| BACKGROUDStroke is one of the most serious diseases of human beings currently. Stroke is a kind of disease with characteristics that include high incidence rate, high disability rate, high recurrence rate, high mortality and high economic burden. In our country, stroke is the first cause of death. The immune system and inflammation play a critical role in the development and subsequent pathobiology of stroke. Inflammation is a defense response to the stimulus, and it plays an important role in the organism. When stimulated by a stimulating factor, the inflammatory response can be induced to repair tissue and resist damage. Inflammation can also have adverse effects on the body, leading to immune system disorders. Inflammation and immunity have been linked to multiple risk factors for stroke which include hypertension, atherosclerosis, diabetes, atrial fibrillation, and tobacco smoke-induced vascular impairment. Of particular interest are the damage-associated molecular pattern molecules (DAMPs) released as a result of a cerebrovascular accident, that promote innate immune responses that contribute to brain damage and ultimately to neurological deficits.To restore cerebral perfusion as soon as possible is the most effective treatment for acute ischemic stroke, such as the application of recombinant tissue plasminogen activator in clinical. The earlier to do thrombolytic therapy, the better of the result will be, however, thrombolytic therapy is restricted by the limited time window、the general condition of patient and many complications such as bleeding after therapy, so only 20% of the patients can be benefit from this therapy. Seeking effective treatment of cerebral protection has become the focus of the study for many years. Decades of research focusing on putative mechanisms of cytoprotection that may permit brain cells to maintain homeostasis both during/after an ischemic stress have uniformly failed to translate into clinically relevant therapies. Nonetheless, the basic and translational efforts of the international stroke community have massively advanced the understanding of the governing dynamics underlying stroke pathobiology.Inflammasomes are multi-protein complexes activated as part of the innate immuneresponse to stressors and/or infections that trigger the maturation of caspase-1 followed by the production of IL-1β and IL-18. Caspase-1 and IL-1β promote inflammation and cell death with IL-1β having been implicated in a number of disease processes including those that unfold after an ischemic injury. In experimental stroke, IL-1β expression increases following brain ischemia and multiple studies have shown that blocking IL-1β can be neuroprotective. Of note, in humans IL-1β levels increase in both the cerebrospinal fluid and blood after an ischemic stroke. In murine models, levels of inflammasome related proteins increase after an ischemic brain injury and the inhibition of inflammasome activity has been shown capable of reducing the extent of such injuries.Cell death and activation of pattern recognition receptors set the stage adaptive immunity. Release of nucleotides (ATP, UTP) from injured cells, including neurons, activates purinergic receptors on microglia and macrophages and leads to production of pro-inflammatory cytokines. Although most of these cytokines are transcriptionally induced, IL-1βand IL-18 are processed from their propeptides by the activity of interleukin-1-converting enzyme (ICE; Caspase 1). ICE is embedded in a multi-protein complex (NLRP3, or inflammasome) and is activated by microglial P2X7 receptors. The cell death of ischemic brain leads to the formation of DAMPs, which activates TLRs, especially TLR2 and TLR4. DAMPs released by ischemia include high-mobility group protein B1, an intracellular DNA binding protein released after cellular injury, heat shock protein 60 andβ-amyloid (Aβ) among others. TLRs, in concert with scavenger receptors such as CD36, up-regulate pro-inflammatory gene expression through the transcription factor nuclear factor-KB. DAMPs are also derived from matrix breakdown by lytic enzymes released from dead cells and by the action of ROS on lipids. The cytokine production and complement activation resulting from these events leads to increased leukocyte infiltration and enhances tissue damage, which, in turn, produces more DAMPs. Antigens unveiled by tissue damage are presented to T cells, setting the stage for adaptive immunity. These recent developments make it necessary to evaluate the role of inflammation and immunity in the pathophysiology of stroke.The Nobel Prize winner of 2009 found that the end of the chromosomes telomeres cap named telomere cap can protect chromosomes from fusion, degradation, and/or recombination. This study is very important to aging and cancer. Mammalian telomeres are composed of repetitive TTAGGG motifs. These motifs are released from dying host cells and serve to down-regulate inflammatory responses that can cause tissue destruction (e.g. as in autoimmune disease).The synthetic oligodeoxynucleotide A151 is composed of four TTAGGG motifs on a phosphorothioate backbone. A151 duplicates the ability of telomeric DNA to modulate inflammation, including the production of IL-6, IL-12, IFNy, MIP-2, and TNFa. A151’s potential as an anti-inflammatory agent has been demonstrated in animal models of arthritis, endotoxic shock, concanavalin A induced hepatitis, ocular inflammation, lupus nephritis, atherosclerosis, and silica-induced pulmonary inflammation. Critically, the pharmacokinetics, pharmacodynamics and safety of phosphorothioate oligodeoxynucleotides have been established in multiple clinical trials.Currently, the effect of A151 on critical processes underlying the pathophysiology of brain ischemia and if A151 is good for brain ischemia are uncertain. Based on the role of IL-1β in critical processes of brain ischemia and A151 is helpful to many diseases by reduce the inflammasomes, If A151 reduces ischemic brain damage, It will provide new highlights of ischemic stroke treatment.When brain ischemia occurs, the main sources of IL-1β are bone marrow derived macrophages (BMDM) and microglia. In addition to perivascular macrophages, monocytes rapidly infiltrate the brain and become macrophages after brain ischemia. We sought to examine the effect of A151 on critical processes (i.e. inflammasome mediated responses) underlying the pathophysiology of brain ischemia via the synergistic use of both oxygen-glucose deprivation (OGD) and lipopolysaccharide (LPS) stimulation in a reductionist in vitro system which is intended to replicate the complex pathobiology that unfolds during an ischemic stroke.The lifetime of Stroke-prone spontaneously hypertensive rats (SHR-SP) is short. SHR-SP rats are pone to stroke. Rats was investigated by means of permanent middle cerebral artery occlusion (pMCAO), which is a widely accepted animal model of ischemic stroke.Phosphorothioate oligodeoxynucleotides (ODN) A151 (5’-TTAGGGTTAGGGTTAGGGTTAGGG-3’) and control ODN C151 (5’-TTCAAATTCAAATTCAAATTCAAA-3’) were synthesized at the FDA CBER Core Facility (Silver Spring, MD).OBJECTIVES1. In vitro, to examine the effect of A151 on critical processes (i.e. inflammasome mediated responses) underlying the pathophysiology of brain ischemia via the synergistic use of both oxygen-glucose deprivation (OGD) and lipopolysaccharide (LPS) stimulation in a reductionist in vitro system which is intended to replicate the complex pathobiology that unfolds during an ischemic stroke. To test that if A151 can reduce the inflammasomes after ischemic stroke and at the same time give protection to the brain.2. In vivo, Application of SHR-SP rats model with permanent middle cerebral artery occlusion surgery, we observe the effects of A151 on stroke rats respectively from the aspects of animal behaviors, histology, immunity and genes, and explore its therapeutic potential, in order to protect the brain further.METHODS1. Cell culture. BMDM were derived from both the femurs and tibias of SHR-SP rats. Bone marrow cells were cultured in DMEM supplemented with 10% FBS and 10 ng/ml rat M-CSF. On day 6, the cells were washed twice with PBS and scraped in the presence of cold HBSS. After centrifugation and resuspension, the cell density was adjusted to 2x105 cells per ml and 2 ml cells were seeded in each well of six-well plates. Iba-1 staining indicated that >99% cells were macrophages.2. Pretreated BMDM with A151、C151 and LPS, the cells were placed inside modular incubator chambers, with anaerobic colorimetric indicator strips that detect a 0.2% oxygen threshold, The chamber was flushed with a gas mixture of 95% N2 and 5% CO2 for 20 min at room temperature at 6 L/min. After flushing, the chambers were sealed and maintained at 37℃ for 18 hours. The supernatant was collected for Elisa, and cells were collected for Westernblot.3. Pretreated SHR-SP rats with A151、C151 and LPS, and then do surgery on the rats, that called permanent middle cerebral artery occlusion(pMCAO).48 hours post-pMCAO, the rats were evaluated using methods that have been described previously, after that the Rats were euthanized. Collect brain、spleen、kidney and blood of the rats for use.4. Some of the brains were removed, frozen on dry ice, and stored at-80℃ for downstream analyses. Frozen brains Frozen brains were sectioned coronally at a thickness of 20-μm. The cut sections were then fixed using paraformaldehyde vapor and stained with cresyl violet. Image J (NIH, Bethesda, MD) was used to quantify the infarct area.5. Frozen rat brain sections were homogenized in Qiazol lysis reagent. Real-time PCR was performed using a QuantiTect SYBR Green PCR kit and the following PCR primers:NLRP3、NLRC4、Aim2.6. Collect blood and treat with anticoagulant, test the chemistry material in blood and count blood cells. Plasma was collected after centrifugation for following Elisa.7. To do Elisa and Western blot, protein concentration of the brains and spleens were measured after the use of lysis buffer.8. Use JC-1 assay to measure mitochondrial membrane potential.RESULTS1. To investigate the immunomodulatory potential of A151 under ischemic conditions, BMDM were treated with A151 or C151, LPS, and OGD. A151 dramatically reduced the levels of IL-1β, IL-la, IL-6, CINC-1, and TNFa in culture supernatants. This was in contrast to the effects of the control ODN C151, which only reduced the levels of IL-6 and CINC-1. Of note, neither ODN altered the levels of CINC-3, IFNγ, IL-10, or TGFβ.We explored the effects of A151 on the survival of oxygen and glucose deprived BMDM. These findings verify results from other groups, which have shown that the anti-inflammatory effects mediated by A151, are not due to an increase in target cell cytotoxicity.2. The effect of A151 on IL-1β expression and maturation was explored further by western blot analysis. A151 treatment of oxygen and glucose deprived BMDM reduced the levels of mature IL-1β in cell culture supernatants. We studied the regulatory potential of A151 on the expression/protein levels of additional inflammasome components. A151 reduced mature caspase-1 and NLRP3, but did not affect ASC, AIM2, NLRP1 or NLRC4.3. Mitochondrial dysfunction has been linked to NLRP3 inflammasome activation and we used the JC-1 assay to study the mitochondrial membrane potential (MMP) and found that A151 reduced the depolarization of MMP.4. The ability of A151 to prevent and/or treat ischemic injury was evaluated in SHR-SP rats using the permanent middle cerebral artery occlusion (pMCAO) model. Compared to treated with C151 group and treated with saline group, the infarct volumes of treated with A1513 days before surgery、1 day before surgery and 3 hours after surgery were reduced apparently.5. We purified total RNA from the brain of SHR-SP rats 48 hours after pMCAO. Compared with saline and 3mg C151-1d treatments, th e expression of NLRP3 was reduced in 3 mg A151 treated rats. Of note, the expression of Aim2 and NLRC4 were not altered by A151CONCLUSIONS1. A151 reduces the release of inflammatory factors and the death of BMDM induced by exposure to LPS and OGD.2. A151 reduces the maturation of IL-1β and caspase-1 and the expression of NLRP3 and iNOS in response to LPS and OGD stimulation.3. A151 reduces the depolarization of mitochondrial membrane potential in BMDM.4. A151 reduces pMCAO induced ischemic brain damage in SHR-SP rats.5. A151 reduced NLRP3 mRNA within the brains of post-ischemic SHR-SP Rats. |
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