Effect Of Extract Of Fructus Cannabis On Neuroinflammation In Aging Rats Induced By D-galactose And Its Mechanism

Objective:With the rapid acceleration of social aging,neurodegenerative diseases have a great impact on the quality of life of the elderly.Therefore,it is of critical significance to explore solutions that defer senescence,improve brain aging and maintain brain function.Several published studies have documented that Fructus Cannabis presents effective anti-inflammatory and anti-aging effects,but its relevant mechanism is still unclear.In this study,extract of Fructus Cannabis(EFC)was used to intervene D-gal induced aging rat model,followed by the observation of the effect of EFC on learning and memory ability in aging rats.Meanwhile,the expressions of proteins and genes associated with neuroinflammation were detected,in an attempt to explore the possible molecular mechanisms in which EFC improves the inflammatory response and cognitive impairment in the central nervous system,hoping to provide more theoretical and experimental basis for the application of Fructus Cannabis.Methods:1.Eighty healthy male Sprague-Dawley rats(250-300 g,3 months old)were randomly divided into 8 groups as follows:(1)control group,in which rats were injected intraperitoneally with saline for 3 months and then received intragastric administration of saline for another 3 months;(2)aging model group,in which rats were injected intraperitoneally with D-gal at a dose of 400 mg/kg/day for 3months and then received intragastric administration of saline for another 3months;(3)pretreatment group,in which rats were pretreated respectively with EFC 400 mg/kg,800 mg/kg and 1600 mg/kg for 3 months and then D-galactose400 mg/kg for another 3 months;(4)treatment group,in which rats were treated with D-galactose 400 mg/kg for 3 months and then respectively with EFC 400mg/kg,800 mg/kg,1600 mg/kg for another 3 months.2.The ability of spatial learning and memory was evaluated by Morris water maze.3.The tumor necrosis factor ?(TNF-?)and interleukin-1?(IL-1?)levels in hippocampus were analyzed by Enzyme-Linked Immunosorbent Assay(ELISA).4.The m RNA expression of glial fibrillary acidic protein(GFAP)gene in the hippocampus in aging rats was detected by quantitative reverse transcription polymerase chain reaction(q RT-PCR).5.Expression of GFAP in the dentate gyrus of hippocampus in aging rats was measured by immunofluorescence.6.The expressions of GFAP and the proteins involved in IKK/I?B/NF-?B signaling pathways in the hippocampus of the rats were detected by western blot.7.Expression of p-p65 in the dentate gyrus of hippocampus in aging rats was measured by immunohistochemical.Results:1.According to the MWM test,as compared with the control group,rats in D-gal group showed longer escape latencies on day 5 of navigation training and less crossing platform numbers in the probe trail(P<0.05);the groups treated and pretreated with EFC(400 mg/kg,800 mg/kg)respectively had shorter escape latencies and more crossing times(P<0.05).2.As compared with the control group,the levels of TNF-? and IL-1? were increased notably in the hippocampus of D-gal group(P<0.05).Interestingly,the levels of TNF-? and IL-1? were decreased significantly in the hippocampus of pretreatment and treatment groups who received EFC(400 mg/kg,800 mg/kg)respectively(P<0.05);there was no remarkable difference between EFC(1600mg/kg)treatment group and D-gal group;however the levels of IL-1? but not TNF-? was found to be down-regulated significantly in the hippocampus of EFC(1600 mg/kg)pretreatment group compared with D-gal group(P<0.05).3.The expression of GFAP m RNA in hippocampus of D-gal induced aging model rats was significantly higher than that of control group.However,the expressions of GFAP m RNA in hippocampus of the groups treated with EFC(400 mg/kg,800 mg/kg)respectively and the three pretreatment groups were significantly decreased than that of aging model group(P<0.05).There was no significant difference between EFC(1600 mg/kg)treatment group and D-gal induced aging model group.4.The relative optical density of GFAP was increased in dentate gyrus of hippocampus of D-gal induced aging group as compared with control group(P<0.05).EFC reduced GFAP immunofluorescence in dentate gyrus of hippocampus of EFC pretreated rats and EFC(400 mg/kg,800 mg/kg)treated respectively rats compared with alone D-gal treated rats(P<0.05).There was no significant difference between EFC(1600 mg/kg)treatment group and D-gal induced aging model.5.Western blot results revealed that there were no differences in total IKK,I?B and p65 levels among groups.The expressions of GFAP,p-IKK,p-I?B and p-p65 protein were increased in D-gal-treated alone rats compared with the saline-treated rats(P<0.05).EFC pretreatment(400 mg/kg,800 mg/kg and 1600mg/kg)and treatment(400 mg/kg,800 mg/kg)significantly reduced the expression of GFAP,p-IKK,p-I?B and p-p65 levels in the D-gal treated rats(P<0.05).There was no significantly difference between EFC(1600 mg/kg)treatment group and D-gal induced aging model.6.Immunohistochemical results of p-p65 also showed that EFC significantly attenuated D-gal induced increased the expression of p-p65 in nuclear in the hippocampus compared with D-gal treated group.Conclusions:1.To a certain extent,EFC could ameliorate the space learning and memory impairment in aging rats.2.EFC attenuates inflammation probably through inhibiting astrocytes activation and reducing inflammatory mediators including TNF-? and IL-1?.3.EFC attenuates D-gal-induced learning and memory impairment through inhibiting NF-?B signaling pathway mediated neuroinflammation in aging rats.