Inflammation ( # 2015) DOI: 10.1007/s10753-015-0211-4
Anti-inflammation Effects of Oxysophoridine on Cerebral Ischemia–Reperfusion Injury in Mice Yong-Sheng Wang,1 Yu-Xiang Li,2 Peng Zhao,1 Hong-Bo Wang,1 Ru Zhou,1 Yin-Ju Hao,3 Jie Wang,4 Shu-Jing Wang,4 Juan Du,1 Lin Ma,5 Tao Sun,5 and Jian-Qiang Yu1,6,7,8
Abstract—Oxysophoridine (OSR) is a bioactive alkaloid extracted from the Sophora alopecuroides Linn. Our aim is to explore the potential anti-inflammation mechanism of OSR in cerebral ischemic injury. Mice were intraperitoneally pretreated with OSR (62.5, 125, and 250 mg/kg) or nimodipine (Nim) (6 mg/kg) for 7 days followed by cerebral ischemia. The inflammatory-related cytokines in cerebral ischemic hemisphere tissue were determined by immunohistochemistry staining, Western blot and enzyme-like immunosorbent assay (ELISA). OSR-treated groups observably suppressed the nuclear factor kappa B (NF-κB), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). OSR-treated group (250 mg/kg) markedly reduced the inflammatory-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8). Meanwhile, it dramatically increased the interleukin-10 (IL-10). Our study revealed that OSR protected neurons from ischemiainduced injury in mice by downregulating the proinflammatory cytokines and blocking the NF-κB pathway. KEY WORDS: oxysophoridine; anti-inflammation; cerebral ischemia; NF-κB.
INTRODUCTION Stroke is the third most common cause of death in human beings [1]. Ischemic stroke comprises about 80 % Yong-Sheng Wang and Yu-Xiang Li contributed equally to this work. 1
Department of Pharmacology, Ningxia Medical University, Yinchuan, 750004, China 2 College of Nursing, Ningxia Medical University, Yinchuan, 750004, China 3 Ningxia Medical University, Yinchuan, 750004, China 4 Medical Sci-Tech Research Center, Ningxia Medical University, Yinchuan, 750004, China 5 Ningxia Key Lab of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Yinchuan, 750004, China 6 Ningxia Hui Medicines Collaborative Innovation Center, Yinchuan, 750004, China 7 Department of Pharmacology, Ningxia Medical University and Ningxia Hui Medicines Collaborative Innovation Center, Yinchuan, Ningxia, China 8 To whom correspondence should be addressed at Department of Pharmacology, Ningxia Medical University and Ningxia Hui Medicines Collaborative Innovation Center, Yinchuan, Ningxia, China. E-mail: [email protected]
of all stroke cases [2]. Ischemic brain injury often leads to the irreversible damage including neuronal injury and death associated with inflammation, oxidative stress, excitotoxicity, etc. [3, 4]. Although pathologic mechanism leading to cerebral ischemic injury remains unclear, it has been emphasized that inflammatory process has a fundamental role in both the etiology of ischemic cerebrovascular disease and pathophysiology of cerebral ischemia [5– 7]. In the early stage of ischemia, neutrophils separate from the blood axis and agglutinate the vessel wall [8]. Endothelial cells actively participate in inflammatory events by regulating the leukocyte recruitment via the expression of inflammation-related genes such as intercellular adhesion molecule-1 (ICAM-1), VCAM-1, interleukin (IL)-6, IL-8, and cyclooxygenase-2 (COX-2) [9, 10]. The transcription factor NF-κB is a master regulator of the cellular responses to injury, inflammation, and other stresses [11–13]. In the central nervous system (CNS), NF-κB activity is detected in both neuronal and glial cells following ischemic injury or other inflammation related diseases [14, 15]. Nitric oxide (NO) is a free radical diatomic gas of low molecular weight with an unpaired electron [16, 17]. High
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Wang, Li, Zhao, Wang, Zhou, Hao, Wang, Wang, Du, Ma, Sun, and Yu concentration of NO inhibits the glycolysis and mitochondrial enzymes, thereby reducing the neuron energy generation. Those effects lead to the neurotoxicity of brain tissue [18]. As the overall process of ischemia reperfusion injury is extremely complex and the desired therapeutic drugs are still not yet found, it requires further studies to search some drugs to treat the diseases involved in ischemia reperfusion injury. Recently, researches on the natural components extracted from Chinese medicinal herbs for the treatment of ischemic cerebral vascular diseases have received increased attention. Oxysophoridine (OSR) is one of the main alkaloids isolated from the seeds of Sophora alopecuroides Linn. S. alopecuroides Linn is a medical plant of Sophora (Leguminosae sp.) (Fig. 1). There are plenty of wildly and artificially planted S. alopecuroides Linn in the Ningxia region [19]. Our previous preliminary study results showed that: (1) OSR had a protective effect on focal cerebral ischemic injury in mice and (2) OSR had significantly protective effects on oxygen–glucose deprivation/ reperfusion-induced neuronal damages in rat primary neuron cultures in vitro [20, 21]. However, there is no antiinflammation mechanistic study to clarify the neuroprotection of OSR. Hence, we investigated the possible antiinflammation mechanism of OSR in the cerebral ischemia–reperfusion injury in mice. Fig. 1. The chemical structure of oxysophoridine (OSR).
MATERIALS AND METHODS
OSR was supplied by the Institution of Chemistry and Chemical Engineering, Ningxia Agricultural College (purity >98 %, lot no. 960368). Nim was served as a positive drug obtained from Bayer Healthcare Company Ltd. Reagents were dissolved in 0.9 % (w/v) NaCl solution respectively.
group, that is, ischemia was induced for 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. The OSR-treated groups were separated into low dosage group (OSR 62.5 mg/kg), moderate dosage group (OSR 125 mg/kg), and high dosage group (OSR 250 mg/ kg). The sixth was the Nim-treated group (6 mg/kg). Before ischemia/reperfusion (I/R), all groups were intraperitoneally pretreated with drug or reagent (0.1 ml/10 g) for 7 consecutive days.
Experimental Design
Surgical Procedure
Male Institute of Cancer Research (ICR) mice weighing 25–30 g were supplied from the Experimental Animal Center of Ningxia Medical University (Certificate No. SYXK Ningxia 2014-0001). The mice were housed in cages for 6 days at room temperature under a controlled 12 h light/dark cycle and allowed access to pellet food and water ad libitum. All experiments were performed as approved by the institutional animal care and use committee. Mice were randomly divided into six groups. The first was the sham-treated group. The second was the vehicle-treated
Focal cerebral ischemia was produced by occluding the left middle cerebral artery (MCAO) according to the intraluminal filament technique as described by Longa and Macrae [22, 23]. Briefly, mice were intraperitoneally anesthetized with 3.5 % chloral hydrate (0.1 ml/10 g). Under sterile conditions, a ventral neck incision was made in the external carotid artery (ECA). The internal carotid artery (ICA) was exposed and carefully isolated. A nylon monofilament (15 mm in length and 0.15 mm in diameter) was inserted in the lumen of the left ECA and ICA to occlude
Reagents
Anti-inflammation Effects of Oxysophoridine the origin of the left middle cerebral artery. The monofilament was removed to restore blood flow after 2 h of MCAO. The sham-operated group was treated identically, except the MCAO after the neck incision. Mice were returned to their cages with free access to water and food. Mice were decapitated to remove the brain for the immunohistochemistry staining, Western blot, and ELISA 24 h after reperfusion. Immunohistochemistry Staining After 24 h reperfusion, animals were intraperitoneally anesthetized with chloral hydrate (350 mg/kg) and transitorily perfused with 150 ml 4 °C cold 0.9 % NaCl followed by 300 ml 4 % paraformaldehyde in phosphate buffered saline (PBS) (0.1 M, PH 7.4). Each brain was rapidly removed and immersed in fixative 10 % formalin for 2 h, then was washed three times with PBS. Ultimately, they were transferred to 30 % sucrose in PBS solution at 4 °C until it sank. Every longitudinal section was cut at 5 μm beginning 1.9 mm caudal to the bregma for immunohistochemistry staining. The antigen retrieval of paraffin sections was performed with the high pressure after dewaxing and dehydration. Brain sections were firstly perforated in 3 % triton solutions for 30 min at room temperature, and then washed three times with PBS for 10 min. The tissue was immersed in 1 % H2O2 for 30 min to quench the endogenous peroxidase. After rinsing with PBS for three times, the sections were incubated with 5 % goat serum for 30 min. Following incubation in serum, they were incubated with rabbit polyclonal antibody against NF-κB (lot no. 00500773, diluted 1:50, Proteintech Group Inc., USA) for 4 °C overnight. After incubation, the tissue was rinsed in PBS 3 times for 5 min and then incubated in a biotinylated anti-rabbit secondary antibody in-door for 1 h. Following another series of washing in PBS, the tissue was incubated for 20 min in an AVIDIN-Bioyin. The sections were washed and then placed in a solution of 0.5 mg/ml diaminobenzidine (DAB) for 5–10 min until the desired staining intensity was achieved. Ultimately, the tissue was washed and mounted onto super frost glass slides and left to dry. The three randomly positive area in each section was photographed under high-power magnification (bar = 20 μm) with microscope Olympus BX51 (Olympus, JP) by a blinded manner. Enzyme Linked Immunosorbent Assay IL-1β (lot no. BO0051), IL-6 (lot no. BO0063), and IL-10 (lot no. BO0067) enzyme linked immunosorbent assay (ELISA) kits were purchased from
ABGENT (San Diego, USA). IL-8 (lot no. 20121208) ELISA kit was purchased from MR Biotech (China). Tumor necrosis factor alpha (TNF-α) (lot no. 118178) ELISA kit was purchased from Ray Biotech (USA). Prostaglandin E2 (PGE2) (lot no. 201211) ELISA kit was purchased from Beijing Xinfangcheng Biotechnology (China). Six mice in each group were deeply anesthetized and decapitated at 24 h after reperfusion. The ischemic hemisphere tissue was quickly removed and grinded completely into brain tissue homogenate with saline (0.9 % NaCl), then centrifugalized at 10,000 rpm for 15 min. The upper limpid liquid was collected and stored at −80 °C to avoid repeated freeze-thaw cycles. The competitive ELISA was performed as previously described [24]. We prepared all standard samples before starting assay procedure. Firstly, we ensured the desired amount of coated wells in the holder, and then added 50 μl standard samples to the appropriate wells of the antibody pre-coated microtiter plate. Secondly, we added 100 μl conjugate to each well. Mix it well and incubate it for 1 h at 37 °C. Microtiter plates were washed 5 times with PBS. Substrates A and B in 50 μl were added to each well. We covered wells and incubated them for 15 min at 25 °C. Stop solution in 50 μl was added to each well. We mixed it and calculated the mean absorbance value of A450 for each set of reference standards and samples. Western Blot Analysis Whole brain was rapidly removed 24 h after reperfusion. Ischemic brain tissue was weighted and homogenized (10 %, w/v) with cold RIPA lysis buffer immediately. The homogenate was centrifuged at 10,000 rpm for 15 min, and then the supernatant was used to detect the level of NF-κB, ICAM-1, COX-2, inducible nitric oxide synthase (iNOS), and the total protein. Total protein concentration was detected by the KEYGEN Total Protein Extraction Kit (Chengen Biotech, China, Lot No. P0013B). Loading buffer was added to each sample. The samples were run on sodium dodecyl sulfate polyacrylamide gels (SDSPAGE) and then transferred to nitrocellulose membrane (NC membrane, Bio-Rad, USA). The membranes were blocked with 5 % non-fat milk in PBS with Tween 20 (PBST) for 2 h and incubated with the following primary antibodies at 4 °C overnight: rabbit polyclonal antibody against NF-κB (lot no. 00500773, diluted 1:1200, Proteintech Group Inc., USA), rabbit polyclonal antibody against ICAM-1 (lot no. 00004818, diluted 1:1500,
Wang, Li, Zhao, Wang, Zhou, Hao, Wang, Wang, Du, Ma, Sun, and Yu Proteintech Group Inc., USA), rabbit polyclonal antibody against COX-2 (lot no. 09000102, diluted 1:400, Proteintech Group Inc., USA), mouse monoclonal against iNOS (lot no. 34403-4, diluted 1:300, Abcam, USA), and mouse polyclonal antibody against β-actin (diluted 1:1000, Santa Cruz Biotechnology, USA). The membranes were washed 3 times with the following secondary antibodies for 90 min at room temperature on the shaker: goat anti-rabbit (diluted 1:5000, Beijing Zhongshan Golden Bridge Biological Technology, China) and goat antimouse (diluted 1:5000, Beijing Zhongshan Golden Bridge Biological Technology, China). Blot was developed by using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, USA) in a dark chamber and imaged by EMCCD in a dark box. The protein bands were quantitatively analyzed by using the Bio-RAD image analysis software (NIS-Elements BR 3.1). All data were normalized at the level of β-actin concerning the level of desired protein. Statistical Analysis All data were analyzed with the SPSS 17.0 software (IBM, USA). Data was expressed as mean ± SD. The two-tailed t test was used to determine the mean differences between groups. Statistical significance was set at p < 0.05.
RESULTS Effect of OSR on NF-κB (p65) Protein Expression In the immunohistochemistry staining, the vehicletreated group showed NF-κB an intense immunoreactivity in cortex after 24 h reperfusion (Fig. 2). The Western blot showed that NF-κB expression was upregulated in the vehicle-treated group after 24 h reperfusion (Fig. 3). However, pretreatment with OSR (250 mg/kg) or Nim (6 mg/kg) observably suppressed the immunoreactivity of NF-κB and significantly decreased the NFκB expression after 24 h reperfusion (p < 0.01). These results remind us that OSR could inhibit the expression of I/R-induced NF-κB as well as protect brain against inflammation. Effects of OSR on ICAM-1, COX-2, and iNOS Protein Production The expressions of ICAM-1, COX-2, and iNOS in the vehicle-treated group ischemic cortex were significantly increased compared with those of the sham-treated group after 24 h reperfusion. Nevertheless, administration with OSR significantly decreased the expressions of ICAM-1, COX-2, and iNOS. The effect of OSR-treated group (250 mg/kg) was similar to the Nim-treated group (6 mg/
Fig. 2. Immunohistochemistry staining of NF-κB expression in ischemic cortex from different groups 24 h after reperfusion (Scale bar = 20 μm). I shamtreated group, II vehicle-treated group, III Nim-treated group (6 mg/kg), (IV–VI) OSR-treated groups (62.5, 125, and 250 mg/kg).
Anti-inflammation Effects of Oxysophoridine
Fig. 3. Effects of OSR on NF-κB expression. a Representative Western blotting bands of NF-κB expression in the ischemic tissue 24 h after reperfusion. b Quantification of NF-κB assessed by Western blotting analysis was normalized to the expression level of endogenous β-actin. All values were expressed as the mean ± SD for each group. Lanes I–VI: sham-treated group, the vehicle-treated group, Nim-treated group (6 mg/ kg), OSR-treated group (250, 125, and 62.5 mg/kg). ##p < 0.01 vs. the sham-treated group. **p < 0.01 vs. the vehicle-treated group (n = 13).
kg) (Figs. 4, 5, and 6). These results imply the antiinflammatory effects of ORS among various proinflammatory cytokines induced by ischemia.
Fig. 4. Effects of OSR on ICAM-1 expression. a Representative Western blotting bands of ICAM-1 expression in the ischemic tissue 24 h after reperfusion. b Quantification of ICAM-1 assessed by Western blotting analysis was normalized to the expression level of endogenous β-actin. All values were expressed as the mean ± SD for each group. Lanes I–VI: shamtreated group, the vehicle-treated group, Nim-treated group (6 mg/kg), OSR-treated group (250, 125 and 62.5 mg/kg). ##p < 0.01 vs. the shamtreated group. **p < 0.01 vs. the vehicle-treated group (n = 13).
inhibit its downstream inflammatory factor PGE2 and TNF-α induced by I/R-induced injury. Effects of OSR on IL-1β, IL-6, IL-8, and IL-10 Protein Expression
Effects of OSR on PGE2 and TNF-α Protein Expression The levels of TNF-α and PGE2 were remarkably enhanced in the vehicle-treated group versus the shamtreated group after 24 h reperfusion (p
Anti-inflammation Effects of Oxysophoridine on Cerebral Ischemia–Reperfusion Injury in Mice Yong-Sheng Wang,1 Yu-Xiang Li,2 Peng Zhao,1 Hong-Bo Wang,1 Ru Zhou,1 Yin-Ju Hao,3 Jie Wang,4 Shu-Jing Wang,4 Juan Du,1 Lin Ma,5 Tao Sun,5 and Jian-Qiang Yu1,6,7,8
Abstract—Oxysophoridine (OSR) is a bioactive alkaloid extracted from the Sophora alopecuroides Linn. Our aim is to explore the potential anti-inflammation mechanism of OSR in cerebral ischemic injury. Mice were intraperitoneally pretreated with OSR (62.5, 125, and 250 mg/kg) or nimodipine (Nim) (6 mg/kg) for 7 days followed by cerebral ischemia. The inflammatory-related cytokines in cerebral ischemic hemisphere tissue were determined by immunohistochemistry staining, Western blot and enzyme-like immunosorbent assay (ELISA). OSR-treated groups observably suppressed the nuclear factor kappa B (NF-κB), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). OSR-treated group (250 mg/kg) markedly reduced the inflammatory-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8). Meanwhile, it dramatically increased the interleukin-10 (IL-10). Our study revealed that OSR protected neurons from ischemiainduced injury in mice by downregulating the proinflammatory cytokines and blocking the NF-κB pathway. KEY WORDS: oxysophoridine; anti-inflammation; cerebral ischemia; NF-κB.
INTRODUCTION Stroke is the third most common cause of death in human beings [1]. Ischemic stroke comprises about 80 % Yong-Sheng Wang and Yu-Xiang Li contributed equally to this work. 1
Department of Pharmacology, Ningxia Medical University, Yinchuan, 750004, China 2 College of Nursing, Ningxia Medical University, Yinchuan, 750004, China 3 Ningxia Medical University, Yinchuan, 750004, China 4 Medical Sci-Tech Research Center, Ningxia Medical University, Yinchuan, 750004, China 5 Ningxia Key Lab of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Yinchuan, 750004, China 6 Ningxia Hui Medicines Collaborative Innovation Center, Yinchuan, 750004, China 7 Department of Pharmacology, Ningxia Medical University and Ningxia Hui Medicines Collaborative Innovation Center, Yinchuan, Ningxia, China 8 To whom correspondence should be addressed at Department of Pharmacology, Ningxia Medical University and Ningxia Hui Medicines Collaborative Innovation Center, Yinchuan, Ningxia, China. E-mail: [email protected]
of all stroke cases [2]. Ischemic brain injury often leads to the irreversible damage including neuronal injury and death associated with inflammation, oxidative stress, excitotoxicity, etc. [3, 4]. Although pathologic mechanism leading to cerebral ischemic injury remains unclear, it has been emphasized that inflammatory process has a fundamental role in both the etiology of ischemic cerebrovascular disease and pathophysiology of cerebral ischemia [5– 7]. In the early stage of ischemia, neutrophils separate from the blood axis and agglutinate the vessel wall [8]. Endothelial cells actively participate in inflammatory events by regulating the leukocyte recruitment via the expression of inflammation-related genes such as intercellular adhesion molecule-1 (ICAM-1), VCAM-1, interleukin (IL)-6, IL-8, and cyclooxygenase-2 (COX-2) [9, 10]. The transcription factor NF-κB is a master regulator of the cellular responses to injury, inflammation, and other stresses [11–13]. In the central nervous system (CNS), NF-κB activity is detected in both neuronal and glial cells following ischemic injury or other inflammation related diseases [14, 15]. Nitric oxide (NO) is a free radical diatomic gas of low molecular weight with an unpaired electron [16, 17]. High
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Wang, Li, Zhao, Wang, Zhou, Hao, Wang, Wang, Du, Ma, Sun, and Yu concentration of NO inhibits the glycolysis and mitochondrial enzymes, thereby reducing the neuron energy generation. Those effects lead to the neurotoxicity of brain tissue [18]. As the overall process of ischemia reperfusion injury is extremely complex and the desired therapeutic drugs are still not yet found, it requires further studies to search some drugs to treat the diseases involved in ischemia reperfusion injury. Recently, researches on the natural components extracted from Chinese medicinal herbs for the treatment of ischemic cerebral vascular diseases have received increased attention. Oxysophoridine (OSR) is one of the main alkaloids isolated from the seeds of Sophora alopecuroides Linn. S. alopecuroides Linn is a medical plant of Sophora (Leguminosae sp.) (Fig. 1). There are plenty of wildly and artificially planted S. alopecuroides Linn in the Ningxia region [19]. Our previous preliminary study results showed that: (1) OSR had a protective effect on focal cerebral ischemic injury in mice and (2) OSR had significantly protective effects on oxygen–glucose deprivation/ reperfusion-induced neuronal damages in rat primary neuron cultures in vitro [20, 21]. However, there is no antiinflammation mechanistic study to clarify the neuroprotection of OSR. Hence, we investigated the possible antiinflammation mechanism of OSR in the cerebral ischemia–reperfusion injury in mice. Fig. 1. The chemical structure of oxysophoridine (OSR).
MATERIALS AND METHODS
OSR was supplied by the Institution of Chemistry and Chemical Engineering, Ningxia Agricultural College (purity >98 %, lot no. 960368). Nim was served as a positive drug obtained from Bayer Healthcare Company Ltd. Reagents were dissolved in 0.9 % (w/v) NaCl solution respectively.
group, that is, ischemia was induced for 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. The OSR-treated groups were separated into low dosage group (OSR 62.5 mg/kg), moderate dosage group (OSR 125 mg/kg), and high dosage group (OSR 250 mg/ kg). The sixth was the Nim-treated group (6 mg/kg). Before ischemia/reperfusion (I/R), all groups were intraperitoneally pretreated with drug or reagent (0.1 ml/10 g) for 7 consecutive days.
Experimental Design
Surgical Procedure
Male Institute of Cancer Research (ICR) mice weighing 25–30 g were supplied from the Experimental Animal Center of Ningxia Medical University (Certificate No. SYXK Ningxia 2014-0001). The mice were housed in cages for 6 days at room temperature under a controlled 12 h light/dark cycle and allowed access to pellet food and water ad libitum. All experiments were performed as approved by the institutional animal care and use committee. Mice were randomly divided into six groups. The first was the sham-treated group. The second was the vehicle-treated
Focal cerebral ischemia was produced by occluding the left middle cerebral artery (MCAO) according to the intraluminal filament technique as described by Longa and Macrae [22, 23]. Briefly, mice were intraperitoneally anesthetized with 3.5 % chloral hydrate (0.1 ml/10 g). Under sterile conditions, a ventral neck incision was made in the external carotid artery (ECA). The internal carotid artery (ICA) was exposed and carefully isolated. A nylon monofilament (15 mm in length and 0.15 mm in diameter) was inserted in the lumen of the left ECA and ICA to occlude
Reagents
Anti-inflammation Effects of Oxysophoridine the origin of the left middle cerebral artery. The monofilament was removed to restore blood flow after 2 h of MCAO. The sham-operated group was treated identically, except the MCAO after the neck incision. Mice were returned to their cages with free access to water and food. Mice were decapitated to remove the brain for the immunohistochemistry staining, Western blot, and ELISA 24 h after reperfusion. Immunohistochemistry Staining After 24 h reperfusion, animals were intraperitoneally anesthetized with chloral hydrate (350 mg/kg) and transitorily perfused with 150 ml 4 °C cold 0.9 % NaCl followed by 300 ml 4 % paraformaldehyde in phosphate buffered saline (PBS) (0.1 M, PH 7.4). Each brain was rapidly removed and immersed in fixative 10 % formalin for 2 h, then was washed three times with PBS. Ultimately, they were transferred to 30 % sucrose in PBS solution at 4 °C until it sank. Every longitudinal section was cut at 5 μm beginning 1.9 mm caudal to the bregma for immunohistochemistry staining. The antigen retrieval of paraffin sections was performed with the high pressure after dewaxing and dehydration. Brain sections were firstly perforated in 3 % triton solutions for 30 min at room temperature, and then washed three times with PBS for 10 min. The tissue was immersed in 1 % H2O2 for 30 min to quench the endogenous peroxidase. After rinsing with PBS for three times, the sections were incubated with 5 % goat serum for 30 min. Following incubation in serum, they were incubated with rabbit polyclonal antibody against NF-κB (lot no. 00500773, diluted 1:50, Proteintech Group Inc., USA) for 4 °C overnight. After incubation, the tissue was rinsed in PBS 3 times for 5 min and then incubated in a biotinylated anti-rabbit secondary antibody in-door for 1 h. Following another series of washing in PBS, the tissue was incubated for 20 min in an AVIDIN-Bioyin. The sections were washed and then placed in a solution of 0.5 mg/ml diaminobenzidine (DAB) for 5–10 min until the desired staining intensity was achieved. Ultimately, the tissue was washed and mounted onto super frost glass slides and left to dry. The three randomly positive area in each section was photographed under high-power magnification (bar = 20 μm) with microscope Olympus BX51 (Olympus, JP) by a blinded manner. Enzyme Linked Immunosorbent Assay IL-1β (lot no. BO0051), IL-6 (lot no. BO0063), and IL-10 (lot no. BO0067) enzyme linked immunosorbent assay (ELISA) kits were purchased from
ABGENT (San Diego, USA). IL-8 (lot no. 20121208) ELISA kit was purchased from MR Biotech (China). Tumor necrosis factor alpha (TNF-α) (lot no. 118178) ELISA kit was purchased from Ray Biotech (USA). Prostaglandin E2 (PGE2) (lot no. 201211) ELISA kit was purchased from Beijing Xinfangcheng Biotechnology (China). Six mice in each group were deeply anesthetized and decapitated at 24 h after reperfusion. The ischemic hemisphere tissue was quickly removed and grinded completely into brain tissue homogenate with saline (0.9 % NaCl), then centrifugalized at 10,000 rpm for 15 min. The upper limpid liquid was collected and stored at −80 °C to avoid repeated freeze-thaw cycles. The competitive ELISA was performed as previously described [24]. We prepared all standard samples before starting assay procedure. Firstly, we ensured the desired amount of coated wells in the holder, and then added 50 μl standard samples to the appropriate wells of the antibody pre-coated microtiter plate. Secondly, we added 100 μl conjugate to each well. Mix it well and incubate it for 1 h at 37 °C. Microtiter plates were washed 5 times with PBS. Substrates A and B in 50 μl were added to each well. We covered wells and incubated them for 15 min at 25 °C. Stop solution in 50 μl was added to each well. We mixed it and calculated the mean absorbance value of A450 for each set of reference standards and samples. Western Blot Analysis Whole brain was rapidly removed 24 h after reperfusion. Ischemic brain tissue was weighted and homogenized (10 %, w/v) with cold RIPA lysis buffer immediately. The homogenate was centrifuged at 10,000 rpm for 15 min, and then the supernatant was used to detect the level of NF-κB, ICAM-1, COX-2, inducible nitric oxide synthase (iNOS), and the total protein. Total protein concentration was detected by the KEYGEN Total Protein Extraction Kit (Chengen Biotech, China, Lot No. P0013B). Loading buffer was added to each sample. The samples were run on sodium dodecyl sulfate polyacrylamide gels (SDSPAGE) and then transferred to nitrocellulose membrane (NC membrane, Bio-Rad, USA). The membranes were blocked with 5 % non-fat milk in PBS with Tween 20 (PBST) for 2 h and incubated with the following primary antibodies at 4 °C overnight: rabbit polyclonal antibody against NF-κB (lot no. 00500773, diluted 1:1200, Proteintech Group Inc., USA), rabbit polyclonal antibody against ICAM-1 (lot no. 00004818, diluted 1:1500,
Wang, Li, Zhao, Wang, Zhou, Hao, Wang, Wang, Du, Ma, Sun, and Yu Proteintech Group Inc., USA), rabbit polyclonal antibody against COX-2 (lot no. 09000102, diluted 1:400, Proteintech Group Inc., USA), mouse monoclonal against iNOS (lot no. 34403-4, diluted 1:300, Abcam, USA), and mouse polyclonal antibody against β-actin (diluted 1:1000, Santa Cruz Biotechnology, USA). The membranes were washed 3 times with the following secondary antibodies for 90 min at room temperature on the shaker: goat anti-rabbit (diluted 1:5000, Beijing Zhongshan Golden Bridge Biological Technology, China) and goat antimouse (diluted 1:5000, Beijing Zhongshan Golden Bridge Biological Technology, China). Blot was developed by using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, USA) in a dark chamber and imaged by EMCCD in a dark box. The protein bands were quantitatively analyzed by using the Bio-RAD image analysis software (NIS-Elements BR 3.1). All data were normalized at the level of β-actin concerning the level of desired protein. Statistical Analysis All data were analyzed with the SPSS 17.0 software (IBM, USA). Data was expressed as mean ± SD. The two-tailed t test was used to determine the mean differences between groups. Statistical significance was set at p < 0.05.
RESULTS Effect of OSR on NF-κB (p65) Protein Expression In the immunohistochemistry staining, the vehicletreated group showed NF-κB an intense immunoreactivity in cortex after 24 h reperfusion (Fig. 2). The Western blot showed that NF-κB expression was upregulated in the vehicle-treated group after 24 h reperfusion (Fig. 3). However, pretreatment with OSR (250 mg/kg) or Nim (6 mg/kg) observably suppressed the immunoreactivity of NF-κB and significantly decreased the NFκB expression after 24 h reperfusion (p < 0.01). These results remind us that OSR could inhibit the expression of I/R-induced NF-κB as well as protect brain against inflammation. Effects of OSR on ICAM-1, COX-2, and iNOS Protein Production The expressions of ICAM-1, COX-2, and iNOS in the vehicle-treated group ischemic cortex were significantly increased compared with those of the sham-treated group after 24 h reperfusion. Nevertheless, administration with OSR significantly decreased the expressions of ICAM-1, COX-2, and iNOS. The effect of OSR-treated group (250 mg/kg) was similar to the Nim-treated group (6 mg/
Fig. 2. Immunohistochemistry staining of NF-κB expression in ischemic cortex from different groups 24 h after reperfusion (Scale bar = 20 μm). I shamtreated group, II vehicle-treated group, III Nim-treated group (6 mg/kg), (IV–VI) OSR-treated groups (62.5, 125, and 250 mg/kg).
Anti-inflammation Effects of Oxysophoridine
Fig. 3. Effects of OSR on NF-κB expression. a Representative Western blotting bands of NF-κB expression in the ischemic tissue 24 h after reperfusion. b Quantification of NF-κB assessed by Western blotting analysis was normalized to the expression level of endogenous β-actin. All values were expressed as the mean ± SD for each group. Lanes I–VI: sham-treated group, the vehicle-treated group, Nim-treated group (6 mg/ kg), OSR-treated group (250, 125, and 62.5 mg/kg). ##p < 0.01 vs. the sham-treated group. **p < 0.01 vs. the vehicle-treated group (n = 13).
kg) (Figs. 4, 5, and 6). These results imply the antiinflammatory effects of ORS among various proinflammatory cytokines induced by ischemia.
Fig. 4. Effects of OSR on ICAM-1 expression. a Representative Western blotting bands of ICAM-1 expression in the ischemic tissue 24 h after reperfusion. b Quantification of ICAM-1 assessed by Western blotting analysis was normalized to the expression level of endogenous β-actin. All values were expressed as the mean ± SD for each group. Lanes I–VI: shamtreated group, the vehicle-treated group, Nim-treated group (6 mg/kg), OSR-treated group (250, 125 and 62.5 mg/kg). ##p < 0.01 vs. the shamtreated group. **p < 0.01 vs. the vehicle-treated group (n = 13).
inhibit its downstream inflammatory factor PGE2 and TNF-α induced by I/R-induced injury. Effects of OSR on IL-1β, IL-6, IL-8, and IL-10 Protein Expression
Effects of OSR on PGE2 and TNF-α Protein Expression The levels of TNF-α and PGE2 were remarkably enhanced in the vehicle-treated group versus the shamtreated group after 24 h reperfusion (p
