| As the development of perinatal medicine and technology, especially in rescuing critically ill neonates, the survival rate of premature infants, were significantly improved. Especially in very low birth weight/extremely low birth weight infants (VLBW/ELBW),whose birth weight is under1500g. But at the same time, extrauterine growth retardation (EUGR), neurodevelopmental disorders and other new issues have become globally problems. Althought the survival rate of the premature infants is greatly improved, but the decline is not proportional to the neurodevelopmental disorders. Survival preterm infants face neurological development problems and long-term problems as cerebral palsy, mental retardation, cognitive impairment and audio-visual defects. Even if there are no injury in preterm infants, they may showed recessive disability (such as:the mood is not controlled, inability to concentrate, reading and writing ability, etc.)Nutrition is an important problem that has caused widespread concern of neurodevelopmental outcomes. Lucas proposed the "nutritional programming" concept, namely in the nutritional status of key developmental or sensitive period of the organ function influence the whole life. Clinical study of Lucas also shows that, given the preterm formula milk in the critical period (high protein) can affect the long-term neurodevelopmental. Singhal found that, although the fortified formula feeding group compared with the standard formula feeding group of neural development is better, but the adolescent hypertension and insulin resistance and metabolic disease incidence rate is high. Therefore, in premature infants, nutritional status and related to the critical period of neurodevelopment delay and risk of metabolic disease. How to balance and reduce these risk are the challenges we face. Brain development is a long process, understand the different stages of nutritional effects on brain development and its mechanism, has important significance to improve the neurological development of preterm infants prognosis and reduce the risk of metabolic diseases.Mammalian target of rapamycin (mTOR) is evolutionarily conserved serine/threonine protein kinase, belongs to the phosphatidylinositol3-kinase kinase (PIKK) protein family members, receptor cell nutrition, energy level and growth factor signals change in mammalian cells. mTOR play an important role in the formation of the formation and development of the brain, neural synapses occurrence, learning and memory.In this study, we established premature rat model. Then the rats were feeded to different protein feed after weaning to6weeks and8weeks, and tested the ability of learning and memory in rats through the Morris water maze test. Analyzed the changes of mTOR/S6K pathway activity in hippocampus by using immunohistochemistry and Western blot technique. Then to explore the effects of different protein intake levels on ability of learning and memory of premature birth rats and its possible mechanism.Objective:To study the influence of protein intake on learning,memory capability and mTOR expression in premature rats.Methods:The neonatal Sprague-Dawley premature rats were obtained through the method of cesarean section. Premature new born rats were lactated to21days by nursing mother. Premature rats and full-term rats were randomly divided into six groups after weaning:①preterm standard protein group(PS).②term standard protein group(TS).③preterm low protein group(PL).④term low protein group(TL).⑤preterm high protein group(PH).⑥term high protein group(TH). After weaning, the low protein groups were fed low protein diets (including8%protein) until the end of experiment, the high protein groups were provided with high protein diets (including30%protein) until the end of experiment, the standard protein groups were fed standard protein diets (content of18%protein) until the end of experiment. Within each group, the rats were respectively tested at6weeks and8weeks. Morris water maze task was performed to assess the learning and cognitive abilities of the premature rats. Immunohistochemistry and western blot were used to observe the distribution of mTOR、S6K、4EBP1in the hippocampus.Results1. Morris water maze task:1.1Directional navigation experiments:1.1.1At the age of6weeks, the escape latencies to find the platform were shortened with increased training times for full-term rats (P<0.05). The escape latencies of the premature rats did not chang significantly in the third and fourth day of the experiment.1.1.2At the age of6weeks, the number of crossing the platform f the premature rats were no significant difference (P>0.05)1.1.3At the age of8weeks,the escape latencies to find the platform were shortened with increased training times for all of the rats (P<0.05)1.1.4At the age of8weeks,the high-protein premature rats spent time at platform and the percent of time spent in target quadrant were significantly greater than the6-week-old’s (P<0.05)1.2. Probe trial test:1.2.1At the age of6weeks, prematuer rats spent significantly less time in target quadrant than full-time rats. And the percent of traveled distance in target quadrant of premature rats were significantly less than full term rats (P<0.05)1.2.2At the age of8weeks, high-protein premature rats spent significantly greater time in target quadrant than other two groups of premature rats.And the percent of traveled distance in target quadrant of high-protein premature rats were significantly higher than other two groups of premature rats (P<0.05)1.2.3At the age of8weeks,the high-protein premature rats spent time in target quadrant and the percent of traveled distance in target quadrant were higher than the high-protein full term rats’, but there was no significant difference (P>0.05)1.2.4At the age of8weeks,the high-protein premature rats spent time at platform and the percent of time spent in target quadrant were significantly greater than the6-week-old’s (P<0.05)1.3Immunohisto chemistry results:1.3.1At the age of6weeks, the number of crossing the platform and the expression of mTOR in hippocampal CA1,CA3region were no significant difference.1.3.2At the age of8weeks, the expression of mTOR in high-protein premature rats’ hippocampal CA1,CA3region were also significantly increased(P<0.05).1.3.3Compared with high-protein premature rats at the age of6weeks, the expression of mTOR in hippocampal CA1,CA3region were also significantly increased in high-protein premature rats at the age of8weeks.1.4Western blot results:1.4.1At the age of6weeks,the expressions of p-p70S6K (Thr389) were seriously inhibited in premature rats.Compare to the term rats, the expressions of4EBP1in premature rats were decreased.1.4.2At the age of8weeks,the expressions of p-p70S6K (Thr389) were increased in premature rats.Compare to the other groups, the expressions of4EBP1in premature low protein feed rats were decreased.1.4.3At the age of6weeks, Phosphorylated levels of p70S6K were significantly decreased in the premature rats(A two-way ANOVA with gestation age and protein level as main effects indicated significant effects of gestation age, Fgestation age=8.233, P=0.014<0.05).1.4.4At the age of8weeks, Phosphorylatedlevels of p70S6K were significantly increased in high protein rats.(A two-way ANOVA with gestation age and protein level as main effects indicated significant effects of protein level, Fprotein level=4.13,P=<0.05;high protein groups vs low protein groups,LSD post-hoc test,P=0.021<0.05;high protein groups vs standard protein groups,LSD post-hoc test,P=0.042<0.05).*P<0.05Conclusion:In the early growth and development process of the brain,premature birth may inhibit mTOR activity, which can lead to the learning and memory ability of the premature rats was impaired.After a long time high protein nutritional intake, high-protein premature rats impaired learning and cognitive ability can get recovery. That may be associated with upregulation of mTOR expression. |
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