Extract Of Fructus Cannabis Improves Learning And Memory Impairment In Aging Rats Via Increasing SIRT1 Expression

Part ? SIRT1 contributes to the effect of extract of fructus cannabis?EFC?on learning and memory impairment in aging ratsObjective:Aging is an inevitable complicated biological process in the body with learning and memory impairment as one of its prominent markers.EFC shows favorable neuroprotective and anti-aging effects.SIRT1?silent information regulator1?plays an important role in many biological processes,such as cell differentiation,aging,apoptosis,transcription regulation,and signal transduction.This study was designed to explore whether SIRT1 is involved in the improvement effect of EFC on the learning and memory impairment in aging rats.Methods:One hundred and ten healthy male Sprague-Dawley rats?250-300g,3months old?were randomly divided into 5 groups which contain 11 subgroups as follows:?1?control group treated with saline as a vehicle for 6 months.?2?Rats treated with D-galactose?D-gal??400 mg/kg?for 3 months.?3?Rats treated respectively with EFC 400 mg/kg?EFC-L?,800 mg/kg?EFC-M?,and 1600mg/kg?EFC-H?alone for 3 months.?4?Rats pretreated respectively with EFC400 mg/kg,800 mg/kg,and 1600 mg/kg for 3 months and then D-gal?400mg/kg?for another 3 months,so pretreatment group including three subgroups respectively:EFC-L+D-gal group,EFC-M+D-gal group and EFC-H+D-gal group.?5?Rats treated D-galactose?400 mg/kg?for 3 months and then respectively with EFC 400 mg/kg,800 mg/kg,1600 mg/kg for another 3 months,as the treatment group including three subgroups respectively:D-gal+EFC-L group,D-gal+EFC-M group,D-gal+EFC-H group.After the 6^thh month of the D-gal and EFC administration,spatial memory was assayed with Morris water maze.Nissle staining was employed to evaluate the damage of hippocampus neuron in aging rats.Immunohistochemistry?IHC?,western blot?WB?and quantitative reverse transcription Polymerase Chain Reaction?qRT-PCR?were used to detect the expression of SIRT1.Results:1.In the place navigation training,there was no notable difference among groups on the first training day.While from Day 2 to Day 5,the post hoc comparisons revealed that the escaping latency in the D-gal group was much longer than that in the control group?P<0.05?.Whereas rats in 400 mg/kg and800 mg/k EFC pretreatment group and treatment group as well as treated EFC alone group,their escaping latency were remarkably reduced compared with these in the D-gal model group?P<0.05?.There was no remarkable difference between 1600 mg/kg EFC pretreatment and treatment as well as treated EFC alone group and D-gal group.2.On the Day 5 navigation training,the swim paths of the rats in D-gal group were distributed randomly in each quadrant.However,rats in 400 mg/kg and 800 mg/kg EFC pretreatment group and treatment group as well as treated EFC alone group improved the searching strategy significantly compared with that in the D-gal group?P<0.05?.There was no dramatic difference between1600 mg/kg EFC pretreatment and treatment improved the searching strategy significantly.3.In the spatial probe trial,the rats in D-gal group crossed less times over the platform quadrant than those in control group?P<0.05?.However,rats in400 mg/kg and 800 mg/kg EFC pretreatment group and treatment group as well as treated EFC alone group crossed over the target platform quadrant more often than D-gal group?P<0.05?.4.Compared with the control group,the neurons in the CA1 region of hippocampus were arranged loosely and showed condensed cytoplasm in the D-gal group,and the normal pyramidal neurons were significantly decreased.Meanwhile,for rats in pretreatment group and treatment group received with low and medium dosage of EFC most of their cells CA1 region was similar to those in the control group,and few cells showed pyknosis of the nuclei.5.The levels of SIRT1 mRNA and protein in the hippocampus of D-gal administration group were significantly lower than those in control group?P<0.05?.However,rats in pretreatment group and treatment group received with400 mg/kg and 800 mg/kg of EFC expression levels of SIRT1 mRNA and protein were significantly higher than those in D-gal group?P<0.05?.Conclusion:1.EFC can improve the performance of rats in the Morris water maze test by ameliorating space learning and memory impairment induced by D-gal in a dose-dependent manner,with 400 mg/kg and 800 mg/kg EFC being the most appropriate dosages.2.EFC can reduce the damage to neurons in the CA1 region of hippocampus in aging rats induced by D-gal.3.EFC with dosage 400 mg/kg and 800 mg/kg can up-regulate the expressions of SIRT1 mRNA and protein and then improve the learning and memory impairment in aging rats.Part ? EFC improve the learning and memory impairment in aging rats via the activation of SIRT1-ADAM10-APP signaling pathwayObjective:One of the pathological features of Alzheimer's disease?AD?is beta-amyloid plaques,which also occur in the process of physiological senescence.SIRT1 can regulate the splicing and processing of amyloid precursor protein?APP?.Meanwhile a disintegrin and metalloproteinase 10?ADAM10?and beta-secretase 1?BACE1?plays an important role in the splicing and processing of APP.Herein,we investigated whether the reduction of A? by SIRT1 is associated with ADAM10 or BACE1.Furthermore,we investigated whether EFC-induced SIRT1 expression contributes to the down-regulation of the A? and improvement of learning and memory impairment in aging rats.Methods:1.Experiments in vivo1.1 According to the results of part ?,we chose the 800 mg/kg as the dosage of EFC to treat the rats.To confirm the effect of EFC-induced SIRT1 expression in aging rats,rats were treated with 100 mg/kg of Resveratrol?Res??SIRT1 activator?or nicotinamide?NA??SIRT1 inhibitor?after D-gal?400mg/kg?was administrated for 3 months,followed by treatment without or with800 mg/kg EFC for 3 months.1.2 Immunohistochemical?IHC?staining was used to evaluate the expression of A?₄₂ in the hippocampus of each group.q RT-PCR and Western blot were performed to detect the expression of SIRT1,BACE1,ADAM10 and APP in the hippocampus of each group.2.Experiments in vitro2.1 The senescence state of SH-SY5 Y cell line was induced by D-gal and EFC was used as an intervention factor.SIRT1 over expression lentivirus?MOI=15?,SIRT1 sh RNA interfering plasmid were transfected cells to induce SIRT1 over expression and expression inhibition respectively.The effects of EFC on senescent cells were investigated as follow.2.2 SA-?-Gal staining was performed to elucidate the senescence state of the cells.2.3 Immunofluorescence?IF?assay,qRT-PCR,western blot were performed to detect the expression of SIRT1,BACE1,ADAM10,and APP in SH-SY5 Y cells.Results:1.Results in vivo1.1 Immunocytochemistry staining showed that the presentation of A?₄₂-positive cells in D-gal group were higher than control group?P<0.05?,after EFC intervention,the number of A?₄₂-positive cells in pretreatment group as well as treatment group were significantly decreased compared to D-gal group?P<0.05?.1.2 The changes in A?₄₂ observed in immunocytochemistry analysis were confirmed by western blotting,A?₄₂ were upregulated in the D-gal model group compared with the control group.There were no significantly differences of BACE1 in each group.However,ADAM10 and SIRT1 in D-gal group were decreased compared with control group?P<0.05?.Conversely,the ADAM10 and SIRT1 in pretreatment group and treatment group were up-regulated compared with D-gal group?P<0.05?.Furthermore,SIRT1 and ADAM10 levels in SIRT1 activator group were rescued and on the contrary A?₄₂ levels were increased.In SIRT1 inhibitor group,SIRT1,ADAM10 and A?₄₂ levels were not significantly different when compared with D-gal group.1.3 There were no significantly of the relative expression levels of m RNA of BACE1 and ADAM10 in each group.The relative expression level of SIRT1 m RNA of rats in D-gal group and inhibitor group were decreased?P<0.05?.After EFC intervention,the relative expression levels of SIRT1 m RNA in pretreatment group and treatment group were up-regulation?P<0.05?.Compared to normal control group,the relative expression levels of APP m RNA of rats in D-gal model group and inhibitor group was elevated?P<0.05?.After EFC intervention,the relative expression levels of APP m RNA in pretreatment group and treatment group were decreased compared with D-gal group?P<0.05?.2.Results in vitro2.1 The number of SA-?-Gal-positive cells in D-gal treated group increased significantly compared to the control cells.Furthermore,the number of SA-?-Gal-positive cells treated with both 200 m M D-gal and 50 mg/ml EFC was significantly lower than the D-gal treated group.2.2 The m RNA levels of SIRT1 of cells treated with D-gal alone were significantly decreased compared to control cells.In cells co-treated with EFC,the levels SIRT1 of cells were rescued compared to the D-gal-treated group and increased in EFC-treated alone cells.To confirm the effect of EFC-induced SIRT1 expression in senescent cells,cells were transfected with SIRT1 over expression lentivirus?MOI=15?and SIRT1 sh RNA interfering plasmid.These results showed that EFC significantly upregulated the levels of SIRT1 in cells co-treated with D-gal and over expression lentivirus.In cells treated with D-gal and SIRT1 sh RNA interfering plasmid followed by EFC the levels of SIRT1 were not significantly different when compared with cells treated with D-gal alone.The levels of APP m RNA and A? in D-gal treatment group were increased compared to control group.The levels of APP m RNA and A?₄₂ in cells co-treated with EFC were significantly reduced compared with D-gal treatment group.ADAM10 levels in D-gal treatment group were down-regulated than control group.EFC significantly upregulated ADAM10 levels in cells co-treated with D-gal and EFC.There were no significantly difference of the levels m RNA and protein of BACE1 and the levels ADAM10 m RNA in each group.Conclusion:1.200 mM D-gal can induce the senescence of SH-SY5 Y cells,while 50mg/ml EFC can protest these cells from senescence.2.EFC may be the direct agonist of SIRT1.3.SIRT1 over-expression has significantly elevated ADAM10.4.EFC appears to suppress A?₄₂ production in a SIRT1-ADAM10-APP dependent manner.Part ? EFC activates the SIRT1-YY1-CREB-BDNF signaling pathway to improve the learning and memory impairment in aging ratsObjective:Synaptic plasticity is the main cellular mechanism for many learning adaption and abnormal synaptic plasticity may partially contribute to the cognitive impairment in aging.Activation of SIRT1-YY1-CREB-BDNF signaling pathway may up-regulate the expression of brain-derived neurotrophic factor?BDNF?and thus enhance the plasticity of neuronal synapses.The aim of this part was to explore to the relationship between EFC and the SIRT1-YY1-CREB-BDNF signaling pathway in aging related cognitive impairment and its potential molecular mechanism.Methods:In this part,the animal models and cell models established in part? and part ? were employed.The relative expression of SIRT1,YY1,CREB and BDNF m RNA,and relative protein levels of the SIRT1,YY1,CREB,p-CREB and BDNF in hippocampus of rats and SH-SY5 Y cells were detected by immunofluorescence,Western blot and q RT-PCR.Results:1.There were no significantly differences in the total protein level of CREB in the rats' hippocampus in each group.The expression of SIRT1 and transcription factor YY1 in the rats hippocampus in D-gal group were remarkably lower than that in the control group?P<0.01?.The phosphorylation levels of CREB?p-CREB?regulated by SIRT1 and YY1 together also decreases?P<0.05?,which results in a decrease in the expression of protein BDNF directly related to the synaptic plasticity?P<0.05?.After EFC intervention,the expression levels of SIRT1 and YY1 can be significantly increased,and then the expression of the downstream substance p-CREB and BDNF can be increased.The expression levels of SIRT1,YY1,p-CREB and BDNF in the rats hippocampus of the EFC pretreatment group and the treatment group were not significantly different compared with those of SIRT1 activator group,but were significantly increased compared with those of the SIRT1 inhibitor group?P<0.05?.2.There were no significant differences of the expression levels of the YY1 and CREB m RNA in hippocampus among groups,but the expression trend of SIRT1 and BDNF m RNA in each groups were similar to the protein levels as detection by western blot above.Compared with control group,the expression levels of SIRT1 m RNA of rats in D-gal group and SIRT1 inhibitor group were decreased.However,the trend of this decrease was reversed after EFC intervention.The trend of expression of BDNF m RNA in the hippocampus of each group was also the same as that of SIRT1.3.To confirm the effect of EFC-induced SIRT1 expression in vivo and vitro,rats were treated with 100 mg/ml Resveratrol?SIRT1 activator?or nicotinamide?SIRT1 inhibitor?after D-gal had been used for 3 months,followed by treatment in the presence or absence of EFC 3 months.These results showed that EFC significantly up-regulated SIRT1 levels in SIRT1 activator.In SIRT1 inhibitor followed by EFC,SIRT1 levels were not significantly different when compared with D-gal group.Taken together,the present data indicated that EFC reversed the downregulation of SIRT1 expression in the aging rats.In addition,to determine the relationship between SIRT1 and YY1,p-CREB,BDNF,cells were transfected with SIRT1 over expression lentivirus and SIRT1 sh RNA interfering plasmid.These results showed that the levels of YY1,p-CREB and BDNF in SIRT1 over expression lentivirus-treated cells were significantly increased compared with D-gal-treated cells.Furthermore,the levels of YY1,p-CREB and BDNF in the group that transfected with SIRT1 sh RNA were significantly decreased compared to cells with D-gal +EFC treatment.Taken together,these data indicated that EFC-induced the expression of p-CREB and BDNF via SIRT1 and YY1.Conclusion:EFC activates the SIRT1-YY1-CREB-BDNF signaling pathway to improve the learning and memory impairment in aging rats.