Changes Of Biologic Clock Protein In HIBD Neonatal Rat And Clock Genes' Expression In PBLC Of HIE Neonates

Background Neonatal hypoxic-ischemic brain damage(HIBD) is induced by perinatal oxygen deficiency.The survivals may show disorders of psychomotor development and sleep-waking cycle problem.However,studies about circadian disturbance in HIBD are so few that the mechanism of circadian disorders in HIBD is indefinite.Circadian rhythms are endogenously generated rhythms with a period length of about 24 hours.In mammalian,the major Pacemaker of the biological clock in the hypothalamic suprachiasmatic nuclei is responsible for the generation of circadian rhythms,and in peripheral blood lymphocytes celI(PBLC),pineal gland,liver,adrenal gland and heart have a biological clock system,named peripheral circadian oscillator.The basic circadian structure is the intracellular circadian oscillator,which is made up of core clock genes.These genes and their expression products form interlocked feedback loops of transcription/translation,two most significant genes among which are Clock and Bmall.The mammalian pineal gland,not only acts as a neuro-endocrine organ,like a central circadian oscillator,but also presents the effect of a peripheral circadian oscillator,so it is considered as a neuro-endocrine translator.The pineal gland regulates the circadian rhythm by synthesizing and secreting melatonin,which reaches to every part of the body through the circulation system and causes 24h phase-shift.Objective To explore the effects of biological clock protein on circadian disorder in HIBD by studying the level of CLOCK,BMAL1 in neonatal rat pineal gland and expression of Clock mRNA,Bmall mRNA in PBLC of neonatal hypoxic-ischemic encephalopathy(HIE).Methods This research included animal studies and clinic studies.In the department of animal studies,7-day-old Sprague-Dawley(SD) rats were randomly divided into 2 groups(36 pups each).HIBD models were set up according to modified Levine euthanized 0,2,12,24,36,48 hours afterwards.Western Blot analysis was used to measure the amount of CLOCK and BMAL1 in rat pineal gland.And in clinic studies, Semi-quantitative Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) analysis for the expression profiles of Clock mRNA and Bmall mRNA in PBLC of neonates (18cases HIE and 18cases normal newborn),respectively.Results 1,In the animal studies,the level of CLOCK 48 hour after HIBD raised significantly than the control groups(P＜0.05).Comparing with each corresponding control group,the level of BMAL1 in HIBD group increased at 48 hours(P＜0.05).There were no difference between 0hr,2hr,12hr,24hr and 36hr(P＞0.05).2,In clinic studies,the amount of Clock mRNA and Bmall mRNA declined after HIE,and displayed significantly lower than the control groups,respectively(P＜0.05).3,The level of CLOCK AND BMAL1 in the HIBD neonatal rat pineal gland and the expression of Clock mRNA and Bmall mRNA in PBLC of the HIE neonates demonstrated opposite tendency.Conclusion HIBD indeed affects the level of CLOCK and BMAL1 in the neonatal rat pineal gland and the expression of Clock mRNA and Bmall mRNA in the PBLC of the HIE neonates.These results reveal the existence of changes of CLOCK and BMAL1 expression patterns and Clock mRNA and Bmall mRNA,suggest the presumed regulatory mechanism of circadian disorder in HIBD.