Inflammation ( # 2015) DOI: 10.1007/s10753-015-0137-x
Triptolide Protects Against Ischemic Stroke in Rats Maolin Hao,1,3 Xianghua Li,2 Jianli Feng,1 and Ning Pan1
Abstract—Inflammatory response plays an important role in the pathogenesis of ischemic stroke and anti-inflammatory agents may provide a choice of treatment. Triptolide is reported to be antiinflammatory. In this study, we investigated the effects of triptolide on cultured neuronal cell line in vitro and experimental ischemic stroke in vivo. Oxygen–glucose deprivation (OGD) and tumor necrosis factor-α (TNF-α) stimulated SH-SY5Y cells were incubated with triptolide. In vivo, rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion for 23 h. Results of this study showed that triptolide treatment reduced the OGD-induced cytotoxicity and apoptosis and blocked TNF-α-induced activation of NF-κB and p38MAPK in SH-SY5Y cells. Intraperitoneal injection of triptolide showed significant neuroprotective actions in stroke rats. Triptolide attenuated neurological deficit, brain infarct volume, and brain water content, and inhibited activation of NF-κB and p38MAPK. These data show that triptolide protects rats against ischemic cerebral injury via inhibiting NF-κB and p38MAPK signaling pathways. KEY WORDS: triptolide; rats; experimental stroke.
INTRODUCTION Stroke, a major cause of death and impairment in humans, is the third leading cause of death in industrialized countries and the most common cause of permanent disability in adults globally [1]. Stroke is a constantly expanding area of research. Surviving patients demonstrate significant risk for the development of new acute neurological symptoms and death [2]. The mechanisms that trigger ischemic brain damage are complex, but inflammatory response has been considered as a key element in ischemic stroke of both animals and human [3, 4]. Triptolide is extracted from Tripterygium wilfordii, which is a traditional Chinese medicine and exhibits immunosuppressive, anti-inflammatory, and anti-tumor activities [5–7]. Triptolide exhibits anti-inflammatory activities in cultured lung cells and in rat models with chlorineinduced acute lung injury [8]. However, to our knowledge, 1
Department of Neurology, Shandong Provincial Hospital Affiliated to Shandong University, #4 Duanxing West Road, Jinan, 250022Shandong, China 2 Department of Neurology, The Sixth People’s Hospital of Jinan, Zhangqiu, 250200Shandong, China 3 To whom correspondence should be addressed at Department of Neurology, Shandong Provincial Hospital Affiliated to Shandong University, #4 Duanxing West Road, Jinan, 250022Shandong, China. E-mail: [email protected]
the effect of triptolide in ischemic stroke has not been investigated yet. In this study, we tested the effects of triptolide on cultured neuronal cell line and rat ischemic stroke.
MATERIALS AND METHODS Materials Triptolide was purchased from Sigma Chemical Co. (St. Louis, MO, USA), with purity of more than 98 %. The human neuroblastoma cell line SH-SY5Y was purchased from American type culture collection (ATCC, Manassas, VA, USA). Cell Culture SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) solutions (Gibco Laboratories, Grand Island, NY, USA), supplemented with 10 % fetal bovine serum (FBS, Gibco Laboratories, Grand Island, NY, USA). Cells were kept at 37 °C in a humidified 5 % CO2 incubator. The dissociated cells were seeded in poly5 2 L-lysine-coated plates at a density of 5 × 10 /cm and cultured in DMEM, supplemented with 10 % fetal bovine serum. The medium was changed twice weekly. After
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Hao, Li, Feng, and Pan 8 days of culture, SH-SY5Y cells were randomly divided into seven groups: normal group (no oxygen–glucose deprivation), oxygen–glucose deprivation group (control group), and oxygen–glucose deprivation + triptolide (dissolved in DMSO, 5, 10, 20, 40, and 80 ng/ml) groups. For oxygen–glucose deprivation, the SH-SY5Y cells were washed twice in glucose-free balanced salt solution (BSS), and incubated in BSS (glucose-free) in a humidified chamber filled with 95 % N2/5 % CO2 for 3 h at 36.5 °C, while the normal group and triptolide group (no oxygen– glucose deprivation) filled with 5 % CO2/95 % O2 and incubated in glucose-free BSS. After oxygen–glucose deprivation, the cultures were replaced into neurobasal medium and incubated with triptolide in a CO2 incubator for 12 h.
reperfusion as described before [10]. The vehicletreated group received 1 % phosphate buffered saline (PBS)/DMSO. Neurological deficits were determined at 24 h after ischemia followed by brain infarct volume examination.
Determination of Cell Viability and Apoptosis
Evaluation of Infarct Volume and Brain Water Content
After oxygen–glucose deprivation for 3 h followed by 12-h incubation with or without triptolide, cell viability was assessed using 3-[4, 5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay (Genebase, Shanghai, China). Apoptotic cells were evaluated using an Annexin-V FITC apoptosis detection kit (Genebase, Shanghai, China). In brief, cells were harvested, washed, and incubated at 4 °C for 30 min in the dark with annexinV FITC and propidium iodide, then analyzed on a FACS Vantage SE flow cytometer (Beckman Coulter, USA).
After 23-h reperfusion, rats were anesthetized with sodium pentobarbital through intraperitoneal injection. The adequacy of anesthesia was monitored by the level and stability of the mean arterial pressure (MAP) and absence of the corneal reflex, and adequate levels of anesthesia and analgesia were ensured with supplemental intraperitoneal injection of pentobarbital sodium given as required, then brains were quickly removed. The total wet weight of the brain was measured accurately. The starting site of the brain was cut at forebrain 3 mm, and each brain was sliced into sections of 2-mm thickness each, then stained with a 2 % solution of tetrazolium chloride (Sigma, St. Louis, MO, USA) in saline at 37 °C for 10 min, and photographed. The images were digitized, and infarct volume was calculated with a Compix system computer. Then, brain water content was determined as an indicator of cerebral edema using a wet/dry method.
Induction of Rat Cerebral Ischemia and Treatments All the experiments were approved by our Animal Care Committee. Male Sprague–Dawley rats (250–280 g) were supplied by the Animal Center of our University and maintained on a 12-h light/12-h dark regime. All rats were provided with food and water ad libitum. The animals were acclimatized to the laboratory conditions for 7 days before surgery. Animals were anesthetized by intraperitoneal injection of 10 % chloral hydrate. Rectal temperature was recorded and maintained at 37 °C throughout the surgical procedure. The rats were subjected to middle cerebral artery occlusion (MCAO) by intraluminal placement of a filament as described previously [9]. Sham-operated rats (n = 10) were subjected only to exposure of the MCA without coagulation. Anesthesia duration was similar in all groups. The mortality rate after the surgery was nearly 20 % and we chose the surviving rats for further experiments. Rats were randomly divided into two groups with ten rats each. Rats received intraperitoneal 1.0 mg/kg triptolide (dissolved in DMSO) after
Evaluation of Neurological Deficits Neurological deficits were evaluated using a 0–5 scoring system [11] by an investigator who was blinded to each experimental group: 0, no neurological deficits (normal); 1, failure to extend left forepaw fully; 2, circling to the left; 3, falling to the left; 4, no spontaneous walking with a depressed level of consciousness, and 5, death.
Evaluation of Blood–Brain Barrier Leakage with Evans Blue Extravasation Determination of Evans blue extravasation was based on a previous method [12]. After reperfusion, 0.1 ml of 4 % Evans blue in saline was administered via tail vein. Twenty-three hours after ischemia, rats were anesthetized with chloral hydrate then perfused with 20 ml 10 U/ml heparinized saline to wash out the blood. The brain was then isolated, weighed, and homogenized in 50 % solution of trichloroacetic acid (Sigma, St. Louis, MO, USA). After centrifugation at 600g for 15 min, the supernatant was
Triptolide in Experimental Stroke spectrophotometrically measured at 490 nm. Cerebral Evans blue was quantified as micrograms of dye per gram of wet weight.
Western Blot For the experiment of tumor necrosis factor-α (TNFα) stimulated SH-SY5Y cell lines in vitro, SH-SY5Y cells (5×106) were pre-incubated with triptolide (40 ng/ml) for 12 h, then incubated with TNF-α (20 ng/ml) for 30 min. Cells were washed twice with ice-cold PBS on ice and lysed in cell lysis buffer (Sigma, Saint Louis, MO, USA). For western blot analysis, brains were harvested and the right hemispheres containing the ischaemic sites were excised and nuclear and cytosolic extracts of cells or tissues were isolated using the NE-PER kit (Life Technologies, Foster City, CA, USA) according to the manufacturer’s instructions. Equal amounts of cell protein (50 μg) were separated by SDS-PAGE and analyzed by Western blot using specific antibodies to NF-κBp65 (polyclonal rabbit, Cat No.
Fig. 1. After 3-h oxygen–glucose deprivation followed by 12-h incubation with triptolide, cell viability (a) and apoptosis (b) were assessed. N normal, C control: oxygen–glucose deprivation. Data are presented as mean ± SEM. n = 10. *P < 0.01 vs. control. All experiments were conducted in triplicate.
SAB4502610, 1:2000), p38MAPK (polyclonal rabbit, Cat No. SAB4500493, 1:2500) and p-p38MAPK(polyclonal rabbit, Cat No. SAB4504095,1:2000) followed by secondary antibodies (1:3000, polyclonal rabbit, Cat No. A9919). All antibodies were all purchased from Sigma, Saint Louis, MO, USA. Optical densities of the bands were scanned and quantified with a Gel Doc 2000 (Bio-Rad Laboratories). Lamin A (polyclonal rabbit, Cat No. L1293, Sigma, Saint Louis, MO, USA) levels are shown as an internal control. Results were expressed as ratio of control.
Statistical Analysis All data were analyzed using SPSS 16.0 software. Neurological deficit scores between groups were analyzed using Mann–Whitney U test. Quantitative data were expressed as mean±SEM. Statistical comparisons were performed by one-way ANOVA for multiple comparisons
Fig. 2. Triptolide inhibited activation of NF-κB and MAPK in cultured SH-SY5Y cells. SH-SY5Y cells were incubated with triptolide (40 ng/ml) for 12 h and then incubated with TNF-α (20 ng/ml) for 30 min. Nuclear NF-κBp65 and p-p38MAPK/p38MAPK expression were analyzed by Western blot. #P < 0.01 vs. blank; *P < 0.01 vs. TNF-α stimulation in the absence of triptolide. All the experiments were conducted in triplicate.
Hao, Li, Feng, and Pan following LSD. P < 0.05 was considered statistically significant.
RESULTS Triptolide Protected Cultured SH-SY5Y Cells Against Oxygen–Glucose Deprivation-Induced Cytotoxicity and Apoptosis The MTT assay was used to assess the effect of triptolide on the oxygen–glucose deprivation-induced cytotoxicity and we found that cell viability was markedly decreased after oxygen–glucose deprivation for 3 h followed by 12-h incubation with neurobasal medium (Fig. 1a). In contrast, the cytotoxicity was significantly attenuated by triptolide (5–80 ng/ml) treatment in a concentration-dependent manner. To further investigate the protective effect of triptolide, apoptosis was estimated by flow cytometric analysis of annexin-V and propidium iodide-labeling cells. The oxygen–glucose deprivation for 3 h followed by 12-h incubation with neurobasal medium significantly increased the apoptosis (Fig. 1b). Triptolide (5–80 ng/ml) for 12 h arrested the apoptosis in a concentration-dependent manner.
Triptolide Inhibited Activation of NF-κB and p38MAPK in Cultured SH-SY5Y Cells After TNF-α (20 ng/ml) stimulation for 30 min, NFκB and p38MAPK were both significantly activated in SHSY5Y cells. We compared the effect of triptolide on the TNF-α-induced (20 ng/ml for 30 min) activation of NF-κB and p38MAPK in the SH-SY5Y cells. As shown in Fig. 1, 40 ng/ml triptolide showed the best effectiveness, and we chose this concentration for the following experiments. We found that 40 ng/ml triptolide pretreatment blocked the TNF-α-induced activation of NF-κB and p38MAPK (Fig. 2). Triptolide Reduced Neurological Deficit Scores, Brain Infarct Volume, and Brain Water Content in Stroke Rats Cerebral ischemic injury results in behavioral disturbance and histological changes in MCAO rats, showing that ischemic region was white and non-ischemic region was red. Compared with the sham-operated rats, neurological deficit scores, the ischemic regions and brain water content were significantly increased in rats with experimental stroke. Our results showed that triptolide (1.0 mg/
Fig. 3. Triptolide treatment reduced the infarct volume, neurological scores, and brain water content in stroke rats. Each brain was stained with a 2 % solution of tetrazolium chloride (a). #P < 0.01 vs. sham; *P < 0.01 vs. vehicle.
Triptolide in Experimental Stroke kg) after reperfusion immediately decreased neurological deficit scores, cerebral infarct volume, and brain water content (Fig. 3). Triptolide Reduced Blood–Brain Barrier Breakdown in Stroke Rats Evans blue extravasation was used to assess blood– brain barrier (BBB) breakdown after cerebral ischemia. Vehicle-treated rats showed elevated Evans blue extravasation compared with the sham-operated rats. In contrast, our results showed that triptolide significantly attenuated Evans blue extravasation (Fig. 4). Triptolide Inhibited NF-κB and p38MAPK Activation in Stroke Rats In order to investigate the molecular mechanism of triptolide, NF-κB and p38MAPK activation was examined. Activation of NF-κB and p38MAPK was significantly increased in rats after cerebral ischemia. However, triptolide treatment reduced nuclear NF-κBp65 and pp38MAPK, indicating that triptolide blocked activation of NF-κB and p38MAPK in stroke rats (Fig. 5).
DISCUSSION It is well established that MCAO is a classical model of cerebral ischemia [13]. In this study, we showed that triptolide improved neurological dysfunction, reduced infarct size, and BBB permeability after MCAO. These protective effects might be mediated via inhibiting NFκB and MAPK signaling pathways.
Fig. 4. Triptolide reduced the cerebral Evans blue extravasation in rats with cerebral ischemia. All the experiments were conducted in triplicate. Data are mean ± SEM, n = 10 for each group. #P < 0.01 vs. sham group; *P < 0.01 vs. vehicle-treated animals.
Fig. 5. Triptolide inhibited activation of NF-κB and MAPK in stroke rats. Nuclear NF-κBp65 and p-p38MAPK/p38MAPK expression were analyzed by Western blot. Data are mean ± SEM, n = 10 for each group. # P < 0.01 vs. sham-operated rats; *P < 0.01 vs. vehicle-treated rats. All the experiments were conducted in triplicate.
A growing body of evidence has implicated inflammatory response as a key role in secondary ischemic brain damage and that inflammatory response leads to ischemic brain damage, occurring within minutes after onset of cerebral ischemia [14, 15]. Accumulating data suggest that NF-κB and p38MAPK are an important transcription factors responsible for inflammatory response in stroke [16, 17]. Anti-inflammatory treatment reduces the ischemic brain diseases via downregulating NF-κB and MAPK signaling pathways [16, 17]. The activation of NF-κB is upregulated in ischemic stroke and contributes to cerebral injury induced by ischemia [18]. Inhibition of NF-κB activation attenuates inflammatory response [19]. Also, p38MAPK pathway may promote BBB disruption with secondary vasogenic edema after ischemia-reperfusion injury and inhibitor of p38MAPK can result in a reduction in BBB disruption, edema formation, and infarct volume [20]. T. wilfordii Hook F (TwHF) has been used in traditional Chinese medicine to treat autoimmune and
Hao, Li, Feng, and Pan inflammatory diseases for centuries [21, 22]. Triptolide, a purified component of TwHF, accounts for its major bioactive effect. Clinical trials have shown the safety and efficacy of triptolide in treating patients with rheumatoid arthritis and other immune-mediated inflammatory diseases [23, 24]. As shown in the present and in previous studies, triptolide inhibited the activation and the expression of NF-κB in vitro and in vivo [25, 26]. NF-κB is a pleiotropic transcription factor that controls the expression of several target genes, mainly of them involved in inflammation. The NF-κB activation led to the increased expression of inflammatory mediators, including pro-inflammatory cytokines (TNF-α, IL-1β), which contributed to the recruitment and activation of inflammatory cells (macrophages, T lymphocytes). Of particular interest in this study is the identification of molecular mechanism by which triptolide inhibits ischemic stroke. Triptolide protects brain against ischemic cerebral injury via alleviating BBB breakdown. In summary, results of this study demonstrate that triptolide reduced neurological deficit scores, brain infarct volume, and brain water content in rats. Further data show that triptolide protects the brain against ischemic cerebral injury via alleviating BBB breakdown, which may be mediated via inhibiting NF-κB and MAPK pathways. Our findings indicate that triptolide can be regarded as a potential candidate for treatment of ischemic stroke.
Conflict of Interests. The authors declare that they have no conflict of interests.
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Triptolide Protects Against Ischemic Stroke in Rats Maolin Hao,1,3 Xianghua Li,2 Jianli Feng,1 and Ning Pan1
Abstract—Inflammatory response plays an important role in the pathogenesis of ischemic stroke and anti-inflammatory agents may provide a choice of treatment. Triptolide is reported to be antiinflammatory. In this study, we investigated the effects of triptolide on cultured neuronal cell line in vitro and experimental ischemic stroke in vivo. Oxygen–glucose deprivation (OGD) and tumor necrosis factor-α (TNF-α) stimulated SH-SY5Y cells were incubated with triptolide. In vivo, rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion for 23 h. Results of this study showed that triptolide treatment reduced the OGD-induced cytotoxicity and apoptosis and blocked TNF-α-induced activation of NF-κB and p38MAPK in SH-SY5Y cells. Intraperitoneal injection of triptolide showed significant neuroprotective actions in stroke rats. Triptolide attenuated neurological deficit, brain infarct volume, and brain water content, and inhibited activation of NF-κB and p38MAPK. These data show that triptolide protects rats against ischemic cerebral injury via inhibiting NF-κB and p38MAPK signaling pathways. KEY WORDS: triptolide; rats; experimental stroke.
INTRODUCTION Stroke, a major cause of death and impairment in humans, is the third leading cause of death in industrialized countries and the most common cause of permanent disability in adults globally [1]. Stroke is a constantly expanding area of research. Surviving patients demonstrate significant risk for the development of new acute neurological symptoms and death [2]. The mechanisms that trigger ischemic brain damage are complex, but inflammatory response has been considered as a key element in ischemic stroke of both animals and human [3, 4]. Triptolide is extracted from Tripterygium wilfordii, which is a traditional Chinese medicine and exhibits immunosuppressive, anti-inflammatory, and anti-tumor activities [5–7]. Triptolide exhibits anti-inflammatory activities in cultured lung cells and in rat models with chlorineinduced acute lung injury [8]. However, to our knowledge, 1
Department of Neurology, Shandong Provincial Hospital Affiliated to Shandong University, #4 Duanxing West Road, Jinan, 250022Shandong, China 2 Department of Neurology, The Sixth People’s Hospital of Jinan, Zhangqiu, 250200Shandong, China 3 To whom correspondence should be addressed at Department of Neurology, Shandong Provincial Hospital Affiliated to Shandong University, #4 Duanxing West Road, Jinan, 250022Shandong, China. E-mail: [email protected]
the effect of triptolide in ischemic stroke has not been investigated yet. In this study, we tested the effects of triptolide on cultured neuronal cell line and rat ischemic stroke.
MATERIALS AND METHODS Materials Triptolide was purchased from Sigma Chemical Co. (St. Louis, MO, USA), with purity of more than 98 %. The human neuroblastoma cell line SH-SY5Y was purchased from American type culture collection (ATCC, Manassas, VA, USA). Cell Culture SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) solutions (Gibco Laboratories, Grand Island, NY, USA), supplemented with 10 % fetal bovine serum (FBS, Gibco Laboratories, Grand Island, NY, USA). Cells were kept at 37 °C in a humidified 5 % CO2 incubator. The dissociated cells were seeded in poly5 2 L-lysine-coated plates at a density of 5 × 10 /cm and cultured in DMEM, supplemented with 10 % fetal bovine serum. The medium was changed twice weekly. After
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Hao, Li, Feng, and Pan 8 days of culture, SH-SY5Y cells were randomly divided into seven groups: normal group (no oxygen–glucose deprivation), oxygen–glucose deprivation group (control group), and oxygen–glucose deprivation + triptolide (dissolved in DMSO, 5, 10, 20, 40, and 80 ng/ml) groups. For oxygen–glucose deprivation, the SH-SY5Y cells were washed twice in glucose-free balanced salt solution (BSS), and incubated in BSS (glucose-free) in a humidified chamber filled with 95 % N2/5 % CO2 for 3 h at 36.5 °C, while the normal group and triptolide group (no oxygen– glucose deprivation) filled with 5 % CO2/95 % O2 and incubated in glucose-free BSS. After oxygen–glucose deprivation, the cultures were replaced into neurobasal medium and incubated with triptolide in a CO2 incubator for 12 h.
reperfusion as described before [10]. The vehicletreated group received 1 % phosphate buffered saline (PBS)/DMSO. Neurological deficits were determined at 24 h after ischemia followed by brain infarct volume examination.
Determination of Cell Viability and Apoptosis
Evaluation of Infarct Volume and Brain Water Content
After oxygen–glucose deprivation for 3 h followed by 12-h incubation with or without triptolide, cell viability was assessed using 3-[4, 5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay (Genebase, Shanghai, China). Apoptotic cells were evaluated using an Annexin-V FITC apoptosis detection kit (Genebase, Shanghai, China). In brief, cells were harvested, washed, and incubated at 4 °C for 30 min in the dark with annexinV FITC and propidium iodide, then analyzed on a FACS Vantage SE flow cytometer (Beckman Coulter, USA).
After 23-h reperfusion, rats were anesthetized with sodium pentobarbital through intraperitoneal injection. The adequacy of anesthesia was monitored by the level and stability of the mean arterial pressure (MAP) and absence of the corneal reflex, and adequate levels of anesthesia and analgesia were ensured with supplemental intraperitoneal injection of pentobarbital sodium given as required, then brains were quickly removed. The total wet weight of the brain was measured accurately. The starting site of the brain was cut at forebrain 3 mm, and each brain was sliced into sections of 2-mm thickness each, then stained with a 2 % solution of tetrazolium chloride (Sigma, St. Louis, MO, USA) in saline at 37 °C for 10 min, and photographed. The images were digitized, and infarct volume was calculated with a Compix system computer. Then, brain water content was determined as an indicator of cerebral edema using a wet/dry method.
Induction of Rat Cerebral Ischemia and Treatments All the experiments were approved by our Animal Care Committee. Male Sprague–Dawley rats (250–280 g) were supplied by the Animal Center of our University and maintained on a 12-h light/12-h dark regime. All rats were provided with food and water ad libitum. The animals were acclimatized to the laboratory conditions for 7 days before surgery. Animals were anesthetized by intraperitoneal injection of 10 % chloral hydrate. Rectal temperature was recorded and maintained at 37 °C throughout the surgical procedure. The rats were subjected to middle cerebral artery occlusion (MCAO) by intraluminal placement of a filament as described previously [9]. Sham-operated rats (n = 10) were subjected only to exposure of the MCA without coagulation. Anesthesia duration was similar in all groups. The mortality rate after the surgery was nearly 20 % and we chose the surviving rats for further experiments. Rats were randomly divided into two groups with ten rats each. Rats received intraperitoneal 1.0 mg/kg triptolide (dissolved in DMSO) after
Evaluation of Neurological Deficits Neurological deficits were evaluated using a 0–5 scoring system [11] by an investigator who was blinded to each experimental group: 0, no neurological deficits (normal); 1, failure to extend left forepaw fully; 2, circling to the left; 3, falling to the left; 4, no spontaneous walking with a depressed level of consciousness, and 5, death.
Evaluation of Blood–Brain Barrier Leakage with Evans Blue Extravasation Determination of Evans blue extravasation was based on a previous method [12]. After reperfusion, 0.1 ml of 4 % Evans blue in saline was administered via tail vein. Twenty-three hours after ischemia, rats were anesthetized with chloral hydrate then perfused with 20 ml 10 U/ml heparinized saline to wash out the blood. The brain was then isolated, weighed, and homogenized in 50 % solution of trichloroacetic acid (Sigma, St. Louis, MO, USA). After centrifugation at 600g for 15 min, the supernatant was
Triptolide in Experimental Stroke spectrophotometrically measured at 490 nm. Cerebral Evans blue was quantified as micrograms of dye per gram of wet weight.
Western Blot For the experiment of tumor necrosis factor-α (TNFα) stimulated SH-SY5Y cell lines in vitro, SH-SY5Y cells (5×106) were pre-incubated with triptolide (40 ng/ml) for 12 h, then incubated with TNF-α (20 ng/ml) for 30 min. Cells were washed twice with ice-cold PBS on ice and lysed in cell lysis buffer (Sigma, Saint Louis, MO, USA). For western blot analysis, brains were harvested and the right hemispheres containing the ischaemic sites were excised and nuclear and cytosolic extracts of cells or tissues were isolated using the NE-PER kit (Life Technologies, Foster City, CA, USA) according to the manufacturer’s instructions. Equal amounts of cell protein (50 μg) were separated by SDS-PAGE and analyzed by Western blot using specific antibodies to NF-κBp65 (polyclonal rabbit, Cat No.
Fig. 1. After 3-h oxygen–glucose deprivation followed by 12-h incubation with triptolide, cell viability (a) and apoptosis (b) were assessed. N normal, C control: oxygen–glucose deprivation. Data are presented as mean ± SEM. n = 10. *P < 0.01 vs. control. All experiments were conducted in triplicate.
SAB4502610, 1:2000), p38MAPK (polyclonal rabbit, Cat No. SAB4500493, 1:2500) and p-p38MAPK(polyclonal rabbit, Cat No. SAB4504095,1:2000) followed by secondary antibodies (1:3000, polyclonal rabbit, Cat No. A9919). All antibodies were all purchased from Sigma, Saint Louis, MO, USA. Optical densities of the bands were scanned and quantified with a Gel Doc 2000 (Bio-Rad Laboratories). Lamin A (polyclonal rabbit, Cat No. L1293, Sigma, Saint Louis, MO, USA) levels are shown as an internal control. Results were expressed as ratio of control.
Statistical Analysis All data were analyzed using SPSS 16.0 software. Neurological deficit scores between groups were analyzed using Mann–Whitney U test. Quantitative data were expressed as mean±SEM. Statistical comparisons were performed by one-way ANOVA for multiple comparisons
Fig. 2. Triptolide inhibited activation of NF-κB and MAPK in cultured SH-SY5Y cells. SH-SY5Y cells were incubated with triptolide (40 ng/ml) for 12 h and then incubated with TNF-α (20 ng/ml) for 30 min. Nuclear NF-κBp65 and p-p38MAPK/p38MAPK expression were analyzed by Western blot. #P < 0.01 vs. blank; *P < 0.01 vs. TNF-α stimulation in the absence of triptolide. All the experiments were conducted in triplicate.
Hao, Li, Feng, and Pan following LSD. P < 0.05 was considered statistically significant.
RESULTS Triptolide Protected Cultured SH-SY5Y Cells Against Oxygen–Glucose Deprivation-Induced Cytotoxicity and Apoptosis The MTT assay was used to assess the effect of triptolide on the oxygen–glucose deprivation-induced cytotoxicity and we found that cell viability was markedly decreased after oxygen–glucose deprivation for 3 h followed by 12-h incubation with neurobasal medium (Fig. 1a). In contrast, the cytotoxicity was significantly attenuated by triptolide (5–80 ng/ml) treatment in a concentration-dependent manner. To further investigate the protective effect of triptolide, apoptosis was estimated by flow cytometric analysis of annexin-V and propidium iodide-labeling cells. The oxygen–glucose deprivation for 3 h followed by 12-h incubation with neurobasal medium significantly increased the apoptosis (Fig. 1b). Triptolide (5–80 ng/ml) for 12 h arrested the apoptosis in a concentration-dependent manner.
Triptolide Inhibited Activation of NF-κB and p38MAPK in Cultured SH-SY5Y Cells After TNF-α (20 ng/ml) stimulation for 30 min, NFκB and p38MAPK were both significantly activated in SHSY5Y cells. We compared the effect of triptolide on the TNF-α-induced (20 ng/ml for 30 min) activation of NF-κB and p38MAPK in the SH-SY5Y cells. As shown in Fig. 1, 40 ng/ml triptolide showed the best effectiveness, and we chose this concentration for the following experiments. We found that 40 ng/ml triptolide pretreatment blocked the TNF-α-induced activation of NF-κB and p38MAPK (Fig. 2). Triptolide Reduced Neurological Deficit Scores, Brain Infarct Volume, and Brain Water Content in Stroke Rats Cerebral ischemic injury results in behavioral disturbance and histological changes in MCAO rats, showing that ischemic region was white and non-ischemic region was red. Compared with the sham-operated rats, neurological deficit scores, the ischemic regions and brain water content were significantly increased in rats with experimental stroke. Our results showed that triptolide (1.0 mg/
Fig. 3. Triptolide treatment reduced the infarct volume, neurological scores, and brain water content in stroke rats. Each brain was stained with a 2 % solution of tetrazolium chloride (a). #P < 0.01 vs. sham; *P < 0.01 vs. vehicle.
Triptolide in Experimental Stroke kg) after reperfusion immediately decreased neurological deficit scores, cerebral infarct volume, and brain water content (Fig. 3). Triptolide Reduced Blood–Brain Barrier Breakdown in Stroke Rats Evans blue extravasation was used to assess blood– brain barrier (BBB) breakdown after cerebral ischemia. Vehicle-treated rats showed elevated Evans blue extravasation compared with the sham-operated rats. In contrast, our results showed that triptolide significantly attenuated Evans blue extravasation (Fig. 4). Triptolide Inhibited NF-κB and p38MAPK Activation in Stroke Rats In order to investigate the molecular mechanism of triptolide, NF-κB and p38MAPK activation was examined. Activation of NF-κB and p38MAPK was significantly increased in rats after cerebral ischemia. However, triptolide treatment reduced nuclear NF-κBp65 and pp38MAPK, indicating that triptolide blocked activation of NF-κB and p38MAPK in stroke rats (Fig. 5).
DISCUSSION It is well established that MCAO is a classical model of cerebral ischemia [13]. In this study, we showed that triptolide improved neurological dysfunction, reduced infarct size, and BBB permeability after MCAO. These protective effects might be mediated via inhibiting NFκB and MAPK signaling pathways.
Fig. 4. Triptolide reduced the cerebral Evans blue extravasation in rats with cerebral ischemia. All the experiments were conducted in triplicate. Data are mean ± SEM, n = 10 for each group. #P < 0.01 vs. sham group; *P < 0.01 vs. vehicle-treated animals.
Fig. 5. Triptolide inhibited activation of NF-κB and MAPK in stroke rats. Nuclear NF-κBp65 and p-p38MAPK/p38MAPK expression were analyzed by Western blot. Data are mean ± SEM, n = 10 for each group. # P < 0.01 vs. sham-operated rats; *P < 0.01 vs. vehicle-treated rats. All the experiments were conducted in triplicate.
A growing body of evidence has implicated inflammatory response as a key role in secondary ischemic brain damage and that inflammatory response leads to ischemic brain damage, occurring within minutes after onset of cerebral ischemia [14, 15]. Accumulating data suggest that NF-κB and p38MAPK are an important transcription factors responsible for inflammatory response in stroke [16, 17]. Anti-inflammatory treatment reduces the ischemic brain diseases via downregulating NF-κB and MAPK signaling pathways [16, 17]. The activation of NF-κB is upregulated in ischemic stroke and contributes to cerebral injury induced by ischemia [18]. Inhibition of NF-κB activation attenuates inflammatory response [19]. Also, p38MAPK pathway may promote BBB disruption with secondary vasogenic edema after ischemia-reperfusion injury and inhibitor of p38MAPK can result in a reduction in BBB disruption, edema formation, and infarct volume [20]. T. wilfordii Hook F (TwHF) has been used in traditional Chinese medicine to treat autoimmune and
Hao, Li, Feng, and Pan inflammatory diseases for centuries [21, 22]. Triptolide, a purified component of TwHF, accounts for its major bioactive effect. Clinical trials have shown the safety and efficacy of triptolide in treating patients with rheumatoid arthritis and other immune-mediated inflammatory diseases [23, 24]. As shown in the present and in previous studies, triptolide inhibited the activation and the expression of NF-κB in vitro and in vivo [25, 26]. NF-κB is a pleiotropic transcription factor that controls the expression of several target genes, mainly of them involved in inflammation. The NF-κB activation led to the increased expression of inflammatory mediators, including pro-inflammatory cytokines (TNF-α, IL-1β), which contributed to the recruitment and activation of inflammatory cells (macrophages, T lymphocytes). Of particular interest in this study is the identification of molecular mechanism by which triptolide inhibits ischemic stroke. Triptolide protects brain against ischemic cerebral injury via alleviating BBB breakdown. In summary, results of this study demonstrate that triptolide reduced neurological deficit scores, brain infarct volume, and brain water content in rats. Further data show that triptolide protects the brain against ischemic cerebral injury via alleviating BBB breakdown, which may be mediated via inhibiting NF-κB and MAPK pathways. Our findings indicate that triptolide can be regarded as a potential candidate for treatment of ischemic stroke.
Conflict of Interests. The authors declare that they have no conflict of interests.
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