| Objective: To explore the effects of Dabuyuan Decoction(DBYJ)on the cognitive function of C57/BL6 aging mice,and to verify the mechanism of inhibiting neuroinflammation and improving neurodegenerative changes and hippocampal neuron apoptosis through PPAR-γ/FoxO3 a signaling pathway.Methods: Mice were randomly divided into negative control group(NC),D-gal group,GW9662 group(PPAR-γ inhibitor),DBYJ group and GW9662+DBYJ group with 15 animals in each group.NC group was given 0.9% saline by subcutaneous injection in the neck and back for 8 weeks,while the other groups were given D-gal(100mg/kg/d).GW9662 group and GW9662+DBYJ group were intraperitoneally injected with 1mg.kg-1·d-1 for a total of 4 weeks.On the 5th to 8th week,the NC group,D-gal group and GW9662 group were given the same volume of normal saline at the dose of 10 m L/mg/kg/d.DBYJ group and GW9662+DBYJ group were given DBYJ aqueous decoction.Morris Water mazes were used to test the learning and memory abilities of mice.Neurodegenerative changes were detected by FJB staining.The loss and apoptosis of hippocampal neurons were detected by Nissl and TUNEL staining.The contents of TNF-α,IL-1β and IL-6 in the hippocampus were detected by ELISA.Immunofluorescence staining was used to examine the levels of IBA-1 and GFAP in the hippocampus.Western blot(WB)was used to detect the protein expression levels of Bcl-2,Bax,Caspase-3,Cleaved caspase-3,PPAR-γ,FoxO3 a,and p-FoxO3 a in the hippocampus of different groups.Results: 1.Biological characteristics of aging model mice,such as weight loss decreased activity,mental fatigue,gray hair and other biological manifestation consistent with aging.There was no aging in the control group,but DBYJ or GW9662+DBYJ group improved the biological manifestations of aging,and the DBYJ group showed significant effects.2.Analysis of Water Maze Experiment: In navigation test,there was no significant change in the escape incubation period of all groups during 3 days of continuous training(p>0.05).From day 4,D-gal group treatment significantly increased the escape latency,which was significantly reversed in the DBYJ group(p<0.01),followed by GW9662+DBYJ group.In the space probe test,compared with the D-gal group,the DBYJ group had a higher number of crossing platforms(p<0.01).Compared with GW9662 group,GW9662+DBYJ group had a higher number of cross platforms(p<0.01),but its effect was lower than that of DBYJ group.There was no significant difference in average swimming speed among all groups(p>0.05).3.The neurodegenerative changes in the hippocampus were detected by FJB staining: compared with the NC group,the number of FJB positive cells in the CA1 and CA3 regions of the hippocampus in the D-gal group was significantly increased(p<0.01).Compared with D-gal group,the number of FJB positive cells in the hippocampus of DBYJ group were significantly decreased(p<0.01).Compared with GW9662 group,the number of FJB positive cells in the hippocampus of GW9662+DBYJ group was decreased,but the effect was lower than that of DBYJ group.4.The contents of TNF-α,IL-1β and IL-6 in cortex were measured by ELISA: compared with NC group,the levels of TNF-α,IL-1β and IL-6in D-gal group were higher(p<0.01);Compared with D-gal group,proinflammatory cytokines were inhibited in DBYJ group(p<0.01).Compared with GW9662 group,GW9662+DBYJ group showed similar results,but the effect was lower than that of DBYJ group.5.Immunity fluorescence staining was used to detect the levels of IBA 1and GFAP: compared with NC group,the fluorescence intensity of IBA 1 and GFAP in D-gal group was higher(p<0.01);Compared with D-gal group,the fluorescence intensity of IBA-1 and GFAP in DBYJ group was decreased(p<0.01).Compared with GW9662 group,GW9662+DBYJ group showed similar results,but the effect was lower than that of DBYJ group.6.Nissl staining: Compared with NC group,the number of neurons in CA1 and CA3 regions in D-gal group decreased(p<0.01);Compared with the D-gal group,the loss of neurons was reduced in the DBYJ group(p<0.01).Compared with GW9662 group,GW9662+DBYJ group showed similar results,but the effect was lower than that of DBYJ group.7.TUNEL staining:Compared with NC group,the number of positive neuron cells in D-gal group increased.Compared with D-gal group and GW9662 group,the number of TUNEL positive neurons in CA1 and CA3 in DBYJ group and GW9662 group was decreased(p<0.05 or 0.01).8.Western blot analysis showed that compared with the NC group,protein levels of Bax,Caspase3,Cleaved caspase3 were up-regulated(p<0.001),and protein levels of Bcl-2 were significantly down-regulated(p<0.001)in the D-gal group.Compared with D-gal group,protein levels of Bax,Caspase3 and Cleaved caspase3 in the hippocampus of DBYJ group were down-regulated(p<0.01),and protein levels of Bcl-2 were significantly up-regulated(p<0.01).Compared with GW9662 group,GW9662+DBYJ group showed similar results,but the effect was lower than that of DBYJ group.9.Mechanism detection results of PPAR-γsignaling pathway: Compared with NC group,PPAR-γ level in D-gal group was significantly down-regulated(p <0.001),while p-FoxO3 a level was significantly up-regulated(p<0.01);Compared with D-gal group,PPAR-γ level in DBYJ group was significantly up-regulated(p<0.001),and p-FoxO3 a level was significantly down-regulated(p<0.01).Compared with GW9662 group,GW9662+DBYJ group showed similar results,but the effect was lower than that of DBYJ group.After addition of GW9662,the therapeutic effect of DBYJ was reversed.Conclusion: DBYJ can alleviate neurodegenerative changes and hippocampal neuronal apoptosis in aging mice by regulating PPARγ/FoxO3 a signaling pathway,and improve their learning and memory ability. |
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