INTRODUCTION

Stroke is the third most common cause of death in human beings [1]. Ischemic stroke comprises about 80 % 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 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 anti-inflammation mechanistic study to clarify the neuroprotection of OSR. Hence, we investigated the possible anti-inflammation mechanism of OSR in the cerebral ischemia–reperfusion injury in mice.

Fig. 1
figure 1

The chemical structure of oxysophoridine (OSR).

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MATERIALS AND METHODS

Reagents

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.

Experimental Design

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 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.

Surgical Procedure

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 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 (SDS-PAGE) 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, 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 anti-mouse (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 EM-CCD 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 vehicle-treated group showed NF-κB an intense immuno-reactivity 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 immuno-reactivity of NF-κB and significantly decreased the NF-κB 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.

Fig. 2
figure 2

Immunohistochemistry staining of NF-κB expression in ischemic cortex from different groups 24 h after reperfusion (Scale bar = 20 μm). I sham-treated group, II vehicle-treated group, III Nim-treated group (6 mg/kg), (IV–VI) OSR-treated groups (62.5, 125, and 250 mg/kg).

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Fig. 3
figure 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).

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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/kg) (Figs. 4, 5, and 6). These results imply the anti-inflammatory effects of ORS among various proinflammatory cytokines induced by ischemia.

Fig. 4
figure 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: 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).

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Fig. 5
figure 5

Effects of OSR on COX-2 expression. a Representative Western blotting bands of COX-2 expression in the ischemic tissue 24 h after reperfusion. b Quantification of COX-2 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 < 0.05 and **p < 0.01 vs. the vehicle-treated group (n = 13).

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Fig. 6
figure 6

Effects of OSR on iNOS expression. a Representative Western blotting bands of iNOS expression in the ischemic tissue 24 h after reperfusion. b Quantification of iNOS 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 < 0.05 and **p < 0.01 vs. the vehicle-treated group (n = 13).

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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 sham-treated group after 24 h reperfusion (p < 0.01). The PGE2 protein concentration in the OSR-treated groups (125, 250 mg/kg) and the Nim-treated group (6 mg/kg) was decreased by 17.04 % (p < 0.05), 42.19 % (p < 0.01), and 45.24 % (p < 0.01) compared with the vehicle-treated group respectively. PGE2 protein concentration was increased slightly in the OSR-treated group (62.5 mg/kg), but it was not statistically significant (Fig. 7a). The TNF-α protein concentration in the OSR-treated groups (62.5, 125, and 250 mg/kg) and the Nim-treated group (6 mg/kg) was decreased by 7.17 % (p < 0.05), 12.41 % (p < 0.01), 15.78 % (p < 0.01), and 15.50 % (p < 0.01) compared with the vehicle-treated group respectively (Fig. 7b). The results infer that ORS may suppress the activation of NF-κB to inhibit its downstream inflammatory factor PGE2 and TNF-α induced by I/R-induced injury.

Fig. 7
figure 7

Effects of OSR on inflammation-mediated cytokines levels in the ischemic tissue 24 h after reperfusion. a The effects of OSR on the expression of PGE2. b The effects of OSR on the expression of TNF-a. c The effects of OSR on the expression of IL-1β. d The effects of OSR on the expression of IL-6. e The effects of OSR on the expression of IL-8. f The effects of OSR on the expression of IL-10. All values were expressed as the mean ± SD for each group from six independent experiments. ## p < 0.01 vs. the sham-treated group. *p < 0.05 and **p < 0.01 vs. the vehicle-treated group (n = 6).

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Effects of OSR on IL-1β, IL-6, IL-8, and IL-10 Protein Expression

The protein concentrations of IL-1β, IL-6, IL-8, and IL-10 were remarkably enhanced in the vehicle-treated group after 24 h reperfusion (p < 0.01). The IL-1β, IL-6, and IL-8 protein concentrations in the OSR-treated group (250 mg/kg) were decreased by 31.12 % (p < 0.01), 36.24 % (p < 0.01), and 51.04 % (p < 0.01) compared with the vehicle-treated group respectively (Fig. 7c–e). Unexpectedly, the IL-10 protein concentration in the vehicle-treated group was upregulated. The OSR-treated group (250 mg/kg) was elevated by 38.49 % compared with the vehicle-treated group (p < 0.01) (Fig. 7f). The elevation of IL-10 protein may be due to the compensatory mechanism for the ischemia stress in mice after 24 h reperfusion. Additionally, our result has confirmed that pretreatment with OSR (250 mg/kg) or Nim (6 mg/kg) further facilitated the IL-10 expression.

DISCUSSION

Chinese herbal medicine has the advantages of low side effects. Our previous preliminary study results have showed the neuroprotection of pretreatment with OSR (250, 125, and 62.5 mg/kg, i.p.) against ischemia-induced injury in mice [20]. The aim of the current study was to further investigate the potential effects of OSR on the inflammatory signaling molecules in the NF-κB pathway and the underlying mechanism. Nim reduces the ischemic neurological deficits via its neuroprotection and is recommended for the management of aneurysmal subarachnoid hemorrhage [25]. Therefore, Nim was served as the positive drug in this experiment.

In the current study, OSR showed protective effect on brain injury induced by I/R in mice via blocking the expressions of inflammation-related cytokines (Interleukin and ICAM-1) and oxidative enzymes such as COX-2 and iNOS. Furthermore, the protective effect of OSR was associated with the downregulation of NF-κB. It is well known that NF-κB is a master regulator of inflammatory signaling pathways accounting for the expressions of proinflammatory cytokines induced by ischemia. The NF-κB pathway has been considered to play a pivotal role in the pathogenesis. It is an important nuclear transcription factor in eukaryotic cells, which exists in almost all the cells [26–28]. Thus, we investigated the potential effects of OSR on the production of proinflammatory cytokines (PGE2, TNF-α, IL-1β, IL-6, and IL-10) as well as the activation of NF-κB.

In the brain, I/R induce the release of reactive oxygen species (ROS). ROS are thought to initiate leukocyte recruitment with resultant inflammatory mediators and brain dysfunction [29]. Since these inflammatory mediators, such as TNF-α, IL-1β, and IL-6, are known to have κB-binding motifs in their promoter regions. The transcriptions are thought to be under the control of NF-κB. Thereby, modulation of NF-κB activity might provide the meaningfulness of reducing inflammatory factors following I/R injury. The reduced activation of NF-κB in this present study might lead to a decrease in COX-2 expression and an alleviated inflammatory injury. Zhu reported that the early increase of COX-2 was consistent with the upregulation of NF-κB target genes at 3–24 h after global ischemic insult [30]. Deleterious role of COX-2 in the global ischemic injury was demonstrated with aggravated brain damage in mice by carrying COX-2 transgene [31]. COX-2 is a primary free radical in ischemic brain [32]. More importantly, the increase of COX-2 and PGE2 directly preceded the onset of histopathological changes in the hippocampus following global cerebral ischemia [33]. It has been demonstrated that selective COX-2 inhibitor as well as COX-2 gene disruption reduced cell damage in CA1 after global ischemia [34]. In the current study, we found that increased COX-2 in ischemic tissue was detected. It suggests that the protective effects of OSR on ischemia-induced injury were partly attributed to inhibition of COX-2.

Studies have demonstrated that inflammation is associated with cerebral ischemic reperfusion injury, which is triggered by ROS accumulation [35]. Proinflammatory cytokines and neutrophils play an important role in the brain inflammatory response to I/R. TNF-α is the proinflammatory cytokines which significantly contribute to brain dysfunction. Neutrophils also induce inflammatory mediators which in turn facilitate neutrophil recruitment in the ischemic reperfusion and then worsen the brain damage [36–38]. Thus, in this study, we examined the protein level of TNF-α. Our data showed that the production of TNF-α was significantly suppressed by treatment with OSR. It indicated that OSR could affect one of the most important factors involved in proinflammatory responses in ischemia tissue, thereby ameliorating the ischemia-mediated brain dysfunction.

NO is a free radical and neurotransmitter. As for its role, NO possesses a duality in the pathophysiological process of cerebral ischemia and reperfusion injury. On one hand, the low concentration of NO can produce the dilation of blood vessels to regulate cerebral blood flow and then improve microcirculation exerting the antiplatelet aggregation action. It confers protective effects on brain tissue [39]. On the other hand, the high concentration of NO inhibits the activity of glycolysis and mitochondrial enzyme. Meanwhile, it reduces the energy generation in neurons and triggers the neurotoxicity in brain tissue [18]. Tissue hypoxia could induce the serious cell damage in focal cerebral ischemia. The ischemic condition accelerates the release of excitatory amino acids, nitric oxide and free radicals. It induces microglial activation and programs cell death [40, 41]. The synthesis of NO depends on the NOS catalysis, and NOS expression is directly related to NO production. So we evaluated the level of NOS to observe the changes of NO. In the central nervous system of organism, NOS has three forms: nNOS, eNOS, and iNOS. eNOS exerts a protective effect on brain tissue by producing a small amount of NO, and iNOS conducts a neurotoxic effect on brain tissue by producing a large of NO. iNOS mainly expresses in astrocytes and activates the microglia after focal cerebral ischemia [42]. A reduced brain damage was observed after ischemia in iNOS knockout mice and the increased expression of iNOS contributes to neuronal injury [43]. In addition, NO increases the formation of free radical and promotes brain damage after stroke by generated iNOS [44]. In this experiment, it showed that the expression of iNOS was increased in the vehicle-treated group compared with the sham-treated group. The result was highly consistent with the previous reports [45]. At the same time, OSR could reduce the expression of iNOS and inhibited the NO synthesis against NO-induced brain damage. We preliminarily concluded that the possible neuroprotective mechanism of OSR on I/R injury may be related to the anti-inflammatory and antioxidant effects.

As to inflammatory cytokines, IL-10 was reported to attenuate brain injury after I/R because of its ability to decrease the amount of inflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-α. In addition, IL-10 inhibits the granulocyte colony-stimulating factor (GCSF) and reduces the neutrophil [46]. In addition, IL-10 is capable of inducing a resistance of the brain cells to ischemia evoked damages in in vivo and in vitro models of the ischemic insults in rats [47]. Interestingly, our data show that the anti-inflammatory property of OSR was evident with the further elevation in the level of anti-inflammatory cytokine IL-10 compared with that in the vehicle-treated group after ischemia.

CONCLUSION

Our study demonstrated that OSR significantly protected neurons from I/R-induced injury by limiting the leukocyte infiltration and attenuating the expression of ICAM-1, IL-1β, IL-6, and IL-8. Meanwhile, OSR promoted the IL-10 expression. OSR also suppressed the upregulation of inflammatory-related oxidative enzymes (iNOS and COX-2) in ischemic brain. In conclusion, the present study showed that OSR plays a potent anti-inflammatory role in cerebral ischemic injury via regulating the NF-κB signal transduction pathway and thus inhibiting inflammation cascade reaction. These findings encourage further pharmacological studies to evaluate whether other underlying complex and interrelated pathways are mediated in the neuroprotection of OSR.