| Objective The etiology and pathogenesis,the drugs using regularity and the rule of treatment of vascular dementia were discussed,and the inhibitory effect on the vascular dementia and possible mechanism of the prescription were studied in activating blood circulation and eliminating phlegm.Methods 1.Theoretical research The relevant theories of dementia from pre-Qin period to modern times sorted out and analyzed using the traditional literature research method;by combining the research progress of traditional Chinese medicine in terms of the syndrome differentiation and treatment of vascular dementia,the etiology and pathogenesis and treatment rules of vascular dementia was discussed;based on the literature research method and by integrating the data mining and association rules,the medicine groups of relevant prescriptions that the doctors of past dynasties used to treat the vascular dementia were sorted out and analyzed,and the drugs using regularity of vascular dementia were explored;from the angle of "theory of qi and blood",based on the preliminary researches of research group,the Naotaifang was modified,and the efficacy of "Modified Naotaifang" in benefiting qi for activating blood circulation and reducing phlegm and possibility of preventing and curing the disease with the drug was discussed.2.Meta-analysis of Yiqi Huoxue Huatan recipe in treating vascular dementia Evaluation of efficacy and safety of Yiqi Huoxue phlegm recipe in treating vascular dementia by meta analysis system,So as to provide comprehensive evidence and decision-making basis for clinicians,and provide the basis for further study of this experiment.3.Experimental study 3.1 animal experiment: the rat model is prepared using the BCCAO,which is interfered with "Modified Naotaifang".The general conditions of rats of all groups are observed.30 days later,the water maze testis performed,and the HE staining is used to observe the changes in the pathomorphism of hippocampus.The immunohistochemistry technology and Western blot were adopted to test the protein expression of SIRT1,NF-κB,IκBα,Bax,Bcl-2 and Caspase-3 of cerebral hippocampus of rats.The nucleic acid amplification test based on real-time fluorescence quantification were performed to examine the expression of gene levels of SIRT1,NF-κB,IκBα,Bax,Bcl-2 and Caspase-3.3.2 In vitro cells: through cultivation of hippocampal nerve cell of primary cultured rat and relevant identifications,the hypoxic model/reoxygenation model was established,and the interference was made with "Modified Naotaifang" and positive-drug serum.MTT method was adopted to test the activities of hippocampal neuron 24 hours after oxygen deprivation and 2 hours of reoxygenation,and the cell viability was calculated;The FCM(flow cytometry)was adopted to test the apoptosis of hippocampal nerve cell;the analysis technology of high content imaging based on the Fluorescent Immunochemistry Stain and Western blot technology were used to test the protein expression of SIRT1,NF-κB,IκBα,Bax,Bcl-2 and Caspase-3 of hippocampal neurons.The real-time fluorescence quantification based nucleic acid amplification test was performed to determine the gene levels of SIRT1,NF-κB,IκBα,Bax,Bcl-2 and Caspase-3.Results 1.Theoretical research: 1.1 The Inner Canon of Huangdi is the theoretical basis of traditional Chinese medicine in understanding the vascular dementia,and the vascular dementia wasn’t proposed as an independent disease until Ming Dynasty.The doctors of Ming and Qing Dynasties found that "the dementia is frequently-occurring disease after stroke",which laid foundations for the differentiation of vascular dementia from the senile dementia;the aged people are more susceptible to the vascular dementia,and the pathogenesis is featured by deficiency in origin and excess in superficiality and the disease is located in brain,with the deficiency of Qi and blood of the five internal organs as the essence and the collaterals blocked by phlegm-stasis as appearance;the weakness,blood stagnation and phlegm are important etiology and pathogenesis.1.2 By exploration and analysis of relevant disease prescriptions that doctors of past dynasties used to treat the dementia,it was found that,the drugs using regularity can be generalized as “taking the qi-tonifying drug,blood-replenishing and activating potion and nerve soothing and resuscitation drugs as the main treatment therapy with phlegmatic hygrosis repelling drugs and drugs of nourishing yin as supplementary medicine.” 1.3 Based on the literature research of ancient times,from the angle of theory of qi and blood,we have combined the progress of modern traditional Chinese medicine in terms of the syndrome differentiation and treatment of vascular dementia,and found that benefiting qi for activating blood circulation and reducing phlegm are important methods of curing the vascular dementia;based on the preliminary researches of research group,we found that the “Jiaweinaotaifang” capable of benefiting qi for activating blood circulation and reducing phlegm can be used to prevent and cure the vascular dementia.2.Meta-analysis of Yiqi Huoxue Huatan recipe in treating vascular dementia Yiqi Huoxue Huatan Recipe or Yiqi Huoxue Qutong Recipe combined with western medicine conventional treatment is superior to the simple western medicine treatment of vascular dementia.3.Experimental result 3.1 General situation: Normal group and sham-operation group: the rat appeared well in terms of hair,diet and spirit.The model group: after the modeling,the rats displayed varying degrees of mental malaise,lags in response,slow in actions and dark hair color.Their lips and nails turned to dark purple,etc;some rats displayed varying degrees of blepharoptosis,decreased palpebral fissure and slight enophthalmos;a small portion of rats displayed irritability,increase activities and unstable movement and they were just keeping turning around and so on.Moreover,the self-cleaning ability of rats decreased,and their hair turned sparse and dry with piloerection.The attack of transient paroxysmal seizure was occasionally observed.Treatment group: after the modeling,in the preliminary stage,the rats were similar with the VD model in terms of the general situation.After drug intervention,compared with the model group,the rats were slowly recovered in terms of diet,spirit and activities,and they were better than rats of model group in general.3.2 Water maze experiment results: compared with model group,the rats’ escape latency in high and low dose group of “Jiaweinaotaifang” significantly shortened,and after removing the platform,the times of traversing the platform significantly increased,which means that the “Jiaweinaotaifang” has a certain improvement effect on the learning and memory of vascular dementia rats. 3.3 The pathological morphology test results: the hippocampal neurons pyramidal cells of rats in model group are sparse;the arrangement is irregular and loose;cell spacing broadened;the volume of hippocampal neurons pyramidal cells became smaller;nuclear volume became smaller;dyeing became darker;nucleoli is unclear;and part of the cells necrotized.Compared with model group,in high and middle dose group of supplemented Naotaifang,the arrangement of hippocampal neurons pyramidal cells is more regular;the cell density increased,the cells’ form is complete and their outline is clear;nuclear volume increased;nucleolus is more clear;there is no evident difference with western medicine Oxiracetam group.3.4 The detection results of immunohistochemical method,Western blot method and real-time fluorescence quantitative detection for nucleic acid amplification show: compared with model group,after the intervention of “Jiaweinaotaifang”,the expression level of SIRT1,IκBα and Bcl-2 in middle and high dose group significantly rose(P < 0.05),while the expression level of Bax,NF-κB and Caspase-3 lowered,and there is a significant difference between middle and high dose group of TCM and model group(P < 0.05).3.5 The detection results of MTT method show that,compared with the blank group,the cell vitality of model group significantly reduced,and the difference has a statistical significance(P < 0.05),suggesting that hypoxia and reoxygenation can have a certain damage effect on hippocampal neurons.Compared with model group,the cell vitality of positive medicine group and supplemented Naotaifang group obviously increased,and the difference has a statistical significance(P<0.05),suggesting that the Supplemented Naotaifang has a certain therapeutical effect on the damage of hippocampal neurons caused by hypoxia and reoxygenation.3.6 The results that FCM detects the cell apoptosis of hippocampus neurons show that,Normal control group,blank serum group,model group,positive drug group,Jiaweinaotaifang group the proportion of apoptosis were 32.17%,70.93%,72.7%,70.93% and 50.87%;and the results of Q3 area living cells show that,compared with the blank group,the living cells of model group significantly decreased,and the difference has a statistical significance(P < 0.05).Compared with the model group,the living cells of positive medicine group and “Jiaweinaotaifang” group obviously increased,and the difference has a statistical significance(P < 0.05). 3.7 The results of fluorescent immunochemistry staining high content imaging analysis technology,Western blot technique and real-time fluorescence quantitative nucleic acid amplification detection show that,compared with the model group,after the intervention of the supplemented Naotaifang drug-containing serum,the expression of SIRT1,IκBα and Bcl-2 significantly rose,while the expression level of Bax,NF-κB and Caspase-3 lowered,and there is a significant difference,P < 0.05.Conclusions 1.Through the theoretical research of TCM and meta-analysis,this paper puts forward that “deficiency,blood stasis and phlegm” are the etiology and pathogenesis of vascular dementia,and Yiqi Huoxue phlegm eliminating method is an important therapy.2.The Supplemented Naotaifang can significantly improve the learning and memory ability of vascular dementia rats.It can significantly improve the expression of SIRT1 protein which is related to learning and memory.On the one hand,it can activate the signal channel of SIRT1/NF-ΚBp65,IκBα expression increased,and NF-κB expression reduced;On the other hand,it can activate the signal channel of SIRT1/Bax,and the expression of Bcl-2 related to the channel significantly increased,but the expression of Bax,Caspase-3 significantly decreased.So it speculated that its possible mechanism is: through improving the expression of hippocampus SIRT1 protein,the Supplemented Naotaifang regulates and controls inflammatory pathways and inhibits the apoptosis of rats’ hippocampal nerve cells,so as to protect neurons and improve the learning and memory ability of rats.3.The Supplemented Naotaifang has a better preventive and therapeutic effect on vascular dementia rats,suggesting that treating vascular dementia from the angle of Yiqi Huoxue and phlegm eliminating is feasible,which is worthy of further exploring. |
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