| Background:A large number of studies,including previous studies in our laboratory,have shown that the drugs for diabetes,especially glucagon-like peptide-1 and its analogues,could effectively inhibit the occurrence and development of neurodegenerative diseases.Geniposide is a key component extracted from the fruit of Gardenia jasminoides Ellis.Utilizing the high-throughput screen,geniposide was identified as an agonist for GLP-1receptor.Geniposide has a good therapeutic effect on diabetes and has a wide range of neuroprotective effects.The mammalian target of rapamycin(m TOR)is a serine/threonine protein kinase that controls cellular growth as well as cellular homeostasis.m TOR is considered to be important to the occurrence and development of aging and aging related diseases and is closely related to the occurrence and development of Alzheimer's disease(AD).m TOR signal pathway is one of the most classical pathways of autophagy.Autophagy is a highly evolutionary conservative process regulated by some related gene to degrade organelles and proteins in eukaryotic cells,which is important to maintain the homeostasis of cells.Autophagy could degrade abnormal proteins.A? and Tau could be degraded through the autophagic lysosome pathway(ALP).Autophagic dysfunction may be a pathogenic factor in the occurrence of AD.Rapamycin could rescue cognitive impairments and ?-amyloid plaques via down regulation of m TOR signal pathway and enhancement of autophagy.Therefore,we speculate that m TOR is a key molecule for GLP-1 and geniposide to play the protective effect of AD.GLP-1 and geniposide may provide the neuroprotective effect of AD via down regulation of m TOR signal pathway and enhancement of autophagy.In the present study,we try to verity the hypothesis in APP/PS1 mice treated by geniposide utilizing the measurement of autophagy marker proteins and the related proteins of m TOR signaling pathway.Methods1.APP/PS1 mice were randomly divided into the geniposide treatment or vehicle group and treated with either geniposide(50mg/kg/d,n = 15)or water(n = 15)respectively,for 6 weeks via intragastric administration and a continuous intragatric administration was performed during 2 weeks behavior test.Age-matched C57BL/6 mice(n = 15)were fed equal volume of water as control.Novel object recognition(NOR)test and Morris water maze(MWM)were applied for behavioral assessment.2.Coranal sections of whole brain tissue were cut and stained with HE to observe the pathologic changes in cortex and hippocampus.3.SPs was shown by immunohistochemiscal staining.4.Soluble A?1-40 and A?1-42 in hippocampus were detected by ELISA.5.The expressions of LC3-?,Beclin1 and p62 in hippocampus were detected by immunohistochemical stainning and Western-blot.6.p-Akt/Akt,p-m TOR/m TOR and p-4E-BP1/4E-BP1 were detected by Western-blot.7.Aktm RNA,m TORm RNA and 4E-BP1 m RNA were detected by Rt-PCR.Results1.The results of NOR test showed that there was no significant difference between RI(recognition index)of the A1 and A2 in the three groups in the training stage(P >0.05).In test phase,the RI of vehicle-treated APP/PS1 mice was lower compared to WT mice(P < 0.01)and the RI of geniposide-treated mice was improved compared to vehicle-treated APP/PS1 mice(P < 0.05).The DI of vehicle-treated APP/PS1 mice was lower compared to WT mice(P < 0.001)and the DI of geniposide-treated mice was improved compared to vehicle-treated APP/PS1 mice(P < 0.05).2.During the navigation test,there was no significant difference in escape latency of the three groups on day 1 and 2(P > 0.05).On day 3,4,5 the escape latency of vehicle-treated APP/PS1 mice was longer compared to WT mice(P < 0.001,P < 0.001,P < 0.001).On day 5,the escape latency in geniposide-treated APP/PS1 mice was shorter compared with that of vehicle-treated APP/PS1 mice(P < 0.05).Geniposide also decreased the path length.Geniposide-treated APP/PS1 mice exhibited a notably shorter path length compared with vehicle-treated APP/PS1 on day 5(P < 0.05).The recorded path track of mice showed that the typical path track of vehicle-treated APP/PS1 mice was disorganized compared to WT mice,APP/PS1 mice treated by geniposide showed shorter path lengths and selective search track.The number of crossing the removed-platform area and the percentage of time spent in the target quadrant of vehicle-treated APP/PS1 mice were significantly reduced compared with that of WT mice(P < 0.001,P < 0.001);by contrast,geniposide significantly increased the number of crossing the removed-platform area(P < 0.01)and the percentage of time spent in the target quadrant(P < 0.05)compared with vehicle-treated APP/PS1 mice.In visible platform test,there was no significant difference in the swimming speed of the three groups(P > 0.05).3.The result of HE stain showed that the neurons in WT mice were normal in shape,closely arranged,with complete nuclei.In vehicle-treated APP/PS1 mice,the volume of the neurons decreased and the staining of the neurons was dark,and shrinkage and necrosis was observed in scattered neurons.Geniposide significantly ameliorated the impairments in the brain tissue of APP/PS1 mice.4.The cortex and hippocampus of WT mice was free of senile plaques(SPs),but abundant SPs was found in vehicle-treated APP/PS1 mice.The area and number of SPs in geniposide-treated APP/PS1 mice were reduced compared with that in vehicle-treated APP/PS1 mice(P < 0.01,P < 0.05).5.The levels A?1-40 and A?1-42 in hippocampus in APP/PS1 mice were significantly reduced by geniposide(P < 0.05,P < 0.05).6.The expressions of LC3-? and Beclin1 in hippocampus in vehicle-treated APP/PS1 mice were decreased compared with WT mice(P < 0.001,P < 0.01),geniposide promoted the expressions of LC3-? and Beclin1(P < 0.05,P < 0.01).The numbers of LC3-?-positive cells and Beclin1-positive cells in hippocampus in vehicle-treated APP/PS1 mice were reduced compared with WT mice(P < 0.001,P <0.001),geniposide increased the numbers of LC3-?-positive cells and Beclin1-positive cells(P < 0.001,P < 0.001).Conversely,the expression of p62 in hippocampus was increased in vehicle-treated APP/PS1 mice compared to WT mice(P < 0.001)and geniposide decreased the expression of p62(P < 0.01).The number of p62-positive cells in hippocampus in vehicle-treated APP/PS1 mice increased compared with WT mice(P< 0.001),geniposide reduced the expression of p62(P < 0.001).The results of Western-blot were consistent with that of immunohistochemiscal staining.7.p-Akt/Akt was remarkably increased in vehicle-treated APP/PS1 mice when compared to WT mice(P < 0.01),geniposide obviously reduced the increment(P < 0.05).The similar influence occurred in p-m TOR/m TOR.p-m TOR/m TOR was remarkably increased in vehicle-treated APP/PS1 mice when compared to WT mice(P < 0.05),geniposide markedly attenuated p-m TOR/m TOR(P < 0.05).p-4E-BP1/4E-BP1 was significantly decreased in APP/PS1 mice than in WT mice(P < 0.001),but geniposide obviously increased its level(P < 0.05).Aktm RNA and m TORm RNA was remarkably increased in vehicle-treated APP/PS1 mice when compared to WT mice(P < 0.001,P <0.001),geniposide obviously reduced the increment(P < 0.05,P < 0.05).4E-BP1 m RNA was significantly decreased in APP/PS1 mice than in WT mice(P < 0.001),but geniposide obviously increased its level(P < 0.05).Conclusions1.Geniposide remarkbly improved the ability of identifying new object and effectively ameliorated learning and memory impairments in APP/PS1 mice.2.Geniposide decreased the neuronal damages in the brain tissue of APP/PS1 mice.3.Geniposide reduced SPs in cortex and hippocampus,and reduced A?1-40 and A?1-42 levels in hippocampus in APP/PS1 mice.4.Geniposide up-regulated the expression of LC3-? and Beclin1,and down-regulated the expression of p62 in hippocampus in APP/PS1 mice.5.Geniposide markedly decreased p-Akt/Akt and p-m TOR/m TOR,remarkably increased p-4E-BP1/4E-BP1 in hippocampus in APP/PS1 mice.6.Geniposide obviously reduced Aktm RNA and m TORm RNA,remarkably increased 4E-BP1 m RNA in hippocampus in APP/PS1 mice.7.Geniposide ameliorated behavioral impairements and pathological changes via down regulation of m TOR signal pathway and enhancement of autophagy in APP?PS1mice. |
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