Vinpocetine Alleviate Cerebral Ischemia/reperfusion Injury By Down-regulating TLR4/MyD88/NF-?B Signaling

Background: Stroke is the second most common cause of death in the world,which is the leading cause of adult disability.Intravenous thrombolysis at 3-4.5h after stroke onset is effective treatment for ischemic stroke by evidence,but narrow time window and poor medical conditions make many patients miss treating.It is urgent to find a new effective prevention and treatment of stroke,stroke pathophysiology is very complex,more and more studies have found that inflammatory response is key point in secondary brain injury of cerebral ischemia.Most scholars believe that anti-platelet aggregation,carotid endarterectomy and carotid stenting can effectively start the secondary prevention of stroke.Except aspirin and hydrochloropyrogrel,other drugs on the treatment of ischemic stroke are not certain.It is to need badly seek new,safer and more effective drugs,in particular,which have a clear anti-inflammatory effect and small side effects.Vinpocetine is the alkaloid extracted from small vinca,the formula: C22H26N2O2,molecular weight 350.5g / mol,soluble in dimethyl sulfoxide,100% ethanol and acetone and other organic solvents,which has applied in the cerebrovascular disease and cognitive impairment,and has accumulated more evidence,its effectiveness and safety has also been confirmed by many clinical trials.The study found that vinpocetine can dilate cerebral vessels and regulate cerebral blood flow.In recent years,the study showed that vinpocetine can not only expand the cerebral vessels,but also play an anti-inflammatory effect.Jeon et al.found that vinpocetine inhibits the presence of NF-?B activation and subsequent inflammatory factor secretion in a variety of cell types,including vascular smooth muscle cells,human umbilical vein endothelial cells,human lung adenocarcinoma cell line A549,macrophages,and observed that vinpocetine can inhibit monocyte adhesion and aggregation.In murine pneumonia,irritated by tumor necrosis factor alpha(TNF-?)or lipopolysaccharide(LPS),vinpocetine inhibits tumor necrosis factor alpha(TNF-?),interleukin-1?(IL-1?)and macrophage Cell inflammatory protein-2,and at the same time inhibit the infiltration of polymorphonuclear leukocytes in the interstitium,more importantly,vinpocetine as a phosphodiesterase inhibitor can directly inhibit IKK(I?B kinase),and not by inhibiting phosphodiesterase and regulate calcium channel.The studies have shown that vinpocetine has a clear anti-inflammatory effect,and by inhibiting NF-?B activation and relating inflammatory factors.TLRs were found in human Toll receptor protein in 1997.More interestingly,it is homologous to the Toll protein of Drosophila,the two belong to type I transmembrane glycoprotein.TLRs initiate immune response mainly through the specific recognition of PAMPs(pathogen-related molecules Mode)and DAMPs(damage-related molecular patterns).It not only constitute an important part of the innate immune system but also play an important role in the acquired immune response signal.In the central nervous system disease(CNS),it can not be ignored that autoimmune response play important role.The study found that TLRs have two classical signal transduction pathways,including Myd88-mediated(Myd88-dependent)signaling pathways and TRIF-mediated(Myd88-independent)signaling pathways.My D88 mediates a variety of TLRs signal transduction(except TLR3),NF-?B and MAPKs(mitogen-activated protein kinases),resulting in many kinds of inflammatory mediators.TRIF only mediates TLR3 and TLR4 signaling,NF-?B activates MAPKs(pathogen-associated molecular patterns),and produces inflammatory mediators.Tumor necrosis factor alpha(TNF-?)and interleukin-1(IL-1)or interferon-[beta](IFN-[beta])can be produced only by TLR4 via MyD88 or through TRIF signaling pathway.The expression of TLR4/MyD88/ NF-?B signaling pathway in peripheral glial cells can promote the expression of inflammatory mediators and promote the apoptosis of neurons.Recent studies have found that regulation of TLR-mediated NF-?B pathway can reduce a variety of organs ischemic injury.The subjects and other scholars found that infarct size is decrease,brain edema and other damage is alleviative in TLR4-knockout(TLR4-/-)mice in the ischemia-reperfusion injury.Pretreatment with TLR4 before ischemia can not only increase the ischemic tolerance,but also can significantly reduce neurological impairment after ischemia.Vinpocetine as a phosphodiesterase inhibitor has played an important role in cerebrovascular disease and cognitive impairment,and has been widely used in clinical practice.Vinpocetine can improve the symptoms of ischemic cerebrovascular disease,there is not enough evidence for vinpocetine as a neuroprotective agent.Previous studies have suggested that vinpocetine has a protective effect on ischemic cerebrovascular disease,but those studies are limited to animal models and do not simulate ischemic cerebrovascular disease from both in vitro and in vivo.The target is relatively simple,and there is no clear specific target cells.It only observe vinpocetine can play anti-inflammatory effect by inhibiting NF-?B,but for NF-?B upstream signaling pathway,such as TLR4,so we presume whether vinpocetine can protect the nerve cells by regulating the TLR4 / MyD88 / NF-?B signaling pathway.We intend to use the model of OGD and MCAO,microglia and neurons were used as target cells.TUNEL staining and FJB staining were used to observe whether vinpocetine had protective effect on ischemic cerebrovascular disease.Flow cytometry,CCK8 and LDH release were used to detect neuron or microglia,which was target cell for vinpocetine in cerebral ischemia.We observed TLR4 signaling molecules and downstream inflammatory factors by using Westerblotting,PT-PCR,which can further illustrate the anti-inflammatory effects and possible mechanisms of vinpocetine.Method:Part 1: Protective effect of vinpocetine on cerebral ischemia-reperfusion injury in mice1.The model of middle cerebral artery occlusion(MCAO)in mice was established.The neuroprotective effect of vinpocetine on cerebral ischemia-reperfusion injury was evaluated by TTC staining and neurological function score.2.The model of middle cerebral artery occlusion(MCAO)in mice was established.The changes of apoptotic cells and neuron degeneration or necrosis were observed by TUNEL staining and FJB staining.Part 2: Vinpocetine play a protective effect on neurons through microglia1.Primary culture of cortical neurons and microglia,and the establishment of oxygen glucose deprivation(OGD)model to determine the optimal time of oxygen sugar deprivation.2.The cell viability,LDH release and the changes of apoptotic cells were obtained after the addition of vinpocetine to the cortical neurons.3.The cell viability,LDH release and the changes of apoptotic cells were obtained after the addition of vinpocetine and microglia supernatant to the cortical neurons.Part 3: Vinpocetine protects microglia by down-regulating TLR4 signaling pathway1.In vivo WB experiment,intraperitoneal injection of vinpocetine was performed before ischemia-reperfusion to observe the changes of TLR4 signaling molecule.2.In vitro WB experiment,microglia cells were added with vinpocetine before oxygen glucose deprivation,and the changes of TLR4 signal molecules were observed.3.The expression of TNF-? and IL-1? in the ischemic brain tissue and microglia cells after the addition of vinpocetine were detected by RT-PCR.Result:Part 1: Protective effect of vinpocetine on cerebral ischemia-reperfusion injury in mice1.Intraperitoneal injection of vinpocetine before ischemia and reperfusion can reduce the volume of cerebral infarction and improve the neurological function score.by using intracranial middle cerebral artery occlusion(MCAO)model,2.TUNEL positive cels and FJB positive cells decreased by intraperitoneal injection of vinpocetine before ischemia and reperfusion.Part 2 : Vinpocetine play a protective effect on neurons through microglia1.There was no change in cell viability,LDH release and apoptotic cell before addition of vinpocetine to the neuronal glucose deprivation(OGD)model.2.Vinpocetine can increased cell activity,decreased LDH release,decreased apoptotic cells after vinpocetine and microglia supernatant were added to neurons.Part 3.: Vinpocetine protects microglia by down-regulating TLR4 signaling pathway1.The expression of TLR4,Myd88 and NF-?B P65 was down-regulated by vinpocetine and had no effect on TRIF.in vivo.2.The expression of TLR4,Myd88 and NF-?B P65 was down-regulated by vinpocetine and had no effect on TRIF.in vitro.3.The expression of TNF-? and IL-1? was down-regulated by vinpocetine both in vivo and in vitro.Conclusion1.Vinpocetine has a protective effect on ischemic brain damage.2.Vinpocetine play a protective effect.through microglia.3.In this study,we found that vinpocetine can down-regulate TLR signaling pathway,consider the effect of vinpocetine on the release of inflammatory factors and the negative feedback of TLR4.