| ObjectiveThe aim was to observe the therapeutic effect of TLJN Oral Solution about learning and memory abilities on AD Rats induced by amyloidβ1-40 injection in bilateral hippocampus tissue and its mechanism, to provide a scientific ground in clinical applications.MethodsMale , Sprague-Dawley(SD) 8～12 weeks Rat(n=140) were enrolled in the study, and they were equally randomly divided into seven groups.There are experiment group (12mg·kg-1·d-1,24mg·kg-1·d-1,48mg·kg-1·d-1),model controls group, positive control group(Donepezil group ) and sham controls group,blank controls group. To induce AD, five groups rats model include control group, positive control group and experiment group (12mg·kg-1·d-1,24mg·kg-1·d-1,48mg·kg-1·d-1) received amyloid-β1-40 sterotaxis injection the bilateral hippocampus,. And the left, one goup served as blank controls, the other as sham controls group wich received NS sterotaxis injection the bilateral hippocampus. TLJN Oral Solution groups was administered 28 days by oral gavage to experiment group (12mg·kg-1·d-1,24mg·kg-1·d-1,48mg·kg-1·d-1) after three days of AD induction. Donepezil Hydrochloride Tablets served as control drug and administered by oral gavage. The learning and memory abilities were evaluated with a series of tasks to find a hidden platform in Morris Water Maze. after treatment for 4 weeks, and all rats were sacrificed at 5 week. The expression levels of AChE, ChAT, NF-κB, CRP and NOS were measured by immunohistochemical SABC techniques. APP, BACE1, Bax and Bcl-2 mRNA expression in brain tissue was assayed by real-time quantitative reverse transcription-PCR. TNFαand IL-1βprotein level in serum were determined using ELISA. The expression of SYP protein and caspase-3 protein in rat brain were estimated by western blotting method. The total activity of MAO,SOD and volume MDA in the brain tissue were detected by ultraviolet-spectrophotometry. The changes in the ultrastructure of the hippocampus of the AD rats were observed With a transmission electron microscope, the content of lipofuscin in the brain tissue was measured by spectrofluorimetry.ResultMorris Water Maze test: On day 2,3,4,5 of place navigation test, the mean escape latencies of the model group were obviously increased compared with control group (p<0.01), in spatial probe test, the frequency of passing through the platform of the model group were obviously decreased as compared with the control group(p<0.01), on day 2,3,4,5 of place navigation test, the mean escape latencies of the positive control group and experiment group(48mg·kg-1·d-1,24mg·kg-1·d-1,12mg·kg-1·d-1) were obviously decreased as compared with model group(p<0.01). In spatial probe test, the frequency of passing through the platform of the positive control group and experiment group(48mg·kg-1·d-1,24mg·kg-1·d-1,12mg·kg-1·d-1) were obviously increased as compared with the model(p<0.01). RT-PCR: The BACE1 mRNA expression was up-regulate expression in model groups, in which 2-ΔΔCt were 4.67±0.52 , compared with the sham group(1.07±0.08,p<0.01). In Donepezil group(1.80±0.23) and experiment groups ,which respectively (2.92±0.22,1.54±0.25 and 0.54±0.11), the mRNA expression was down-regulate expression compared with the model group (p<0.01). The APP mRNA expression was up-regulated in model groups, in which APP mRNA 2-ΔΔCt value were 5.88±0.70, compared with the sham group (1.57±0.16,p<0.01). The APP mRNA expression in experiment group (respectively 4.24±0.44, 2.93±0.49, 2.53±0.43) and Donepezil group (3.99±0.52) was down-regulated compared with the model group (p<0.01).The Bax mRNA expression was up-regulate expression in model groups, in which 2-ΔΔCt were 4.17±0.38, compared with the sham group(1.79±0.13,p<0.05). In experiment groups ,which respectively (2.52±0.68,1.89±0.38 and 1.58±0.22), the mRNA expression was down-regulate expression compared with the model group (p<0.05). The Bcl-2 mRNA expression was down-regulate expression in model group(0.12±0.02)compared with the sham group (0.52±0.10). In experiment groups (respectively 0.16±0.03, 0.29±0.04, 0.31±0.05) the Bcl-2 mRNA expression being up-regulated compared with the model group (p<0.05)..Immunohistochemistry. The levels of CRP expression from the model group (8.95±0.57) was significantly highter than that in the sham group(3.10±0.39) (p<0.05), and that from the experiment groups (respectively 6.36±0.53, 5.57±0.34, 4.76±0.31) was significantly lower than in the model group (p<0.05). The levels of NF-κB protein expression from the model group (6.90±0.80) was significantly highter than that in the sham group(3.01±0.19) (p<0.05), and that from the experiment groups (respectively 5.71±0.49, 4.58±0.34, 4.57±0.35) was significantly lower than in the model group (p<0.05). The levels of Nitric oxidase 1 protein expression from the model group (2.38±0.30) was significantly lower than that in the sham group(5.74±0.39) (p<0.05), and that from the experiment groups (respectively 3.57±0.30, 3.80±0.24, 4.99±0.31) was significantly highter than in the model group (p<0.05). The levels of Somatostatin protein expression from the model group (2.99±0.14) was significantly lower than that in the sham group(8.45±0.97) (p<0.05), and that from the experiment groups (respectively 4.22±0.20, 5.63±0.25, 5.93±0.30) was significantly highter than in the model group (p<0.05). The levels of Acetylcholinesterase expression from the model group (6.42±0.38) was significantly lower than that in the sham group(6.74±0.47) (p<0.05), and that from the 24mg/kg/d and 48mg/kg/d TLJN Oral Solution groups (respectively 4.37±0.33, 3.20±0.40) was significantly lower than in the model group (p<0.05). The levels of Choline Acetyltransferase expression from the model group (2.81±0.15) was significantly lower than that in the sham group(6.58±0.58) (p<0.05), and that from the experiment groups (respectively 3.83±0.22, 4.44±0.23, 4.31±0.28) was significantly highter than in the model group (p<0.05).Western blotting. The western blot analysis show that the levels of Synaptic protein expression from the model group (0.59±0.03) was significantly lower than that in the sham group(0.94±0.03) (p<0.05), and that from the experiment groups (respectively 0.68±0.04, 0.71±0.04, 0.81±0.05) was significantly highter than in the model group (p<0.05). The levels of caspase-3 protein expression from the model group (2.98±0.17) was significantly highter than that in the sham group(0.86±0.05) (p<0.05), and that from the experiment groups (respectively 2.44±0.19, 1.62±0.16, 1.69±0.11) was significantly lower than in the model group (p<0.05).Biochemical Test. The SOD activity from the model group (52.43±3.25U/h·mg-1·prot-1) was significantly lower than that in the blank group(69.86±6.52U/h·mg-1·prot-1,p<0.05), and that from the experiment groups(respectively 61.48±3.05, 64.04±4.64, 66.99±2.75 U/h·mg-1·prot-1)was significantly higher than in the model group (p<0.05). The MDA activity from the model group (9.09±0.41 U/h·mg-1·prot-1) was significantly higher than that in the blank group(5.37±0.65)U/h·mg-1·prot-1,p<0.05), and that from the experiment groups (respectively 5.71±0.58, 6.39±0.38, 7.12±0.29 U/h·mg-1·prot-1) was significantly lower than in the model group (p<0.05). The MAO activity from the model group (7.88±0.76U/h·mg-1·prot-1) was significantly higher than that in the blank group(5.17±0.33U/h·mg-1·prot-1,p<0.05), and that from he experiment groups(respectively 7.07±0.81, 7.13±0.53, 6.36±0.36U/h·mg-1·prot-1) was significantly lower than in the model group (p<0.05). The content of lipofuscin in brain from the model group (5.42±0.24μg/g) was significantly higher than that in the blank group(4.29±0.33μg/g) (p<0.05), and that from the experiment groups (respectively 4.89±0.17, 4.52±0.20, 4.23±0.35) was significantly lower than in the model group (p<0.05). Transmission electron microscope investigations. By the light microscopy and electron microscopy observation,the neurocyte decreased and deformed in model group rats. TLJN Oral Solution can ameliorate this pathological changes.ELISA. Protein level of TNFαand IL-1βin serum was raised in model groups, in which TNFαwere 85.43±6.55 and IL-1βwere 68.29±4.37, compared with the sham group(TNFαwere 85.43±6.55 ,IL-1βwere 68.29±4.37,p<0.01). In experiment groups ,which TNFαrespectively were (100.08±9.29,99.97±6.09 and 85.90±4.40) and IL-1βrespectively were (77.56±6.97,77.85±3.81 and 67.87±2.38) , protein level of TNFαand IL-1βwas reduced compared with the model group (p<0.01).Conclusion1,These results suggest that AD rat model could be induced by amyloid-β1-40 sterotaxis injection the bilateral hippocampus. 2,TLJN Oral Solution can ameliorate this pathological changes of AD rat model. 3,TLJN Oral Solution improve the ability of learning and memory of AD model rats and its related mechanisms least relevant to that it possesses a certain improvement the protein expression of the ChAT, nNOS, SS, and synapsin, decreased the protein expression of AChE. 4,TLJN Oral Solution Possesses a certain up-regulating expression of Bcl-2 mRNA and down-regulating Bax mRNA and caspase-3 protein, it can decrease apoptosis, so 5TLJN Oral Solution has the neuro-protective effect against Alzheimer disease. 5,TLJN Oral Solution can alleviate the damage of free radical by decrease MAO and MDA activity and increase SOD activity. 6,TLJN Oral Solutioncould decrease BACE1 and APP mRNA expression. Accordingly it can decrease the amount of Aβin hippocampus. 7,TLJN Oral Solutionmight suppress the inflammatory by decreasing protein expression of TNFα,IL-1β, CRP and NK-κB. |
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