A Study On The Relationship Between HIF-1α With C1772T Polymorphism And Neonatal Hypoxic Ischemic Encephalopathy

Objective: Neonatal hypoxic-ischemic encephalopathy (HIE) is brain injury of fetal or neonatal caused by a variety of factors concerning perinatal asphyxia.With the improvement of technology and treatment, the survival rate of this disease has been greatly improved, but the neonates with moderate and severe HIE are still very likely to cause acute death and chronic nervous system damage.In current times, supportive and symptomatic treatment still are the main means of HIE,and there is no treatment for the basic mechanism of hypoxia. New treatment methods, such as mild hypothermia therapy, hyperbaric oxygen and neural stem cell transplantation, etc.their exact effect remain further explored.Hypoxia-inducible factor-1α(HIF-1α) is a key transcription factor under hypoxic / ischemic terms.It's essential for both the cell and the organism's survival in whatever normoxic and hypoxic conditions.Animal experiments confirmed that the expression of HIF-lαprotein in brain tissue of hypoxic-ischemic rat increased within 1 hour and reached its peak level within 24-48 hours.HIF-lαraised the transcription activity of a series of target genes,such as vascular endothelial growth factor,erythropoietin,glucose transporter protein -1 through hypoxia responsive element, activated apoptosis-related genes such as p53, BINP3, NIX, and so that made the brain adapt to hypoxia. It has been reported that the C1772T single nucleotide polymorphism (SNP) of HIF-1αgene in exon 12 can increase the expression and change the function of HIF-1αin recent years. It is still unknown whether the change of HIF-1αC1772T polymorphism can raise the newborns'susceptibility to hypoxic-ischemic brain damage. Insulin-like growth factor -1 (IGF-1) is a neurotrophic factor, and plays an important role in nerve cell repair after injury with nutritional support.Related clinical trials confirmed that IGF-1 in blood decreased in acute stage of HIE, and the decline is in the same degree of severity, so it is considered as one of the marks to reflect neonatal hypoxic-ischemic injury.Myelin basic protein (MBP) is one of the main components of nervous system myelin.When the severe hypoxia-ischemia occurs,myelin comes into damage, MBP is released into the blood via the blood-brain barrier with increased the permeability, so that the serum MBP level is elevated with the parallel relationship, MBP can be used as one of a marker of nervous system injury, especially the white matter damage. While there is no related studuies on the interaction and mechanism between the HIF-lαwith its SNP and IGF-1, MBP in the neonatal hypoxic-ischemic encephalopathy.By detecting serum HIF-1α, MBP, and IGF-1 levels in neonates with HIE during acute and recovery stages and analyzing HIF-1αC1772T single nucleotide polymorphisms,we discuss①the relationship between serum HIF-1αlevels and the degree of brain damage,②the relationship between serum HIF-1αlevels and serum IGF-1, MBP levels,and further elaborate the role of HIF-1αin the pathogenesis of neonatal hypoxic-ischemic encephalopathy and its possible mechanism③Analyze the relationship between HIF-1αC1772T SNP and serum levels,investigate the relationship between the polymorphisms and susceptibility to hypoxia,and also the pathogenesis of neonatal HIE,provide experimental basis for the clinical diagnosis and treatment of HIE.Methods: we selected 55 HIE cases admitted to The 4th Hospital of Hebei Medical University and Department of Neonatology, Central Hospital of Cangzhou.According to the diagnosis and sub-degree standard developed by the Conference of changsha in 2004,we divided the HIE neonate into mild HIE group of 20 cases, moderate group of 20 cases and severe group of 15 cases.Control group were selceted from neonates who were admitted in the same time without hypoxia, producing injuries and infection,who were diagnosd as swallowing syndrome, neonatal wet lung.the total number was 60.Record the gestational age, age, weight and other relevant clinical data to conduct comparative analysis.For all newborns,2ml peripheral venous blood was taken in the first 2-3 days, 7-10 days after birth, then 0.2ml of the blood was used to extract DNA and DNA was stored in -30℃refrigerator for inspection; about 0.5ml serum was extracted from the rest 1.8ml blood by centrifuge (3000 r / min,15min) and were placed in -20℃refrigerator for inspection.Detected the serum HIF-1α,MBP levels by ELISA mathod,measured IGF-1 levels by radioimmunoassay,and analyzed the polymorphism by PCR-RFLP method respectively. Measurement data was showed by (x±s).The experimental data was analyzed with the SPSS 17.0 package (t test, one-way ANOVA,rank sum test,χ2 test,Fisher exact test,linear correlation, Hardy-Weinberg genetic equilibrium tests, etc.). Two side test and test level(α=0.05) were taken.When P<0.05,it was considered statistically significant difference.Results:1. Changes in serum HIF-1αlevels of HIE group.1.1 During acute phase, serum HIF-1αlevels of HIE group were significantly higher (P<0.001)than that of control group; and HIF-1αlevels of severe HIE group were significantly higher than those of mild, moderate HIE group and control group, the differences were statistically significant (P <0.001).the differences among mild, moderate,severe HIE groups and control group were significant.1.2 During recovery phase, HIF-1αlevels of severe HIE group were still significantly higher than those of moderate, mild and control group, the difference was statistically significant (P<0.001); the differences among mild, moderate,severe HIE groups were significant.1.3 The comparison of serum HIF-1αlevels between acute and recovery phase.The HIF-1αlevels of morderate, severe HIE group at acute phase were higher than that at recovery phase.2. Distribution of HIF-1αpolymorphismIn this experiment, two kinds of genotypes: C/C-type and the C/T type were detected, and we did not find T/T type; the C/C type were concidered as the wild type,whereas C/T for the mutation. The control group and the HIE groups were consistent with Hardy-Weinberg genetic equilibrium tests, and with good comparability. the overall mutation rate of T allele: 4.78%.2.1 Distribution of HIF-1αpolymorphism in the control group. In this study, among 60 cases there were 6 cases confirmed as C/T genotype and 54 cases as C/C genotype. the mutation rate of T allele:5%.2.2 Distribution of HIF-1αpolymorphism in the HIE group.In this study, among 55 cases there were 5 cases confirmed as C/T genotype and 50 cases as C/C genotype. he mutation rate of T allele:4.55%.2.3 The distribution of HIF-1αpolymorphisms in the control group and the HIE group had no statistical (P>0.5).2.4 The serum HIF-1αlevels of C/T genotype were slightly higher than that of C/C genotype in control group,but there was no statistical significance.2.5 The serum HIF-1αlevels of C/T and C/C genotypes in HIE group were higher than control group(P<0.001).At acute phase,the serum HIF-1αlevels of C/T genotype were higher than that of C/C genotype in HIE group,and the same situation took places at recovery phase.But there were no statistical significance of the two phase.3. Changes of serum IGF-1 and MBP levels in HIE group.3.1 At acute phase,the serum IGF-1 levels of severe HIE group were much lower than those of mild, moderate groups and control group (P<0.01). compared with the control group,each of the mild, moderate and severe groups had a statistical significance(P<0.001).At acute phase, the serum MBP levels of severe HIE group were much higher than those of mild, moderate groups and control group(P<0.001),but there was no significant difference among the mild,morderate groups and control group.3.2 At recovery phase, the serum IGF-1 levels of severe HIE group were still lower than those of mild, moderate HIE groups(P<0.05);At recovery phase, the serum MBP levels of severe HIE group were still higher than those of mild, moderate groups(P<0.001);but there was no significant difference among the mild,morderate groups.3.3 The comparison of serum IGF-1 and MBP levels between acute and recovery phase.The differences among mild, moderate and severe HIE group were statistically significant.Refered to MBP,The IGF-1 levels of severe HIE group was statistically significant between the two phases.4. The correlation with serum IGF-1 and HIF-1αlevels in HIE group. At acute phase, serum HIF-1αand IGF-1 levels showed a negative correlation, P<0.001;but at recovery phase,there was no correlation between the two.5. The evaluation of Serum HIF-1α, IGF-1 and MBP levels in HIE.The sensitivity and specificity comparisons of HIF-1α, IGF-1 and MBP levels in HIE group by using the ROC (Receiver operating characteristic curve) curve.At acute phase,comparison of area under the ROC curve in HIE group: IGF-1>HIF-1α>MBP;comparison of the sensitivity in HIE group: IGF-1>HIF-1α>MBP;comparison of specificity in HIE group: HIF-1α> MBP > IGF-1.At recovery phase, comparison of area under the ROC curve in HIE group: HIF-1α>IGF-1 >MBP;comparison of the sensitivity in HIE group: HIF-1α>IGF-1> MBP;comparison of specificity in HIE group: HIF-1α> MBP> IGF-1Conclusion:1. The HIF-1αlevels of HIE neonates was significantly higher than that of control group,and the paralleled with the degrees of HI damage.2.In HIE group,the HIF-1αlevels of C/T genotype were higher than that of C/C genotype,implying that the C1772T polymorphism is concerned with HI damage.But deep analysis are needed by increasing sample size.3.Compaired with acute phase,the HIF-1αlevels declined in recovery phase,but the HIF-1αlevels were still higher than that of control group.the high levels of HIF-1αmight have a relationship with the induction by IGF-1,and the induction by IGF-1 might patticipate in the repairment of HI damage.4.Compaired with the mild,morderate HIE groups and control group,the levels of serum MBP in severe HIE group was much higher at acute phase,and lasted till the recovery phase.It was implied that MBP as a mark for the white matter damage can be considered to reflect the damage degrees of white matter in severe HIE group.