Effect Of Extract Of Fructus Cannabis On Spatial Learning And Memory In An Aging Rats Model Induced By D-galactose And Its Mechanism

Since the 1990 s,the decline in the population birth rate and the extension of human lifespan resulted in a rapid growth of aging population in China.The brain aging is accompanied by different degrees of memory decline in the elderly.In addition,cognitive dysfunction caused by neurodegenerative diseases such as Alzheimer's disease has become a major problem in older people.Therefore,it is important to study the mechanism related to cognitive aging and explore effective interventions to slow down brain aging as well as to achieve healthy aging and improve the life quality of the elderly.The extract of Fructus Cannabis(EFC)was extracted from the dried and matured fruit of hempseed.Its main components are saturated fatty acids and polyunsaturated fatty acids.In recent years,studies have shown that hemp kernels have wide pharmacological effects on the central nervous system,cardiovascular system,digestive system and immune system as well as positiveeffects on anti-oxidation,anti-inflammation,anti-thrombosis and decreasing blood lipids.However,it is not clear whether hemp kernels play a role in the protection against cognitive impairment associated with aging.Objective: To establish a subacute aging rats model induced by D-galactose(D-gal),and evaluate the spatial learning as well as memory by Morris water maze.Moreover,in order to explore the molecular biological mechanism of cognitive decline in senescence,the expression of genes and proteins associated with brain aging was detected at the m RNA and protein level.Furthermore,the aging of mouse embryonic fibroblasts(NIH/3T3)was induced by D-gal,and the effect of EFC on cellular senescence was observed at the celluar level.In summary,the present study sought to define the underlying mechanism by which the extract of EFC rotects against spatial learning and memory impairment in aging rats induced by D-gal.Method1.The male rats were randomly divided into 6 groups with 8 rats for each: 1)control group;2)D-gal group;3)EFC pretreatment group(200 mg/kg);4)EFC pretreatment group(400 mg/kg);5)D-gal + EFC(200 mg/kg)group,and 6)D-gal + EFC(400 mg/kg)group.To establish the animal model of aging,rats in the D-gal group were injected intraperitoneally with D-gal at a dose of 400mg/kg/day for 14 weeks.Rats in D-gal plus EFC group were injected intraperitoneally by D-gal 400mg/kg/day and simultaneously received intragastric administration of EFC for 14 weeks.Meanwhile,the rats in control group received an injection of equal volume of saline consecutively for 14 weeks.In EFC pretreatment group,rats received intragastric administration of EFC for 14 weeks and then were injected intraperitoneal injection with D-gal for another 14 weeks.2.The ability of spatial learning and memory was evaluated by the Morris water maze test.3.Hematoxylin-eosin(HE)staining was used to evaluate the damage of hippocampus neuron in aging rats.4.SOD activity and MDA level in the brain tissue were analyzed by WST-1 and TBA method.5.Detected organ indices,including heart,liver,thymus and spleen.6.Expression of ?-H2 AX and GFAP in the dentate gyrus of hippocampus in aging rats were measured by immunofluorescence.7.The m RNA expressions of APP,Tau and PS1 genes in the hippocampus were detected by quantitative reverse transcription polymerase chain reaction(q RT-PCR).8.The expression of total Tau,p-Tau,PS1 protein in the hippocampus of the rats was detected by western blotting.9.The senescence of NIH/3T3 cells was induced by D-gal and EFC was used as an intervention factor.The effects of EFC on aging cells were investigated as followed:9.1 The intracellular ROS level was quantified by FACS flow cytometry after ROS generation was detected by DHE fluorescent probe.9.2 The expression of SA-?-galactose(SA-?-gal)was detected by senescence detection kit.9.3 Immunofluorescence assay was used to detect the expression of ?-H2 AX and p-ATM which is the markers of DNA damage.9.4 The expression levels of SIRT1,p53 and p21 were detected by western blotting.The findings are as followed:Part ? Effect of EFC on the protection against oxidative damage induced by D-gal in aging ratsResults:1.Compared with the control group,the activity of SOD was decreased significantly and the MDA levels were increased notably in the brain tissue of D-gal group(P<0.05).However,the activity of SOD was increased significantly and the MDA levels were decreased notably in the brain tissue of D-gal plus EFC(400 mg/kg)group and EFC(400 mg/kg)pretreatment group,compared with the D-gal group(P<0.05).2.The organ indices,including heart,liver,thymus and spleen,in D-gal model group were significantly lower than those of the control group(P < 0.05).However,these parameters turned toward normal after EFC administration.3.Compared with the control group,the pyramidal neurons in CA1 region of hippocampus were arranged loosely in the D-gal group,and the normal pyramidal neurons were significantly decreased,meanwhile the pyknotic nucleus were significantly increased and presented a densely staining.However,in D-gal plus EFC(400 mg/kg)group,the pyramidal neurons in CA1 region arranged orderly and the number of pyknosis cells was significantly decreased.The morphology of pyramidal neurons in CA1 region was normal in EFC(400mg/kg)pretreatment group,but the number of pyramidal cells was less than that of control group.Conclusion: 1.EFC can alleviate the signs related to aging by improving the antioxidant capacity of rats.2.EFC can delay senescence of rats by enhancing the immune function with increasing spleen and thymus indices.3.EFC can reduce the damage to pyramidal neurons in CA1 region of hippocampus in aging rats induced by D-gal.Part ? Effect of EFC on spatial learning and memory impairment in aging rats induced by D-galResults:1.There was no difference in escape latencies and distances to hidden platform between groups on Day 1 training.However,from Day 2 to Day 5,the post hoc comparisons revealed that D-gal model group showed longer escape latencies and distances to find the target platform than the control group(P <0.05).Whereas rats in D-gal plus EFC treatment(400 mg/kg)group and EFC(400 mg/kg)pretreatment group had shorter latencies and distances to find the hidden platform than the D-gal group(P < 0.05).2.On the fifth day of navigation training,the rats in D-gal group took a tortuous swimming path to find the hidden platform and the swimming trace was distributed randomly in each quadrant.However,the rats in D-gal plus EFC(400mg/kg)group and EFC(400 mg/kg)pretreatment group improved the searching strategy significantly and the swimming trace was mostly distributed in the quadrant or adjacent quadrant of the previous platform.3.In the probe trial,the rats in D-gal group spent less time in the target quadrant than those in control group and crossed less times over the platform quadrant(P < 0.05).However,the rats in D-gal plus EFC(400 mg/kg)group and EFC(400 mg/kg)pretreatment group spent more time in the platform quadrant and crossed over the target quadrant more often than D-gal treated rats(P <0.05).4.The number of GFAP and ?-H2 AX positive cells in dentate gyrus of hippocampus in D-gal treated rats was higher than that in the control group(P <0.05).However,EFC reversed these changes in D-gal plus EFC(400 mg/kg)group and EFC(400 mg/kg)pretreatment group.Conclusion:1.EFC can improve the performance of rats in Morris water maze test by ameliorating space learning and memory impairment induced by D-gal.2.EFC can protect the hippocampal neurons and improve the ability of spatial learning and memory by inhibiting the activation of astrocytes and reducing the positive expression of GFAP in dentate gyrus of hippocampus in rats.3.EFC can improve the ability of spatial learning and memory by reducing DNA damage and ?-H2 AX foci in dentate gyrus of hippocampus in rats.Part ? Effect of EFC on the expression of APP,Tau and PS1 genes in aging rats induced by D-galResults:The relative expression of APP,Tau and PS1 m RNA in hippocampus of D-gal treated rats were significantly higher than that of control group(P < 0.05).However,the expression of APP,Tau and PS1 m RNA in hippocampus of D-gal plus EFC(400 mg/kg)group and EFC(400 mg/kg)pretreatment group were significantly decreased than that of D-gal group.Conclusion:EFC can delay the neurodegeneration lesions and improve the cognitive function by down-regulating the expression of APP,Tau and PS1 m RNA in hippocampus of aging rats induced by D-gal.Part ? Effect of EFC on the expression of total-Tau,p-Tau,PS1 protein in aging rats induced by D-galResults:The levels of PS1 and the phosphorylation of tau in the hippocampus of D-gal administration group were significantly higher than those in control group(P< 0.05).However,in D-gal plus EFC(400 mg/kg)group and EFC(400 mg/kg)pretreatment group,expression of PS1 and phosphorylated tau were significantly lower than those in D-gal group(P<0.05),but there was no significant difference in total tau level in all of the groups.Conclusion:EFC can improve the spatial learning and memory by down-regulating the levels of PS1 and phosphorylation of tau in aging rats induced by D-gal.Part ? Effect of EFC on the expression of SIRT1 and p53-p21 pathway and its mechanismResults:1.ROS fluorescence signal was significantly higher in D-gal(400m M)treated cells than that of control group,meanwhile ROS fluorescence signal decreased notably after EFC(50?g/ml)pretreatment for 24 h.The results in FACS flow cytometry quantified test show that the percentage of ROS production in D-gal treated group was up to 45.2%,and the percentage of ROS production in control group,D-gal plus EFC group and EFC group were 1.4%,3.2%,2.5% respectively.2.Senescence cells increased significantly in D-gal treated group,40% of3T3 cells were stained positively with SA-?-gal.Compared with D-gal treated group,the number of SA-?-gal staining positive cells was significantly decreased in D-gal plus EFC group and EFC group.3.Immunofluorescence staining showed that the number of ?-H2 AX foci was increased significantly and the immunoreactivity of p-ATM was enhanced after the treatment with D-gal(400m M).However,the number of ?-H2 AX foci was increased significantly and the immunoreactivity of p-ATM was decreased in D-gal plus EFC group and EFC group.4.Compared with the control group,the expression of SIRT1 was decreased significantly and the expression of p53 and p21 in D-gal treated group was higher than that in control group(P <0.05).However,the expression of Sir T1 was up-regulated while the expression of p53 and p21 was decreased significantly after EFC pretreatment for 24h(P <0.05).Conclusion:1.EFC can inhibit the production of ROS by increasing the antioxidant capacity of NIH/3T3 cells,and plays a role in down-regulating SA-?-gal expression to delay cellular aging.Furthermore,EFC can improve the ability to repair DNA damage and decrease the expression of ?-H2 AX as well as p-ATM associated with DNA damage.2.EFC can increase the expression of p53 and p21 by up-regulating the expression level of SIRT1 in NIH / 3T3 cells which would result in delaying cellular senescence.