlingame, CA, USA). The images were analyzed using Magna Fire SP 2.1 B software (USA).
4.15.
Statistical analysis
All data were expressed as mean7S.E.M, and the statistical significance was determined using one-way ANOVA. Statistical analysis was conducted using SPSS 11.5 (Chicago, Illinois, USA). A P-value of less than 0.05 was considered to indicate statistical significance.
Acknowledgments This work was supported by the Province Science Foundation of China (No. 090413120), Natural Science Foundation of the Higher Education Institutions of Anhui Province, China (KJ2007B147), and Medical Scientific Research Foundation of Anhui Province (2010B003) to Qiqiang Tang.
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Available online at www.sciencedirect.com
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Research Report
Neuroprotective effects of tanshinone IIA and/or tetramethylpyrazine in cerebral ischemic injury in vivo and in vitro Qiqiang Tanga,1, Ruodong Hana,1, Han Xiaoa, Jilong Shenb, Qingli Luob, Jun Lic,n a
Department of Neurology Disease, Province Affiliated Hospital, Anhui Medical University, Hefei 230022, Anhui, China Institute of Clinical Pharmacology, Anhui Medical University, the Key Laboratories of Zoonoses and Pathogen Biology, Hefei 230022, Anhui, China c School of Pharmacy, Anhui Medical University, No. 69 Meishan Road, Hefei 230032, Anhui, China b
art i cle i nfo
ab st rac t
Article history:
The present study compared the potential neuroprotective effects of tanshinone (Tan) IIA
Accepted 21 September 2012
monotherapy, tetramethylpyrazine (TMP) monotherapy, and Tan IIAþTMP combination
Available online 11 October 2012
therapy in adult rat subjected to cerebral ischemic injury using the permanent middle
Keywords:
cerebral artery occlusion (MCAO) model and in primary cortical neuron culture exposed to
Tanshinone
oxygen-glucose deprivation (OGD) model. Male Sprague Dawley rats (n¼ 84) were randomly
Tetramethylpy
divided into sham-operated, MCAO, cmc-Na (sodium carboxymethyl cellulose), TMP, Tan
Middle cerebral artery
IIAþTMP, and Tan IIA groups. In agreement with the in vivo experiment, primary cortical
occlusion (MCAO)
neuron culture was prepared from one-day-old SD rats and grouped according to exposure:
Neuroprotection
normoxia control (NC), OGD, dimethyl sulfoxide, TMP, Tan IIAþTMP, and Tan IIA groups. The neurological deficits and infarct volume were evaluated at 24 h after the MCAO models. Oxidative stress (malondialdehyde, glutathione, and superoxide dismutase) and intracellular [Ca2þ]i concentration were measured through spectrophotometric analysis. Neurocyte apoptosis and viability were respectively evaluated through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay, respectively. Apoptosis factors (Bax, Bcl-2, caspase-3, and trmp-7) were analyzed using western blot and immunohistochemistry. The results suggest that Tan IIAþTMP combination therapy was more effective than TMP monotherapy but not Tan IIA monotherapy. Tan IIA monotherapy is more effective than TMP monotherapy in protecting the neuron against hypoxia/ischemia both in vitro and in vivo. Interestingly, Tan IIA significantly increased the phosphorylation of AKT in primary cortical neuronal culture exposed to OGD, which was abolished by PI3K inhibitor LY294002. The PI3K/AKT signaling pathway may be involved in the neuroprotective mechanism of Tan IIA on primary cortical neurons. & 2012 Elsevier B.V. All rights reserved.
n
Corresponding author. Fax: þ86 551 5161001. E-mail address: [email protected] (J. Li). 1 Co-first authors. (Qiqiang Tang); (Ruodong Han).
0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.09.034
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1.
brain research 1488 (2012) 81–91
Introduction
Stroke is one of the most serious diseases that threaten the human health. The number of patients who suffer from new stroke cases is estimated to reach more than 200 million every year and almost 150 deaths annually. Cerebral ischemia accounts for 87% of all stroke cases (Rosamond et al., 2008) and is one of the major causes of death and subsequent sequela (Rathore et al., 2002; Suethanapornkul et al., 2008; Urban et al., 2010). Various therapeutic strategies have been used to prevent the neuronal damage of ischemic brain injury. In traditional Oriental medicine, Tanshinone IIA (Tan IIA) and tetramethylpyrazine (TMP) are widely used in the neurovascular and cardiovascular diseases such as ischemic stroke by Chinese herbalists. Recent studies have shown that these two drugs have been proposed as potential therapeutic agents in heart diseases, tumor, disease of heart head blood-vessel, liver diseases (Dai et al., 2012; Dong et al., 2011; Gao et al., 2012; Peng et al., 1992; Xu et al., 2007). Tan IIA is a lipid-soluble active compound extracted from the rhizome of herb miltiorrhiza. Similarly, TMP is one of the most important active ingredients in traditional Chinese herbal medicine. Synergistically, the combined therapy using Tan IIA and TMP is commonly applied in the treatment of ischemic stroke in traditional Chinese herbal medicine. Tan IIA and TMP have been separately proven to protect the brain from ischemic injury (Dong et al., 2009; Liao et al., 2004), which may be associated with their potential anti-inflammatory, anti-oxidant, and anticell apoptotic properties (Chen et al., 2007; Hintz and Ren, 2003; Liu et al., 2005). However, whether the neuroprotective effect of combination therapy (Tan IIA plus TMP) on brain injury is superior to monotherapy (Tan IIA or TMP) is yet to be determined. The exact mechanism of its neuroprotective effect in cerebral ischemic diseases remains unclear. PI3K/AKT plays an important role in facilitating cellular processes by activating various anti-apoptotic pathways. The overexpression of AKT can markedly reduce infarct volume
(Ohba et al., 2004). Furthermore, the activation of AKT has been demonstrated to provide protection against ischemic injury (Pignataro et al., 2008). Numerous neuroprotectants, including vascular endothelial growth factor, glial cell line-derived neurotrophic factor, human neuronal glial, and erythropoietin (Xu et al., 2008), exert their protective effects through the PI3K/AKT pathway. Considering the neuroprotective role of the PI3K/AKT pathway in ischemic brain injury, we speculated that PI3K/AKT signaling is involved in the neuroprotective mechanisms of Tan IIA. In this study, the protein expressions of AKT and p-AKTwere explored in oxygen-glucose deprivation (OGD) models. Moreover, we explored the neuroprotective effect of TMP monotherapy, Tan IIA monotherapy, and Tan IIAþTMP combination therapy against brain injury induced by middle cerebral artery occlusion (MCAO) and OGD models.
2.
Results
2.1.
Neurological deficits
Neurological deficits were assessed at 24 h after sham operation. The sham-operated rats achieved a score of 0 in the four-score system. The neurological deficit score was significantly higher in the MCAO rats (2.9370.30) than in the shamoperated ones (Po0.01). The groups treated with 40 mg/kg TMP, 40 mg/kg TMPþ30 mg/kg Tan IIA, and 30 mg/kg Tan IIA exhibited remarkably reduced neurological deficit scores from 2.9370.30 to 2.4870.10, 2.0570.19, and 2.1970.37, respectively (Fig. 1A).
2.2.
Infarct volume
The effects of TMP (40 mg/kg/day) and/or Tan IIA (30 mg/kg/ day) on brain infarct volume are shown in Fig. 1B. No increase in infarct volume was observed in the sham-operated rats. A significantly higher infarct ratio was noted after 24 h of
Fig. 1 – In the MCAO model, treatment with Tan IIA and/or TMP (with a dose of 30 and 40 mg/kg/day respectively, i. p. 3 days prior to induction of operation and continued for next 1 day following the operation) significantly improve the neurological function (A. n ¼14 per group, Po0.05) and attenuated the infarct compared ratio (B. n¼ 4 per group, Po0.05). Values given are the mean 7S.E.M. np-value o0.05 as with sham-operation, #p-valueo0.05 as compared with MCAO, and p-value o0.05 as compared with TMP.
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suffering from ischemia in the MCAO group (38%70.82%). The treatment with TMP, TMPþTan IIA, and Tan IIA significantly decreased the infarct volume to 34.75%70.95%, 27.25%70.96%, and 27.50%71.29%, respectively, compared with those in the MCAO groups (Po0.05).
2.3.
Oxidative stress level
We further investigated the effect of TMP (40 mg/kg/day) and/ or Tan IIA (30 mg/kg/day) on oxidative stress by evaluating malondialdehyde (MDA) content, glutathione (GSH) activity, and superoxide dismutase (SOD) activity in the brain (Fig. 2A–C). In contrast to the sham-operated rats, a significant increase in MDA contents was observed in the MCAO rats (Po0.05). The activity levels of GSH and SOD in the MCAO rats significantly decreased compared with the shamoperated rats (Po0.05). In the TMP- and/or Tan IIA-treated groups after MCAO for 24 h, the MDA level was significantly decreased, whereas the GSH and SOD activity was increased (Po0.05).
2.4.
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Intercellular free [Ca2þ]i concentration
Compared with sham-operated rats, the concentration of intercellular free [Ca2þ]i in MCAO increased from 391.87 74.64 to 774.07112.38 (Fig. 2D; Po0.05). The concentration of [Ca2þ]i in MCAO was then decreased to 573.6761.68, 446.2742.81, and 432.2749.91 in the groups treated with TMP (40 mg/kg/day), TMP (40 mg/kg/day)þTan IIA (30 mg/kg/ day), and Tan IIA (30 mg/kg/day), respectively (Po0.05 as compared with MCAO rats).
2.5. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The apoptotic cells caused by MCAO were determined using the TUNEL assay. As shown in Fig. 3E, the increased number in TUNEL positive cell death induced by MCAO (79.273.27) was significantly reduced after treatment with TMP (38.271.48), TMPþTan IIA (3272.92), and Tan IIA (3071.58).
Fig. 2 – In the MCAO model, effect of Tan IIA and/or TMP treatment (with a dose of 30 and 40 mg/kg/day respectively, i. p. 3 days prior to induction of operation and continued for next 1 days following the operation) on the levels of (A) MDA content, (B) SOD activity, (C) GSH activity and (D) Intercellular free [Ca2þ]i concentration. Values expressed as mean 7S.E.M, n¼ 5 per group. np-value o0.05 as compared with sham-operation, #p-valueo0.05 as compared with MCAO, and p-value o0.05 as compared with TMP.
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Fig. 3 – In the MCAO model, effect of Tan IIA and/or TMP treatment (with a dose of 30 and 40 mg/kg/day respectively, i. p. 3 days prior to induction of operation and continued for next 1 days following the operation) on the expression levels of Bax, Bcl-2, Caspase-3, TRPM-7 proteins by western blot analysis (Fig. 3A–D) and IHC (Fig. 3F and G). In Fig. 3-F and G, the picture shows hippocampal region of brain tissue cells in MCAO model, of which the positive cell was presented brown in membrane or cytoplasm. Datas were expressed as mean7S.E.M, Significance of differences was evaluated by one-way analysis of variance. n¼ 5 per group. np-value o0.05 as compared with sham-operation, #p-valueo0.05 as compared with MCAO, and p-valueo0.05 as compared with TMP.
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Quantitative analyses of cell death in different culture models were performed using the MTT assay (Fig. 4E). The neurons exposed to OGD indicated only 42.2% viability (% of NC group). TMP and/or Tan IIA treatment protected the neurons against cell death induced by OGD from 42.2%77.94% to 79.8%73.03%, 89.80%73.11%, and 91.4%7 3.84% in the groups treated with 10 mmol TMP, TMP (10 mmol)þTan IIA (8 mmol), and 8 mmol Tan IIA, respectively (Po0.05 as compared with the OGD group).
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2.6. Western blot analysis for Bax, Bcl-2, Caspase-3, and TRPM-7 The western blot results of MCAO-induced cerebral tissues are shown in Fig. 3A–D. Tan IIA can function in various cancer types as one of the underlying mechanisms in the antiapoptotic effect by inhibiting Bax and/or increasing the levels of anti-apoptotic Bcl-2 (Sukumari-Ramesh et al., 2011). The anti-apoptotic role of Tan IIA was also observed in this study.
Fig. 4 – In primary cortical neurons culture models, expression of (A)Bax, (B)Bcl-2, (C)Caspase-3 and (D)TRPM-7 proteins were determined by western blotting, with results normalized relative to b-actin. Cell viability were evaluated by MTT assay. Datas were expressed as % NC. n¼ 5 per group. np-valueo0.05as compared with sham-operation, #p-valueo0.05 as compared with MCAO, and p-value o0.05 as compared with TMP.
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That is, the MCAO-induced ischemic brain injury was accompanied by a decrease in the expression of Bcl-2 and an increase in the Bax expression (Po0.05 compared with the sham-operated rats). Treatment with TMP and/or Tan IIA inhibited the decrease in Bcl-2 expression but suppressed the increase in Bax expression. We also measured the protein expression of TRPM-7 and caspase-3. Furthermore, MCAO significantly increased the expression of active caspase-3 (17 kDa) and TRPM-7 (216 kDa) compared with the levels obtained from the sham-operated group (Po0.05). TMP and/or Tan IIA treatment can attenuate the increased caspase-3 and TRPM-7 induced by MCAO.
2.7.
Immunohistochemistry
The results from the immunohistochemistry experiments revealed a significant increase in the immunoreactivity of Bax, caspase-3, and TRPM-7 protein, whereas a decrease in Bcl-2 was observed in the ischemic penumbral region of the MCAO rats (Po0.05; compared with the sham-operated rats). Treatment with TMP and/or Tan IIA significantly reduced the immunoreactivity of Bax, caspase-3, and TRPM-7. However, an increased immunoreactivity in Bcl-2 was observed (Fig. 3G and F). Similar to the results of western blot, Tan IIA monotherapy indicated the remarkable effect compared with the other treatments. TRPM-7 was found residing on the body and associated processes in cortical neurons, whereas Bax and Bcl-2 were found in the neuronal cytolemma rather than the cytoplasm.
2.8. Western blot analysis for Bax, Bcl-2, caspase-3, and TRPM-7 The expression levels of Bax, Bcl-2, caspase-3, and trmp-7 protein were examined through western blot of the following groups: NC, OGD, TMP, TMPþTan IIA, and Tan IIA treatment
groups. Relative to the NC group, the expression of pro-apoptotic markers (Bax and caspase-3) in the OGD group was significantly increased, whereas that of the antiapoptotic marker (Bcl-2) was reduced. Neuronal cultures were incubated with TMP and/or Tan IIA 30 min before and 2 and 4 h after exposure to OGD. This procedure was performed to address the possible role of TMP and/or Tan IIA in the regulation of Bax, Bcl-2, and caspase-3. As shown in Fig. 4A–D, Bax, Bcl-2, caspase-3, and TRPM-7 proteins could be significantly affected by the TMP and/or Tan IIA treatment. Recently, TRPM-7 has been reported capable of mediating the dominant damage mechanism of antiexcitotoxic therapy in unmasked anoxic neurons (Aarts et al., 2003). Increased TRPM-7 was also observed in the OGD group (Po0.05; compared with the NC group). Interestingly, the increased TRPM-7 induced by OGD could be reduced with TMP and/or Tan IIA treatment. Tan IIA monotherapy elicited a better effect than the other treatments (Fig. 4A–D).
2.9.
Recently, TRPM-7 has been recognized as a nonselective cation channel and has been reported to play a dominant role in anoxic neuronal damage. PI3K/AKT pathways have been reported to participate in regulating the gene expression of apoptotic and anti-apoptotic molecules against ischemia (Saito et al., 2004; Tabakman et al., 2005). We demonstrated that Tan IIA monotherapy is more effective than TMP monotherapy (Po0.05) and that no significant difference was found between Tan IIA monotherapy and TMPþTan IIA combination therapy (P40.05). We also explored whether the PI3K/AKT signaling pathway contributed to the protective effect of Tan IIA in cortical neuronal cultures after OGD. The inhibitor LY294002 (10 mmol) was separately co-incubated and solo incubated with Tan IIA in the OGD group for 30 min. The expressions of AKT and p-AKT proteins were evaluated using western blot in cultures (Fig. 5A and B). The protein expression of p-AKT in the OGD group was markedly decreased compared with that in the NC group (Po0.05). Tan IIA with a concentration of 8 mmol significantly increased p-AKT protein expression compared with the OGD group (Po0.05). However, the effect of Tan IIA on p-AKT protein could be reversed with the treatment of PI3K inhibitor LY294002. The p-AKT protein level in the OGDþLY294002 group was also reduced by the PI3K inhibitor LY294002 (Po0.05; compared with the OGD group). Otherwise, the p-AKT protein expression in the OGDþTan IIAþLY group was higher than that in the OGDþLY group (Po0.05; Fig. 5A and B).
3.
Fig. 5 – In primary cortical neurons cultures, the effects of LY294002 (8lM) inhibitor on AKT and p-AKT proteins by western blotting and densitometric analysis gave the related ratio of b-actin. Datas expressed as mean 7S.E.M. n p-value o0.05 as compared with NC group, # p-value o0.05 as compared with OGD groups. and p-valueo0.05 as compared with TSA (8 lM) groups.
PI3K/AKT in neuronal culture
Discussion
The present study systematically evaluated the therapeutic neuroprotective effect of Tan IIA monotherapy, TMP monotherapy, and Tan IIAþTMP combination therapy in rats with ischemic brain injury induced by MCAO (with 30 mg/kg/day of Tan IIA and 40 mg/kg/day of TMP) and neuronal cultures exposed to OGD (8 mmol of Tan IIA and 10 mmol of TMP). Two new findings were revealed from this study. First, Tan IIAþTMP combination therapy was more effective than TMP
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monotherapy but not Tan IIA monotherapy. Tan IIA monotherapy is superior to TMP monotherapy in protecting the neuron against hypoxia/ischemia both in vitro and in vivo. Second, the neuroprotective role of Tan IIA monotherapy may be mediated by regulating the PI3k/AKT signaling pathway because of an increase in p-AKT phosphorylation levels. Measurement of infarct volume is one of the tools in evaluating the functional outcome after suffering from stroke. In rats, administering both Tan IIA and TMP can decrease the infarct volume in a dose-dependent manner in MCAO models (Liao et al., 2004). In addition, neurological scores can also improve as ischemia progresses (Dong et al., 2009). In the present study, the increased infarct volume and improved neurological scores induced 24 h after MCAO was significantly reversed with TMP and/or Tan IIA treatment (Po0.05; compared with the MCAO group). Furthermore, TMPþTan IIA combination therapy is superior to TMP monotherapy but not Tan IIA monotherapy. Tan IIA monotherapy resulted in a more significant decrease in infarct volume and neurological scores (Po0.05; compared with TMP monotherapy; Fig. 1A and B). The potential mechanism of ischemic brain injury remains unclear. However, neuronal apoptosis is one of the major pathogenic mechanisms that govern ischemic brain injury. The increased activation of oxidative stress, sustained increase in [Ca2þ]i levels, and a series of apoptosis-related proteins contributed to the pathogenic processes of neuronal death following ischemia, which ultimately lead to cell death (Mattson, 2000). Various drugs have been used in the treatment of ischemic brain injury in MCAO rats or in neurons exposed to OGD to elicit effects on oxidative stress, [Ca2þ]i, apoptotic factors, and neuronal apoptosis. In the present study, the increased MDA content as well as the decreased GSH and SOD activities induced in MCAO were significantly reversed with the treatment of Tan IIA monotherapy, TMP monotherapy, and Tan IIAþTMP combination therapy (Po0.05; compared with the MCAO group). This finding indicates that Tan IIA and TMP may exhibit a direct effect on oxidative stress. The increased [Ca2þ]i and the number of neuronal apoptosis induced by ischemic brain injury were markedly decreased with TMP monotherapy, Tan IIA monotherapy, and TMPþTan IIA combination therapy in vivo (Po0.05; compared with MCAO). Caspase-3, as one of the caspase family members, plays an important role in medicating cell apoptosis (Watanabe et al., 2004). However, Bcl-2 decreases cell apoptosis by inhibiting caspase-3 activity. Bcl-2 is enzymolied to shorter fragments (22 kDa), and its role as an anti-apoptotic agent is reversed to apoptotic while caspase-3 becomes activated (Fu and Fan, 2002). Obviously, an association exists between Bcl-2 and caspase-3 family in modulating apoptosis. In the present study, TMP and/or Tan IIA treatment can also markedly interfere the increased pro-apoptosis factors Bax and caspase-3, the decreased anti-apoptosis factor Bcl-2, and the increased TRPM-7 protein induced by ischemic brain injury in vivo and in vitro (Po0.05; compared with MCAO/OGD). Similar to previous findings, the results of the present study demonstrate the potential character of TMP and Tan IIA to induce anti-apoptotic effects (Dong et al., 2009; Liao et al., 2004). TRPM-7 is a ubiquitous divalent-selective channel with its own kinase domain, and the knockdown of TRPM-7 in cultured neurons can lead to anoxic cell death in cortical neurons (Aarts et al., 2003; Kraft and Harteneck, 2005). Wei et al. demonstrated the
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expression of TRPM-7 channels in neurons and identified them as key mediators of OGD-induced neuronal death (Sun et al., 2009). The finding may lead to new insights for further development of neuroprotective therapies. The results further suggest that TRPM-7 may be involved in the neuroprotective effects of Tan IIA and TMP. The Bcl-2 protein family members are potent regulators of apoptosis. The anti-apoptotic Bcl-2 and pro-apoptotic Bax also belong to the Bcl-2 protein family. Bcl-2/ Bax ratio is often used to represent the extent of apoptosis (Gallogly et al., 2010). Tan IIAþTMP combination therapy produced no significant effect on increasing Bcl-2 compared with TMP monotherapy. A more significant effect on increasing the level of Bax/Bcl-2 ratio was demonstrated in Tan IIAþTMP combination therapy (Po0.05; compared with the TMP monotherapy). In traditional Chinese herbal medicine, a recommended strategy in the management of ischemic stroke is the combination therapy rather than monotherapy. However, our results demonstrated a different concept. In the present study, a series of bioindices was evaluated, including oxidative stress (MDA content, SOD activity, and GSH activity), apoptotic factors (Bax, Bcl-2, Bax/Bcl-2, and caspase-3), [Ca2þ]i content, neuronal apoptosis, and viability in both MCAO and OGD models. Tan IIAþTMP combination therapy was more effective than TMP monotherapy but not Tan IIA monotherapy. PI3K/AKT pathways play important roles in the regulation of cell survival and death (Arai and Lo, 2010; Langenbach and Rando, 2002; Zhang et al., 2010). They also participate in the regulation of apoptotic and anti-apoptotic molecules against ischemic injuries (Jiang et al., 2008). The results of the present study demonstrate that the neuroprotective effect of Tan IIA relies on the increased levels of phosphorylated AKT. The PI3K inhibitor LY294002 decreased the Tan II-mediated protection. This result indicates that the PI3K/Akt pathway plays a role in the neuroprotective effect of Tan II. Several issues could be addressed in this study. First, we speculated that extensive insights may be obtained from the ischemic reperfusion MCAO model with prolonged therapy because the neuroprotective effects of TMP and/or Tan IIA treatment were evaluated 24 h after permanent MCAO. Second, the therapeutic effect observed in stroke patients in clinical settings may directly provide evidence. Last, the characteristics of the metabolic pathway, half-life period, and medication may also contribute to the results of the present study. These issues should be addressed in future studies. The present study is the first to reveal that Tan IIAþTMP combination therapy is more effective than TMP monotherapy but not Tan IIA monotherapy. In addition, Tan IIA monotherapy exhibits a more effective neuroprotection than TMP monotherapy against hypoxia/ischemia both in vitro and in vivo. The present study also proves that PI3K/AKT signaling pathway is involved in the protective mechanism of Tan IIA on primary cortical neurons.
4.
Experimental procedures
4.1.
Experimental materials
Adult male rats (Sprague Dawley) weighing 200–220 g (Vital River Laboratory Animal Technology Co., Ltd. China) were
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used in this study. The experiments were conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, China. All experimental protocols were approved by the University Animal Care and Use Committee.
4.2.
MCAO model
Permanent cerebral ischemia was performed through the intraluminal suture occlusion based on previously reported procedures (Foley et al., 2010). In brief, the animals were anesthetized with 10% chloral hydrate. The temperature was maintained at 37.0 1C and controlled using a thermostatic blanket. A 6-0 nylon monofilament with a round tip (0.24–0.26 mm) was inserted into the right internal carotid artery via an external carotid artery stump, whereas the common carotid artery was occluded. Successful occlusion was verified using a laser Doppler flowmetry. The local cerebral blood flow in the right MCA territory declined to approximately 10–15% of the baseline. The sham-operated rats were treated similarly, except that the middle cerebral arteries were not occluded after the neck incision.
4.3.
4.5.
Oxygen-glucose deprivation model
OGD was induced as previously described (Aarts et al., 2003; Chen et al., 2011) with slight modification. The cells were fed with glucose-free DMEM and infused with 5% CO2 and 95% N2 at 37 1C for 1 h. The maintenance medium was used to terminate OGD. Primary NC neurons were maintained in DMEM containing 5% CO2 and 95% air. In all experiments, the pH of the medium was maintained at 7.2 under OGD conditions.
4.6. Treatments and study design in neuronal culture models
Study design in animal models
In this study, 84 rats were randomly grouped into six groups (n ¼14, each group): group 1, sham-operated (control); group 2, MCAO rats; group 3, cmc-Na treatment (MCAO rats treated with cmc-Na); group 4, TMP (MCAO rats treated with 40 mg/ kg/day TMP); group 5, Tan IIAþTMP (MCAO rats treated with 30 mg/kg/day Tan IIAþ40 mg/kg/day TMP); group 6, Tan IIA (MCAO rats treated with 30 mg/kg/day Tan IIA). In all animals, the treatments were given via intragastric administration. The drugs were dissolved in 0.5% cmc-Na and administered once a day for 5 d [i.e., 3 d before operation and 2 twice after operation (include treated 4 h and 24 h after surgery]. MCAO surgery was performed on day 3, 1 h after TMP and/or Tan IIA administration. The MCAO group received an equal volume of cmc-Na.
4.4.
arabinoside and maintained for 48 h. The medium was replaced with fresh neurobasal medium after 24 h, and half of the culture medium was changed every 2 d. Arabinosylcytosine (5 mg/mL) was added on the third day of incubation to prevent non-neuronal cell growth. The cultures were used for 8 d to 10 d in vitro. All drugs were diluted in DMSO and administered 30 min before and 2 and 4 h after exposure to OGD in neuronal culture. The OGD group received an equal volume of DMSO.
Cell culture and treatment
Primary culture of cortical neurons was isolated and cultured as previously described (Jiang et al., 2008). Briefly, cerebral cortical neurons were prepared from the cerebrum of oneday-old Sprague Dawley rats (Qi et al., 2010) and dissected in ice cold dissection buffer. After the meninges of the cortex were peeled off and separated from the olfactory bulb and hippocampus, the dissected cortical tissues were treated in supplemented Hank’s balanced salt solution for 25 min at 37 1C, followed by mechanical dissociation. The cells were digested with trypsin. Subsequently, the cells were centrifuged at 1000g for 10 min. The cell pellets were resuspended and cultivated in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) suplemented with 15% fetal bovine serum, 5% horse serum (Sigma, St. Louis, MO), and 100 U/mL penicillin/ streptomycin. Cortical neurons at a density of 1 105/mL were seeded onto plates pre-coated with poly-l-lysine (25 mg/mL) and placed in a humidified incubator (37 1C with 5% CO2). After 3 d, the cells were cultured with 1 mM cytosine
Primary cortical neuron cultures were grouped into six (n ¼4, each group): group 1, NC (cells exposed to normoxia control); group 2, OGD (cultures exposed to OGD); group 3, DMSO (cultures exposed to OGD and incubated with DMSO); group 4, TMP (cultures exposed to OGD and treated with 8 mmol TMP); group 5, Tan IIAþTMP (cultures exposed to OGD and treated with 8 mmol Tan IIAþ10 mmol TMP); group 6, Tan IIA (cultures exposed to OGD and treated with 8 mmol Tan IIA). In culture models, the drugs were diluted in DMSO of the same volume and administered 30 min before and 2 and 4 h after exposure to OGD in the neuronal culture. The OGD group received cortical neurons with an equal volume to DMSO. The regulatory effect of the PI3K/AKT pathway on Tan IIA neuronal protection in cortical neuronal cultures after OGD was assessed. The expressions of AKT and p-AKT proteins were evaluated in the culture before conducting the following experiments (five groups): group 1, NC (cells were exposed to normoxia control); group 2, OGD (cultures exposed to OGD); group 3, LY294002 (cultures exposed to OGD and incubated with 10 mmol LY294002 for 30 min); group 4, Tan IIA (cultures exposed to OGD A and incubated with 8 mmol Tan IIA for 30 min); group 5, Tan IIAþLY294002 (cultures exposed to OGD and incubated with 8 mmol Tan IIAþ10 mmol LY294002 for 30 min). These levels were normalized to NC (incubated in DMEM high-glucose medium and maintained at 5% CO2 and 95% N2 at 37 1C). Whole proteins were then isolated from cortical neuronal cultures.
4.7.
Neurological deficit examination
Neurological deficits were carried out 24 h after MCAO or sham-operation as previously reported (Longa et al., 1989) with slight modifications. A 0–4 point scale was used to evaluate the neurological deficits observed in all groups (rats subjected to sham operation or MCAO procedure): 0, no neurological symptoms; 1, unable to stretch the upper
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extremities completely; 2, rotation to the left; 3, unable to walk without help; and 4, no spontaneous motor activity.
4.8.
Evaluation of Infarct volume
2,3,5-Tripenyltetrazolium chloride (TTC) staining was carried out to evaluate the infarct volume. The rat’s brains were quickly removed and sectioned into 2 mm thick slices. Subsequently, cornel slices were immersed in 0.25% TTC at 37 1C for 30 min and then fixed in 10% phosphate-buffered formalin for 45 min (Liao et al., 2004). Areas of the infarcted regions were captured using a digital camera (AV-P960C, JVC, Japan) and analyzed with an image C morphology analysis system (Chansan Instrument Co. Shanghai, China). The results were represented as the percentage of the total volume.
4.9.
TUNEL staining
TUNEL methods were used to detect and quantify apoptotic cell death on paraffin-embedded sections using an In Situ Cell Death Detection Kit (Roche, Germany). According to the manufacturers’ instructions, the rats were sacrificed 24 h after MCAO. Subsequently, brain sections (10 mm) were prepared using the same methods as in immunohistochemistry. The cells displaying brown stain within the nucleus were counted as TUNEL-positive cells. The number of TUNELpositive cells was determined in three randomly selected microscopic eyeshots and expressed as percentage under high-power magnification. The apoptotic index¼ TUNELpositive cell number/the number of nucleated cells 100%.
4.10.
[Ca2þ]i measurement
Intercellular free [Ca2þ]i was determined using the fluorescence of fura-2 in a spectrofluorometer as described in a previous study (Vinje et al., 2002). Briefly, the cortical neurons were dissected and washed thrice with Hank’s solution. The tissues were homogenized and then centrifuged for 3 min at 3000g and the supernatant for 30 min at 15000g. The formed pellet was two-layered and diluted in calcium-free hepes-buffered medium (HBM) for the released experiments (suspension for the evaluation of [Ca2þ]i and the supernatant for MDA content, GSH activity, and SOD activity). Fura-2/AM was added to the suspension of tissue cell (whereas the neural cells were incubated in HBM containing fura-2/AM) to a final concentration of 5 mmol for 30 min at 37 1C (neural cells were incubated in CO2 at 37 1C for 30 min). Then, the cells were centrifuged, and the supernatant was washed twice with Hank’s solution containing 0.2% bovine serum medium and resuspended into 1 106/mL cell concentration. The fluorescence light (F) was monitored using a Hitachi Fluorescence Spectrophotometer as previously reported (Vinje et al., 2002). Maximum (F max) and minimum (F min) fluorescence were obtained after 0.5% sodium dodecyl sulfate and 7.5 mM EGTA were added, respectively. The level of [Ca2þ]i was calculated according to the following equation: [Ca2þ]i ¼ [(FF min)/(F maxF)] kd (224 nM) is the dissociation constant of fura-2 with calcium.
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4.11. Evaluation of oxidative stress (MDA, GSH-Px, and SOD) The suspension was used to evaluate [Ca2þ]i, and the supernatant was used to assess oxidative stress (MDA, GSH-Px, and SOD) with a spectrofluorometer according to the manufacturer’s instructions (Bora and Sharma, 2010). (1) The MDA content was evaluated by measuring the content of thiobarbituric acid (TBA) reactant substances according to the TBA test method with slight modifications. The optical density of the supernatant was determined using a spectrophotometer at l¼532 nm. The MDA content was expressed as nmol/mg tissue. (2) The activity of GSH was measured using the method described by Jollow et al. (1974). Equal quantity of brain homogenate was mixed with 10% trichloroacetic acid and centrifuged at 1200g for 15 min. Phosphate buffer (0.1 M) and 5-50 -dithiobis[2-nitrobenzoic acid] (100 mM in 0.1 M phosphate buffer) were added to the mixture. The mixture was vortexed, and the absorbance was determined at l ¼412 nm. The concentration of GSH was expressed as nmol/g of tissue. (3) The activity of SOD in the cerebral tissues of all the rats was estimated according to a previous study (Kakkar et al., 1984) based on the inhibition of the formation of nicotinamide adenine dinucleotide, phenazine methosulphate (PMS), and nitroblue tetrazolium (NBT) formazan. In brief, 10 mL of the homogenate was mixed with 90 mL of 30 mM sodium tetrapyrophosphate buffer (pH 8.3), 30 mL of 0.3 mM NBT, 10 mL of 0.96 mM PMS, and 40 mL of doubledistilled water. Absorbance was obtained at l¼ 560 nm. A single unit of enzyme was expressed as 50% inhibition of NBT reduction/min/mg protein. The results were expressed as a unit of SOD/mg protein.
4.12.
MTT assay
MTT solution was added to 96-well plates at a final concentration of 500 mg/mL and then incubated at 37 1C for 4 h. After the supernatant was removed, DMSO was added into each well, which was shaken for 10 min. The absorbance was obtained at 570 nm, and the survival rate (%) was determined and compared with the control group.
4.13.
Bax, Bcl-2, Caspase-3, and TRPM-7 western blot
The total proteins were separately extracted from the cortical neuronal tissues or cultures according to previous methods (Jiang et al., 2008; Mattsson et al., 2003). The protein concentration was measured using a BCA protein assay kit (Cwbio Ltd., China). Equal amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. After the membranes were blocked with 5% nonfat dry milk, they were incubated overnight at 4 1C with rabbit anti-b action (1:1000, cell signaling), rabbit anti-Bcl-2 (1:1000, cell signaling), rabbit anti-Bax (1:1000, cell signaling), rabbit anti-caspase-3 (1:1000, cell signaling), and mouse anti-trmp-7 (1:1000, Abcam, Hong Kong). HRP conjugate goat anti-rabbit IgG and HRP conjugate goat anti-mouse IgG were used as secondary antibodies (1:2500, Promega, USA). The reaction was developed using a chemiluminescent substrate (Thermo Scientific), and signal
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density was measured using Alpha Image. Equal protein loading was confirmed by measuring b-actin. Densitometric analysis was performed using NIH Image J analysis software (USA). The values were normalized with b-actin.
4.14. Bax, Bcl-2, Caspase-3, and TRPM-7 immunohistochemistry Immunohistochemistry analysis for the in situ expression of Bax, Bcl-2, caspase-3, and TRPM-7 proteins were performed as previously described (Kaundal and Sharma, 2011). The sections were dewaxed with xylene, rehydrated through an alcohol gradient, and heated with 10 mm/L Na citrate (pH 6) in a microwave for 15 min to retrieve the antigenic determinants. The resulting product was treated with 1% H2O2 to inactivate endogenous peroxidase activity and was blocked with 5% goat serum in phosphate at 4 1C with the primary antibody in phosphate-buffered saline/2% goat serum. The following primary antibodies were used: anti-b-actin (1:1000, cell signaling), anti-Bcl-2 (1:200, Boster, China), anti-Bax (1:200, Boster, China), anti-caspase-3 (1:500, cell signaling), anti-TRPM-7 (1:200, ABCam, Hong Kong). The sections were treated with Vecta stain ABC kit (Vector Labs, Burlingame, CA, USA). The images were analyzed using Magna Fire SP 2.1 B software (USA).
4.15.
Statistical analysis
All data were expressed as mean7S.E.M, and the statistical significance was determined using one-way ANOVA. Statistical analysis was conducted using SPSS 11.5 (Chicago, Illinois, USA). A P-value of less than 0.05 was considered to indicate statistical significance.
Acknowledgments This work was supported by the Province Science Foundation of China (No. 090413120), Natural Science Foundation of the Higher Education Institutions of Anhui Province, China (KJ2007B147), and Medical Scientific Research Foundation of Anhui Province (2010B003) to Qiqiang Tang.
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