TLR4/NF-?B Signaling Pathway Mediates Autophagy Of Hippocampal Neurons In The Immune Inflammatory Damage After Hypoxic-ischemic Encephalopathy

Neonatal hypoxic-ischemic encephalopathy(HIE)is one of the main causes of neonatal death,secondary mental retardation,cerebral palsy and other disabilities,which is caused by partial or complete hypoxia of brain tissue,decrease or pause of cerebral blood flow.Up to now,there is a lack of HIE specific diagnostic markers and effective treatment.Therefore,in-depth study of its mechanism is of great clinical value and significance for optimizing the early intervention treatment scheme and reducing the mortality and disability rate of neonatus.Immune inflammatory response plays an important role in the pathological mechanism of HIE,but the specific mechanism is still unclear.TLR4 is a natural immune pattern recognition receptor,which plays an important role in the induction and regulation of immune inflammation.Autophagy is an important regulatory mechanism for cell metabolism to maintain homeostasis.It has been found that TLR4 can be used as an environmental receptor for autophagy and induce autophagy activation.However,whether there is TLR4/NF-?B signaling pathway mediated autophagy and inflammatory cytokine release in HIE is not clear.The purpose of this study is to explore the role of TLR4/NF-?B signaling pathway mediated autophagy in hypoxic-ischemic encephalopathy and its specific molecular mechanism.Part ? The role of TLR4/NF-?B signaling pathway and autophagy of neuron in the injury of hippocampal immune inflammation in hypoxic-ischemic brain damage of neonatal ratsObjective:To observe the temporal changes of TLR4/NF-?B signal pathway and autophagy related protein expression level of neurons in the process of hippocampal immune inflammation injury in HIBD neonatal rats,and to analyze whether there is consistency in the temporal changes of the two levels.Methods:7-day-old neonatal Sprague-Dawley(SD)rats were randomly divided into Sham-operated group and HIBD group.The HIBD model of neonatal rats was established by ligation of left common carotid artery and hypoxia for 2.5h with Rice-vannucci method.The following indexes were observed after hypoxia ischemia:1.Neurobehavioral function was measured by Negative geotaxis test,Righting reflex test and Feeney score of Beam walking test to evaluate the short-term and long-term neurobehavioral damage of neonatal rats;2.The volume of cerebral infarction was measured by TTC staining;3.Gross morphological damage of brain tissue observed by naked eye;4.HE staining was used to observe the pathomorphological changes of left hippocampus;5.Immunohistochemistry was used to detect the expression of TLR4,p-NF-?B,Beclin-1 and LC3 protein positive cells in left hippocampal CA1 region;6.Western blot was used to detect the protein levels of TLR4,p-NF-?B,Beclin-1 and LC3 in left hippocampus;7.The levels of TNF-? and IL-1? in the downstream of TLR4/NF-?B signal pathway were measured by ELISA;8.To analyze the consistency of TLR4/NF-?B signal pathway and autophagy related protein expression level of neuron in the time trend.Result:1.Short-term and long-term neurobehavioral function test:the rats in the Sham-operated group had normal left and right limb activities,could quickly turn over the body,grasped the balance beam effectively,passed the balance beam quickly without falling,and had good coordination ability of left and right limb movements.In HIBD group,the right limb was obviously weak,the time of turning over body was obviously prolonged,the balance beam was slowly passed,the balance beam could not be grasped,and even fell or fell down.The test result was significantly lower than that in Sham-operated group(P<0.05),the left and right limb movement coordination and integration ability were poor.2.TTC staining:after TTC staining,the left and right hemispheres of the neonatal rats in the Sham-operated group showed uniform red;the left brain tissue of the neonatal rats in the HIBD group had large area of white infarct(P<0.05).3.Gross morphological observation of brain tissue:in the Sham-operated group,the left and right hemispheres of the neonatal rats were symmetrical,regular in shape,transparent and clear in brain tissue,without edema,turbidity and liquefying necrosis.In HIBD group,the bilateral cerebral hemispheres were asymmetric,the left cerebral hemispheres were plump,and there were edema,turbidity and liquefying necrosis in different degrees.4.Pathological changes of hippocampal tissue:in the Sham-operated group,the left hippocampal tissue structure was clear,the neuron cells arranged orderly and closely,and the cell morphology was normal.In HIBD group,24 hours after hypoxia and ischemia,the left hippocampal structure was disordered,the arrangement of neurons was loose and uneven,the cells were edematous,a small number of nuclei were pyknosis,fragmentation and nerve cell necrosis;48 hours later,the damage was further aggravated,and there were obvious pyknosis,fragmentation,cell gap expansion,vascular expansion and a large number of nerve cells necrosis.5.The expression of TLR4,p-NF-?B,Beclin-1 and LC3 positive cells was detected by immunohistochemistry:TLR4 was mainly stained by cytoplasm and membrane,p-NF-?B was mainly stained by nucleus,Beclin-1 and LC3 by cytoplasm.The expression of TLR4,p-NF-?B,Beclin-1 and LC3 in the left hippocampal tissue of the neonatal rats in the Sham-operated group was only a small amount;the expression of TLR4,p-NF-?B,Beclin-1 and LC3 in the left hippocampal tissue of the HIBD group was significantly increased(P<0.05).6.Western blot and ELISA analysis of TLR4/NF-?B signal pathway and autophagy related protein level time expression trend:the expression of TLR4,p-NF-?B,TNF-?,IL-1?,Beclin-1 and LC3 in the left hippocampal tissue of the Sham-operated group was at a low level;the expression of TLR4,p-NF-?B,TNF-?,IL-1?,Beclin-1 and LC3 in the left hippocampal tissue of the HIBD group increased gradually after HIBD,peaked at 48h and decreased gradually at 72h(P<0.05),and the trend of the change was basically the same in time.Conclusion:1.TLR4/NF-?B signaling pathway and autophagy of neurons are involved in the process of hippocampal immune inflammatory injury in neonatal rats after HIBD;2.The trend of TLR4/NF-?B signal pathway and autophagy related protein expression in hippocampal of HIBD neonatal rats is consistent in time.TLR4/NF-?B signal pathway may be the key promoter of autophagy activation.Part ? Effects of TAK-242 on autophagy of hippocampal neurons in HIBD neonatal rats via TLR4/NF-?B signaling pathwayObjective:To observe the effect of TAK-242,a TLR4 antagonist,on autophagy of hippocampal neurons in HIBD neonatal rats,and to elucidate the molecular mechanism of TLR4/NF-?B signaling pathway mediated autophagy in regulating hippocampal immune inflammatory injury after HIBD.Methods:7-day-old neonatal SD rats were randomly divided into Sham-operated group,HIBD+Vehicle group,HIBD+TAK-242(0.75 mg/kg)group,HIBD+TAK-242(1.5 mg/kg)group and HIBD+TAK-242(3 mg/kg)group.The HIBD model of neonatal rats was established by Rice-vannucci method.The following indexes were observed after hypoxia ischemia:1.Neurobehavioral function was assessed by Neurological deficit score,Rotarod task and Beam walking test;2.Determination of infarct volume in neonatal rats by TTC staining;3.Determination of water content in brain tissue by dry wet specific gravity to evaluate the degree of brain edema;4.Gross morphological damage of brain tissue observed by naked eye;5.HE staining was used to observe the pathomorphological changes of left hippocampus;6.The expression of TLR4 signaling pathway and autophagy related protein TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 positive cells in CA1 region of the left hippocampus of neonatal rats were detected by immunohistochemistry;7.Western blot was used to detect the changes of TLR4 signaling pathway and autophagy related proteins TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 in the left hippocampus of neonatal rats;8.The levels of TNF-? and IL-1? in the left hippocampal tissue were measured by ELISA.Result:1.Neurobehavioral function test:the left and right limbs of rats in the Sham-operated group had good motor integration and coordination ability,and ran rapidly without falling.The results of neurobehavioral function test in HIBD+Vehicle group were significantly lower than that in Sham-operated group,showing obvious weakness of right limbs,poor motor integration and coordination ability(P<0.05).Compared with the rats in HIBD+Vehicle group,the rats in HIBD+TAK-242 group had a significant improvement in neurological deficit,a significant increase in the wheel time,a significant decrease in the travel time of the balance beam,and a better motor integration and coordination ability,among which the high dose group(3 mg/kg)had the most significant effect(P<0.05).2.TTC staining:after TTC staining,the left and right hemispheres of the rats in the Sham-operated group showed uniform red,without obvious infarct;the left brain tissues of the rats in the HIBD+Vehicle group had large area of white infarct;compared with the H1BD+Vehicle group,the left brain tissue infarct volume of the neonatal rats in the HIBD+TAK-242 group decreased significantly,especially in the high dose group(3 mg/kg)(P<0.05).3.Determination of brain edema degree:the water content of brain tissue in the Sham-operated group was constant;the water content of brain tissue in the HIBD+Vehicle group was significantly increased(P<0.05);compared with the HIBD+Vehicle group,the content of brain edema in the HIBD+TAK-242 group was significantly reduced(P<0.05).4.Gross morphological changes of brain tissue:in the Sham-operated group,the left and right hemispheres of the neonatal rats were symmetrical,the brain tissue was transparent and clear,without edema,turbidity and liquefaction necrosis;In the HIBD+Vehicle group,the cerebral hemispheres on both sides of the neonatal rats were asymmetric,and the left cerebral hemispheres were obviously full,and there were edema,turbidity and liquefying necrosis in different degrees of brain tissue;In the HIBD+TAK-242 group,the left and right hemispheres of the neonatal rats were basically symmetrical,and the left brain tissue was in a state of mild edema and turbidity,and the degree was significantly reduced,the scope was significantly reduced,and there was no obvious brain tissue liquefaction necrosis.5.Pathological changes of hippocampus:in the Sham-operated group,the left hippocampal structure of the neonatal rats was clear,the neuron cells were arranged orderly and closely,and the cell morphology was normal.In HIBD+Vehicle group,the structure of hippocampal tissue was disordered,the arrangement of neuron cells was loose and uneven,the cell edema,nuclear pyknosis,nuclear fragmentation and necrosis of nerve cells were observed.In HIBD+TAK-242 group,the changes of hippocampal structure were lighter,only a few neurons appeared edema and necrosis,and the degree was significantly lighter than that in HIBD+Vehicle group.6.The expression of TLR4 signaling pathway and autophagy related protein positive cells was detected by immunohistochemistry:Beclin-1 and LC3 were mainly stained by cytoplasm,MyD88 and TRIF also by cytoplasm.The expression of TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 positive cells in the left hippocampus of the Sham-operated group were only a small amount;the expression of TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 positive cells in the left hippocampus of the HIBD+Vehicle group were significantly increased 48 hours after HIBD(P<0.05),but the expression of TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 positive cells in the left hippocampus were significantly decreased after the intervention of TAK-242(P<0.05).7.Western blot analysis of TLR4 signaling pathway and autophagy related protein level:TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 in the left hippocampal tissue of the Sham-operated group were expressed at a low level;TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 in the left hippocampal tissue of the HIBD+Vehicle group were significantly increased(P<0.05);Compared with HIBD+Vehicle group,the expression of TLR4,p-NF-?B,MyD88,TRIF,Beclin-1 and LC3 in HIBD+TAK-242 group decreased significantly(P<0.05).8.The level of inflammatory cytokines was detected by ELISA:the expression of TNF-? and IL-1? in the left hippocampal tissue of the Sham-operated group was low;the level of TNF-? and IL-1?in the left hippocampal tissue of the HIBD+Vehicle group was significantly higher(P<0.05);compared with the HIBD+Vehicle group,the level of TNF-? and IL-1? in the HIBD+TAK-242 group was significantly lower(P<0.05).Conclusion:1.TLR4 signaling pathway mediates autophagy of neurons in the process of hippocampal immune inflammatory injury after HIBD in neonatal rats;2.TAK-242 may play a neuroprotective role after HIBD by blocking TLR4/NF-?B signaling pathway,inhibiting autophagy of neurons and reducing the release of downstream inflammatory cytokines.Part ? Expression of TLR4 signaling pathway and autophagy related protein in peripheral blood of neonates with hypoxic-ischemic encephalopathyObjective:To observe the expression of TLR4 signaling pathway and autophagy related protein in the peripheral blood of neonates with HIE,analyze the correlation between TLR4 signaling pathway,autophagy and neonatal behavioral neurological assessment(NBNA),and explore the relationship between TLR4 signaling pathway,autophagy and neonatal HIE severity and recent prognosis,so as to evaluate the value of clinical condition judgment and prognosis evaluation.Method:1.21 neonates with HIE admitted to NICU in Affiliated Hospital of Yangzhou University from July 2018 to July 2019 were collected.According to the severity of HIE,the patients were divided into Mild HIE group,Moderate HIE group and Severe HIE group,and healthy neonates were the Control group;2.Western blot was used to detect the expression of TLR4,Beclin-1 and LC3 in peripheral blood monocytes;3.ELISA was used to determine the levels of TNF-? and IL-1? in serum;4.The NBNA scores of neonates were measured;5.The correlation between TLR4,Beclin-1,LC3 protein expression level and NBNA scores was analyzed.The relationship between TLR4,Beclin-1,LC3 level and HIE severity and short-term prognosis was evaluated.Result:1.The levels of TLR4,Beclin-1,LC3,TNF-? and IL-1? in the peripheral blood of HIE group were significantly higher than those of the Control group(P<0.05),and increased with the severity of HIE(P<0.05).2.The NBNA score of HIE group was significantly lower than that of Control group,and decreased with the severity of HIE(P<0.05).3.The expression of TLR4,Beclin-1,LC3 protein in peripheral blood of HIE neonates was negatively correlated with NBNA score(P<0.05).Conclusion:1.TLR4,Beclin-1 and LC3 are sensitive indexes for HIE severity judgment and short-term prognosis evaluation;2.TLR4 signaling pathway and autophagy may play an important role in the pathogenesis of HIE.