| Research Background Neonatal hypoxic-ischemic brain damage(HIBD)is caused by a variety of perinatal asphyxia,clinical manifestations of a series of encephalopathy,It is still one of the main causes of perinatal neonatal death and Child disability.But there is still no ideal treatment,so forced to study the damage mechanism of HIBD and to find an effective treatment strategy.Hypoxia-inducible factor-1alpha(HIF-1?)is a highly specific regulatory factor of cellular oxygen partial pressure,which plays a crucial role in the pathophysiology of hypoxic brain injury,involved in cell survival-related cell cycle stability and energy metabolism.It plays a crucial role in cell survival,apoptosis,inflammatory response,autophagy activation and angiogenesis after hypoxia-ischemia injury.However,there are few reports about the expression change and its role and mechanism of HIF-1? in the early stage of HIBD in neonatal rats in China and abroad,and the conclusion is not consistent.Therefore,this study established the HIBD model of neonatal rats to observe the dynamic expression of HIF-1? in the early stage of HIBD,and further explore its possible role and mechanism for clinical research and treatment of neonatal HIBD to provide a new therapeutic target to provide theoretical support.Research purposes This study was to investigate the expression of HIF-1? m RNA and protein in the early stage of HIBD in neonatal rats,to observe the time-dependent dynamic changes of HIF-1? m RNA and protein in hypoxic-ischemic brain tissue,and to determine the time point of the highest expression level.Application of HIF-1? inhibitor 2-methoxyestradiol(2ME2)inhibited the up-regulated expression of HIF-1?,to observe the pathological changes of brain tissue,brain water content changes,the rate of cell apoptosis and its downstream apoptotic target gene expression of BNIP3,and to explore the possible role and mechanism of HIF-1? in the early stage of HIBD.Research methods 1 Laboratory animals and grouping The first day of birth was P0,99 SD rats aged P7,male and female,weight 14.0~18.0 g,SPF grade,provided by Experimental Animal Center of Henan Province.Experiment 1.Thirty-six postnatal day 7 SD rats were divided into Sham group(n = 6)and model group(HIBD,n = 30),the rats in the model group were divided into 5 subgroups according to the time of sacrifice after HIBD(6 h?12 h?24 h?48 h?72 h,n = 6).The expression levels of HIF-1? m RNA and protein were detected by q RT-PCR and Western blot.Experiment 2: Sixty-three postnatal day 7 SD rats were randomized into three groups: Sham group(n = 21),HIBD group(n = 21),HIBD+2ME2 group(n = 21).According to experiment 1,Three groups were sacrificed to the brain at the 24 hours after HIBD,The expression levels of HIF-1? and BNIP3 protein were detected by Western blot,immunofluorescence,HE staining,brain water content and cell apoptosis rate were detected.2 Model establishment and drug intervention All procedures of the neonatal HIBD model were adapted from the Rice-Vannucci Model:The left common carotid artery was ligated and hypoxic for 2.5 h in a mixture of nitrogen and oxygen(8 % O2 + 92 % N2,1.5 L/min),surgery time for each pup did not exceed 5 min.Sham group only free the left common carotid artery,not ischemic hypoxia treatment.Rats in the HIBD+2ME2 group were immediately injected intraperitoneally with 2ME2 after HIBD in a dosage of 15 mg/kg(2ME2 was dissolved in DMSO and then diluted with PBS).Sham group and HIBD group were given the same volume of DMSO and PBS 3 Specimen preparation Preparations of q RT-PCR and Western blot specimens: Rats were anesthetized with 10 % chloral hydrate,and the left brain tissue was quickly removed on ice plate and divided into 2 samples,which were packed into sterile and enzyme-free tubes,frozen in liquid nitrogen and stored in-80 degrees Celsius refrigerator.Paraffin section preparations: After 10 % chloral hydrate anesthesia and fixation,rats were transcardially perfused with 50 ml saline followed by 50 ml 4 % paraformaldehyde solution.Brain tissues were then removed and fixed by immersion in the same solution at 4 °C for 24 h.Brain tissue from the optic chiasm to the optic dome was selected for dehydration,paraffin embedding and sectioning,each slice being about 3 ?m thick.Sections were dewaxed,rehydrated,and then processed for immunofluorescence,HE staining and TUNEL staining.4 q RT-PCR q RT-PCR was used to detect the expression of HIF-1? m RNA in Sham group and HIBD group at different time points.5 Western blot Western blot was used to detect the expression of HIF-1? protein in Sham group and HIBD group at different time points,and the expression of HIF-1? and BNIP3 protein after 2ME2 intervention.6 HE staining Paraffin sections were dewaxed and rehydrated,stained with hematoxylin for 3 ~ 5 min,stained with eosin for 1 ~ 2 min,then sealed with neutral gum,and the pathological changes of the brain tissues were observed under optical microscope.7 Immunofluorescence The distribution and expression of HIF-1? protein were detected by immunofluorescence.Double immunofluorescence was used to detect the co-localization of HIF-1? with BNIP3 and Neu N in hypoxic-ischemic brain tissue 8 Brain water content measurement Brain water content was evaluated by a common wet/dry method.Briefly,at 24 h post-HIBD,rats were anesthetized and decapitated.The brains were removed and immediately separated into ischemic ipsilateral and contralateral hemispheres and the cerebellum and wet weighed.The cerebellum was used as an internal control.Brain specimens were dried in an oven at 100 °C for 24 h to obtain the dry weight.Calculate the brain water content.Brain water content=([wet weight] – [dry weight])/(wet weight)× 100 %.9 TUNEL fluorescence staining At 24 h after HIBD,TUNEL staining was performed with an in situ apoptosis detection kit,according to the manufacturer's instruction.Three slices were taken from each brain specimen,five non-overlapping high magnification fields(× 400)of ischemic cortex were randomly selected from each slice,and the number of TUNEL positive cells and the total number of cells were counted by Image-Pro Plus 6.0 image analysis software.The apoptosis rate was calculated.10 Statistical analysis Data are shown as mean ± SD.Statistical analysis was performed using SPSS 21.0.One-way analysis of variance(ANOVA)followed by least significant difference(LSD-t)tests with multiple comparisons.The statistically significant level was P < 0.05.Research results 1 q RT-PCR results The expression level of HIF-1? m RNA in the Sham group was low,HIF-1? in HIBD group significantly increased at 6 h after HIBD,peaked at 24 h(3.38 ± 0.21),then decreased gradually,72 h to(1.53 ± 0.16).The expression of HIF-1? m RNA at each time point after HIBD was significantly higher than that in Sham group(P < 0.01),and the expression of HIF-1? m RNA in 24 h group was significantly higher than that in other time points and Sham group(P < 0.05).2 Western blot results(1)The expression level of HIF-1? protein in the Sham group was low,HIF-1? in HIBD group gradually increased at 6 h after HIBD,peaked at 24 h(2.81 ± 0.36),then decreased gradually,72 h to(1.37 ± 0.19).The expression of HIF-1? protein was higher at each time point after HIBD compared with that in Sham group,and significantly increased at 12 h,24 h and 48 h groups(P < 0.01).The expression of HIF-1? protein was the highest in 24 h.(2)there were significant differences in HIF-1? and BNIP3 protein expression levels between Sham group,HIBD group and HIBD+2ME2 group(P < 0.01).The expression of HIF-1? and BNIP3 protein in HIBD group and HIBD+2ME2 group was significantly higher than that in Sham group(P < 0.05).Compared with HIBD group,the expression of HIF-1? and BNIP3 protein in HIBD+2ME2 group was significantly decreased,the difference was statistically significant(P < 0.05).3 HE staining results(1)Sham group: the brain tissue structure is clear,the cells are neatly arranged,the morphology is normal,the nucleus is intact and clear,and the dyeing is even.(2)HIBD group: brain tissue structure disorder,severe interstitial edema,cytoplasmic loose,light staining,nuclear pyknosis,fragmentation,dissolution,nucleolus disappearance,necrosis of some nerve cells,glial cells increased,with varying degrees of inflammation cell infiltration,cortical and hippocampal regions of the nerve cells decreased,especially in the hippocampus reduced more obviously.(3)HIBD+2ME2 group: the structure of the brain is fuzzy,the arrangement is still rules,interstitial edema,accompanied by a small amount of inflammatory cell infiltration,degeneration and necrosis of the nerve cells can be seen,but the degree of lesions is lighter than the HIBD group.4 Immunofluorescence results There were significant differences in the number of HIF-1? staining positive cells/mm2 between Sham group,HIBD group and HIBD+2ME2 group(P < 0.01).In the Sham group,the number of HIF-1? positive cells/mm2 was less and mainly expressed in cytoplasm.In the HIBD group,the number of HIF-1? positive cells/mm2 was significantly higher than that in Sham group(P < 0.01)and mainly expressed in nucleus.In the HIBD+2ME2 group,the number of HIF-1? positive cells/mm2 was significantly lower than that in HIBD group(P < 0.05).Double immunofluorescence showed that HIF-1? and neuronal marker Neu N were co-localized,and HIF-1? was co-localized with pro-apoptotic protein BNIP3.5 Brain water content measurement results There were significant differences in the brain water content of the ipsilateral hemisphere between Sham group,HIBD group and HIBD+2ME2 group(P < 0.01).Compared with Sham group,the brain water content of HIBD group and HIBD+2ME2 group were significantly increased(P < 0.01).Compared with the HIBD group,the brain water content of HIBD+2ME2 group was significantly decreased(P < 0.01).The contralateral cerebral hemisphere and cerebellum of the brain water content in the three groups were no significant differences.6 Apoptosis rate test results There were significant differences in the apoptosis rate between Sham group,HIBD group and HIBD+2ME2 group(P < 0.01).Compared with Sham group,the apoptosis rate of HIBD group and HIBD+2ME2 group was significantly increased(P < 0.01).Compared with HIBD group,the apoptotic rate of neurons in HIBD+2ME2 group was significantly decreased(P < 0.01)Conclusion 1 HIBD can activate the expression of HIF-1? in hypoxic-ischemic brain tissue,the expression level of HIF-1? m RNA and protein were increased first and then decreased,and the expression levels of its m RNA and protein were the highest at 24 h after HIBD.2 The HIF-1? protein was transferred from the cytoplasm to the nucleus after HIBD,and it was mainly expressed in hypoxic-ischemic neurons.3 In the early stage of HIBD,cell apoptosis,Pathological morphology and cerebral edema were aggravated with the up-regulation of HIF-1? and BNIP3 in the hypoxic-ischemic brain tissue.Inhibition of HIF-1? expression,BNIP3 expression also decreased,brain damage induced by hypoxia ischemia alleviated.HIF-1? may mediate brain damage by regulating the expression of its downstream target gene BNIP3... |
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