Sinomenine relieves oxygen and glucose deprivation-induced microglial activation via inhibition of the SP1/miRNA-183-5p/IκB-α signaling pathway

Studies have shown that the inflammatory activation of miroglia (MG) and nuclear factor kappa B ( NF-κB ) play a dominant role in inflammatory response. Previous studies have shown that sinomenine, an anti-inflammatory agent extracted from Sinomenium acutum, can directly protect neurons against cerebral ischemia injury. However, there are no reports on its effect on ischemia/reperfusion-induced inflammatory activation of MG. In the present study, an in vitro ischemia/reperfusion model was developed with mouse BV-2 microglia cells, a model of oxygen-glucose deprivation/reperfusion (OGD/R), and the inhibitory effect of sinomenine pretreatment on inflammatory activation was confirmed through measurement of inflammatory indicators. Mechanistically, sinomenine suppressed OGD/R-induced inflammatory activation through the SP1/miRNA-183-5p/IκB-α pathway. In conclusion, this study shows that sinomenine effectively inhibits OGD/R-induced inflammatory activation in MG by suppressing the activation of transcription specificity protein 1 (SP 1). This finding is of significance for the clinical use of sinomenine in treating cerebral ischemia/reperfusion injury.


Introduction
During ischemic brain injury, which is characterized by high morbidity, high mortality, and high disability, the rupture or blockage of blood vessels causes hypoxic injury to cerebral cells and tissues (1)(2)(3). Although the current clinical treatment for ischemic brain injury is based on timely recovery of blood supply protects neurons from injury, the ischemia/reperfusion process inevitably aggravates the pathological injury in tissues affected by ischemia. It is therefore important to control inflammation for post-operation recovery of patients (4)(5)(6).
Inflammatory activation of MG is a marker of diseases involving brain nerve injury (7,8). MG exist extensively in the central nervous system, accounting for 20 % of the total number of brain gliocytes, a number comparable to that of neurons. MG secrete a variety of factors under different conditions, thus playing a dual role in neural injury and recovery. Malfunction of MG is always involved in inflammatory nerve injury. It is of significance to study the association between MG and nerve injury, and to improve MG function, for improving therapy and recovery of injured nerves (9). In the normal brain, MG, with multiple branches, are at a resting status, but become activated during ischemic brain injury. It is generally understood that microglia are derived from marrow mononuclear cells or hematopoietic stem cells, which explains their immune celllike characteristics. In fact, MG do exhibit the biological functions of mononuclear macrophages (10). During brain ischemia, MG are rapidly activated and migrate to the injured area, where they secrete multiple cytokines. Some of these cytokines are protective factors for nerve cells, reducing the number and degree of necrotic neurons, while others are pro-inflammatory factors which induce a cascade of responses and aggravate the damage to brain tissues and cells. Chronic inflammation induced by excessive activation of microglia usually leads to damage to nerve cells or brain tissues, and eventually to degenerative diseases. Therefore, the development of anti-inflammatory drugs targeted at MG is always a hotspot in research on ischemia-induced brain injury (11). Acute inflammation has been demonstrated to play a critical role in secondary brain injury induced by cerebral ischemia/reperfusion. Cell injury mediated by inflammatory cells is close to delayed neuronal death with respect to. Drugs that block every link to inflammation can be used to treat delayed damage caused by cerebral ischemia (12,13). Studies have shown that the inflammatory activation of MG and transcription factor NF-κB play a dominant role in the inflammatory response. NF-κB is found almost in all cell types. It has been long considered to be involved in various disease pathogenesis. P65, RelB, cRel, P50, and P52 are five homologous subunits of NF-κB, which always exist as homodimers and heterodimers. In the classic NF-κB pathway, various pathogens, including reactive oxygen species, endotoxin, bacterial tumor necrosis factor alpha, etc, are considered as common stimuli for incurable inflammation (14). Heterodimers of p65 and p50 are the most abundant in cells. The IκB family includes several proteins, one of the most important regulators of mammalian NF-κB is IκB-α. In unstimulated cells, NF-κB is sequestered in an inactive form in the cytoplasm bound to inhibitory IκB-α protein (15). Under the action of stimulatory signals, IκB-α undergoes phosphorylation, ubiquitination, and finally degradation in the cytoplasm. At the same time, NF-κB also undergoes phosphorylation, then translocates to the nucleus and activates the transcription of its target genes. Phosphorylation of NF-κB plays an important role in the dissociation of NF-κB from inhibitory proteins, nuclear translocation of NF-κB, and binding to DNA (16). Activation of NF-κB pathway is a major trigger of inflammation, in which NF-κB regulates inflammation-induced cytokines, such as TNF-α and IL-1β, through binding sites in their promoters. Thus regulation of NF-κB has been considered as a potential therapeutic for inflammatory diseases (17,18). In NF-κB pathway, IκB-α can be activated by IκB kinase complex through ubiquitinated and degraded process, and then promoted translocation of the p65/p50 complex into the nucleus and activated the expression of downstream genes, thus the expression of IκBα was the classic negative feedback of NF-κB activation (19). SP1, a well studied transcription factor, is critical for many biological processes, including cellular growth and differentiation (20), there are few studies related to SP1 about inflammation. Interestingly, in this study, we found that SP1 was activated by OGD/R, promotes the expression of miRNA-183-5p, which, binding to the 3'-untranslated region (3'-UTR) of IκB-α mRNA, inhibits its expression, and thus activates NF-κB, resulting in inflammation. Sinomenine suppresses OGD/R induced inflammatory activation in MG through the SP1/ miRNA-183-5p/IκB-α pathway.
Sinomenine, extracted from the stem and root of Sinomenium acutum, is soluble in ethanol, acetone, chloroform and diluted base, and slightly soluble in water, diethyl ether and benzene. According to the theory of traditional Chinese medicine, sinomenine can help to ease pain, relieve cough, lower blood pressure, and resist inflammation; it is used to treat rheumatic arthritis and neuralgia. For cerebral ischemia, sinomenine is able to inhibit nerve cell apoptosis and inflammation induced by ischemia, while promoting cell proliferation. However, no studies have been reported on its role in the inflammatory activation of MG induced by cerebral ischemia/reperfusion (21,22). The primary objective of this study was to investigate the inhibitory action of sinomenine on OGD/R-induced inflammatory activation in BV-2 cells (a murine microglial cell line), and the regulating pathway involved. This was with a view to laying a theoretical basis for applying sinomenine in resisting inflammatory activation of MG, and in treatment of cerebral injury caused by ischemia/reperfusion.

Cell culture
BV-2 cells purchased from the Cell Bank of the Chinese Academy of Sciences (CBCAS, Shanghai, China), were maintained in DMEM (Invitrogen, CA, USA) supplemented with 10 % fetal calf serum (FCS) (Invitrogen, CA, USA) at 37 ºC in an atmosphere of 5 % CO 2 . The cells were passaged at the coverage of 70 % using 0.25 % Typsin (Invitrogen, CA, USA). Cells subjected to OGD/R treatment were first cultured under normal conditions for 24 h, followed by 2 h culturing under 5 % CO 2 and 95 % N 2 in the serum and glucose free medium, and then culturing for 24 h under normal conditions. At the same time, 293 cells obtained from ATCC (MD, USA) were maintained in DMEM supplemented with 10 % FCS at 37 ºC under 5 % CO 2 .

Sinomenine source and toxicity test
Analytically pure sinomenine (Shenzhen Sendi Biotech Company, ShenZhen, China) was dissolved in dimethyl sulphoxide (DMSO, Sigma, CA, USA) to prepare a 100 mM stock solution, which was diluted in culture medium to final concentrations of 25, 50, and 100 μM used for cell treatment. The BV-2 cells in logarithmic phase were seeded in 96-well plates at a density of 1×10 5 cells/well, and cultured at 37 ºC in an atmosphere of 5 % CO 2 overnight. Three groups of cells were used: control group, vehicle (DMSO) group, and sinomenine treatment group. The latter group was divided into 5 sub-groups on the basis of levels of sinomenine administered (5, 25, 50, 100, and 200 μM). After overnight culture, the medium was replaced and sinomenine was added to final concentrations of 5, 25, 50, 100, and 200 μM. The cells were cultured under normal conditions for 24 h, following by CCK-8 assay for cell proliferation. Each well was treated with 10 μl CCK-8 solution (DOJINDO, Japan), and the plates were incubated at 37 ºC under 5 % CO 2 for 4 h. Thereafter, the absorbance of each well was read at 450 nm in Multiskan GO (Thermo, MI, USA). The maximum concentration of sinomenium that produced no significant cytotoxicity was calculated and used for subsequent experiments.

Determination of the effect of sinomenine on mRNA and protein expressions of IκB-α
BV-2 cells in logarithmic phase were seeded in 6-well plates at a density of 3×10 5 cells/well, and cultured overnight at 37 ºC under 5 % CO 2 . The cells were divided into four groups: control group, 25 μM sinomenine group, 50 μM sinomenine group, and 100 μM sinomenine group. The cells were incubated for 24 h under normal conditions, and total RNA and protein were extracted for determination of IκB-α mRNA and protein expressions using RT-PCR and western blotting, respectively. out. Cells (293) were transfected with pcDH1-SP1 and pGL3-wp-183 or pGL3-mp-183, and harvested 48 h later for luciferase activity assay. The transfection experiment was carried out in 24-well plates, following the instructions for Lipofectamine 2000 (Invitrogen, CA, USA). Each well was transfected with PGL-TK (100 ng) (Promega, WI, USA) as an internal reference for the luciferase assay.

Measurement of TNF-α, IL-1β and IL-6 by ELISA
Total protein was extracted and quantified using the bicinchoninic acid (BCA) method, and intracellular TNF-α, IL-1β and IL-6 were measured using appropriate mouse ELISA kits (Invitrogen, CA, USA) according to the manufacturer's instructions. The levels of TNF-α, IL-1β and IL-6 were calculated according to the A450.

Determination of IκB-α mRNA contents
Total RNA was reverse-transcribed into cDNA with Oligo dT18, and 2 ΔΔCt method was used to analyzed the results of PCR with beta actin as the internal reference. The PCR primers were: β actin-forward: 5'-AACCCTAAGGCCAACCGTGAAAAG-3', β actinreverse: Determination of the binding site of mmu-miRNA-183-5p on IκB-α 3'UTR by luciferase reporter assay TargetScan was used to predict the theoretical target (seed region) of mmu-miRNA-183-5p in the mRNA sequence of IκB-α. In this process, 293 cells were transfected with miRNA-183-5p-mimics, inhibitor, or NC, as well as pGL-wt-IκB-α and pGL-mt-IκB-α using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instructions. The cells were harvested 48 h after transient transfection, and luciferase assays were performed. The relative luciferase activities (ratios of firefly and renilla luciferase activities) of lysates were measured using a dual luciferase reporter assay system (Promega, WI, USA).

Statistical analysis
All data are expressed as mean ± SD, and analyzed by one-way ANOVA. Least Significant Difference (LSD) was used for multiple comparisons between any two means. Values of p < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS 13.0 software.

Toxicity and anti-inflammatory activity of sinomenine in BV-2 cell model of OGD/R
Results from CCK-8 assay showed that 200 μM sinomenine significantly inhibited cell proliferation (p < 0.01), but there was no statistically significant difference between 100 μM sinomenine and control (p > 0.05) (Fig. 1A). BV-2 cells pretreated with sinomenine for 2h were subjected to OGD/R. Following 24 h reoxygenation, the inflammatory factors were assayed to confirm that the model was established, and to determine the effect of sinomenine on the model. ELISA assay showed that TNF-α, IL-1β and IL-6 levels in the supernatant from the OGD/R model were significantly increased (p < 0.01), when compared with the control group, indicating inflammatory activation of BV-2 cells. Pretreatment with 100 μM sinomenine significantly and dose-dependently decreased the levels of TNF-α, IL-1β and IL-6 in the culture supernatant, relative to the model group (p < 0.01, Fig. 1B). It is generally understood the inflammatory activation is ascribed to inactivated regulation of NF-κB. Therefore, phosphorylated p65 and p50 and IκB-α were assayed for. The data showed that OGD/R treatment stimulated the phosphorylation of p65 and p50, and reduced IκB-α (p < 0.01). These effects were reversed in a significant and dose-dependent manner by sinomenine pretreatment, with reduction in phosphorylated p65 and p50 levels, and enhancement in IκB-α level (p < 0.01, Fig. 1C).

Effect of sinomenine on mRNA and protein expressions of IκB-α
Sinomenine suppressed inflammatory activation in BV-2 cells by inhibiting NF-κB activation, and the process was directly associated with increased IκB-α expression (Fig. 1). In order to determine whether the increased IκB-α expression was regulated at the transcriptional or post-transcription level, cells were exposed to sinomenine at different concentrations for 24 h, and IκB-α mRNA and protein were assayed. The results showed that there was no apparent difference in IκB-α mRNA level between cells treated with sinomenine and untreated cells (p > 0.05), but IκB-α protein was higher in the group treated with sinomenine (p < 0.01, Figures  2A and 2B). These results suggest that IκB-α may mediate the inhibition of NF-κB by sinomenine, and the increase in IκB-α by sinomenine was through a posttranscription regulation. To elucidate how sinomenine regulates the expression of IκB-α, attention was focused on post-transcription by miRNA. Thus, miRNAs with the potential to bind to the 3' UTR of mouse IκB-α were searched for using TargetScan Human 7.1, and changes in the miRNAs were determined through RT-PCR. It was found that miRNA-183-5p was negatively correlated with IκB-α protein levels, that is, sinomenine pretreatment increased miRNA-183-5p (p < 0.01, Fig. 2C).

Genetic intervention through a lentiviral approach
For BV-2 cells, conventional transfection methods can hardly achieve a high gene delivery efficiency: lipofection transfection can hardly apply to G0 cells, and eletro-transfection damages gliocytes. Thus, a lentiviral system was selected to solve overcome this problem so as to achieve efficient and stable intervention. As mentioned earlier, miR-183-5p promoter sequence was get, software "JASPAR" was used to predict the transcription factors that may have binding sites on the miRNA-183-5p promoter. The results indicated that SP1 had a theoretical binding site on the miRNA183-5p promoter.

TFBS of SP1 in miRNA-183-5p promoter
The promoter sequence of miRNA-183-5p gene was cloned from mouse genomic DNA. To verify whether the promoter was active, we cloned this promoter sequence upstream of the plasmid carrying the fluorescent marker green fluorescent protein (GFP), followed by transfection of 293 cells. 48 hours after transfection, a large amount of GFP was expressed in the cells under fluorescence microscopy, indicating that this promoter is fully capable of directing transcription of downstream genes (Fig. 5A).
Bioinformatics analysis showed the theoretical binding site of SP1 on the miR-183-5p promoter (Fig. 5B). Luciferase activity assay showed that overexpression of SP1 increased the activity of luciferase in cells transfec- (C) miRNA-185-5p relative content was studied using RT-PCR, with β-actin as reference. The relative content of miRNA-183-5p was analyzed using the 2 ΔΔCt method. Tests were carried out on three biological triplicates, and data are expressed as mean ± SD, **p < 0.01, *p < 0.05 vs. BV-2 or DMSO group. ted with the wild-type reporter gene expression vector (p < 0.01), but had no apparent effect on the reporter vector carrying a mutant promotor (p > 0.05, Fig. 5B), indicating that SP1 did bind to the miRNA-183-5p through the TFBS and did positively regulate its transcription.

Specificity of the SP1/miRNA-183-5p/IκB-α pathway
Based on data obtained so far, it was posited that there might be a SP1/miRNA-183-5p/IκB-α pathway in the inhibition of OGD/R-induced inflammatory activation of BV-2 cell by sinomenine. If the pathway did exist, it was important to confirm whether it played a dominant role in the inhibition. So recombinant viruses Lv-SP1, Lv-miR183-5p and Lv-shRNA-IκBα were constructed, respectively, and transfected BV-2cells to overexpress SP1, miR-183-5p or silence IκBα, then the resultant changes in SP1, IκB-α and phosphorylation of p65 and p50 were studied to investigate the role of the pathway in sinomenine protection against ischemia reperfusion. The results showed that OGD/R significantly increased SP1 protein, reduced IκB-α, and promoted the phosphorylation of p65 and p50 (p < 0.01). Pretreatment with 100 μM of sinomenine inhibited the increases in SP1 protein, as well as increases in phosphorylated p65 and p50, and the decreases in IκB-α (p < 0.01). Overexpression SPI increased SP1 protein content (p < 0.01), but miRNA-183-5p overexpression or IκB-α silencing had no observable effect on SP1 (p > 0.05). The expressions of SP1 and miRNA-183-5p and silencing of IκB-α both significantly decreased IκB-α expression (p < 0.01), and increased the levels of phosphorylated p65 and p50 (p < 0.01, Fig. 6).

Discussion
Ischemic brain injury is one of the common diseases in the aged: it causes almost 200 deaths in China every hour. During ischemic brain injury, the rupture or blockage of blood vessels causes obstructed circulation and hypoxia injury in cerebral cells and tissues (2). The   main therapeutic approach is focused on the recovery of blood supply combined with multiple cytokines, which, if applied in time, will effectively improve the patient's symptom. However, clinical statistics have revealed some adverse effects of this regimen. Reperfusion, which is inevitable in recovery of circulation, often leads to inflammation in MG. Activated MG release a lot of inflammatory factors and mediators, thereby exposing the brain neurons to inflammation and apoptosis (4,11). The control of ischemia/reperfusion-induced inflammatory activation of MG is important for reducing the loss of brain nerve function caused by ischemia, and for improving prognosis.
Microglia account for about one-fifth of total gliocytes in brain. They have some characteristics of immunocytes, and are very biologically active: they secrete a variety of factors under different conditions, thus playing dual roles in neural injury and recovery. Since MG and neurons are interwoven in the brain, inflammatory nerve injury is closely related to MG malfunction. During ischemia, activated MG move to the damaged areas and secrete multiple nutritional factors so as to maintain the basic functions of neurons. However, reperfusion leads to excessively activation of MG and secretion of more inflammatory factors, resulting in a cascade inflammatory response, which may aggravate neuronal malfunction and exacerbate neuronal injury (23,24). So far, brain injury caused by cerebral ischemia/reperfusion can only be relieved: there are no effective treatments. The development of efficient drugs for treatment cerebral ischemia/reperfusion-induced injury is always a subject of immense interest in cerebral ischemia studies.
Studies have shown that, in general, inflammatory response of MG in mice occurs 3 h after ischemia/reperfusion, and peaks at 48 h. According to a long clinical monitoring study, MG activation starts at 72 h after cerebral ischemia/reperfusion, and lasts for over one month. Therefore, the essence of ischemia/reperfusion treatment is to effectively inhibit inflammatory activation of MG (25). The development of anti-inflammation drugs targeted at MG remains a major objective of research on ischemia-induced brain injury. Acute inflammation plays a critical role in secondary brain injury induced by cerebral ischemia/reperfusion, and cell injury mediated by inflammatory cells resembles delayed neuronal death in terms of timing. Drugs that block all inflammation links could be used to treat delayed damage caused by cerebral ischemia (26,27). So far, the drugs that resist inflammatory activation of MG after cerebral ischemia are pro-inflammatory factor inhibitors: a non-selective adenosine receptor agonist 2-Cado can effectively suppress the expression of TNF-α in ischemia-injured tissue and cells, and brazilein has been shown to effectively protect brain tissue by inhibiting TNF-α and IL-6 expressions (28,29). Mechanistic studies have shown that NF-κB, a classical regulator of inflammatory response, plays a dominant role in ischemia-reperfusioninduced MG activation. Therefore, the suppression of excessive activation of NF-κB is an important step in the treatment of ischemia-reperfusion injury (30).
An important way for developing new drugs is to search for lead compounds, improve their activities, and reduce their toxicities through structural modifica-tions. Sinomenine is an isoquinoline alkaloid with antiinflammatory, immune-strengthening, pain-relieving, and blood pressure-lowering properties. Resistance to inflammation is an important property of sinomenine which has been used in the clinical treatment of rheumatic arthritis. Indeed, studies have shown that sinomenine downregulates IL-1, TNF-α, and IL-6 expressions in arthritic mice (31). In addition, mechanistic studies have shown that sinomenine activity may be associated with the inhibition NF-κB and expression of IκB-α (32,33). Therefore, it is worthwhile to investigate the effect of sinomenine in resistance to inflammation. Although a few studies have been reported on its role in cerebral ischemia-reperfusion, none has focused on the inflammatory activation of MG.
In the present study, an in vitro OGD/R model was created using BV-2 cells, and used to study the effect of sinomenine on inflammatory activation of MG. Based on assessments of NF-κB activation and IκB-α expressions, the anti-inflammatory activity of sinomenine relies on the suppression of NF-κB activation by upregulation of IκB-α. Results from quantitative PCR and western blotting revealed that sinomenine upregulated IκB-α in a post-transcription manner. From bioinformatical analysis and validation experiments, it can be concluded that sinomenine resists inflammatory activation of BV-2 cells via the SP1/miRNA-183-5p/IκB-α, that is, it inhibits the transcription of miRNA-183-5p, and thus increases the expression of IκB-α. This, in turn limits the phosphorylation of p65 and p50, and the activation of NF-κB, thereby suppressing OGD/R-induced inflammation. Further investigations on the specificity of the pathway showed that it dominates anti-inflammatory activity of sinomenine. This is considered of significance in the use of sinomenine for treatment of ischemia-reperfusion injury and development of sinomenine derivatives targeting SP1. However, the mechanism involved in the sinomenine-induced suppression of SP1 expression needs further studies. Moreover, it is not clear whether SP1 is a direct target of sinomenine. Based on current data and bioinformatical analysis, a preliminary hypothesis on how sinomenine suppresses SP1 expression can be proposed: sinomenine binds to SP1, exposes its ubiquitination site, and thus induces its ubiquitination. However, this hypothesis needs to be verified.
In conclusion, the present study has elucidated the role and mechanism of sinomenine in resisting OGD/Rinduced inflammatory activation of MG. These findings are significant because they can help develop new sinomenine-based regimens for treating ischemia-reperfusion, and they are of benefit for prospective studies on sinomenine and gene combination therapy. Moreover, the findings can help in the modification of sinomenine to derivatives with potent drug properties. Thus, these findings provide a solid theoretical base for the role of sinomenine in resisting inflammation and improving nerve injury in ischemic brain injury.