Study On The Effect Of Human Umbilical Cord Mesenchymal Stem Cells On Learning And Memory Ability Of APP/PS1 Mice And Its Mechanism

A progressive neurodegenerative disorder,Alzheimer’s Disease(AD)is marked by memory impairment and other cognitive impairments that become more pronounced with time.The pathology of AD is characterized byβ-amyloid(Aβ)deposition to form extracellular plaques,neurofibrillary tangles(NFTs).Neuroinflammation is the third core neuropathological feature in the AD brain besides Aβor NFTs.It is thought that chronic neuroinflammation mediated by oligomerized domain-like receptor protein 3(NLRP3)inflammasomes specifically activated by microglia plays an important role in the pathogenesis of AD.At present,there is no cure for AD,and conventional treatments only relieve the symptoms of the disease and cannot prevent the progression of AD.After the successful acquisition of human stem cells,the application of stem cell therapy has become a new direction of AD treatment.Pluripotent stem cells,derived from human umbilical cord tissue,are known as human umbilical cord mesenchymal stem cells(h UC-MSCs),which make them ideal seed cells for the treatment of AD due to their advantages of high cell yield,convenient material collection,no ethical restrictions,low immunogenicity,directional migration and rapid expansion,secretion of a variety of bioactive factors and non-tumorigenicity.Many animal AD models have confirmed that its treatment is effective,but the mechanism of action has not been fully elucidated,and it may play a therapeutic role through the secretion of neurotrophic factors,pro-angiogenesis,immunomodulation,anti-inflammatory and other effects.The gradual increase in Aβ-deposited plaques is the initiating factor and key link leading to pathological changes in AD.Neuroinflammation is one of the important pathological factors of AD.Aβdeposition induced the activation of NLRP3 inflammasomes,which then matured interleukin-1β(IL-1β)precursors and IL-18 precursors into IL-1βand IL-18 and promoted the secretion of both,causing inflammatory reactions in the brain and aggravating brain tissue damage.The NLRP3 inflammasome,composed of NLRP3,a speck-like protein associated with apoptosis,with a CARD(ASC),and Caspase-1,a cysteine-containing proteinase,is highly expressed in neurons and has a significant role in AD neuroinflammation.When the NLRP3 inflammosome is activated,it can promote the occurrence of AD in two ways.First,it regulates IL-1β,producing neurotoxins that cause neuronal degeneration.Second,it can cause an increase in Aβdeposition,induce a positive feedback loop of Aβself-sustainment,and eventually lead to the occurrence of AD.Aβdeposition and activation of NLRP3 may be the result of interaction.The neurogenic plaques of AD recruit microglia to phagocytose Aβ,which then stimulates NLRP3 inflammosome activation,followed by the release of pro-inflammatory cytokines IL-1β,IL-18,and potential neurotoxins.This release enhances the neurotoxic effect of Aβand aggravates the pathological process of AD.APP/PS1 double-transgenic mice have been widely used in AD studies expressing chimericβ-amyloid precursor protein(app)and mutated human progerin 1(ps1),both of which are localized to central nervous system neurons,and both mutations are associated with early-onset AD.APP/PS1 double-transgenic mice can show typical features of AD and pathophysiological features such as Aβdeposition,microglia activation and increased pro-inflammatory cytokines.To sum up,both in vitro and in vivo experiments have demonstrated that NLRP3 inflammasome activation is a key initiating element in the Aβ-induced inflammatory signaling cascade,a pathway that is linked to AD progression and could be a novel target for treating this illness.Recent studies suggest that inhibition of the expression levels of NLRP3inflammasome-related proteins and genes may be key targets for neuroprotection and intervention of AD.Previous experiments have also shown that glia-like cells from human mesenchymal stem cells(h MSCs)ultimately improve viability and proliferative capacity and reduce toxicity by reducing the activation of NRLP3 inflammatory bodies in Aβ-induced neural stem cells.No literature exists to assess the NLRP3 neuroinflammatory pathway’s effect on the learning and memory of mice,thus this project will concentrate on the effect and mechanism of h UC-MSCs on APP/PS1 double-transgenic mice,in order to construct a theoretical basis for the clinical utilization of h UC-MSCs.Objective:By studying the effects of h UC-MSCs on NLRP3,Casepase-1 and ASC in the NLRP3 neuroinflammatory pathway in the brain of APP/PS1 double-transgenic mice,the expression of pro-inflammatory factors IL-1βand IL-18,and the learning and memory ability of APP/PS1 mice,the mechanism of h UC-MSCs on learning and memory of APP/PS1 double-transgenic mice was studied,which laid a theoretical foundation for the clinical application of h UC-MSCs.Methods:1.12 C57BL/6 wild-type female mice were employed as the wild control group(Group C,C57BL/6 mice)in the experiment;24 APP/PS1female mice were then divided into 12 model groups(M group,APP/PS1mice)and 12 h UC-MSCs treatment groups(T group,APP/PS1 mice+h UC-MSCs)using the random number table method.The T group of mice were administered h UC-MSCs via the tail vein every two weeks,with 200μL(2×10~6 cells)injected,while the other groups were given normal saline in equal amounts,each injected twice,for a total of four weeks of treatment.Zoological experiments are then conducted.In addition,4 APP/PS1 mice were killed by injecting h UC-MSCs into the tail vein at 24 and 72 hours,while 4 APP/PS1 mice were killed by injecting h UC-MSCs/GFP into the tail vein at 24 and 72 hours,respectively,to observe the survival of h UC-MSCs in brain tissue.2.Morris water maze was employed to ascertain the learning and memory aptitude of mice.3.Observation of h UC-MSCs/GFP survival in brain tissue using frozen section method4.HE,Nissl staining and pathological morphological observation.5.Detection of NLRP3,Casepase-1,ASC m RNA levels in the hippocampus by RT-q PCR.6.Detection of NLRP3,Casepase-1,ASC,Aβ,IL-1β,and IL-18proteins in the hippocampus by Western blotting.7.Detection of serum IL-1βexpression by ELISA method.Results:1.Comparison of Morris water maze behavior in three groups of miceGroup C’s evasion incubation period was the shortest on the initial to second days of the positioning navigation experiment,T group’s was longer,and M group’s was the longest;no statistical distinctions were observed between the groups.Group M’s average escape latency on days 3-5 was greater than that of group C,and the average escape latency of mice in group T was significantly lower than that of group M,with a statistically significant difference(P<0.05).Observing the positioning voyage trajectory chart,it can be seen that the swimming trajectories of each group of mice in the early stage of training were mainly based on the edge of the swimming pool,and then tended to the surroundings of the platform,which proved that each group of mice had learning ability.The difference is that the mice in group C can reach the platform in a short time from day 2 and have the strongest learning ability;Even if the mice in group M can reach the platform after learning,the path is the longest and most complex;The mice in the T group were more purposeful than the mice in the M group during the learning process.Group C’s platform exploration period saw a significant decrease in the platform quadrant of group M,with a statistically significant difference(P<0.01).In contrast,group T’s platform quadrant of mice experienced a significantly greater time spent than group M,with a statistically significant difference(P<0.05).The trajectory diagram of the platform’s exploration period reveals that the majority of mice in group C have trajectories in the quadrant where the platform is situated,while the majority of the M group’s trajectories are in the starting quadrant and rarely in the quadrant where the platform is located.The T group’s trajectory is notably higher than the M group’s,and its purpose is more powerful.Group C mice hippocampal neurons were closely arranged,with a large number of neuronal structures and clear structures.Compared with group C,the distribution of hippocampal neurons in group M were sparse,some neurons were necrosis,and changes such as nucleus contraction and fragmentation appeared.Compared with the M group neurons,the T group have a relatively close arrangement of neurons,an increased number,and a smaller number of necrotic neurons.2.Activity of h UC-MSCs/GFP in the brainAfter 24 hours of tail vein injection of h UC-MSCs,no green fluorescence signal was found in the brain tissue,after 24 and 72 hours of tail vein injection of h UC-MSCs/GFP,green fluorescence signals can be observed in brain tissue,indicating that tail vein injection of h UC-MSCs can reach brain tissue and survive.3.HE staining results of hippocampal DG region of mice in each groupGroup C mice hippocampal neurons were closely arranged,with a large number of neuronal structures and clear structures.Compared with group C,the distribution of hippocampal neurons in group M were sparse,some neurons were necrosis,and changes such as nucleus contraction and fragmentation appeared.Compared with the M group neurons,the T group have a relatively close arrangement of neurons,an increased number,and a smaller number of necrotic neurons.4.Nissl staining results of hippocampus in each group of miceThe Nissl bodies of hippocampal neurons in the brain tissue of group C mice were blue purple in color;The number of neurons in the brain tissue of group M mice was significantly lower than that of group C,and the arrangement of neurons was disordered;Compared with group M,the number of Nissl bodies in the brain tissue of group T mice was significantly increased,the degree of staining was significantly deeper,and the arrangement of neurons was more orderly.5.Relative expression levels of hippocampal NLRP3,Casepase-1,and ASC m RNA in each groupCompared with Group C,NLRP3,Casepase-1 and ASC m RNA Significantly,the hippocampus of M group mice saw an increase,and the differences were statistically significant(P<0.01).In contrast,the T group’s hippocampus showed a significant decrease in NLRP3,Casepase-1 and ASC m RNA,and the differences were also statistically significant(P<0.05).6.Aβ,NLRP3,Casepase-1,ASC in NLRP3 neural pathways,pro-inflammatory factor IL-1βand IL-18 protein levels in the hippocampus of mice in each groupGroup M’s hippocampus saw a marked rise in Aβ,NLRP3,Casepase-1,ASC,IL-1βand IL-18 proteins when compared to group C,with the differences being statistically significant(P<0.05).The T group’s hippocampus exhibited a decrease in NLRP3,Casepase-1,ASC,Aβ,IL-1βand IL-18 proteins when compared to the M group,with the differences being statistically significant(P<0.05).7.Expression of pro-inflammatory factor IL-1βin serum of mice in each groupThe expression of pro-inflammatory factor IL-1βin the serum of mice in group M was significantly higher than that of group C,and the differences were statistically significant(P<0.01).In contrast,the expression of pro-inflammatory factor IL-1βin serum of mice in T group was significantly lower than that of M group,and the differences were statistically significant(P<0.01).Conclusions:HUC-MSCs treatment reduced the expression of Aβin the hippocampus,NLRP3,Casepase-1 and ASC in the neuroinflammatory pathway of NLRP3and the pro-inflammatory factors IL-1βand IL-18 in the brain of APP/PS1double-transgenic mice,and improved the learning and memory ability of APP/PS1 mice by inhibiting the NLRP3 inflammasome signaling pathway.