Effects And Mechanisms Of Metrnl On Cognitive Function In Mice

BackgroundCognitive function refered to the psychological behavior or process in which organisms acquire knowledge through thinking,experience or sensory organs.Along with the progress of aging,Alzheimer's disease,the leading cause of cognitive function decline,has become an urgent problem for the elderly.The decline of cognitive function not only brought pain to patients themselves and their families,but also caused a heavy economic burden to society.However,so far,seldem ideal drugs for improvement of cognitive function were available.Metrnl was a novel adipokine reported by our laboratory in 2014 and it has been found many different functions.Metrnl was highly expressed in central nervous system of rodents in embryonic stage,while declining after birth.According to the case report of human Mild ring 17 syndrome by Surace C et al.in 2009,the mild cognitive dysfunction of the disease was caused by the loss of four genes including METRNL near the long arm telomere during the cyclization of chromosome 17.This made us interested in whether Metrnl was related to cognitive function.Whereas,as a novel secretory protein,the function of Metrnl in central nervous system has not been fully elucidated.In particular,whether it can affect the cognitive function of rodents has not been reported.ObjectiveWe aimed to estabilish and evalvate the in vivo and in vitro D-galactose-induced models.In addition,we explored the effects and mechanisms of Metrnl on cognitive function in mice.Methods1.We established the D-galactose-induced mice model and explained its possible mechanisms in use.We then evaluated the learning and memory functions of Metrnl knockout mice by Morris water maze test under physiological and D-galactose-induced model conditions.Memory-related protein molecules and inflammatory factors in brain tissues were detected to explore the mechanism of Metrnl action.2.We established the D-galactose-induced model in vitro and explained its possible mechanisms in use.The effects of Metrnl overexpression,knockdown and Metrnl recombinant protein on neurons,astrocytes in the hippocampus of mice and BV-2 cell lines under the three different conditions were then studied under D-galactose-induced models in vitro.P Memory-related protein molecules or inflammatory factors were detected to further explore the mechanism of Metrnl.Results1.Establishment and evaluation of D-galactose-induced model(1)To a certain extent,there was gender difference in the learning and memory functions of 2-month-old mice.Male mice were slightly better than female mice.Sex distinction should be paid attention to in the experimental process.(2)The learning and memory functions of D-galactose-induced mice model were impaired.The protein levels of brain-derived neurotrophic factor,glial fibrillary acidic protein,Synaptophysin and postsynaptic density~95 kDa were significantly reduced while the level of Trk-B was increased.Meanwhile,the content of caspase-3 in the brain was significantly increased,indicating that the level of neuronal apoptosis was high.The levels of glutathione peroxidase and malondialdehyde were also significantly increased,suggesting that the accumulation of D-galacose metabolites resulted oxidative stress injury in the brain.(3)In D-galactose-induced model in vitro,hippocampal neurons showed obvious cytotoxic reaction and oxidative stress injury at 10 mg/mL for 48 hours without changes in morphology;hippocampal astrocytes showed the same changes at 3~10 mg/mL for 48 hours.BV-2 cell line showed obvious inflammatory reaction at 3~10 mg/mL for 24 hours.D-galactose could induce neurons and astrocytes to secrete brain derived neurotrophic factor in a protective manner.In the relative higher doses,D-galactose could induce morphological changes of the three different kinds of cells.2.Effects and mechanisms of Metrnl on cognitive function of mice(1)Under physiological condition,Metrnl expressed higher in hippocampus than cortex in adult mice.Whereas,Metrnl knockout(KO)mice showed no significant impairment of learning and memory functions compared with wild type(WT)mice,although the content of glial fibrillary acidic protein in the brains of KO mice was significantly lower than those of WT mice.(2)Under D-galactose-induced model in vivo,the learning and memory functions of KO mice were significantly decreased.Further studies showed that Metrnl could regulate the levels of brain-derived neurotrophic factor and other memory-related protein molecues in hippocampus at very early stage(7 d).However,no significant changes of the N-methyl-D-aspartate receptor(NMDAR)and proteins related to JAK-STAT3 pathway were detected in two groups.(3)Experiments conducted on D-galactose-induced model in vitro showed that Metrnl did not directly act on hippocampal neurons but has significantly neurotrophic effect on hippocampal astrocyte in vitro.Meanwhile,Metrnl affected many inflammatory factors both in hippocampal astrocyte and BV-2 cell line in vitro and alleviate the inflammatory responses,which might also serve as one of the mechanisms of its protective function.ConclusionMetrnl expressed higher in hippocampus than cortex in adult mice under physical condition,but the effect of Metrnl on the learning and memory functions in vivo was not obvious.Interestingly,the level of glial fibrillary acidic protein in brain of KO mice decreased significantly.In D-galactose-induced model,Metrnl could alleviate the learning and memory dysfunction in vivo,functioning in maintaining the levels of memory-related protein molecules in the hippocampus.This effect could begin in the early stage of D-galactose-induced model(7 d).However,so far,the neurotrophic effect of Metrnl on hippocampal neurons has not been observed.Current studies showed that Metrnl has significant neurotrophic effect in D-galactose-induced model of hippocampal astrocyte in vitro.Meanwhile,Metrnl could regulate inflammation factors both in hippocampal astrocyte and BV-2 cell line in vitro and allayed the inflammatory response under D-galactose-induced model condition,which might be one of the reasons that Metrnl alleviated the decline of the learning and memory functions in mice D-galactose-induced model.In conclusion,the expression and special functions of Metrnl in hippocampus might be a novel target for the alleviation of early-stage Alzheimer's disease.