The Expression Of NLRC4 Inflammasome Pathway In Hypoxic-ischemic Brain Injury In Neonatal Rats And The Effect Of RF On It

Research background Neonatal hypoxic-ischemic encephalopathy(HIE)refers to a disease in which asphyxia caused by multiple factors in the perinatal period then leads to neonatal Hypoxic-ischemic brain injury(HIBI),which can lead to irreversible neurological dysfunction and even neonatal death.The pathogenesis of HIBI is complex and involves multiple pathophysiological processes,and studies have shown that neuroinflammatory responses mediated by multiple immune cells after hypoxia ischemia is an important mechanism leading to brain tissue damage.NLRC4 inflammasome is a multiprotein complex that plays an important inductive and regulatory role in the immune inflammatory response.NLRC4 activates the NLRC4 inflammasome by recognizing damage-related molecular patterns(DAMPs)and pathogen-associated molecular patterns(PAMPs),causing the maturation and release of the downstream pro-inflammatory factors IL-1β and IL-18,leading to the occurrence of an inflammatory cascade.Currently,there is no definitive treatment for HIBI other than hypothermia and combining some adjuvant treatments.This subject speculates that the NLRC4 inflammasome may play a role in HIBI.It is also speculated that targeting NLRC4 mediated inflammatory response may contribute to the therapeutic progression of HIBI.Riboflavin(RF),also known as vitamin B2,is a water-soluble vitamin present in animal innards,milks,eggs,soy products and vegetables,which is one of the indexes for evaluating human growth and development and nutritional status,participating in body redox reactions and has a wide range of physiological effects.Riboflavin sodium phosphate injection is a water-soluble preparation of riboflavin(vitamin B2)for the same purposes as riboflavin,is more water-soluble than riboflavin,and is convenient to use.In recent years,riboflavin has been shown to play a therapeutic role in a variety of diseases,and studies have shown that riboflavin can counteract tissue damage caused by ischemia-reperfusion and play an obvious therapeutic role in ischemic stroke.However riboflavin has been less studied in terms of brain injury in HIBI rats.Therefore,by establishing an animal model of hypoxic-ischemic brain injury in sprague-Dawley rats,which is the most commonly used animal model for the study of hypoxic-ischemic brain injury,this experiment explores the role of NLRC4 inflammasomes in HIBI,and the effects of riboflavin on the NLRC4 inflammasome pathway after HIBI rats,and then discovers new clinical uses of riboflavin and provides new ideas for HIBI drug treatment.Research objective To investigate and analyze the occurrence and development process of NLRC4 inflammatory pathway in neonatal rats with hypoxic-ischemic brain injury,and the effect of riboflavin sodium phosphate injection on it.Research methods 1(1)A total of 120 clean grade Sprague-Dawley(SD)7 days neonatal rats were selected and randomly divided into three group:Sham group,HI model group,and riboflavin groups,with 40 rats in each group.Divided into five time points to measure the indicators,6 h,24 h,48 h,72 h,7d after the model is manufactured,and each group is randomly divided into 8 newborn rats at each time point.(2)The HI group and the RF group were treated with modified Rice-Vannuncci method,and after the left common carotid artery was ligated,the8 % oxygen and 92 % nitrogen mixture was treated with hypoxia for 2.5 h to create a HIBI animal model.Sham group only blunt separated the left common carotid artery from the vagus nerve,without ligation and hypoxia.The RF group received intraperitoneal riboflavin sodium phosphate injection 0.5 ml / 10 g each 2 h before and 30 min after successful modeling.The same volume of saline was used as control in Sham group and HI group.(3)The heart perfusion fixation was performed at 6 h,24 h,48 h,72 h and 7 d after the fabrication of the model,and the brain tissues were taken.Detection methods HE staining microscope was used to observe the histopathological changes of neonatal rat brain microscopically;The extent of neuronal damage and the number and color of Nissl bodies in brain cells were assessed microscopically with Nissl staining.Immunohistochemistry detected the expression levels of NLRC4,Caspase-1,IL-1β and IL-18 in brain tissue at different experimental time points,and the expression of NLRC4 at mRNA level was measured by real-time PCR(q RT PCR)at each time point.Experimental results(1)The general performance : there were no skin color and consciousness state changes in sham group,and their behavioral abilities,such as balance and muscle tone,were normal;The experimental mice in the HI group and the RF group under hypoxia for about 15-20 min showed skin color changes from ruddy to bruised and pallor,cyanosis of the extremities,restlessness,frequent shaking of the head,deep and fast breathing,1.5 h appeared in the box of chaos,spontaneous tail and left rotation,not easy to turn,standing unsteadily,limb spasm convulsions,2.5 h after mental atrophy,decreased autonomy,shallow breathing,weak response to external stimuli,urinary and urinary incontinence,convulsive coma,etc.(2)HE staining:Sham group: the structure of neonatal SD rat brain tissue at each time point was clear,and the cells in the cerebral cortex were distributed neatly,with normal morphology,intact envelopes,clear nucleoli,and no manifestations of nerve cell degeneration and necrosis.HI group:Six hours after HI,a relatively slight reduction in cell layer number and a relatively loose and disorganized cell arrangement in the cortex were observed;At 24 h after HI,the number of cell layers in the cerebral cortex was reduced,and the arrangement of cells was loose and disordered;At 48 h after HI,the cerebral cortex showed a reduced number of cell layers and a loose and disorganized arrangement of cells;At 72 h after HI,the focal cells in the cerebral cortex were seen to be vacuolated,degenerated and necrotic,with cell fragmentation and lysis;At 7d after HI,the brain tissue was congested,and some gliosis was observed.RF group: at all time points,the structural changes of the brain were observed,and the degree of neuronal damage was less severe in the HI group,while the number of neurons was higher,the structure was still regular,and the degree of cellular damage was less severe than that in the HI group.(3)Nissl staining: Sham group: the cytoplasm and nuclei of neuronal cells were clear,and the number and color of Nissl bodies were not significantly changed.HI group: at 6h after HI,occasional neurons were found to be damaged,with slightly skewed nuclei and relatively pale Nissl bodies staining;At 24 h after HI,neurons were more damaged and Nissl bodies stained sparsely and lighter;At 48 h after HI,neuronal damage was obvious,vacuolization showed that Nissl bodies staining was sparse and pale,and central Nissl bodies disappearance was partly present;At 72 h after HI,neurons were found to be significantly damaged,some nuclei were dissolved or mislocalized and appeared vacuolated,Nissl staining was pale and sparse,and some central Nissl bodies were absent;At 7d after HI,gliosis was observed in the cerebral cortex.RF group: structural brain changes,neuronal cell damage,and a reduction in the number of Nissl bodies were observed at all time points,but to a lesser extent than in the HI group.(4)Immunohistochemistry results:In Sham group:NLRC4,Caspase-1,IL-1 β,IL-18 were less expression and no significant change in expression at each time point,which was not statistically significant by statistical software analysis(P > 0.05).In HI group: NLRC4 、Caspase-1、IL-1 β、IL-18 began to increase at 6 h,peaked at 72 h,and decreased at 7 d compared with before,which was statistically significant(P < 0.05)at each time point by statistical software analysis.In RF group: Compared with the time points in the sham surgery group,the expression of NLRC4,Caspase-1,IL-1β,and IL-18 was statistically significant(P<0.05),and compared with the time points in the HI group,NLRC4,Caspase-1,IL-1β,and IL-18 all decreased,and the difference was significant(P<0.05),which was statistically significant.(5)q RT-PCR: In Sham group:there was no significant change in the expression of NLRC4 at the mRNA level at various time points,which was not statistically significant.In HI group : at 6h,24 h,48h,72 h and 7d,the expression of NLRC4 at the mRNA level was statistically higher than that in the Sham group,and the difference was statistically significant compared with the time points in the Sham group.In RF group : Compared with the corresponding time points in the RF group and the HI group,the expression of NLRC4 at the mRNA level decreased,which was statistically significant.Conclusions 1.NLRC4 protein is activated in hypoxic-ischemic brain tissue,causing downstream factors caspase-1,IL-1β 、 IL-18 expression was elevated,suggesting that the NLRC4 inflammasome pathway is involved in the development of neonatal hypoxic-ischemic brain injury.2.Riboflavin sodium phosphate injection may inhibit the activation of NLRC4 inflammasome pathway and decrease its downstream factors caspase-1,IL-18,IL-1β expression,alleviating the damage of neuroinflammation in HIBI,may have a certain protective effect on brain cell injury caused by HIBI.