| Background:Hypoxia-ischemia encephalopathy(HIE)is widely regarded as a serious nervous system disease,and it is also the main cause of neonatal acute death and chronic nervous system diseases,which have a high incidence,mortality and permanent disability rate all over the world.At present,the research on neonatal HIE focuses on the protective mechanism of central nervous system.In clinical research,it is found that the distribution of lymphocyte subsets in children with HIE is abnormal,which leads to immune dysfunction and aggravates HIE injury.However,whether brain injury affects the development of neonatal immune system,its mechanism and approach are still unclear.Thymus is a necessary place for lymphocyte differentiation,development and maturation.Adult thymus gradually shrinks,the ability to secrete mature T cells gradually declines,and the immune function played by thymus weakens.In the study of adult stroke,it is found that cerebral ischemia can cause changes in immune organs such as thymus and spleen,induce immune response of central nervous system,and thus lead to neuroinflammation.The hypothalamic-pituitary-adrenal(HPA)axis is an important way for the brain to send signals to the target cells of the immune system,and it is involved in regulating the development of immune cells and various stages of immune response.In adult stroke,it is found that cerebral ischemia can activate HPA axis,make it dysfunctional,lead to immune suppression,increase the risk of infection,and affect long-term rehabilitation.The thymus in infancy is relatively large and developed in the stage of rapid development,but it is easily affected by external factors(hunger and disease)to reduce the thymus volume.This experiment mainly explores the influence of HIE on the development of the thymus of neonatal rat,and whether this effect is realized through HPA axis,which provides new ideas and research basis for immunotherapy or comprehensive treatment of HIE in clinic.Objective:1.Observe thymic development,thymic cell apoptosis and autophagy occurrence in HIE neonatal rats,and analyze the relationship between thymic changes and the degree of brain injury;2.To clarify the dysfunction of the HPA axis in HIE neonatal rats and its mechanism;3.To clarify the mechanism by which HIE activates hypothalamic HMGB1/TLR4/NF-κB signaling and affects the HPA axis to regulate apoptosis and autophagy in thymic cells.Methods:The HIE model of Sprague Dawley(SD)newborn rats(7 d)was established by Rice-Vannucci method,regardless of sex.In order to observe the effects of hypoxia and ischemia(HI)on HMGB1/TLR4/NF-κB signal,HPA axis and thymus,animals were randomly divided into two groups:Sham group and HI group,and the samples were taken from SD newborn rats at five time points on the 1st,3rd,7th,14th and21st day after HI operation.Brain index and thymus index were used to evaluate brain injury and thymus development at different time points,and the correlation between them was analyzed.Apoptosis and CD3~+T lymphocyte ratio were detected by flow cytometry at different time points after HI operation,and the most significant time point of thymocyte apoptosis was selected.Western Blot was used to detect the activation markers of hypothalamic microglia and the expression of HMGB1/TLR4/NF-κB signaling pathway related proteins and thymocyte autophagy signaling pathway related proteins on the 7th day after HI operation.The expression levels of CORT and ACTH in peripheral blood and CRH hormone,TNF-αand IL-1βin hypothalamus were detected by ELISA kit at different time points after HI operation,and the function of HPA axis was evaluated.In order to prove the effect of glucocorticoid on thymus,animals were randomly divided into three groups:Sham+Oil group,HI+Oil group,HI+10 mg/kg RU486(glucocorticoid receptor antagonists)group,and the materials were collected from SD newborn rats 7 days after HI operation.Western Blot was used to detect thymocyte apoptosis and autophagy molecules on the 7th day after HI operation.Hypothalamic CRH and TNF-αexpression levels were determined by ELISA,and thymocyte apoptosis and CD3~+T cell percentage were determined by flow cytometry.In order to prove the effect of HMGB1 on HPA axis and thymus of HIE newborn rats,the animals were randomly divided into two groups,HI+DMSO group and HI+20 mg/kg GLY(HMGB1 inhibitor)group,and the materials were collected from SD newborn rats 7 days after HI operation.The activation markers of hypothalamic microglia,molecules related to HMGB1/TLR4/NF-κB signaling pathway and proteins related to thymocyte apoptosis and autophagy were detected by Western Blot on the 7th day after HI operation,and the expression levels of CORT and ACTH in peripheral blood and CRH in hypothalamus were detected by ELISA,and thymocyte apoptosis and the percentage of CD3~+T cells were measured by flow cytometry.Results:1.The body weight of HIE newborn rats decrease slightly on the 1st,3rd,and 7th day,and increased lightly on the 14th and 21st day,but there was no remarkable statistical difference.The brain index showed an increasing trend on the 1st day after HI(P<0.05),no obvious difference on the 3rd day,and a significant decrease in brain index on the 7th,14th and 21st days(P<0.05);the thymus index decreased be clear at a glance on the 1st,3rd,7th and 14th days(P<0.05),and there was no striking change on the 21st day.2.The brain index and thymus index of HIE newborn rats were negatively correlated on the 1st and 3rd days(P<0.05)and positively correlated on the 7th day(P<0.05),14th and 21st(P<0.01).3.The apoptosis of thymocytes in neonatal rats with HIE was the most striking on the 7th day compared with the 1st,3rd,7th,14th and 21st day,with a significant statistical different(P<0.01);the proportion of CD3~+T lymphocytes tended to decrease on days 1,3,7,14 and 21 after HI,and the proportion of CD3~+T lymphocytes decreased notably on day 7(P<0.01).4.The expression levels of pro-apoptotic proteins Cleaved-caspase 3 and Bax in the thymocytes of HIE newborn rats showed an increasing trend on the 1st,3rd,7th,14th and 21st day,with a prominently statistical difference on the 7th day(P<0.05),and the anti-apoptotic protein Bcl-2 and the ratio of Bcl-2/Bax tended to decrease on the 1st,3rd,7th,14th and 21st day,with a markedly statistical difference on the 7th day(P<0.05).The autophagy protein Beclin 1 showed an increasing trend on the 1st,3rd and 7th day,and reached the peak on the 7th day(P<0.05),and then decreased.And the change of LC3 II to I ratio also had a similar change trend.It was verified that the expressions of Cleaved-caspase3(P<0.01)and Beclin 1(P<0.001)proteins in thymus distinctly increased,the ratio of Bcl-2/Bax(P<0.001)was significantly reduced,and the ratio of LC3 II/I(P<0.001)was overtly increased,then inhibition of the activation of the negatively regulated PI3K/Akt/m TOR autophagy signaling pathway in thymic(P<0.01).5.The hypothalamic index of HIE newborn rats increased significantly on the 1st,3rd and 7th day(P<0.05)and tended to recover on the 14th and 21st day(P<0.05);the pituitary index decreased obviously on the 1st and 3rd day(P<0.05),and increased prominently on the 7th,14th and 21st day(P<0.05);the adrenal index dramatically elevated on the 1st,3rd,7th,14th and 21st day(P<0.05).Correspondingly,the content of CRH in hypothalamus increased obviously on the 1st,3rd and 7th day and tended to decrease on the 14th and 21st day,the content of ACTH in peripheral blood did not have meaningful discrepancy on the 1st and 3rd day,but lessened significantly on the 7th day(P<0.05),increased greatly on the 14th and 21st day(P<0.05);and the content of CORT in peripheral blood increased transparently on the 1st,3rd,7th,14th and 21st day(P<0.05).The expression of GR in thymocytes was strikingly reduced(P<0.05)while nuclear translocation distinctly increased(P<0.001).6.On the 1st,3rd,7th,14th and 21st day,the expression levels of IL-1βand TNF-αwere distinctly increased(P<0.05);CD11b protein expression(P<0.001),HMGB1(P<0.01)and TLR4(P<0.001)and phosphorylation levels of P65-NF-κB protein in the hypothalamus of HIE newborn rats were increased as plain as print(P<0.01);7.RU486 significantly increased the brain index(P<0.05),hypothalamic index(P<0.01)and thymus index(P<0.05),and reduced the content of CRH(P<0.01)and TNF-α(P<0.05)in hypothalamus.At the same time,it greatly reduced the percentage of thymocyte apoptosis(P<0.01)and increased the percentage of CD3~+lymphocytes,reduced the expression of Cleaved-caspase 3,Bax,Beclin 1,LC3 II/I(P<0.05),and increased the expression of Bcl-2 and m TOR.8.GLY prominently increased the brain index of HIE newborn rats(P<0.05),dramatically decreased the microglia marker CD11b protein expression(P<0.01)and TNF-αcontent(P<0.01),and down-regulated HMGB1(P<0.001),TLR4(P<0.01)and the phosphorylation levels of P65-NF-κB protein(P<0.001)in hypothalamus.9.GLY significantly decreased the hypothalamic index(P<0.05),the content of CRH in hypothalamus(P<0.05),and ACTH(P<0.05)and CORT(P<0.05)in peripheral blood,while significantly increased the expression of GR protein in thymocytes(P<0.001).10.GLY significantly decreased the thymus index(P<0.05),the expression of Cleaved-caspase 3,Bax,Beclin 1 and LC3 II/I proteins in the thymus(P<0.05),while increased the expression of Bcl-2 protein and upregulated strikingly the phosphorylation level of m TOR(P<0.001).Conclusion:1.Brain injury in HIE neonatal rats induced thymic atrophy,and promoted thymic cell apoptosis and cellular autophagy,in which process the PI3K/Akt/m TOR signaling pathway may be involved.2.HIE induces microglial activation and proinflammatory cytokine expression in the neonatal rat hypothalamus,and this effect may be achieved through the activation of the HMGB 1/TLR4/NF-κB signaling pathway.3.HIE-induced neuroinflammation in the hypothalamus may lead to hyperactivation of the HPA axis,which then causes GC action on the GR within thymocytes,causing thymic atrophy. |
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