Introduction

Stroke, a global health issue, causes financial, emotional and other heavy burdens for families and societies worldwide. Ischemic stroke, occlusion of blood vessel in brain, accounts for a majority of stroke cases (about 87 % statistically) (Mozaffarian et al. 2015). Inflammation is the key risk factor to neuron damage in stroke (Tuttolomondo et al. 2009) and neurodegenerative diseases (Block et al. 2007; Block and Hong 2005). Microglia, the brain-resident macrophage, becomes activated within a few hours after brain ischemia (Block et al. 2007; Chew et al. 2006; Lee et al. 2014; Nimmerjahn et al. 2005). Although microglia activation is required for host defense and neuron survival (Kettenmann et al. 2013), the overactivation of microglia produce excess neurotoxic products such as superoxide, nitrite oxide (NO) or tumor necrosis factor α (TNF-α) (Colton and Gilbert 1987; Moss and Bates 2001; Liu et al. 2002; Lee et al. 1993) causing cell death.

Lipopolysaccharide (LPS), the polysaccharide component of the cell walls of gram-negative bacteria, is the most frequently used model to investigate the inflammatory responses of microglia (Gao et al. 2002; Zheng et al. 2008; Le et al. 2001). LPS can be recognized by the pattern recognition receptors (PRRs) of microglia and then binds to the Toll-like receptor 4 evoking a complex array of intracellular signaling pathways (Anderem and Ulevitch 2000; Jung et al. 2009). In addition, stimulation of microglia with LPS induces the release of tumor necrosis factor-α (TNF-α) and facilitates TNF-α- or NO-mediated neuron death in vitro (Hemmer et al. 2001). Inducible NO synthase (iNOS) is quickly transcribed and expressed in microglia after stimulation with bacterial LPS and cytokines (Kim et al. 2000; Choi et al. 2008). COX-2 (cyclooxygenase-2) also plays a predominant role in inflammation (Minghetti 2004). LPS induces PGE2 production in human microglia through COX-2 induction (Hsu and Wen 2002). The molecular mechanisms of inflammation induced by LPS in microglial cells have been well reported (Wang et al. 2002; Bhat et al. 1998; Woo et al. 2003; Pawate et al. 2004; Xie et al. 2004). NF-κB pathway, one of the most important mechanisms in inflammatory diseases, is the key transcription factor in the regulation of proinflammatory cytokines, iNOS and COX-2 (Baeuerle and Henkel 1994; Baldwin 2001). At the molecular level, NF-κB induces activation of cytoplasmic proteins and the nuclear translocation of p65, p50 subunit of NF-κB (Karin and Ben-Neriah 2000; Delhase et al. 2000). Previous studies have shown that LPS causes the nuclear translocation of p65 subunit of NF-κB through IκB degradation (Baeuerle and Baltimore 1988; Henkel et al. 1993).

Cinnamon powder, one of the most famous spices, is made from the inner bark of cinnamon trees and can be found in many types of recipes and foods. The extract from cinnamon bark has also been used as a traditional medicine. Some studies have found that cinnamon may have beneficial health properties, such as lowering blood sugar and cholesterol levels, and reducing inflammation (Bernardo et al. 2015; Shalaby and Saifan 2014; Pahan 2015). Cinnamaldehyde, an essential oil in cinnamon powder, is the source of the distinct smell and flavor of cinnamon (Adams et al. 2004). Trans-cinnamaldehyde displays diverse pharmacological properties including anti-inflammatory activity (Wu et al. 2012; Lee et al. 2002; Kim et al. 2007; Amalaradjou et al. 2010; Shahverdi et al. 2007). Our preliminary data showed that cinnamon powder significantly reduced ischemia/reperfusion (I/R)-induced cerebral infarction (data not shown). The purposes of the present study were to examine the neuroprotective effect of trans-cinnamaldehyde in I/R-induced brain injury in vivo and anti-inflammatory mechanisms in vitro by LPS-induced insults in BV-2 cells.

Materials and Methods

Chemicals

LPS (0111:B4), MTT, Griess reagent and trans-cinnamaldehyde were purchased from Sigma-Aldrich (St Louis, MO, USA).

Animal Cerebral Ischemia Model

Adult male C57BL/6 mice (weight 22–28 g), purchased from National Laboratory Animal Center, were used in this study. Animals were fed with standard chow and housed in standard cages at a constant room temperature of 22 ± 1 °C. Relative humidity of 55 ± 5 % was maintained with 12-h inverted light–dark cycle for 1 week at least prior to the experiment. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC), China Medical University (Permit Number: 101-148-N). The minimum number of animals and duration of observations required to obtain reliable data are used. For infarct volume studies, the animals were divided into four groups: the I/R group (ischemia/reperfusion induction, as a control group) and TCA treatment groups (10, 20, 30 mg/kg, orally administration 60 min before ischemia surgery). Animals were subjected to two-vessel ligation. All surgical procedures were performed by sterile/aseptic techniques. The mice were anesthetized with chloral hydrate (0.4 g/kg, ip). Ligation of the right middle cerebral artery (MCA) and right common carotid arteries (CCA) was performed by methods described previously, with slight modifications (Yang et al. 1997). The right CCA was clamped with non-traumatic arterial clip. The right MCA was ligated with 10-0 nylon suture. After 120 min of ischemia, the suture on the MCA and arterial clip on CCA were removed to allow reperfusion. Core body temperature was monitored with a thermostat probe and maintained at 37 °C with a heating pad during anesthesia. After recovery from anesthesia, body temperature was maintained at 37 °C with a heat lamp (Ding et al. 2007).

Evaluation of Neurological Deficits and Measurement of Infarction Area

Twenty-four hours following reperfusion, neurological function of mice was assessed as previously described (Hattori et al. 2000). The degree of neurological deficits were divided 0–4 (higher score is for more severe neurological deficits) as follows: 0 = no deficit; 1 = forelimb weakness; 2 = circling to affected side; 3 = partial paralysis on affected side and 4 = no spontaneous motor activity. After completion of neurological evaluation, mice were deeply anesthetized by an intraperitoneal injection of chloral hydrate (0.8 g/kg) followed by an intracardiac perfusion and decapitation. After decapitation, the brain was removed and sectioned into 2-mm-thick slices and then immersed for 15 min into 2 % TTC solution at 37 °C. Animals with extended hemorrhage in their skull base were excluded from the study. The area of the ischemic damage of the hemispheres was measured for each brain slice using an Image-Pro Plus 6.0 (Media Cybernetics, MD, USA). Complete lack of TTC staining was defined as core (Bederson et al. 1986). Purple–red brain tissue was penumbra positioned between core, and viable brain tissue was stained red (Gill et al. 1995). Total infarction area was the sum of core and penumbra areas. Results were given as the means of SEM for the different treatment groups.

iNOS and COX-2 Protein Expression Level in Ischemic Brain Tissue

After completion of neurological evaluation, mice were deeply anesthetized, decapitated, and the brains were removed for protein extraction. Tissue lysates were prepared in lysis buffer, and protein concentrations in the tissue lysates were determined by Bio-Rad protein assay kit (Richmond, CA, USA). Samples of protein (50 μg) were electrophoresed using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane. The iNOS, COX-2 were assayed by specific antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA, and Cell Signaling Technology, Danvers, MA, USA). Immunodetection was done using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ, USA).

Cell Culture

Murine BV-2 microglial cells were kindly provided by Professor Jau-Shyong Hong (Neurobiology laboratory/Neuropharmacology group, NIEHS/NIH, NC, USA). BV-2 microglial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS, 100 units/ml penicillin and 100 mg/ml streptomycin, and kept at 37 °C in a humidified incubator with 5 % CO2 and 95 % air. A scraper transferred confluent cultures every 2–3 days.

MTT Assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay determined cell viability. The BV-2 microglia cells were cultured at the density of 5 × 104 cells/well in 96-well plate. BV-2 microglia cell cultures were treated with TCA (12.5, 25, 50 μM) and LPS (0.5 μg/ml) for 24 h. MTT was added to each well, and the cells were incubated for 1 h at 37 °C. After culture media were discarded, DMSO was added to dissolve the formazan dye. The optical density was measured at 570 nm.

Nitrite Quantification

NO generation was measured by the accumulation of nitrite in the culture medium. The colorimetric assay was used to determine nitrite with Griess reagent. BV-2 cells (5 × 104 cells/well) in 96-well plates in 200 μl culture medium were treated with LPS (0.5 μg/ml) for 24 h. Hundred microliters of isolated supernatant was added with an equal volume of Griess reagent in 96-well plates for 10 min at room temperature and light avoidance. Standard solution of sodium nitrite prepared in cell-culture medium was used to determine nitrite concentrations. The absorbance at 570 nm was read using an Elisa reader (Triad LT, DYNEX Technologies Inc, VA, USA). Each experiment was duplicated three times.

Western Blot Analysis

Cell lysates were prepared in lysis buffer. Protein concentrations in the cell lysates were determined by Bio-Rad protein assay kit (Richmond, CA, USA). Samples of protein (50 μg) were electrophoresed using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane. The iNOS, COX2, p53, phospho-IκBα, IκBα, phospho-NF-κBp65, NF-κBp65 and NF-κBp50 were assayed by specific antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA Cell Signaling Technology). Immunodetection was done using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ, USA) (Huang et al. 2011).

Nuclear Protein Extraction and mRNA Analysis by Quantitative Real-Time PCR

Cytoplasmic proteins and nuclear protein was isolated by the ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas). Total RNA was isolated and purified from BV-2 microglial cells using the TRIzol method and ChargeSwitch® Total RNA cell kits (Invitrogen, CA, USA). Using Thermo Scientific Verso cDNA Synthesis Kit reverse-transcribed RNA to generate cDNA in a Bio-Rad’s iQ5 multicolor real-time PCR according to the manufacturer’s instructions. The generated cDNA was used to quantify real-time PCR through real-time analysis using Invitrogen’s SYBR® GreenER™ qPCR SuperMix. The primer sets used were purchased from Dharmacon and were

  • GAPDH, forward primer: 5′-GGCTGGCATTGCTCTCAA-3′ and reverse primer: 5′-GCTGTAGCCGTATTCATTGTC-3′;

  • TNF, forward primer: 5′-AACTCCAGGCGGTGCCTAT-3′ and reverse primer: 5′-GGAGGCCATTTGGGAAC-3′;

  • COX2, forward primer: 5′-TAGCAGATGACTGCCCAACT-3′ and reverse primer: 5′-GAATCAGGAAGCTCCTTA-3′;

  • iNOS, forward primer: 5′-CGTGAAGAAAACCCCTTGTG-3′ and reverse primer: 5′-GATTCTGGAACATTCTGTGCTG-3′.

Thermal cycles were programed according to the manufacturer’s instructions for each primer. To normalize the data for each experiment, GAPDH was used as a reference gene. Using ΔΔC T calculations determined fold expression of gene.

Statistics

Statistical analysis was performed using the software SPSS version 10.0. The values given were mean ± SE from 3 to 4 experiments of each group. Statistical analysis between two samples was performed using Student’s t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA) with Duncan’s test. A P value <0.05 was considered statistically significant.

Results

TCA Ameliorated Ischemia/Reperfusion Brain Injury and Reduced iNOS and COX-2 Protein Expression Level in I/R-Induced Brain Injury of Mice

TCA (10, 20 and 30 mg/kg, p.o.) significantly reduced the infarction area in I/R-induced brain injury (Fig. 1a, b) and neurological deficit score (Fig. 1c) in mice in a dose-dependent manner (P < 0.001). The reduction rates of TCA (10, 20, 30 mg/kg, p.o.) on infarction area are 60, 68 and 80 %, respectively. TCA (10, 20, 30 mg/kg, p.o.) demonstrated a 13, 40 and 50 % reduction of neurological deficit score, respectively. Moreover, TCA also significantly reduced iNOS and COX-2 protein expression levels in ischemic mice brain (Fig. 2a–c) in a dose-dependent manner (P < 0.001). The reduction rates of TCA (10, 20, 30 mg/kg, p.o.) on iNOS protein expression level are 70, 88 and 95 %, respectively. TCA (10, 20, 30 mg/kg, p.o.) illustrated a 37, 55 and 73 % reduction of COX-2 protein expression level.

Fig. 1
figure 1

Effects of trans-cinnamaldehyde (TCA) on ischemia/reperfusion (I/R)-induced brain injury in mice. Coronal section of brain after ischemia for 120 min and followed by reperfusion for 24 h. Then, staining with TTC, the infarction areas appeared white and non-infarction areas appeared red–purple in color. S1 → S4 slices from frontal lobe. a TTC of control mice and mice treated with TCA (10, 20, 30 mg/kg, p.o.) 60 min before I/R. b Statistical bars of cerebral infarction area. c Neurological deficit score of mice 24 h after I/R. Scale bar 1 cm. Data are mean ± SE from more than three independent experiments. ***P < 0.001 compared with the control (I/R) group (Color figure online)

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Fig. 2
figure 2

Effects of trans-cinnamaldehyde (TCA) on protein expression level of iNOS and COX-2 in I/R-induced brain injury mice. a Western blot of iNOS and COX-2 in control group and mice treated with TCA (10, 20, 30 mg/kg, p.o.) 60 min before I/R injury. b Statistical bars of protein expression level of iNOS. c Statistical bars of protein expression level of COX-2. Data are mean ± SE from more than three independent experiments. ***P < 0.001 compared with the control (I/R) group

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TCA Inhibited NO Production, Reduced Protein Expression Level of iNOS, COX-2, But Increased Expression of p53 in LPS-Challenged BV-2 Microglial Cells

Results of BV-2 cells treated with TCA (12.5, 25, 50 μM) plus LPS (0.5 μg/ml) or LPS (0.5 μg/ml) alone for 24 h was showed in Fig. 3a. TCA did not induce cytotoxicity. It significantly inhibited NO production at concentration of 50 μM after 24 h treatment compared with the LPS-only group (P < 0.001) (Fig. 3b). TCA (12.5, 25, 50 μM) displayed a 10, 17 and 53 % reduction of NO production, respectively. TCA also reduced cytosolic protein levels of iNOS, COX-2 (Fig. 4a, b) and attenuated inhibition of p53 elicited by LPS (Fig. 4c). The reduction rates of TCA (12.5, 25, 50 μM) on iNOS protein expression level are 9, 26 and 35 %, respectively, and 50 μM of TCA showed a 13 % reduction of COX-2 protein expression level in LPS-induced BV-2 microglia cells.

Fig. 3
figure 3

Effects of trans-cinnamaldehyde (TCA) on NO production in LPS-challenged BV-2 microglial cells. Trans-cinnamaldehyde significantly reduced NO production (b) without affecting the BV-2 cell viability (a) induced by LPS (0.5 μg/ml). Data are mean ± SE from more than three independent experiments. ### P < 0.001 compared with untreated group. *P < 0.05, **P < 0.01, ***P < 0.001, compared with LPS alone

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Fig. 4
figure 4

Effects of trans-cinnamaldehyde (TCA) on expression of iNOS, COX-2 and p53 in LPS-challenged BV-2 microglial cells. Trans-cinnamaldehyde attenuated expression of iNOS (a), COX-2 (b) proteins, while increased expression of p53 (c) protein BV-2 cells, which were simultaneously treated different concentrations of trans-cinnamaldehyde (12.5, 25, 50 μM) and LPS (0.5 μg/ml) for 24 h. Protein level of iNOS, COX-2 and p53 was analyzed by Western blotting. β-actin was used as a control of protein loading. Data are mean ± SE from more than three independent experiments. ### P < 0.001 compared with untreated group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with LPS alone

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TCA Reduced NF-κB Activation and IκB Degradation Induced by LPS

In Fig. 5a, b, it showed that treatment with LPS for 15 min stimulated nuclear translocation of NF-κB, while TCA at 12.5–50 μM significantly suppressed the nuclear translocation of p65 and p50. The effect of TCA on the expression of cytosol IκBα by Western blot analysis showed that it increased the level of IκBα (Fig. 5c), but had no obvious significance on total IκB (Fig. 4d) compared to LPS-challenged-only group.

Fig. 5
figure 5

Effects of trans-cinnamaldehyde (TCA) on NF-κB and IκB induced by LPS. Trans-cinnamaldehyde curbed NF-κBp65 (a) and NF-κBp50 activation (b) and IκBα degradation (c, d) induced by LPS. BV-2 cells were simultaneously treated different concentrations of trans-cinnamaldehyde (12.5, 25, 50 μM) and LPS 0.5 μg/ml for 15 min. Protein levels of NF-κB (nuclear protein) and IκBα (cytosol and total proteins) were analyzed by Western blotting. Actin and H3 were used as the control of protein loading. ### P < 0.001 compared with untreated group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with LPS alone

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TCA Reduced mRNA Expression Levels of iNOS, COX-2 and TNFα Induced by LPS

Data in Fig. 6a–c showed that TCA reduced mRNA expression of iNOS, COX-2, and TNFα in BV-2 cells treated with LPS. Significant effects on mRNA levels were observed after 4 and 12 h of TCA incubation.

Fig. 6
figure 6

Effects of trans-cinnamaldehyde (TCA) on mRNA expression levels of iNOS, COX-2 and TNFα induced by LPS. Trans-cinnamaldehyde reduced mRNA expression of iNOS (a), COX-2 (b) and TNFα (c) in cells treated with LPS. BV-2 cells were treated with LPS (0.5 µg/mL) or trans-cinnamaldehyde (50 μM) for 12 h or 4 h, and total RNA was subjected to quantitative RT-PCR. The RT products were labeled with SYBR Green dye. Relative iNOS, COX-2 and TNFα mRNA expressions (2 – ΔC t ) were determined by real-time PCR and calculated by subtracting the C t value for iNOS, COX-2 and TNFα from GAPDH mRNA, respectively. ΔC t  = C t COX-2 − C t GAPDH. Each value represents the mean ± SE of three independent experiments. ## P < 0.01, ### P < 0.001 compared with untreated group. **P < 0.01, ***P < 0.001 compared with LPS alone

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Discussion

Inflammation plays an important role in the pathogenesis of ischemic stroke, and the activation of microglia is the key factor contributing to neuroinflammation (Tuttolomondo et al. 2009). LPS activates NF-κB resulting in the induction of several inflammation-related early genes (Baeuerle 1991; Yamamoto and Gaynor 2001) particularly gene expression of iNOS (Griscavage et al. 1996) and COX-2 (Umezawa et al. 2000). LPS activates NF-κB resulting in the induction of several inflammation-related early genes (Baeuerle 1991; Yamamoto and Gaynor 2001) particularly gene expression of iNOS and COX-2 (Umezawa et al. 2000). LPS induces cellular inflammation by up-regulating pro-inflammatory factors such as iNOS, COX-2 and TNFα (Block et al. 2007). Those genes are regulated by NF-κB (Mattson and Camandola 2001). Trans-cinnamaldehyde, an essential oil in cinnamon powder, has anti-inflammatory effects (Kim et al. 2007). In our data, it showed that trans-cinnamaldehyde significantly ameliorated the infarct volume in mice brain subjected to ischemia and reduced the neurological deficit score in ischemia mice revealed that trans-cinnamaldehyde had neuroprotection against cerebral ischemia in vivo (Fig. 1). In addition, trans-cinnamaldehyde markedly attenuated protein expression level of iNOS and COX-2 in ischemia brain tissue that revealed its anti-inflammatory activity in vivo. The effect of trans-cinnamaldehyde has not only showed neuroprotection in a mouse ischemic stroke model, but also revealed anti-inflammatory activity in activated BV-2 microglial cells elicited by an LPS challenge. The anti-inflammatory mechanisms of trans-cinnamaldehyde is via the inhibition of inflammatory molecules including production of NO and expression of iNOS, COX-2, NF-κB, IκB, mRNA of iNOS, TNFα and the increasing p53 levels.

Cerebral ischemia involves both innate and adaptive immunity (Famakin 2014). Microglia originates from the hematopoietic system and has been regarded as the innate immune cells taking responsibility for immune surveillance in the CNS (Ginhoux et al. 2010). However, once activated by inflammatory stimuli, such as LPS, the cell wall component of gram-negative bacteria, microglia can become overactivated and cause neurotoxicity (Block et al. 2007). LPS can trigger an immune response, and it has been a technique to activate microglia (Gao et al. 2002; Gibbons and Dragunow 2006). BV-2 cells derived from raf/myc-immortalized murine neonatal microglia are a recognized model of primary microglia (Henn et al. 2009; Laurenzi et al. 2001). In the present study, we determined whether trans-cinnamaldehyde would be neuroprotective in BV-2 cells incubated with LPS and in a mouse model of cerebral ischemia.

Trans-cinnamaldehyde reduced production of nitric oxide and expression of iNOS mRNA and iNOS protein stimulated by LPS-induced inflammation in BV-2 cells. These results are similar to earlier studies showing that trans-cinnamaldehyde reduced the expression of iNOS and COX-2 in different models, such as 6-OHD (Pyo et al. 2013), carrageenan-induced mouse paw edema (Liao et al. 2012) and LPS-stimulated mouse macrophage (RAW264.7) (Liao et al. 2012; Lee et al. 2005).

The anti-inflammatory effect of trans-cinnamaldehyde is attributed to its pharmacological action and not due to cytotoxicity. Cell viability was similar in LPS (0.5 μg/ml) treatment alone or co-treatment with LPS (0.5 μg/ml) and trans-cinnamaldehyde (12.5–50 μM). Previous studies have reported that activated microglia increase levels of COX-2 and TNF-α (Mozaffarian et al. 2015; Zhao et al. 2007). COX-2, a rate-limiting enzyme for the synthesis of prostaglandins, is a characteristic of neuroinflammation (Hoozemans et al. 2002). TNF-α, the proinflammatory cytokine, is known to up-regulate the transcription of COX-2 and iNOS genes (Hoozemans et al. 2002). Trans-cinnamaldehyde at 50 μM inhibited the LPS-induced increase in COX-2 protein levels and the expression of COX-2 mRNA and TNF-α mRNA. These data indicate that trans-cinnamaldehyde diminishes production of pro-inflammatory molecules induced by LPS. LPS stimulates inflammation via the activation of the NF-κB signaling pathway (Anderson 2000; Jung et al. 2009). NF-κB is an important transcription factor in responding pro-inflammatory stimuli, such as LPS. NF-κB regulates transcription of several genes such as iNOS, COX-2 and TNF-α, whose proteins products involve in inflammatory pathways (Baldwin 1996). Our present data showed that trans-cinnamaldehyde reduced p65 and p50 activation and IκBα. The anti-inflammatory effect of trans-cinnamaldehyde is consistent with previous reports (Tung et al. 2008, 2010; Ballabeni et al. 2010). Trans-cinnamaldehyde suppressed age-related NF-κB activation and its targeting inflammatory genes iNOS and COX-2 in rat kidney (Kim et al. 2007).

The p53 and NF-κB pathways are the two main pathways that respond to cell stress. p53, the major tumor suppressor, is activated by intrinsic stress signals (e.g., DNA damage, oncogene expression, spindle poisoning), while NF-κB is responding to extrinsic stress signals (e.g., the presence of infectious agents) (Gudkov et al. 2011). Several reports suggest that cells expressing NF-κB inhibit p53 activity and p53 responses, and, inversely, cells with up-regulated p53 suppress NF-κB activity (O’Prey et al. 2010; Hudson et al. 1999; Yonish-Rouach et al. 1991). Therefore, agents that inhibit NF-κB can activate p53 (Gurova et al. 2005). There is an abundance of evidence showing that p53 is an inhibitor of inflammation due to its antagonism of NF-κB (Gudkov et al. 2011; Komarova et al. 2005; Yamanishi et al. 2002; Okuda et al. 2003). We found that trans-cinnamaldehyde significantly inhibited both p53 reduction (Fig. 4c) and NF-κB activation (Fig. 5a, b) in LPS-stimulated BV-2 cells, and those findings are in agreement with those earlier studies (Youn et al. 2008).

In conclusion, we demonstrated that trans-cinnamaldehyde, an essential oil in cinnamon powder, significantly reduced infarct area in cerebral ischemia mouse model. It inhibited nitric oxide production and inflammation which were by down-regulating gene expression of iNOS, COX-2 and TNF-α, and suppressing the NF-κB and p53 pathways (showed as Fig. 7). These in vivo and in vitro results are strong evidence that trans-cinnamaldehyde can be a potential therapeutic agent to reduce inflammation associated with ischemic stroke.

Fig. 7
figure 7

Proposed action mechanism of trans-cinnamaldehyde (TCA) on neuroinflammation and neuroprotection. Trans-cinnamaldehyde inhibited nitric oxide production and inflammation which were by down-regulating gene expression of iNOS, COX-2 and TNF-α, and suppressing the NF-κB pathway

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