| BackgroundAlzheimer's disease is a neurodegenerative disease and which is characterized by senile plaques(SP)formed by over-deposition of soluble ?-amyloid and neuofibillary tangles(NFTs)in the brain.The brain A?1-42 fragment is the longest,most toxic,most likely to gather in the process of neurodegeneration strongest.Chinese medicine Rehmannia is Marrow-filling and marrow-nourishing medicine,Schisandra can tranquilize the mind,replenish qi and product jing,tonify the kidney to relieve mental strain,traditional chinese medicine think that the brain is the material basis of intellectual activity,and brain depend on Kidney Essence nourishing.Schisandra and Rehmanniae drugs alone have anti-aging,puzzle,improve cognitive function and protect the role of nerve cells,but Schisandra-prepared radix rehmanniae combined treatment of AD has not been reported.PurposeCompared with the single drug Schisandrins or prepared radix rehmanniae,the effects of combined treatment of Schisandra chinensis and prepared radix rehmanniae on the learning ability and memory impairment of the AD model rats were observed,and the mechanism of action was further explored.Methods1 Alzheimer's disease model replication and model success criteria.Male SD rats(220±20 g)were mounted on a stereotaxic apparatus and incisions were made.The approximately 1.5 cm incision was made along the sagittal line,two coordinate points were determined at a distance of 2.1 mm on the two sides of the anterior malleolus about 3 mm,using dental drill drilled a small hole at two coordinate points and used a Microsyringe to inject the needle 3 mm vertically into thehippocampal CA1 area.The cerebral hippocampal CA1 area was injected with 5 ?l of A?1-42 solution,and the sham group was injected with 0.9% normal saline 5 ul.On the next day after waking up,the rat was subcutaneously injected with a 25% D-galactose solution 150 mg/kg subcutaneously on the back of the neck.The sham group was injected subcutaneously with the same volume of saline once a day for 35 consecutive days.New object recognition test and Morris water maze test were performed on the28 th day after A?1-42 injection.The criteria the success of the model: Compared with the average evasion latency of sham-operated rats,the escape latency of Morris water maze in each model rat was significantly prolonged,and the residence time in the quadrant of the platform was significantly reduced,and the number of platform crossings was significantly reduced(all P<0.01).Moreover,the appearance of "dementia" symptoms such as apathetic,unstable gait,unresponsiveness,low activity,and low dietary drinking water indicate that the AD model was successfully prepared.2 Alzheimer's disease model success animal was grouped and balance test between group and administrationThe number of horizontal movements of successful model rats were recorded by open-field experiments.The times of horizontal movements of successful model rats was sorted numbers from highest to lowest,and the number was mapped to the random number table.Finally,Model rats were divided into 7 groups by randomized block design method: model group,Huperzine A(0.3 mg/kg),Schisandra chinensis 1.6 g/kg group,prepared radix Rehmannia 1.6 g/kg group,and combination drug 3.2 g/kg.Kg group,1.6 g/kg group,0.8 g/kg group,10 in each group.The average of horizontal exercise times was counted in the above-mentioned each group rats,the results showed no significant difference(all P<0.05),followed by administration and maded subsequent trials.Gavage was started on the third day after surgery,once a day for 28 consecutive days.3 Effect of Schisandra-Rehmannia glutinosa granule improve learning and memory in Alzheimer's disease model ratsAfter the gavage was completed,learning and memory capability of rats was detected by Morris water maze and The Novel Object Recognition Test.(1)Morris water maze spatial memory capacity determination: 1 to 5 days fornavigation test,recording the escape latency of rats.The sixth day is the space exploration test,the platform is removed,which the time and distance the rats swimming in the quadrant where the platform is located were recorded and analyzed.(2)new object recognition non-spatial memory ability test: the old and new object substitution method to test the contact time of rat contact time,a total of 3 days,rat new and old objects of the inquiry time were recorded as Tn and Tf.The recognition and memory ability of rats exposed to old and new objects was evaluated by the discrimination index DI =(Tn-Tf)/(Tn + Tf)× 100%.4 Effect of Schisandra-Rehmannia granule on Cerebral Pathology in Alzheimer Disease Model RatsThe rats were euthanized and stained with hematoxylin-eosin(HE)to observe the damage of hippocampal neurons and Nissl body.Nissl staining was used to observe the damage of Schisandra-Rehmannia granule to hippocampal neurons and Nissl body.Finally,Pathological morphology were ameliorated,and analysis the differences between the indicators of the group.5 Mechanism of Schisandra-Rehmannia granule improving Learning and Memory in Alzheimer Disease Model RatsThe total protein was extracted from the hippocampus and the expression of BDNF-TrkB-CREB protein and synaptic functional protein PSD95 and SYP were detected by Western blotting.The contents of acetylcholinesterase(AChE)and choline acetyltransferase(ChAT)in hippocampus were measured by ELISA kit.6 Effect of Schisandra-Rehmannia granule on A? and TAU Synthesis in AD Model RatsBlood was collected from the abdominal aorta to separate the serum,and the cerebral cortex and hippocampus tissues were separated to determine the contents of A?1-42 and A?1-40 in the serum and the hippocampus.The hippocampus was separated and the expression of AKT,GSK3?,A?1-42,APP and TAU in Alzheimer's disease were detected by Western blot,Immunohistochemistry was used to inspect the expression of A?1-42 protein in hippocampus.Results1 Schisandra-Rehmannia granule improved the AD model rat's constitution and weight better than single drugCompared with the sham-operated group,all the rats in the model group had slowresponse to varying degrees,decreased diet,decreased water intake,reduced urine and urine,lack of luster,yellow hair,and manifest drowsiness.Compared with model group rats,The above symptoms of Schisandra chinensis 1.6 g/kg group,Rehmannia 1.6 g/kg group,combined drug 0.8 g/kg group,1.6 g/kg Group and 3.2 g/kg group were significantly ameliorated.Compared with the sham group,body weight of the model group rats was significantly decreased at the third,fourth,and fifth week after administration(P<0.01).Compared with the model group,the body weights of the rats in the 0.8 g/kg combination group,1.6 g/kg group and 3.2 g/kg group were significantly increased(all P<0.05);Compared with Schisandra 1.6 g/kg group or Rehmannia glutinosa 1.6 g/kg group,the body weight of the combined drug 0.8 g/kg group and 1.6g/kg group significantly increased(all P <0.05).2 Schisandra-Rehmannia granule improve learning and memory ability of AD model rats better than single drug1 The Positioning navigation test compared with the sham-operated group,the escape latency of the model group rats on the second,third,fourth,and fifth days was significantly prolonged(all P<0.01),indicating that A?1-42 Significantly reduce the spatial memory ability of rats.Compared with the model group,the escape latency of combination drug 1.6 g/kg group and 3.2 g/kg group was significantly shortened on the above daily(P<0.01).Compared with Schisandra 1.6 g/kg group or Rehmannia glutinosa1.6 g/kg group,the escape latency of combined drugs 1.6 g/kg group and 3.2 g/kg group was significantly shortened on the above daily(all P<0.01).It shows that the combination drugs 1.6 g/kg and 3.2 g/kg can significantly improve the escape latency of AD model rats,and the combined use of drugs is better than single drug.2 The space exploration test there was no significant difference in mean swimming speed between groups(P>0.05).Compared with the sham group,the time and number of passing through the platform of the model group were significantly decreased,and the distance between the platform and the target quadrant of the model group decreased significantly,The dwell time in the platform area and the quadrant crossing of the target quadrant were also significantly reduced(P<0.01).Indicating that A?1-42 can significantly reduce the spatial memory retention in rats.Compared with the model group,The above indicators of Schisandra 1.6 g/kg group,the Rehmanniae1.6 g/kg group,the combined drug 0.8 g/kg group,1.6 g/kg group and 3.2 g/kg group were significantly increased(P<0.01).Compared with Schisandra 1.6 g/kg group orRehmannia glutinosa 1.6 g/kg group,the combined drug 1.6 g/kg group were significantly increased(all P <0.05).3 Schisandra-Rehmannia granule improved Cerebral Pathological Structure in AD Model Rats better than single drugHE staining and Nissl staining sections were observed under the light microscope,Neurons in the hippocampus of rat model in the model group were disordered,the size and shape of Nissl body were irregular,the number was small,the distribution was uneven,the whole was not clear enough,the outline was fuzzy,and even a large amount of Nissl body dissolved and disappeared.Disorganized vertebral cells,vacuolar degeneration of some cells,a large number of cells shrinkage necrosis.Compared with the model group,Schisandra 1.6 g/kg group,the Rehmanniae 1.6 g/kg group,the combined drug 0.8 g/kg group,1.6 g/kg group and 3.2 g/kg group of rat brain tissue hippocampal CA1 and CA3 area tissue staining was shallow,Clear cell boundaries,neatly arranged,the cell structure and light nucleolus;neurons arranged sparse disorder,Nisshin staining darker,more number,clear outline,more rounded shape.Brain slices of the combined drug 1.6 g/kg group,3.2 g/kg group tend to sham operation group,indicate that the combined drug 1.6 g/kg group,3.2 g/kg group can significantly improve AD model rats brain pathology,the effect is better than single medicine.4 The Mechanism of Schisandra-Rehmannia granule improved learning and memory is related to memory protein in AD model ratsCompared with the sham group,the expression of BDNF,TrkB,CREB,SYP,and PSD95 in the hippocampus of the model group rats were significantly decreased(P<0.01);Compared with the model group,The expressions of BDNF,TrkB,CREB,SYP and PSD95 in Schisandra chinensis 1.6 g/kg group,Rehmannia 1.6 g/kg group,combinated drug 0.8 g/kg group,1.6 g/kg group and 3.2 g/kg group were significantly increased(P<0.05);Compared with the Rehmanniae 1.6 g/kg group,The expressions of BDNF and SYP in the three combination dosage groups were significantly increased.the expression of PSD95 in the combination drug 1.6 g/kg group was significantly increased.The expression of CREB protein was also increased in the combinated drug0.8 g/kg group and 1.6 g/kg group,while the expression of TrkB protein was significantly decreased in the combination drug 3.2 g/kg group(all P<0.05).Compared with the Schisandra chinensis 1.6 g/kg group,the expression of BDNF protein was significantly increased in the three dose groups of combined drugs,and the expressionof TrkB protein was significantly decreased in the combined drug 3.2 g/kg group(P<0.05).Compared with the sham group,the ChAT content in the hippocampus of the model group rats was significantly reduced,and the AChE content was significantly increased(all P<0.01).Compared with the model group,the ChAT content in the hippocampus of the rats in the Schisandra chinensis 1.6 g/kg group,Rehmannia 1.6 g/kg group,combinated drug 0.8 g/kg group,1.6 g/kg group and 3.2 g/kg group increased significantly.The AChE content was significantly decreased(all P<0.05);Compared with Schisandra chinensis 1.6 g/kg or Rehmannia radix 1.6 g/kg group,the ChAT content in the hippocampus of the combined drug 1.6 g/kg group was significantly increased,the AChE content were significantly reduced(all P <0.01).5 Effect of Schisandra-Rehmannia Granule on Mechanism of A? and TAU Protein Synthesis in AD Model RatsCompared with the sham operation group,the levels of A?1-42 and A?1-40 in the hippocampus and serum of the model group were significantly increased(P<0.01);compared with the model group,The levels of A?1-42 and A?1-40 in serum and the hippocampus were all significantly decreased(P<0.05).Compared with Schisandra chinensis 1.6 g/kg or Rehmannia radix 1.6 g/kg group,The content of A?1-42 in the hippocampus of all the combined drug groups was significantly decreased(P<0.05),and the combined drug 1.6 g/kg group also significantly reduced A?1-42,A?1-40 levels in the serum and A?1-40 levels in the hippocampus(P<0.05).Compared with sham operation group,AKT protein expression was significantly decreased and GSK3?,APP,TAU and A?1-42 protein expressions were increased(P<0.01).Compared with the model group,the expression of AKT protein in hippocampus of rats in each group was significantly increased(P<0.05),the expression of GSK3?,TAU,APP and A?1-42 protein was decreased(P<0.05).Compared with Schisandra 1.6 g/kg group or Rehmannia glutinosa 1.6 g/kg group,The combinated drug 0.8 g/kg and 1.6 g/kg group significantly increased the AKT expression(P<0.05),The combinated drug 1.6 g/kg group significantly reduced GSK3?,APP and A?1-42 protein expression(P<0.05),the combination drug of three doses group significantly reduced the expression of TAU protein(P<0.05).Immunohistochemistry also has similar results.Conclusions1 Constitutional effect observation Conclusion: the combination drug 0.8 g/kg ~3.2g/kg can significantly improve Shenpi fatigue,stupid reaction,dullness,physical Poor,rough coat,poor diet,less defecation or loose stools and other conditions in the AD model rats.And it can significantly increase the weight of rats.2 Effect of memory observed Conclusion: The combined drug of 1.6 g/kg~3.2 g/kg significantly increased the relative discrimination index,significantly improved AD model rats memory recognition.Shortening the escape latency,increasing the frequency and time of crossing the platform,extending the target quadrant dwell time and the target quadrant distance,increasing the platform area dwell time and the target quadrant crossing frequency significantly improved the spatial memory ability of AD model rats.3 Morphological observation Conclusion: The combined drug 0.8 g/kg~3.2 g/kg can significantly promote the repair of Nissl's corpuscles in the brain of AD rats and reduce neuronal cell damage.4 Compared with the same dose of single schisandra or single rehmannia,the combinated drug can improve the physical constitution of AD rats,increase the body weight of rats,enhance the learning and memory abilities of rats in positioning navigation and space exploration experiments,and improve brain tissue damage.It's effect is better than single drug.5 The conclusion of mechanism: The combined drug 0.8 g/kg~3.2 g/kg can significantly increase SYP,PSD95,BDNF,TrkB and CREB protein expression related to learning and memory,significantly increase the choline acetyltransferase Content,and significantly reduce the content of acetylcholinesterase,increase the expression of AKT protein,decrease the expression of GSK3?,APP,TAU and A?1-42 protein,and reduce the synthesis of A? and TAU protein in the AD model rat brain.And the content of A?1-40 and A?1-42 of hippocampus and serum association with pathological A? and TAU protein synthesis is significantly decreased in the AD model rat brain. |
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