Study On The Improvement Of Learning And Memory And Synaptic Plasticity In Aged MCI Rats With Phlegm-turbidity Syndrome By Ditan Decoction

ObjectiveThe theoretical research part of this thesis aims at discussing the etiology and pathogenesis of mild cognitive impairment（MCI）in the elderly from the perspective of"phlegm evil is the cause of amnesia and dementia",and clarifying the theoretical basis of the classic Traditional Chinese Medicine prescription"Ditan Decoction"for the treatment of elderly MCI with"strengthening spleen and eliminating phlegm,invigorating qi and resolving turbidity,awakening brain and opening resuscitation"as the basic treatment principles.In the experimental study,we used general observation,behavioral and biochemical methods to observe and analyze the effects of Ditan Decoction on the manifestation of phlegm-turbidity syndrome,learning and memory ability,lipid metabolism and oxidative stress index in the aged MCI rat model of phlegm-turbidity syndrome,and further used histomorphology,neuroelectrophysiology and protein detection methods,aiming at exploring and analyzing the possible mechanism of Ditan Decoction in improving learning and memory impairment in model rats from the perspective of synaptic plasticity.MethodTheoretical ResearchIn the theoretical research part,this paper reviews and summarizes the records and understanding of TCM on MCI’s name,location and symptoms,and emphatically explores and analyses the etiology and pathogenesis of MCI from the perspective of"phlegm is pathogenic".It also summarizes and analyses the TCM foundation,clinical and epidemiological studies of MCI.Combined with the analysis of the prescription of"Ditan Decoction"and the preliminary study of the tutor’s team,the paper provides a theoretical basis for the theory of"Ditan Decoction"in treating senile MCI of Phlegm-Turbid syndrome from the perspective of treating phlegm.Experimental StudyThe experimental study was carried out in two parts.13-month-old male SD rats of SPF grade were randomly divided into old control group,model group,western medicine group,high-dose group of Ditan Decoction and low-dose group of Ditan Decoction.Three-month-old male SD rats of SPF grade were chosen as young control group.An experimental animal model of aged mild cognitive impairment with phlegm turbidity syndrome was established by subcutaneous injection of D-galactose(50mg·kg^-1)into the back of the neck for 8 weeks and with semi-high fat diet for 4 weeks（in the first four weeks only injecting D-galactose and from the start of the fifth week,adding semi-high fat diet）.Starting from the5th week,each group began to be given intragastric administration for 4 weeks,10 ml·kg^-1,once a day.The western medicine group was given donepezil hydrochloride,0.9mg·kg^-1.The dosage of Ditan Decoction in low and high dosage groups was converted into 1 or 2times of human dosage（4.275g·kg^-11 and 8.55g·kg^-11 respectively）,and the other three groups were given distilled water of equal volume.In the first half of the experiment,Morris water maze and shuttle box were used to test and evaluate the learning and memory ability of rats in each group.Blood lipid levels were measured by biochemical analyzer.T-AOC and GSH-PX contents in hippocampus were measured by colorimetry.The effects of Ditan Decoction on learning and memory,lipid metabolism and oxidative stress were observed and discussed.In the second half of the experiment,Nissl staining was used to observe and analyze the number of hippocampal neurons in each group,Golgi staining was used to observe and analyze the morphology,number and composition of dendritic spines in hippocampal tissue of each group.Neuroelectrophysiological method was used to detect the long-term potentiation effect of hippocampal CA1 area in each group.Western blot was used to detect the levels of synaptic-related proteins such as SYP,PSD95,NR2B,etc.To explore the possible mechanism of Ditan Decoction in improving learning and memory impairment in model rats from the perspective of synaptic plasticity.ResultTheoretical ResearchMCI belongs to the category of amnesia and dementia of Traditional Chinese Medicine.Its location is in the brain,and its onset is related to the five Zang organs.Its clinical manifestations are characterized by deficiency and excess,and both deficiency and excess.The pathogenesis of"dementia"caused by phlegm evil is its important pathogenesis.Whether it is the traditional theories of Chinese medicine,such as"more phlegm for dementia"and"treating phlegm as a treatment for dementia",or the existing research on the basis of traditional Chinese medicine,clinical and syndrome,all provide a detailed basis for the treatment of MCI from phlegm.From the perspective of treating phlegm syndrome,Ditan Decoction takes"strengthening spleen and eliminating phlegm,invigorating qi and resolving turbidity,awakening brain and opening resuscitation"as the basic method to treat cognitive impairment.It has good curative effect in basic and clinical research.All the medicines in Ditan Decoction prescription contain beneficial components for cognitive function.It may play a multi-target combined role through synergy among them to enhance the overall therapeutic effect.Experimental Study1.The effects of Ditan Decoction on learning and memory function,lipid metabolism and oxidative stress of aged MCI with Phlegm-Turbid syndrome model rats:（1）General observation:The model group rats showed the characteristics of Phlegm-Turbid syndrome,such as dirty fur,depression,laziness,poor appetite and pasty stools.Ditan Decoction could improve the Phlegm-Turbid syndrome of model rats.（2）Morris water maze experiment:the latency of positioning navigation evasion in model group was significantly prolonged,the space exploration experiment showed that time consumed for crossing underwater hidden platform for the first time was significantly prolonged,number of trips across the platform was significantly reduced,and the exploration time of target quadrant was significantly reduced,compared with the control group,there was a significant difference（P<0.01）;compared with model group,the escape latency of traditional Chinese medicine Ditan Decoction group and Western medicine donepezil hydrochloride group were significantly prolonged.Time consumed for crossing platform for the first time were shortened significantly,number of trips across the platform were increased significantly,and the exploration time of the target quadrant were significantly prolonged（P<0.01 or P<0.05）.（3）Shuttle box experiment:Compared with the control group,the number of active avoidance was significantly reduced,the number of electric shocks was significantly increased,the latency of active avoidance and the duration of electric shocks were significantly prolonged in the model group（P<0.01）;after administration,compared with the model group,the number of active avoidance in the high,low dose of Ditan Decoction group,and Western medicine group were significantly increased,the number of electric shocks were significantly reduced,the latency of active avoidance and the duration of electric shock were significantly prolonged（P<0.01 or P<0.05）;there was no significant difference between the high-dose Ditan Decoction group and the western medicine group（P>0.05）;there was significant difference between the high-dose and the low-dose of Ditan Decoction group in the number of active avoidance,the number of electric shocks and the duration of electric shocks（P<0.05）.（4）Blood lipid test:Compared with the control group,TC（total cholesterol,TC）,TG（triglyceride,TG）,LDL-C（low density lipoprotein cholesterol,LDL-C）in the model group increased（P<0.01 compared with the young group,and P<0.05 compared with the old group）;compared with the model group,TC,LDL-C,TG in the high-dose group of Ditan Decoction decreased（P<0.05）,TG decreased significantly（P<0.01）,and TG in the low-dose group of Ditan Decoction decreased（P<0.05）.There was no significant change in donepezil hydrochloride group（P>0.05）.（5）Detection of T-AOC and GSH-PX contents:Compared with the control group,the contents of T-A0C and GSH-PX in hippocampus of model group rats decreased significantly（P<0.01）;compared with model group,the contents of T-A0C and GSH-PX in hippocampus of model rats were significantly increased by Ditan Decoction and Donepezil Hydrochloride（P<0.01 or P<0.05）.2.The effect of Ditan Decoction on synaptic plasticity in aged MCI model rats with Phlegm-Turbid syndrome:（1）Nissl staining:Compared with the control group,the number and morphology of Nissl bodies in the model group decreased,and the number of neurons in the model group decreased significantly（P<0.01）;compared with the model group,the number of neurons in the high-dose Ditan Decoction group increased significantly（P<0.05）.（2）Golgi staining:Dendritic spines in model group were irregular in shape,loosely arranged and scarce in number.Compared with the elderly control group,the total number of dendritic spines and the number of mushroom-like dendritic spines were significantly reduced（P<0.01）;the total number of dendritic spines and mushroom-like dendritic spines in the high-dose group of Ditan Decoction and the western medicine donepezil hydrochloride group were increased,with significant difference（P<0.05）.（3）Electrophysiological LTP test:The rats in model group could not induce stable LTP smoothly.Compared with the control group,the amplitude of LTP was lower and the slope of EPSP decreased significantly（P<0.01）;compared with model group,the slope of EPSP and the amplitude of LTP in high-dose Ditan Decoction group increased significantly（P<0.01）,while there was no significant change in western medicine group and low-dose Ditan Decoction group（P>0.05）.（4）Western blot analysis:Compared with the control group,the expression of synaptic related proteins SYP,PSD95 and NR2B in hippocampus of the model group was significantly decreased（P<0.01）;compared with the model group,the expression of PSD95 and NR2B in the low-dose group of Ditan Decoction and the western medicine Donepezil Hydrochloride were significantly increased（P<0.01）,and the expression of NR2B was also increased（P<0.05）;the expression of PSD95 and NR2B in the high-dose group of Ditan Decoction was increased significantly（P<0.01）,and the expression of SYP also increased（P<0.05）.Conclusion1.MCI belongs to the category of amnesia and dementia of Traditional Chinese Medicine.Its location is in the brain,and its onset is related to the five Zang organs."Phlegm evil"is an important pathogenesis of MCI.There is substantial evidence to treat MCI based on the theory of phlegm syndrome.Ditan Decoction may play an integral therapeutic effect on MCI through the synergistic effect of various drugs in its prescription.2.D-galactose subcutaneous injection combined with semi-high fat diet was successfully used to construct a model of mild cognitive impairment in the elderly with Phlegm-Turbid syndrome rats.The model rats showed Phlegm-Turbid syndrome,spatial learning and memory impairment,reduced avoidance response ability,lipid metabolism disorder and oxidative stress injury.3.Ditan Decoction can improve the syndrome of phlegm turbidity,alleviate lipid metabolism disorder and oxidative stress injury,which may lead to ameliorate hippocampal-dependent learning and memory impairment in model rats.4.Pathological changes of synaptic plasticity were observed in model rats,which were as follows:loss of hippocampal neurons,hypofunction,damage of synaptic microstructures and long-term potentiation effect,and decrease of synaptic-related protein expression.5.Ditan Decoction may improve learning and memory impairment by repairing damaged hippocampal neurons and synaptic microstructures,enhancing long-term potentiation effect,up-regulating the expression of synaptic related proteins SYP,PSD95 and NR2B,which may improve synaptic structure and functional impairment and lead to restore the damaged synaptic plasticity to a certain extent in model rats.