| Objectives:To investigate the protective effect of bFGF on neurons after hypoxic-ischemic brain damage(HIBD)in neonatal rats and its related molecular mechanisms,and provide new evidence for potential therapeutic targets for Neonatal hypoxic-ischemic encephalopathy(NHIE).Methods:In vivo:HIBD model of neonatal 7-day Sprague-Dawley(SD)rats was established,and observed the degree of brain injury in the acute phase(24h)of rats after hypoxic-ischemic(HI)through brain gross view,TTC staining,Zea-longa score,HE staining and immunofluorescence staining.The long-term(60d)neurological dysfunction of rats after HI was detected by NSS scoring,rotating rod,water maze,open field and Y maze,including the ability of exercise,spatial memory,self-exploration and learning and memory were detected.The changes of mRNA and protein expression of bFGF in brain tissue at 24 h after HI were detected by Quantitative Rea-time-PCR(qRT-PCR)and Western Blot.In vitro,neonatal rat primary cortical neurons were cultured and the HI model was established by oxygen-glucose deprivation(OGD)treatment,and the expression level of bFGF mRNA was verified by qRT-PCR.The three effective fragments of chemically synthesized bFGF-siRNA were transfected into PC 12 cells,and the most effective interference fragment was screened by qRT-PCR.Then the most effective fragments were transfected into primary cortical neurons.The expression of bFGF was successfully silenced,and cell viability was detected by MTT.Bright field and Immunofluorescence staining was used to evaluate the effect of bFGF silencing on the number of primary cortical neurons,axon length and cell area before and after OGD.The GENEMANIA website was then used to predict the closely related molecules of bFGF.The expression changes of bFGF-related molecules were detected by qRT-PCR and Western Blot.Results:In vivo:1.Compared with the sham group,brain swelling and the infarct size was enlarged,zea-longa score was increased(the exercise capacity was seriously weakened)and the number of cortical neurons was decreased in the rats at 24 hours after HI.The difference was statistically significant(P<0.05).2.After 24 hours of HI,HE staining showed vacuolization,nuclear pyknosis,disordered arrangement and cytoplasm loosening in the right(infarcted)cortical cells.3.At 60 days after HI,the NSS scores were higher than Sham group;the time on the rotating stick became shorter than Sham group;the time of found the original platform area hidden under the water became longer than Sham group,the number of times across the original platform area were decreased compared with Sham group,the time and distance of stayed in the original platform quadrant area became shorter than Sham group;the standing time and the grooming time in the open field became shorter than Sham group;the correct rate and time of finding the food arm in the Y maze were decreased compared to Sham group,the error rate and time of staying in the wrong arm were increased compared to Sham group.The difference was statistically significant(P<0.05).4.The mRNA and protein levels of bFGF in the right cortex of rats were increased 24 h after HI(P<0.05).In vitro:1.Compared with the normal group,the relative expression of bFGF mRNA was significantly increased 24 h after hypoxia(Hypoxia)and 24 h after OGD(P<0.05).2.Compared with the negative control group(NC group),the mRNA expression levels of the F3 fragments of the three specific bFGF siRNA fragments-F1,F2 and F3 were the lowest(P<0.05).3.Transfection of F3 fragments into primary cortical neurons revealed that the relative expression of bFGF mRNA was decreased compared with NC group under normal conditions and OGD conditions(P<0.05).4.The number of neurons and the cell viability were decreased,the cell body was increased and the axon length was shortened in the bFGF-si and OGD groups,there were significantly differences compared with the NC group and the Normal group(P<0.05).5.Compared to the OGD+NC group,the OGD+bFGF-si group further reduced the number of cells and increased the cell body size(P<0.05),but did not reduce the axon length.6.The molecules closely related to bFGF were predicted by GENEMANIA,namely nerve growth factor(NGF)and interleukin-1β(IL-1β).7.The relative expression of NGF and IL-1β mRNA was down-regulated in the bFGF-si group compared with the NC group under normal conditions(P<0.05),and the relative expression of mRNA and protein in the OGD group was up-regulated compared with the Normal group(P<0.05).However,in the OGD+bFGF-si group,the mRNA and protein relative expression levels of NGF were down-regulated and IL-1β was up-regulated compared to the OGD+NC group(P<0.05)Conclusions:The expression level of bFGF was elevated in the right cortex at 24h after HI.Under OGD conditions,elevated bFGF was essential for maintaining the normal number and morphology of primary cortical neurons,and may be associated with up-regulation of NGF and down-regulation of IL-1β.The results of this study will provide new insights and molecular foundations for treatment of HIE in the future. |
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