| Background:The nervous system consists of central and peripheral nervous system,the central nervous system consists of brain and spinal cord,is the main part of human nervous system.The first two years after infant birth is the most rapid period of infant brain development,because it is the most important period for neurons functional maturation,synaptic growth and axon myelin.The retina is the outspread part of the brain,is the first place to form thevisual information.Although genetic factors determine the visual nerve function is already rudiment when the baby was born,but the nutrition,environment and visual experience regulation can still result in significant changes of visual function of the nervous system after the baby births,so the development of the visual system is shaped by genetics,nutrition,and environment.Infantile neurological development requires a lot of nutrients,and during this period,breast milk is the main source of nutrients for infants.Breast milk is the best source of nutrition in the early development of infants and young children,because it can provide all the nutrition that infant needs for normal growth and development.In addition,the breast milk is also rich in some other nourishment compositions,may provide infants more benefits.Breast milk is rich in abundant oligosaccharides,human milk oligosaccharides(HMOs)are an important fraction in human milk,consisting of neutral and acidic oligosaccharides.Acidic oligosaccharides,such as sialyllactose(SL),which containing sialic acid(Sia)residues;Neutral oligosaccharides,which include galacto oligosaccharides and fructo-oligosaccharides.In breast milk,73.8%of sialic acid is bound with oligosaccharides,one of the main oligosaccharide is 3'-SL and 6'-SL.The concentration of the 6'-SL in colostrum is the highest(250-1300 mg/L),Then gradually decreased,and the concentration of 3'-SL is relatively stable(76-300 mg/L).SL is thought to be playing an important role in the development of newborn babies,because they play an important role in against pathogens,promote the intestinal mature,and the development of immune function and cognition.Meanwhile,SL can be digested in the intestines,providing necessary nutrients to neural development-sialic acid.Sialic acids(Sia)are a diverse family of sugars based on the parent compound,neuraminic acid,a nine-carbon sugar acid,they can be found in all vertebrates and involved in many bioactivities.So far have found more than 50 kinds of sialic acid.Sialic acid general located at the end of the large molecules of glycoprotein and glycolipids,is an important part of cell membrane of animals.Sialic acid has many biological functions such as Cell proliferation,differentiation and identification;With the interaction of pathogenic microorganisms,participate in the immune response,promotes the nervous system development,facilitation of memory formation,improve the learning ability.In the brain,Sialic acid is mainly present in the ganglioside.Ganglioside is one of the most complex sheath glycolipids.Ganglioside in the biological activity of the brain is mainly reflected in its regulating synaptic plasticity.Synaptic plasticity is an important mechanism for learning and memory and other advanced neural activity.Polysialic acid(polySia)is a carbohydrate composed of a linear homopolymer of a-2,8-linked sialic acid residues and is mainly attached to the neural cell adhesion molecule(NCAM).PolySia plays important roles in intercellular adhesion,cell migration,formation and remodeling of the neural system through regulation of the adhesive property of NCAM.Although polySia is lost from NCAM in most adult tissues,various tumors are known to re-express polySia.Based on these potential benefits,we hypothesized that SL and sialic acid can act on the nervous system,maybe is a very important nutrients that can promote infants'early brain development and visual development,and also can improve the cognitive function of the infants.Part one:Dietary Sialyllactose Supplementation improves Early Neurodevelopment and Cognition in Piglet newborn pigletsObjective:We used newborn piglets as animal model,through the dietary intervention of sialyllactose,carry out the eight-arm maze behavior training and testing,to assess their learning and memory ability,and analyzed the expression levels of sialic acid and its related brain development pathways of the brain areas,to explore the influence of SL on brain development and the learning and memory of piglets,and to find out the molecular mechanism.Methods:28 male piglets were allocated to SL treatment group(n=14)and the control group(n=14),and adding a certain dose of SL for 35 days.Fed sow milk replacer supplemented with SL at a dose level of 250 mg/Kg/d(SL treatment group)and 40 mg/Kg/d(control group).Eight-arm maze behavior training and testing were carried between 23-36days,to assess their learning and memory ability.The euthanasia were carried out in age of 39 days,take the related brain area to do the sialic acid level analysis,using PCR-array technology to analyse brain development pathway of the hippocampus(including 64 synaptic plasticity genes,55 neurotrophic factor pathways genes,20 of GABA and glutamate neurotransmitter pathways genes,64 neurogenesis pathways genes,and 29 genes which related to metabolism of sialic acid),and use qRCR,Western blot and ELISA experiment research on the affected gene transcription,translation and activation of downstream genes,to explore the influence of SL on brain development and learning and memory,thus speculated the molecular mechanism of SL that affect the brain development and cognitive function.Results:Dietary sialyllactose supplementation improves(1)no significant effect on body weight and brain weight of piglets;(2)no significant effect on the stress hormone concentration of Cortisol and ACTH in plasma;(3)no significant effect on the expression of sialic acid in plasma and red blood cell;(4)significant effect on the expression of ganglioside-bound and protein-bound sialic acids in the hippocampus and prefrontal of the piglets;(5)reduce the mistakes of piglets made in eight-arm maze training and testing and improves the learning speed of piglets,also promoted the formation of piglet 5-min-Short-term memory,and 40-min,16 h and 3 h,48 h of long-term memory;(6)affects the expression of 37 genes which related to brain development,there are 16 genes have significant difference(P<0.05)and fold change>1.1,including,4 genes of GABA and glutamate neurotransmitter pathways,4 sialic acid metabolism related genes,2 genes of neurotrophic factor pathways,5 genes of neurogenesis pathways and 1 gene of synaptic plasticity.There are 21 genes which fold change>1.1 but the difference is not obvious(0.05<P<0.1),including 3 of synaptic plasticity,4 sialic acid metabolism related genes,6 of neurotrophic factor pathway and 8 gens of neural pathways;(7)significantly increased the expression level of the NTF-3 in the hippocampus,activate the expression of receptors of NFT-3:TrkB and TrkC.Thus promotes the downstream factor-CREB's phosphorylation,which can improve the cognitive ability of the piglets.Further illustrates that sialyllactose is a key nutrients that can promote brain development and cognitive function.Conclusions:Dietary sialyllactose had no significant effect on increast the weight and brain weight of piglets,stress hormone concentration and content of sialic acid in plasma and red blood cells,but can significantly improve the level of sialic acid in hippocampus and prefrontal of piglets,also can improve the learning speed and the formation of short-term memory and long-term memory.It can affect to rise multiple genes which are related to brain development in the hippocampus of the piglets,and significantly improve the protein expression level of the NTF-3 in the hippocampus,activate the expression of receptors of NFT-3:TrkB and TrkC.Thus promotes the downstream factor-CREB's phosphorylation,which can improve the cognitive ability of the piglets.Further illustrates that sialyllactose is a key nutrients that can promote brain development and cognitive function.Part two:Dietary of Sialyllactose supplementation can influence the retinal development and sialic acid expression in the retina of pigletsObjective:We used newborn piglets as animal model,through the dietary intervention of sialyllactose,to find out whether dietary of SL supplementation can influence the retinal development and sialic acid expression in the retina of piglets.Methods:28 male piglets were allocated to SL treatment group(n=14)and the control group(n=14),and adding a certain dose of SL for 35 days.Fed sow milk replacer supplemented with SL at a dose level of 250 mg/Kg/d(SL treatment group)and 40 mg/Kg/d(control group).Eight-arm maze behavior training and testing were carried between 23-36days,to assess their learning and memory ability.The euthanasia were carried out in age of 39 days,take the retina and optic nerve to analyze the sialic acid expression level,the differences of the retina structures,expression of polySia-NCAM,and the mechanism of synthesis and metabolism of sialic acid.To study how dietary of SL supplementation influence the retinal development and sialic acid expression in the retina of piglets.Results:Dietary of SL supplementation can influence the retinal thickness of the piglets,can significantly increase the expression level of sialic acid in the retina,and can improve the expression level of polySia-NCAM in the retina,which is closely related to the neurological development.And also it can increase the expression level of mRNA of ST8Sia?,ST8SiaIII,ST8SiaIV,NCAM and Neuropilin-2,but there is no difference between groups.This may be because this research is nutrient intervention,rather than the drug intervention,so did not essentially change mechanisms,but still has a promoting effect on the retina development.At the same time,we use immunofluorescence method to stain polySia-NCAM and GFAP,PolySia-NCAM and NeuN,the results shows that polySia-NCAM expressed through all the cell layers of retina,but mainly on GCL and IPL layer,which is consistent with previous reports.Conclusions:Dietary of SL supplementation can influence the retinal thickness of the piglets,can significantly increase the expression level of sialic acid in the retina,and can improve the expression level of polySia-NCAM in the retina.And also it can increase the expression level of mRNA of ST8SiaII,ST8SiaIII,ST8SiaIV,NCAM and Neuropilin-2.PolySia-NCAM expressed through all the cell layers of retina,but mainly on GCL and IPL layer. |
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