Lycium Barbarum Polysaccharide Protects Hippocampal Neural Stem Cells From Injury And Promotes Their Proliferation And Neuronal Differentiation

Part1: Chronic exposure to methylmercury chloride does harm to the memory andlearning in newborn rats which are prevented by lycium barbarum polysaccharidesObjective: To study the harmful effects on the ability of learning and memory andhippocampal neurogenesis of newborn rats when exposed to low-dose methylmercurychloride (MMC), the environmental contaminants. Subsequent aim is to explore the protectioneffect of LBPs from MMC-induced injury and find a new way to resist environmentalcontaminants methylmercury chloride. Method:(1)60SD rats were divided randomly intocontrol, model and protective groups with20cases in each. The injury model was establishedby subcutaneous injection of MMC (4mg/kg/d) for28days. The animals in control groupwere injected with the same volume of physiological saline. LBPs (2g/kg/d) wereadministrated by feeding in the intervention group with. At3,6and9weeks afteradministration, Morris equipement was used to assess the ability of learning and memory ofanimals by the indexes escape latency of the determination of newborn rats and the number ofcross-flat. DCX immunohistochemical staining was adapted to detect the number and growthof newborn neurons in the dentate gyrus of hippocampus.(2) NSCs acquired from embryonichippocampus were treated by different concentration of MMC (100pmol/L、1nmol/L、10nmol/L、100nmol/L、1μmol/L) and then the activity of them was assessed by MTT assay todeternine the harmful effect on NSCs. To determine the protection effect of LBPs, LBPs wereadded into the media before addition of MMC. Immunofluorescence was used to observe themorphology of hippocampal neural stem cell proliferation, differentiation and neuritedevelopment. Results:(1) Compared with the control group, anminals in model groupshowed significantly longer escape latency and less number of cross-flat than the controlgroup. The escape latency and the collapse of the number in the intervention group showed nosignificant difference with the control group, while the escape latency was significantly shorter compared with the model group and the number of cross flat was increasedsignificantly. The number of DCX-positive cells in the dentate gyrus in the intervention groupwas not significantly different with the control group. Compared with control group, theDCX-positive cells in model group decreased markedly. However, the number ofDCX-positive cells in the dentate gyrus in the intervention group with LBPs administrationwas much more than that in the model group and increased in a time-dependent manner.(2)MMC treatment decrease the survival of hippocampal NSCs in a concentration-dependentmanner. MMC with over100nmol/L almostly destroyed all of the hippocampal NSCs.However, addition of LBPs promoted the survival of hippocampal NSCs significantly andneuronal differentiation. The neurite also grew well after LBPs treatment. Conclusion:(1)Chronic exposure to low-dose MMC lead to damage of the newborn rat spatial learning andmemory and the reduction of hippocampal DCX-positive neurons of the dentate gyrus;(2)MMC has strong neurotoxic effects on hippocampal neural stem cells;(3) LBPs has aprotective effect on the NSCs from MMC-induced injury which provides a new theoreticalbasis and means for treatment of neurodegenerative diseases. Part2: Study of the effects of lycium barbarum polysaccharide on the neuronaldifferentiation of hippocampal neural stem cellsObjective: To investigate the effects of lycium barbarum polysaccharide (LBP) combinedwith the fimbria fornix-transected (denervated) hippocampal extracts in inducing thehippocampal neural stem cells (NSCs) to differentiate into neuron. Method: Hippocampuswas dissociated from16-day embryos of SD rats for NSC specific cultures.5-6days later, thepurified hippocampal NSC neurospheres were prepared into single cell suspension andincubated in the24-well culture plate. Wells were randomly divided into blank control group(DMEM/F12medium), denervation group (DMEM/F12medium with denervationhippocampal extract), LBPs group (DMEM/F12medium with LBPs), combination group(DMEM/F12medium with denervated hippocampal extract and LBPs). At10days afterculture, MAP-2/NeuN immunofluorescence staining was used to detect the differentiation ofhippocampal NSCs in each group into positive neurons and the ratio of neuronaldifferentiation were analyzed statistically between groups. Results: Compared with controlgroup, the neuronal differentiation rate of hippocampal NSCs and the circumference ofneurons showed significant differences in other groups. The neuronal differentiation rate ofcontrol group was4.579±0.2926%(n=10) and the average circumference of neurons was644.1±38.25μm (n=100). While the neuronal differentiation rate of denervation hippocampalextract group:9.133±0.2835%(n=10) and the average circumference of neurons was1163±42.30μm (n=100); The neuronal differentiation rate of LBPs group was7.919±0.1827%(n=10) and the average circumference of neurons1057±31.15μm (n=100); The neuronaldifferentiation rate of combination group was11.29±1.008%(n=10) and the averagecircumference of neurons was1547±47.25μm (n=100). Among the four groups, theneuronal differentiation rate of combination group is highest and the average circumference isthe longest. Conclusions: The denervated hippocampal extract combinated with LBPs can better promote hippocampal NSCs to differentiate into neurons and neurite growth.