Tongxinluo Right Ventricles Of The Brain β-amyloid Protein In Alzheimer's Disease Model Rats Induced By Injection Of Intervention Research

Objective: Usingβ-amyloid peptide (Aβ1-42) inject to intracerebroventricular establish AD rats model, using Tongxinluo to intervene, through observing the activity of learning and memory of rats, the indicators change of oxidative stress in the serum and brain tissue, and cholinergic in the brain tissue, to explore the therapeutic effects of Tongxinluo on Alzheimer's disease, and provide experimental evidence for the treatment of Tongxinluo on Alzheimer's disease in clinical.Methods:1. The effects of Tongxinluo on behavior in AD model rats 70 healthy male SD rats, which was 250 to 300 g, were randomly divided into seven groups: (1) normal control group; (2) sham-operated group; (3) Tongxinluo low-dose group(0.6g/kg.d); (4) Tongxinluo middle dose group(1.2g/kg.d; (5) Tongxinluo high-dose group(2.4g/kg.d); (6) Ginaton group(0.032g/kg.d); (7) model control group. Usingβ-amyloid peptide (Aβ1-42) inject to intracerebroventricular establish AD rats model, the sham-operated group inject physiological saline,using drugs to intervene after three days of operation ,give physiological saline to normal control group, sham operation group, model group. 21 days after gavage, using Morris water maze to test the learning and memory capacity of rats in each group, a total of 6 days.2. The effects of Tongxinluo on MDA, SOD, and GSH-Px in AD model ratsThe packet and the administration were the same to the experiment one, 30 days after gavage, take the blood from the rats carotid artery, separate the serum standby. Decapitated ,remove the brain ,cut the hippocampus , homogenate, centrifugal , Using xanthine oxidase assay to detect SOD activity, Using sulfur on behalf of the barbituric acid (TBA) colorimetric assay to detect MDA content, Using chemical colorimetric assay to detect GSH-Px activity.3. The effects of Tongxinluo on the activity of ChE, AchE in AD model ratsThe packet and the administration were the same to the experiment one, 30 days after gavage, remove the brain, cut the hippocampus, homogenate, centrifugal, using thio-D-choline assay to detect ChE activity, using colorimetric detection to detect AchE Activity.4. The effects of Tongxinluo on APP, Aβexpression of hippocampus in AD model ratsThe packet and the administration were the same to the experiment one, 30 days after gavage , remove the brain after cardiac perfusion , fixed with 4%paraformaldehyde (PH=7.4), conventional gradient alcohol dehydration , Xylene transparent, Dipping wax ,Paraffin-embedded , Cortex and hippocampus of serial sectioning , the thickness of Slice were 3μm, paste it into the glass ,which was treated by the poly-lysine-treated , using immunohistochemistry to detect the expression of APP.Slice was dewaxing to water, Congo red dye liquor stained , with alkaline ethanol solution differentiation differentiation, washed, counterstained with hematoxylin, dehydrated, transparent, zonal isolation, detect the expression of Aβin the hippocampus.5. The effects of Tongxinluo on Tau protein expression of hippocampusin in AD model ratsThe packet and the administration were the same to the experiment one, 30 days after gavage , emove the brain ,cut the hippocampus quickly , dgrinding, centrifugation.After gel electrophoresis, remove the gel, cut membrane ,according to the protein molecular weight, add Tau,β-actin primary antibody respectively, overnight incubation at 4℃, add two resistant and incubated at room temperature 1h. Light color with ECL, photo togel imaging system, using quantity one software to analysis the results, scanning optical density (OD) values, the protein expression levels relative was expressed with the ratio of tau protein and theβ-actin.Results:1. The changes of water maze behavior in different groups1.1 Latency(s): During the experiment, compared with the normal group,the escape latency time of the model group was prolonged significantly,there had statistical significance (P<0.05, P<0.01), compared with the normal group,there was no significant difference in the sham group(P>0.05).In the first day, there had no significant difference between the each treatment group and the model group (P>0.05); In the second day, compared with the model group, the latency of The Tongxinluo high-dose group, the Tongxinluo low-dose group and the Ginaton group were shorter significantly, and the difference has statistically significant (P<0.05,P<0.01), while the Tongxinluo middle-dose group has no significant difference (P> 0.05); the first 3-6 days, compared with the model group, there had significant difference in each treatment group (P <0.05, P <0.01).1.2 Total distance (cm): During the experiment, compared with the normal group,the total distance of the model group was prolonged significantly,there had statistical significance (P<0.05, P<0.01), compared with the normal group,there was no significant difference in the sham group(P>0.05).In the first day, there had no significant difference between the each treatment group and the model group (P> 0.05); In the second day, the total distance of the Ginaton group was significantly shorter than the model group(P<0.01); In the third day, the total distance of the Tongxinluo middle dose group and the Ginaton group were significantly shorter than the model group (P <0.05); In the fourth and the fifth day ,the total distance of the Tongxinluo high-dose group, the Tongxinluo middle dose group, and the Tongxinluo low-dose group were significantly shorter than the model group (P <0.05 , P <0.01), In the sixth day , the total distance of each treatment group were shortened, compared with the model group, there had significant statistically difference in the Tongxinluo middle-dose group,the Tongxinluo low-dose group and the Ginaton group. (P <0.05, P <0.01)2. The changes of SOD activity, MDA content, GSH-Px activity in different groups2.1 The changes of SOD activity, MDA content, GSH-Px activity in the brain in different groupsCompared with the normal group , the SOD activity, the GSH-Px activity were significantly decreased ,the MDA content was significantly increased in the model group , the differences had statistically significant (P<0.01),there were no significant difference betwwen the sham-operated group and the normal group (P> 0.05);Compared with the model group , the SOD activity were significantly increased , MDA content were significantly decreased in each treatment group , the differences had statistically significant (P<0.05,P<0.01); compared with the model group , the GSH-Px activity were significantly increased in Tongxinluo large, medium-dose group, the differences had statistically significan(tP<0.01), while the Tongxinluo low-dose group, the Ginaton group showed no significant difference (P> 0.05).2.2 The changes of SOD activity, MDA content, GSH-Px activity in serum in different groupsCompared with the normal group , the SOD activity, the GSH-Px activity were significantly decreased ,the MDA content was significantly increased in the model group , the differences had statistically significant (P<0.01),there were no significant difference betwwen the sham-operated group and the normal group (P> 0.05);Compared with the model group , the SOD activity were significantly increased , MDA content were significantly decreased in each treatment group , the differences had statistically significant (P<0.05,P<0.01);while on the GSH-Px activity, only see the Tongxinluo high-dose group had significantly higher, the difference had statistically significant ( P <0.01).3. The changes of the ChE, AchE activity in different groupsCompared with the normal group ,the ChE, AchE activity were significantly increased in the model group , there had statistically significant (P<0.01), there were no significant difference betwwen the sham-operated group and the normal group (P> 0.05);Compared with the model group , the ChE, AchE activity were significantly decreased in each treatment group, the difference had statistically significant (P <0.01).4. The changes of the expression of APP, Aβin hippocampus in different groups4.1 The changes of the expression of APP in hippocampus in different groupsIn the Normal group, there had few positive cells of APP, and the staining was weak, the expression was low, there had a small amount of the distribution in the hippocampus and cerebral cortex part. In the sham-operated group, there had few positive cells of APP, and the staining was weak, the expression was low, there had a small amount of the distribution in the hippocampus and cerebral cortex part .In the Model group there had a large number positive cells of APP, and the staining was strong positive, there had widely distributed in the hippocampus, there had a small amount of the distribution in the cerebral cortex part. In the Tongxinluo large, middle, low-dose groups, there had positive cells of APP, and the staining was strong positive, there had widely distributed in the hippocampus, there had a small amount of the distribution in the cerebral cortex part. In the Ginaton group, there had a large number positive cells of APP, and the staining was strong positive, there had widely distributed in the hippocampus, there had a small amount of the distribution in the cerebral cortex part.4.2 The changes of the expression of Aβin hippocampus in different groupsThe normal group: Congo-red staining negative, clear and rich in Negri bodies. The sham group: Congo red staining negative, clear and rich in Negri bodies. The model group: Congo red staining showed positive reaction, neuron Negri bodies with pale color, structural is disorder and not clear. The Tongxinluo high-dose group: Congo red staining showed positive reaction, neuron Negri bodies with pale color. The Tongxinluo middle dose group: Congo red staining showed positive reaction, neuron Negri bodies with pale color. The Tongxinluo low-dose group: Congo red staining showed positive reaction, neuron Negri bodies with pale color. The Ginaton Group: Congo red staining positive neurons Negri bodies with pale color, structural is disorder and not clear.5. The changes of Tau protein expression in Hippocampus of rats in different groupsCompared with normal group, OD values of phosphorylated Tau protein in model group was significantly higher, and there is statistical significance (P <0.01); while the sham-operated group compared with the normal group showing no significant difference (P> 0.05); compared with model group, the phosphorylated Tau protein OD values of medication groups were significantly reduced, and there is statistical significance (P <0.05, P <0.01).Conclusion:1. Collateral disease theory is the theoretical characteristics of integral part of Traditional Chinese MedicineYuanqi deficiency of blood network is unable to transport blood, blood circulation slow, blood coagulation astringent, then Collateral blood Stagnation. Collateral blood Stagnation was the key factor and important pathological aspect of the incidence and increased dementia Stagnation. Qi, blood and body fluid run abnormaly, and in vivo physiological or pathological product could not be timely removed, accumulation in vivo, and so generate blood stasis.Blood stasis in Collateral blood,"Blood moistures it" functional disorders, which occurred dysfunction at the end of the blood and supply, fluid and blood exchange, nutrition metabolic, so it can not provide material basis for qi-Collateral functional activity. Qi-Collateral lose blood and other nutrients, then increased qi-Collateral damaged, which warmth nursed back to health, and information communication dysfunction, the emergence of dementia syndromes.2. Tongxinluo can significantly improve the learning and memory function obstacle to the AD model rats , its role were achieved by decreasing the oxidative stress , which was caused by the body free radicals , decreasing the activity of cholinesterase to protect the function of the cholinergic system , decreasing the Aβdeposition , which was caused by APP over-expression , decreasing the degeneration and necrosis of neurons , which was caused by phosphorylation of tau protein over-expression .