Anti-inflammatory Mechanism Of Triptolide On Ischemic Brain Injury

BackgroundCerebral ischemia caused by cerebral blood circulation disorder(ischemia or hemorrhage)is a common neurological disease.Stroke is one of the most life-threatening neoplasia in the world with high incidence and mortality rates.Recently,studies on the immune inflaumatory response between glial cells in cerebral ischemia have become very important.Microglia cells are the "macrophages" in the brain;their activation state is paramount to alleviating neuronal apoptosis and promoting neurogenesis in the wake of cerebral ischemia.CSF1R,known as the colony stimulating factor 1 receptor,is a transmembrane tyrosine kinase,which is expressed in all microglia and mainly responsible for regulating the proliferation and differentiation of microglia.Ki20227,a specific inhibitor of CSF1R,inhibits CSF1R phosphorylation and affects microglia activity and regulates the morphology and density of microglia in the whole cerebral ischemia model and further regulate the density of dendritic spines of neurons.Changes in the number of microglia or in the M1/M2 phenotype could modulate the inflammatory response in cerebral ischemia lesions.Triptolide(TP),an anti-inflammatory drug that is extracted from the root of tripterygium wilfordii,is used for the treatment of various autoimmune diseases.TP exhibits neuroprotectiveness in central nervous system diseases through regulating autophagy and oxidative stress.TP inhibited nucleotide-binding domain,leucine-rich repeat pyrin domain containing 3(NLRP3)inflammatory response by regulating microglia activation.Previous studies have shown TP to significantly reduce the cerebral infarction area in cerebral ischemic rats by decreasing NF-?B inflammatory and autophagy response.Microglia autophagy is involved in synaptic differentiation and social behavior regulation.The inhibition of autophagy in microglia results in impaired synaptic microsomal degradation and altered connections between brain regions.Thus,microglia autophagy plays an important role in synaptic development and neural behavior regulation.Elimination of microglia or inhibition of microglia autophagy provides a powerful tool for the study of microglia function after cerebral ischemia.That said,TP's regulatory mechanism in cerebral ischemia model,in the event of microglial cell dysfunction,is currently unclear.Objectives1.To explore the effects of elimination of microglia or inhibition of microglia autophagy on behavior,microglia and neurons in the cerebral cortex of cerebral ischemic mice.2.To investigate the effects of TP on mice behavioral,microglia cells and neurons in the cerebral cortex of elimination of microglia or inhibition of microglia autophagy cerebral ischemic mice.Materials and Methods1.Two-photon in-vivo imaging method was used to detect the changes in the number of microglia at different times of Ki20227 drug administration.2.Rose-Bengal(10 mg/mL)peritoneal injection was used to induce cerebral ischemia.3.2,3,5-triphenyltetrazolium chloride(TTC)staining was used to detect infarct size after Rose-Bengal induced cerebral ischemia.4.Based on the Cre-loxp system,the double-positive offspring of CX3CR1-Cre and ATG5flox/flox were used in subsequent experiments.5.Electrophysiological techniques were used to detect the excitatory changes in the pyramidal neurons in the cerebral cortex of the transgenic mice.6.The behavioral changes of mice in each group before and after cerebral ischemia modeling were detected by Rota-Rod test,body weight,neurobehavioral assessment and open field test.7.Immunofluorescence and Western blot were used to detect the expression of various proteins in the cerebral cortex of cerebral ischemic mice.8.The qRT-PCR method was used to detect the mRNA expression of various factors in the cerebral cortex of cerebral ischemic mice.ResultsPart one1.Two-photon in-vivo imaging and Ibal immunofluorescence results showed that the number of microglia gradually decreased with the prolongation of Ki20227 administration.Following 7 days of Ki20227 administration,there was about 13.7%dissipation of microglia when compared to the control group.2.TTC staining results showed 15%infarct area of the total area after Rose-Bengal induced cerebral ischemia,implying the successful establishment of cerebral ischemic mice model.3.Behavioral test showed that Ki20227 treatment had no effects on the balance ability,body weight and neurobehavioral score of mice.After stroke,decrease of stay time in Rotarod Treadmill and the neurobehavioral score of were observed in Ki20227 treated mice.TP promoted the decrease of the stay time in Rotarod Treadmill and the neurobehavioral score of Ki20227 treated mice.4.The number of microglia in the penumbra area of the mouse brain was significantly increased after stroke with inflammatory factors(TNF-?,IL-10 and IL-6)and surface receptors(CD86,Arg-1,CXCL1).TP accelerated the number of microglia decrease and the down-regulation of TNF-? and IL-6 in Ki20227 treated stroke mice.5.Ki20227 treatment inhibited NF-?B expression after intervention.After the stroke,the expression of NLRP3 inflammatory protein and NF-?B were increased.However,TP pretreatment decreased both NF-?B expression and NLRP3 inflammatory response.6.The number of neurons,density of dendritic spines and expression of synaptic proteins,PSD95 and SYN,were significantly decreased after stroke.Ki20227 alone did show any effect;however,TP pretreatment increased the number of neurons,the density of dendritic spines and the expression of synaptic proteins,PSD95 and SYN.7.Increased expression of LC3II,P62 and decreased P62,Beclinl and BDNF-AKT pathway proteins in the cerebral cortex injury site were observed after stroke in control and Ki20227 treatment groups.TP pretreatment expedited autophagy protein expression decrement and BDNF-AKT expression increment in the cerebral cortex injured area of stroked-mice with Ki20227 drug intervention.Part two1.Microglia autophagy-deficient mice were successfully constructed.Inhibition of atg5 gene in microglia significantly inhibited autophagy in microglia.The inhibition of microglia autophagy did not affect the motor ability of mice.2.After stroke,the time of stay on Rotarod Treadmill test was significantly reduced,and the behavioral score was significantly increased in microglia autophagy-deficient mice.However,TP pretxeatment reversed these phenomena.3.Inhibition of autophagy in microglia accelerated the increase of microglia number and enhanced mRNA expression of microglia related factors such as TNF-? in the cerebral cortex after stroke.TP pretreatment significantly reduced the number of microglia and the expression of inflammatory cytokines such as TNF-? and IL-10.4.Inhibition of autophagy in microglia accelerated the decrease of neuronal number,density of dendritic spines of neurons and expression of synaptic related proteins,PSD95 and SYN.All these decrements were reversed following TP pretreatment;thus,increased neuronal number,density of dendritic spines and expression of dendritic related proteins.5.Inhibited autophagy in microglia increased the number of action potential emission of pyramidal neurons in the cerebral cortex.TP significantly increased the number of neuronal action potential emission in CX3CR1+/-ATG5flox/flox stroked-mice.6.Inhibited autophagy in microglia aggravated caspase-1 activation and NLRP3 inflammatory response in the cerebral cortex of stroked-mice.TP treatment significantly reduced the expression of NLRP3 and caspase-1 activation.7.Inhibited microglial autophagy aggravated the decrease of BDNF expression and increased LC3? expression to inhibit the activation of AKT signal and over-activate the autophagy level in the cerebral cortex of stroked-mice.TP pretreatment significantly increased the expressions of BDNF,AKT and p-ERK,and inhibited the expression of LC3II autophagy-related proteins.ConclusionsDecreasing microglia number in stroke exhibited neuroprective effect by reducing the expression of inflammatory factors such as TNF-? and NF-?B.Inhibition of microglia autophagy in stroke promoted the expression of microglia inflammatory cytokines and NLRP3 inflammatory complex,inhibited the BDNF-AKT pathway and damaged the density of dendritic spines and synaptic proteins in neurons.After stroke,TP pretreatment decreased microglia numbers and the expression of inflammatory factors,inhibited NLRP3 inflammatory complex and the overall level of autophagy,activated of BDNF-AKT signaling pathway and increased the density of dendritic spines,synaptic protein expression and number of neurons,ultimately enhanced the recovery of neurobehavioral functions.