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Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury
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Hongxin Wang a,∗ , Kan Zhang b , Lan Zhao a , Jiangwei Tang a , Luyan Gao a , Zhongping Wei a a b
Department of Neurology, Affiliated Fourth Centre Hospital of Tianjin Medical University, Tianjin 300140, China Department of Neurology, Affiliated Hospital of Binzhou Medical University, Shandong 256603, China
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We set a rat model of cerebral ischemia–reperfusion injury. We have shown the time-dependent expression of NF-B and TNF-␣ in this model. Examine the expression of TNF-␣ mRNA in cytoplasm by in-situ hybridization. Extract nuclear protein and examine the NF-B P65 protein’s expression in nucleus. We demonstrate the ability of vinpocetine to suppress these inflammatory pathways.
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Article history: Received 17 October 2013 Received in revised form 9 February 2014 Accepted 24 February 2014
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Keywords: Vinpocetine Reperfusion injury Inflammation NF-B TNF-␣
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Introduction
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Objective: The restoration of blood flow to the brain after ischemic stroke prevents further, extensive damage but can result in reperfusion injury. The inflammation response is one of many factors involved in cerebral ischemia–reperfusion injury. This study investigated the use of vinpocetine, a drug used to treat cognitive impairment, to explore its effects on inflammation in a rat model of cerebral ischemia–reperfusion. Methods: Wistar rats were randomly assigned to a control group, (n = 40) a cerebral ischemia–reperfusion group (n = 52) and a vinpocetine cerebral ischemia–reperfusion group (n = 52). A model of middle cerebral artery occlusion was induced for 2 h followed by reperfusion and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 6 h, 24 h, 3 days, and 7 days after reperfusion. The dry–wet weight method was used to measure brain water content and evaluate the extent of brain edema. Immunohistochemistry and in-situ hybridization were used to detect the expression of NF-B and TNF-␣. Results: The NF-B levels in ischemic brain tissue increased 6 h after reperfusion and the TNF-␣ levels increased at 24 h, both reached their peaks at day 3 then decreased gradually, but remained above the controls at day 7. Vinpocetine decreased the levels of NF-B and TNF-␣ 24 h and 3 days after reperfusion. Conclusion: NF-B and TNF-␣ is associated with changes in brain edema and infarct volume. Vinpocetine decreases the expression of NF-B and TNF-␣ and inhibits the inflammatory response after cerebral ischemia–reperfusion. © 2014 Published by Elsevier Ireland Ltd.
During the treatment of stroke the restoration of blood to the ischemic brain is vital in preventing further damage. Unfortunately in some cases this reperfusion can cause further problems. Cerebral
∗ Corresponding author. Tel.: +86 22 26183237. E-mail address: [email protected] (H. Wang).
ischemia–reperfusion injury is caused by many processes such as oxidative stress, inflammation and apoptosis [8]. Among these mechanisms the inflammatory response is a critical factor. In [8] progression of the inflammation after ischemia–reperfusion injury, inflammatory cytokines activate nuclear transcription factor B (NFB) through toll-like receptor 4 (TLR4). NFB signal transduction pathways promote target gene activation and induce neuronal apoptosis, and even necrosis, ultimately aggravating the cerebral disease [2,4].
http://dx.doi.org/10.1016/j.neulet.2014.02.045 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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NF-B is implicated in the amplification and continuation (cascade effect) of the inflammatory response [3]. Cytoplasmic NF-B is in a complex with the inhibitor protein(IB␣).Activated NF-B is translocated into the nucleus where it binds to specific sequences of DNA called response elements (RE) [7,21]. The DNA/NF-B complex then recruits other proteins such as coactivators and RNA polymerase for mRNA transcription. NF-B can efficiently induce varieties of cell factors such as cytokines and chemokines, which in turn activates further NF-B expression. These all sustain and amplify the inflammatory response [17]. Vinpocetine was developed as a prospective treatment for cerebrovascular disorders and cognitive impairment [20]. Its main pathways involve [12]: (1) it restrains brain phosphodiesterase activity, diastolic vascular smooth muscle, increases the brain blood supply; (2) it improves brain metabolism, by promoting utilization of glucose and synthesis of adenosine triphosphate; (3) it is effective in reducing blood viscosity and inhibiting platelet gathering [10,12]. Current areas of neurological research include nerve protection and improvement of cognitive impairment by inhibition of the inflammatory pathway [1,15,16,19,20]. So vinpocetine may have huge potential for multiple uses in the treatment of neurological diseases [14]. Studies suggest that vinpocetine has an inhibitory phosphodiesterase activity, which regulates the cAMP/cGMP ratio, thus it may have a role in regulating the inflammatory response [9,10]. In prior work, many studies have chosen in vitro cultured cells to determine the molecular targets and their association with pro-inflammatory pathways such as NF-B and TNF-␣. In the current study, we have shown the time-dependent expression of NF-B and TNF-␣, enchaledema and cerebral infarct volumes in a rat model of ischemia–reperfusion injury. Importantly we demonstrate the ability of vinpocetine to suppress these inflammatory pathways, suggesting the therapeutic use of this agent in ischemia–reperfusion injury.
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Materials and methods
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144 healthy Wistar male rats 220–250 g in weight, were provided by the Tianjin Medical University laboratory animal centre. The rats were randomly divided into three groups, the control group (n = 40), the ischemia reperfusion group (model group, n = 52) and the vinpocetine treated group (vinpocetine group, n = 52). For each group, five rats were sacrificed at 6 h, 24 h, 3 days and 7 days to measure brain water content, another five were sacrificed for immunohistochemical and Western blot analysis of NF-B and TNF-␣ and in-situ hybridization of TNF-␣. For the vinpocetine group and the model group, the volume of cerebral infarction was measured by the TTC staining technique at each time point (n = 3).
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The model of cerebral ischemia–reperfusion injury
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The middle cerebral artery was occluded according to the Longa method. One end of nylon wire (diameter of 0.24 mm) was fired smooth and polished into the same size. All rats were anesthetized by intraperitoneal injection of chloral hydrate (10%, 3 ml/kg). The neck’s skin was cut to expose the common carotid artery on the right side, the external carotid artery branch and the pterygopalatine artery were ligated. The proximal end of the common carotid artery was ligated and a small hole was cut at the telecentric end, the nylon wire was inserted into the internal carotid artery until the cerebral middle artery (18 ± 0.5 mm). After 2 h the line was removed to achieve a reperfusion model. A successful model was judged to be when the rats woke up demonstrating left foreleg
buckling when lifting the tail, climbing in a circle to the left, and so on. Rats that showed excessive bleeding, dyspnea, subarachnoid hemorrhage and early death were excluded. For the control group, rats were ligated without inserting a nylon line. For the vinpocetine group, vinpocetine [5,6] (10 mg/kg) was given by intraperitoneal injection 1 h after inserting the nylon wire. Measurement of brain water content The water content of the brain tissue were measured by the dry–wet weight method. Rats were sacrificed at each time point after reperfusion injury. The cerebellum was removed with the brainstem and diencephalons and the weight of the wet cerebrum was measured. Then the brain was dried at 110 ◦ C for 24 h in an electrically heated drying oven and the weight of the cerebrum was measured. The following formula was used to evaluate the water content: water content = (wet weight − dry weight)/wet weight×100%. Volume of cerebral infarction After the rats were sacrificed, the cerebrum was removed and put in a −4 ◦ C refrigerator for 20 min; it was then sliced into five uniform coronal sections. The sections were placed in 2% 2,3,5triphenyltetrazolium chloride (TTC, Sigma) at 37 ◦ C in a water bath, then fixed with 4% paraformaldehyde. The normal brain tissue was dyed pink while infarction area was pale. The infarction area of every five sections was measured by using image analysis software (Scion Image) and the volume of cerebral infarction was estimated by cerebral infarction = the infarction area × thickness/2. Immunohistochemistry of NF-B and TNF-˛ Deeply anesthetized rats were beheaded and the brain was removed. After fixing in 4% paraformaldehyde for 48 h, 2 mm coronal sections were made from the front of the optic chiasma then dehydrated and immersed in wax to cut several 4 m slices. The dewaxed and hydrated slices were then probed with a mouse monoclonal primary antibody to NF-B or TNF-␣ (1:200 dilution, Santa cruz, USA) and incubated at 4 ◦ C for 12 h. A biotinylated antibody was then incubated at 37 ◦ C for 30 min and the sections were stained with DAB and restained with hematoxylin. PBS was used as a negative control. The cells were counted in a random 200 magnification view, 10 positive views were counted for every slice: 5 positive views were chosen from the brain cortex and the striate area. The positive cells in a grid were counted 3 times, then the mean value was calculated. In-situ hybridization of TNF-˛ in ischemic brain tissue Slice preparation for in-situ hybridization was as same as for immunohistochemistry, but the section thickness was 10 m and all the equipment used was inactivated for RNA-enzymes by highly compressed steam [13]. The TNF-␣ probe sequences were: (1) 5 -AAAGC ATGAT CCGAG ACGTG GAACT GGCCG AGGAG-3 ; (2) 5 CTGTA CCTCA TCTAC TCCCA GGTCC TCTTC AAGGG-3 ; (3) 5 -GAGTC TGGGC AGGTC TACTT TGGAG TCATT GCCCT-3 . After incubation with 3% pepsase every slice was incubated with 20 l of prehybridization solution (formamide 5 ml, 20 × SSC 2.5 ml, dextran sulfate 1 g, 100 × Denhardt’s 0.5 ml, 10%SDS 0.5 ml, 10 g/l sperm DNA 0.1 ml, H2 O 1.4 l) at 37 ◦ C for 2 h, then with 20 l hybridization solution containing the TNF-␣ probe at 37 ◦ C overnight. The slices were immersed serially in 2×, 0.5×, 0.2× SSC for 10 min each then blocked and then incubated with a biotinylated mouse antidigoxin antibody at 37 ◦ C for 1 h. After washing in PBS, with the
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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streptavidin–biotin complex (SABC) at 37 ◦ C for 20 min, biotin peroxidase at 37 ◦ C for 20 min they were finally stained with DAB and restained with hematoxylin. Prehybridization solution was used as negative control.
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Western blotting for NF-B P65 protein
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The following procedures were performed at 4 ◦ C. (1) Cerebral homogenate was suspended in 100 l buffer A (10 mmol/l HepesNaOH (pH7. 8), 15 mmol/l KCl, 1 mmol/l MgCl2 . 0.1 mmol/l EDTA, 1 mmol/l DTT, 1 mmol/l PMSF, l g/ml Leupeptin) and left to swell on ice for 15 min, then 10 l 10% NP-40 was added and the suspension was vortexed for 10 s; (2) centrifuged for 20 s 10,000 × g. The supernatant was removed and the pellet was mixed with 50–100 l Buffer B (20 mmol/L Hepes-NaOH (pH7. 9). 1.5 mmol/l MgCl2 , 0.42 mol/l NaCl, 0.2 mmol/l EDTA, 25% glycerin, 0.5 mmol/l DTT, 0.5 mmol/l PMSF, 1 g/ml Leupeptin) and placed in an icebath for 30 min. (3) Centrifuged for 4 min, 12,000 × g. Nucleoprotein was in the supernatant. After electrophoresis (separation on a 10% SDS-PAGE gel) and transfer to PVDF membranes (200 mA and 2.5 h) the membranes were then incubated with blocking solution (5% nonfat dried milk powder dissolved in TBST buffer-pH 7.5, 10 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, washed three times with TBST, and incubated with a mouse monoclonal anti-NF-B antibody (1:100 dilution, Santa Cruz, USA) for 24 h at 4 ◦ C in a refrigerator. The membranes were washed three times with TBST buffer and then incubated with the secondary antibody (1:1000 dilution, Santa Cruz, USA) for 1 h followed by washing four times. Signal detection was performed with an enhanced chemiluminescence kit (Beyotime, China). Histone was chosen as the Internal Control [18]. The band intensities were determined using Image-Pro Plus 6.0.
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Statistical analysis
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All descriptive statistics, including mean ± SD, were performed. Repeated-measures of two-way ANOVA was used to test differences among the model and vinpocetine groups. For the main effect of time, when the P value is statistically significant, comparison procedure was applied by using the post hoc test. ANOVA for multigroup analysis, and comparison among groups used a Q test. The water content and NF-B expression of the ischemic area of the cerebrum was analyzed with linear correlation analysis. SPSS 13.0 for Windows software was used for statistical analyses with a P value of 0.05). The comparisons between the control group and the vinpocetine group at each time point also showed no statistical differences (both P > 0.05). The results are shown in Fig. 3. TNF-˛ mRNA expression levels Compared with the control group the expression of TNF-␣ mRNA in both the model group and the vinpocetine group increased 24 h after reperfusion (both P < 0.05). Both groups reached a peak at 3 days (both P < 0.05). Compared with the model group the expression of TNF-a mRNA in the vinpocetine group was lower at 24 h,
Fig. 2. Change in brain water content. The amount of water contained within the brain are shown for the control group, the ischemia–reperfusion group (model) and the vinpocetine-treated group (vinpocetine).Values indicate mean ± SD,* P < 0.05 vs. control group; # P < 0.05 vs. model group; P < 0.05 vs. other time points in the control group, the model group and the vinpocetine treated group regarded separately.
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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Fig. 3. Expression of TNF-˛ of NF-B after ischemia–reperfusion and their inhibition treated with vinpocetine. (A) The inhibition TNF-␣ of 3 days after reperfusion is shown at ×100 magnification in immunohistochemistry experiment. (B) The inhibition NF-B of 3 days after reperfusion is shown at ×100 magnification in immunohistochemistry experiment. The levels of TNF-␣ are shown in A and the levels of NF-B are shown in B for three groups at 12 h, 24 h, 3 days, 7 days. Values indicate mean ± SD,* P < 0.05 vs. control group; # P < 0.05 vs. model group; P < 0.05 vs. other time points in the model group and the vinpocetine-treated group regarded separately.
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3 days and 7 days after reperfusion (all P < 0.05). The results are shown in Fig. 4.
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At the 24 h time point, compared with the control group the expression of NF-B in both the model group and the vinpocetine group increased after reperfusion (both P < 0.05). There was a statistically significant difference between the model group and the vinpocetine group at 24 h (P < 0.05), confirming that vinpocetine inhibited the expression of NF-B. Compared with other time points of the model group, the expression of NF-B at 24 h and 3 days has statistical significance (both P < 0.05), showing the change in expression of NF-B over 7 days. The results are shown in Fig. 5.
Fig. 4. Expression of TNF-˛ mRNA after ischemia–reperfusion and its inhibition treated with vinpocetine. The in-situ hybridization of the brain cells for tumor necrosis factoralpha (TNF-␣) mRNA are shown at ×200 magnification at 24 h after reperfusion. The estimated expression levels of TNF-␣ are shown for the three groups at 12 h, 24 h, 3 days, 7 days. Values indicate mean ± SD,* P < 0.05 vs. the control group; # P < 0.05 vs. the model group; P < 0.05 vs. other time points in the model group and the vinpocetine-treated group regarded separately.
Relationship between brain water content in ischemic brain tissue and the expression of NF-kB Comparison showed there was a positive correlation between the brain water content in ischemic brain tissue after reperfusion and the expression of NF-B (r = 0.4324, P < 0.01).
Discussion This study used a rat model to investigate cerebral ischemia–reperfusion, and if treatment with vinpocetine could improve their outcome. The inflammatory response was the main process of interest and the rat model demonstrated that expression levels of NF-B began to rise 6 h after reperfusion increasing to a peak after 3 days, 7 days later expression levels were still high. There was a significant increase in TNF-␣ and TNF-␣ mRNA expression 24 h after reperfusion with a peak at 3 days and expression remained high after 7 days. This suggests that NF-B expression occurs first and then mediates transcription of TNF-␣. Vinpocetine intervention significantly reduced the expression of NF-B and TNF-␣ by 24 h after reperfusion and for the 7 days studied. The expression levels of NF-B were fairly constant throughout the time of the study in the vinpocetine-treated group with no statistical differences between them. After 3 days the vinpocetine-treated group showed less infarction volume and cerebral edema than the untreated rat model group. Cerebral edema and NF-B expression correlated significantly, suggesting that vinpocetine blocks the inflammatory response pathway and does this by lowering NF-B and TNF-␣ expression levels. Prior research has suggested that vinpocetine has a role in reducing the inflammatory response. Vitro experiments have shown that in a wide range of cells vinpocetine can reduce TNF-␣ induced activation of NF-B [6]. RT-PCR analysis found that vinpocetine can reduce the expression of TNF-␣, IL-1, vascular endothelial cell adhesion molecule 1 (VCAM-1) mRNA during inflammation [11]. In our positive expression NF-B is mainly located in the cell nucleus, while TNF-␣ is mainly in the cytoplasm. In cerebral ischemia–reperfusion injury IB and NF-B dissociate exposing RE and NF-B moves into nucleus. This series of events initiates transcription regulation thus leading to the transcription of TNF-␣ mRNA and so TNF-␣ is highly expressed. We found the nuclear NF-B levels in the vinpocetine-treated group were lower than in the untreated rat model so we speculated that vinpocetine
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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Fig. 5. Western blot analysis for nuclear factor-kappa B. (A) Western blot are shown by ECL chemiluminescence imaging. The estimated expression levels of Nucleus p65 protein at 24 h time point are shown for three groups; (B) Western blot are shown by ECL chemiluminescence imaging. The estimated expression levels of Nucleus p65 protein are shown for the ischemia–reperfusion group (model) at 12 h, 24 h, 3 days. Values indicate mean ± SD, * P < 0.05 vs. the control group; # P < 0.05 vs. the model group; P < 0.05 vs. other time points in the model group and the vinpocetine treated group regarded separately.
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suppressed the movement of NF-B from the cytoplasm to the nucleus. But whether this occurs by inhibiting IkB phosphorylation, by decreasing IB levels in the cytoplasm, or by preventing IB and NF-B dissociation needs further study. In conclusion vinpocetine blocks the inflammatory response pathway after ischemia–reperfusion injury of rats; it can delay necrosis and reduce cerebral infarction volume. With further research, vinpocetine should become an effective antiinflammatory drug in the treatment of neurological diseases.
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Acknowledgement
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This research was supported by Tianjin Health Bureau and approved by the Ethics Committee of the Institute. The authors declare that they have no conflicts of interest in the research.
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References
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Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury
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Hongxin Wang a,∗ , Kan Zhang b , Lan Zhao a , Jiangwei Tang a , Luyan Gao a , Zhongping Wei a a b
Department of Neurology, Affiliated Fourth Centre Hospital of Tianjin Medical University, Tianjin 300140, China Department of Neurology, Affiliated Hospital of Binzhou Medical University, Shandong 256603, China
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h i g h l i g h t s • • • • •
We set a rat model of cerebral ischemia–reperfusion injury. We have shown the time-dependent expression of NF-B and TNF-␣ in this model. Examine the expression of TNF-␣ mRNA in cytoplasm by in-situ hybridization. Extract nuclear protein and examine the NF-B P65 protein’s expression in nucleus. We demonstrate the ability of vinpocetine to suppress these inflammatory pathways.
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Article history: Received 17 October 2013 Received in revised form 9 February 2014 Accepted 24 February 2014
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Keywords: Vinpocetine Reperfusion injury Inflammation NF-B TNF-␣
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Objective: The restoration of blood flow to the brain after ischemic stroke prevents further, extensive damage but can result in reperfusion injury. The inflammation response is one of many factors involved in cerebral ischemia–reperfusion injury. This study investigated the use of vinpocetine, a drug used to treat cognitive impairment, to explore its effects on inflammation in a rat model of cerebral ischemia–reperfusion. Methods: Wistar rats were randomly assigned to a control group, (n = 40) a cerebral ischemia–reperfusion group (n = 52) and a vinpocetine cerebral ischemia–reperfusion group (n = 52). A model of middle cerebral artery occlusion was induced for 2 h followed by reperfusion and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 6 h, 24 h, 3 days, and 7 days after reperfusion. The dry–wet weight method was used to measure brain water content and evaluate the extent of brain edema. Immunohistochemistry and in-situ hybridization were used to detect the expression of NF-B and TNF-␣. Results: The NF-B levels in ischemic brain tissue increased 6 h after reperfusion and the TNF-␣ levels increased at 24 h, both reached their peaks at day 3 then decreased gradually, but remained above the controls at day 7. Vinpocetine decreased the levels of NF-B and TNF-␣ 24 h and 3 days after reperfusion. Conclusion: NF-B and TNF-␣ is associated with changes in brain edema and infarct volume. Vinpocetine decreases the expression of NF-B and TNF-␣ and inhibits the inflammatory response after cerebral ischemia–reperfusion. © 2014 Published by Elsevier Ireland Ltd.
During the treatment of stroke the restoration of blood to the ischemic brain is vital in preventing further damage. Unfortunately in some cases this reperfusion can cause further problems. Cerebral
∗ Corresponding author. Tel.: +86 22 26183237. E-mail address: [email protected] (H. Wang).
ischemia–reperfusion injury is caused by many processes such as oxidative stress, inflammation and apoptosis [8]. Among these mechanisms the inflammatory response is a critical factor. In [8] progression of the inflammation after ischemia–reperfusion injury, inflammatory cytokines activate nuclear transcription factor B (NFB) through toll-like receptor 4 (TLR4). NFB signal transduction pathways promote target gene activation and induce neuronal apoptosis, and even necrosis, ultimately aggravating the cerebral disease [2,4].
http://dx.doi.org/10.1016/j.neulet.2014.02.045 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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NF-B is implicated in the amplification and continuation (cascade effect) of the inflammatory response [3]. Cytoplasmic NF-B is in a complex with the inhibitor protein(IB␣).Activated NF-B is translocated into the nucleus where it binds to specific sequences of DNA called response elements (RE) [7,21]. The DNA/NF-B complex then recruits other proteins such as coactivators and RNA polymerase for mRNA transcription. NF-B can efficiently induce varieties of cell factors such as cytokines and chemokines, which in turn activates further NF-B expression. These all sustain and amplify the inflammatory response [17]. Vinpocetine was developed as a prospective treatment for cerebrovascular disorders and cognitive impairment [20]. Its main pathways involve [12]: (1) it restrains brain phosphodiesterase activity, diastolic vascular smooth muscle, increases the brain blood supply; (2) it improves brain metabolism, by promoting utilization of glucose and synthesis of adenosine triphosphate; (3) it is effective in reducing blood viscosity and inhibiting platelet gathering [10,12]. Current areas of neurological research include nerve protection and improvement of cognitive impairment by inhibition of the inflammatory pathway [1,15,16,19,20]. So vinpocetine may have huge potential for multiple uses in the treatment of neurological diseases [14]. Studies suggest that vinpocetine has an inhibitory phosphodiesterase activity, which regulates the cAMP/cGMP ratio, thus it may have a role in regulating the inflammatory response [9,10]. In prior work, many studies have chosen in vitro cultured cells to determine the molecular targets and their association with pro-inflammatory pathways such as NF-B and TNF-␣. In the current study, we have shown the time-dependent expression of NF-B and TNF-␣, enchaledema and cerebral infarct volumes in a rat model of ischemia–reperfusion injury. Importantly we demonstrate the ability of vinpocetine to suppress these inflammatory pathways, suggesting the therapeutic use of this agent in ischemia–reperfusion injury.
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Materials and methods
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Animals
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144 healthy Wistar male rats 220–250 g in weight, were provided by the Tianjin Medical University laboratory animal centre. The rats were randomly divided into three groups, the control group (n = 40), the ischemia reperfusion group (model group, n = 52) and the vinpocetine treated group (vinpocetine group, n = 52). For each group, five rats were sacrificed at 6 h, 24 h, 3 days and 7 days to measure brain water content, another five were sacrificed for immunohistochemical and Western blot analysis of NF-B and TNF-␣ and in-situ hybridization of TNF-␣. For the vinpocetine group and the model group, the volume of cerebral infarction was measured by the TTC staining technique at each time point (n = 3).
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The model of cerebral ischemia–reperfusion injury
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The middle cerebral artery was occluded according to the Longa method. One end of nylon wire (diameter of 0.24 mm) was fired smooth and polished into the same size. All rats were anesthetized by intraperitoneal injection of chloral hydrate (10%, 3 ml/kg). The neck’s skin was cut to expose the common carotid artery on the right side, the external carotid artery branch and the pterygopalatine artery were ligated. The proximal end of the common carotid artery was ligated and a small hole was cut at the telecentric end, the nylon wire was inserted into the internal carotid artery until the cerebral middle artery (18 ± 0.5 mm). After 2 h the line was removed to achieve a reperfusion model. A successful model was judged to be when the rats woke up demonstrating left foreleg
buckling when lifting the tail, climbing in a circle to the left, and so on. Rats that showed excessive bleeding, dyspnea, subarachnoid hemorrhage and early death were excluded. For the control group, rats were ligated without inserting a nylon line. For the vinpocetine group, vinpocetine [5,6] (10 mg/kg) was given by intraperitoneal injection 1 h after inserting the nylon wire. Measurement of brain water content The water content of the brain tissue were measured by the dry–wet weight method. Rats were sacrificed at each time point after reperfusion injury. The cerebellum was removed with the brainstem and diencephalons and the weight of the wet cerebrum was measured. Then the brain was dried at 110 ◦ C for 24 h in an electrically heated drying oven and the weight of the cerebrum was measured. The following formula was used to evaluate the water content: water content = (wet weight − dry weight)/wet weight×100%. Volume of cerebral infarction After the rats were sacrificed, the cerebrum was removed and put in a −4 ◦ C refrigerator for 20 min; it was then sliced into five uniform coronal sections. The sections were placed in 2% 2,3,5triphenyltetrazolium chloride (TTC, Sigma) at 37 ◦ C in a water bath, then fixed with 4% paraformaldehyde. The normal brain tissue was dyed pink while infarction area was pale. The infarction area of every five sections was measured by using image analysis software (Scion Image) and the volume of cerebral infarction was estimated by cerebral infarction = the infarction area × thickness/2. Immunohistochemistry of NF-B and TNF-˛ Deeply anesthetized rats were beheaded and the brain was removed. After fixing in 4% paraformaldehyde for 48 h, 2 mm coronal sections were made from the front of the optic chiasma then dehydrated and immersed in wax to cut several 4 m slices. The dewaxed and hydrated slices were then probed with a mouse monoclonal primary antibody to NF-B or TNF-␣ (1:200 dilution, Santa cruz, USA) and incubated at 4 ◦ C for 12 h. A biotinylated antibody was then incubated at 37 ◦ C for 30 min and the sections were stained with DAB and restained with hematoxylin. PBS was used as a negative control. The cells were counted in a random 200 magnification view, 10 positive views were counted for every slice: 5 positive views were chosen from the brain cortex and the striate area. The positive cells in a grid were counted 3 times, then the mean value was calculated. In-situ hybridization of TNF-˛ in ischemic brain tissue Slice preparation for in-situ hybridization was as same as for immunohistochemistry, but the section thickness was 10 m and all the equipment used was inactivated for RNA-enzymes by highly compressed steam [13]. The TNF-␣ probe sequences were: (1) 5 -AAAGC ATGAT CCGAG ACGTG GAACT GGCCG AGGAG-3 ; (2) 5 CTGTA CCTCA TCTAC TCCCA GGTCC TCTTC AAGGG-3 ; (3) 5 -GAGTC TGGGC AGGTC TACTT TGGAG TCATT GCCCT-3 . After incubation with 3% pepsase every slice was incubated with 20 l of prehybridization solution (formamide 5 ml, 20 × SSC 2.5 ml, dextran sulfate 1 g, 100 × Denhardt’s 0.5 ml, 10%SDS 0.5 ml, 10 g/l sperm DNA 0.1 ml, H2 O 1.4 l) at 37 ◦ C for 2 h, then with 20 l hybridization solution containing the TNF-␣ probe at 37 ◦ C overnight. The slices were immersed serially in 2×, 0.5×, 0.2× SSC for 10 min each then blocked and then incubated with a biotinylated mouse antidigoxin antibody at 37 ◦ C for 1 h. After washing in PBS, with the
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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streptavidin–biotin complex (SABC) at 37 ◦ C for 20 min, biotin peroxidase at 37 ◦ C for 20 min they were finally stained with DAB and restained with hematoxylin. Prehybridization solution was used as negative control.
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Western blotting for NF-B P65 protein
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The following procedures were performed at 4 ◦ C. (1) Cerebral homogenate was suspended in 100 l buffer A (10 mmol/l HepesNaOH (pH7. 8), 15 mmol/l KCl, 1 mmol/l MgCl2 . 0.1 mmol/l EDTA, 1 mmol/l DTT, 1 mmol/l PMSF, l g/ml Leupeptin) and left to swell on ice for 15 min, then 10 l 10% NP-40 was added and the suspension was vortexed for 10 s; (2) centrifuged for 20 s 10,000 × g. The supernatant was removed and the pellet was mixed with 50–100 l Buffer B (20 mmol/L Hepes-NaOH (pH7. 9). 1.5 mmol/l MgCl2 , 0.42 mol/l NaCl, 0.2 mmol/l EDTA, 25% glycerin, 0.5 mmol/l DTT, 0.5 mmol/l PMSF, 1 g/ml Leupeptin) and placed in an icebath for 30 min. (3) Centrifuged for 4 min, 12,000 × g. Nucleoprotein was in the supernatant. After electrophoresis (separation on a 10% SDS-PAGE gel) and transfer to PVDF membranes (200 mA and 2.5 h) the membranes were then incubated with blocking solution (5% nonfat dried milk powder dissolved in TBST buffer-pH 7.5, 10 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, washed three times with TBST, and incubated with a mouse monoclonal anti-NF-B antibody (1:100 dilution, Santa Cruz, USA) for 24 h at 4 ◦ C in a refrigerator. The membranes were washed three times with TBST buffer and then incubated with the secondary antibody (1:1000 dilution, Santa Cruz, USA) for 1 h followed by washing four times. Signal detection was performed with an enhanced chemiluminescence kit (Beyotime, China). Histone was chosen as the Internal Control [18]. The band intensities were determined using Image-Pro Plus 6.0.
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Statistical analysis
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All descriptive statistics, including mean ± SD, were performed. Repeated-measures of two-way ANOVA was used to test differences among the model and vinpocetine groups. For the main effect of time, when the P value is statistically significant, comparison procedure was applied by using the post hoc test. ANOVA for multigroup analysis, and comparison among groups used a Q test. The water content and NF-B expression of the ischemic area of the cerebrum was analyzed with linear correlation analysis. SPSS 13.0 for Windows software was used for statistical analyses with a P value of 0.05). The comparisons between the control group and the vinpocetine group at each time point also showed no statistical differences (both P > 0.05). The results are shown in Fig. 3. TNF-˛ mRNA expression levels Compared with the control group the expression of TNF-␣ mRNA in both the model group and the vinpocetine group increased 24 h after reperfusion (both P < 0.05). Both groups reached a peak at 3 days (both P < 0.05). Compared with the model group the expression of TNF-a mRNA in the vinpocetine group was lower at 24 h,
Fig. 2. Change in brain water content. The amount of water contained within the brain are shown for the control group, the ischemia–reperfusion group (model) and the vinpocetine-treated group (vinpocetine).Values indicate mean ± SD,* P < 0.05 vs. control group; # P < 0.05 vs. model group; P < 0.05 vs. other time points in the control group, the model group and the vinpocetine treated group regarded separately.
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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Fig. 3. Expression of TNF-˛ of NF-B after ischemia–reperfusion and their inhibition treated with vinpocetine. (A) The inhibition TNF-␣ of 3 days after reperfusion is shown at ×100 magnification in immunohistochemistry experiment. (B) The inhibition NF-B of 3 days after reperfusion is shown at ×100 magnification in immunohistochemistry experiment. The levels of TNF-␣ are shown in A and the levels of NF-B are shown in B for three groups at 12 h, 24 h, 3 days, 7 days. Values indicate mean ± SD,* P < 0.05 vs. control group; # P < 0.05 vs. model group; P < 0.05 vs. other time points in the model group and the vinpocetine-treated group regarded separately.
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3 days and 7 days after reperfusion (all P < 0.05). The results are shown in Fig. 4.
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Western blot analysis of NF-B P65 protein
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At the 24 h time point, compared with the control group the expression of NF-B in both the model group and the vinpocetine group increased after reperfusion (both P < 0.05). There was a statistically significant difference between the model group and the vinpocetine group at 24 h (P < 0.05), confirming that vinpocetine inhibited the expression of NF-B. Compared with other time points of the model group, the expression of NF-B at 24 h and 3 days has statistical significance (both P < 0.05), showing the change in expression of NF-B over 7 days. The results are shown in Fig. 5.
Fig. 4. Expression of TNF-˛ mRNA after ischemia–reperfusion and its inhibition treated with vinpocetine. The in-situ hybridization of the brain cells for tumor necrosis factoralpha (TNF-␣) mRNA are shown at ×200 magnification at 24 h after reperfusion. The estimated expression levels of TNF-␣ are shown for the three groups at 12 h, 24 h, 3 days, 7 days. Values indicate mean ± SD,* P < 0.05 vs. the control group; # P < 0.05 vs. the model group; P < 0.05 vs. other time points in the model group and the vinpocetine-treated group regarded separately.
Relationship between brain water content in ischemic brain tissue and the expression of NF-kB Comparison showed there was a positive correlation between the brain water content in ischemic brain tissue after reperfusion and the expression of NF-B (r = 0.4324, P < 0.01).
Discussion This study used a rat model to investigate cerebral ischemia–reperfusion, and if treatment with vinpocetine could improve their outcome. The inflammatory response was the main process of interest and the rat model demonstrated that expression levels of NF-B began to rise 6 h after reperfusion increasing to a peak after 3 days, 7 days later expression levels were still high. There was a significant increase in TNF-␣ and TNF-␣ mRNA expression 24 h after reperfusion with a peak at 3 days and expression remained high after 7 days. This suggests that NF-B expression occurs first and then mediates transcription of TNF-␣. Vinpocetine intervention significantly reduced the expression of NF-B and TNF-␣ by 24 h after reperfusion and for the 7 days studied. The expression levels of NF-B were fairly constant throughout the time of the study in the vinpocetine-treated group with no statistical differences between them. After 3 days the vinpocetine-treated group showed less infarction volume and cerebral edema than the untreated rat model group. Cerebral edema and NF-B expression correlated significantly, suggesting that vinpocetine blocks the inflammatory response pathway and does this by lowering NF-B and TNF-␣ expression levels. Prior research has suggested that vinpocetine has a role in reducing the inflammatory response. Vitro experiments have shown that in a wide range of cells vinpocetine can reduce TNF-␣ induced activation of NF-B [6]. RT-PCR analysis found that vinpocetine can reduce the expression of TNF-␣, IL-1, vascular endothelial cell adhesion molecule 1 (VCAM-1) mRNA during inflammation [11]. In our positive expression NF-B is mainly located in the cell nucleus, while TNF-␣ is mainly in the cytoplasm. In cerebral ischemia–reperfusion injury IB and NF-B dissociate exposing RE and NF-B moves into nucleus. This series of events initiates transcription regulation thus leading to the transcription of TNF-␣ mRNA and so TNF-␣ is highly expressed. We found the nuclear NF-B levels in the vinpocetine-treated group were lower than in the untreated rat model so we speculated that vinpocetine
Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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Fig. 5. Western blot analysis for nuclear factor-kappa B. (A) Western blot are shown by ECL chemiluminescence imaging. The estimated expression levels of Nucleus p65 protein at 24 h time point are shown for three groups; (B) Western blot are shown by ECL chemiluminescence imaging. The estimated expression levels of Nucleus p65 protein are shown for the ischemia–reperfusion group (model) at 12 h, 24 h, 3 days. Values indicate mean ± SD, * P < 0.05 vs. the control group; # P < 0.05 vs. the model group; P < 0.05 vs. other time points in the model group and the vinpocetine treated group regarded separately.
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suppressed the movement of NF-B from the cytoplasm to the nucleus. But whether this occurs by inhibiting IkB phosphorylation, by decreasing IB levels in the cytoplasm, or by preventing IB and NF-B dissociation needs further study. In conclusion vinpocetine blocks the inflammatory response pathway after ischemia–reperfusion injury of rats; it can delay necrosis and reduce cerebral infarction volume. With further research, vinpocetine should become an effective antiinflammatory drug in the treatment of neurological diseases.
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Acknowledgement
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This research was supported by Tianjin Health Bureau and approved by the Ethics Committee of the Institute. The authors declare that they have no conflicts of interest in the research.
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References
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Please cite this article in press as: H. Wang, et al., Anti-inflammatory effects of vinpocetine on the functional expression of nuclear factor-kappa B and tumor necrosis factor-alpha in a rat model of cerebral ischemia–reperfusion injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.02.045
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