| With cognitive and memory dysfunction as the main clinical manifestations,accompanied by personality and affective disorders,Vascular Dementia(VD)is caused by sustained brain damage that brought about by cerebrovascular diseases.VD is the second most common type of dementia,and its incidence is increasing with the rapid development of population aging,turning into a social and economic problem.Fundamental characteristics of VD is reduction of blood flowing to brain resulting in cerebral ischemia and hypoxia,causing irreversible damage to neurons in functional brain area closely related to learning and memory ability.Therapeutic angiogenesis promotes angiogenesis and vascular remodeling,increases blood supply,improves tissue ischemia and hypoxia injury,becomes the focus of VD treatment.Promoting angiogenesis by modern medicine is limited by biosafety and operation complexity.While,characterized by slight side effects and economic convenience,Traditional Chinese Medicine shows unique advantage in therapeutic angiogenesis.Qinzhi Zhudan prescription was proposed by research team which the doctoral candidate belongs to,based on the pathogenesis theory of "damage of brain collateral by toxin",with effects of clearing heat,resolving phlegm,removing blood stasis,detoxifying and dredging collaterals.Previous studies showed that Qinzhi Zhudan prescription could help inhibit inflammatory in central nervous system,regulate immune response,improve hemodynamic indexes,relieve ischemia and hypoxia injury,showing the potential of angiogenesis intervention to VD.This dissertation combined theoretical analysis and experimental verification to study the effect and mechanism of Qinzhi Zhudan prescription on angiogenesis of VD,so as to provide support for the clinical promotion and application of Qinzhi Zhudan prescription for treatment of VD..Objective:Clatify the prescription idea of Qinzhi Zhudan prescription by analyzing and summarizing distribution of VD syndrome.Observe therapeutic effect of Qinzhi Zhudan prescription on VD through behavioral monitoring and histopathological changes.Study on effect of Qinzhi Zhudan prescription on angiogenesis of VD by comparing microvessel density and angiogenesis related factors.Verify effect of Qinzhi Zhudan prescription on angiogenesis related signal transduction pathway by network pharmacology and experiment,explain the mechanism of Qinzhi Zhudan prescription on angiogenesis after VD.Methods:(1)Combination of disease and syndrome with corresponding prescription and syndrome is the right way for proper treatment based on syndrome differentiation.Employing data mining method,the TCM syndrome distribution of VD was studied and summarized,providing the syndrome basis for treatment of VD by Qinzhi Zhudan prescription.(2)Adopting the improved bilateral common carotid artery ligation to prepare VD model of rats,rats were randomly divided into blank group,model group,Qinzhi Zhudan group and nimodipine group.Qinzhi Zhudan group was given Qinzhi Zhudan decoction,nimodipine group was given nimodipine,and blank group and model group were given the same volume of physiological saline,once a day,4 weeks in a row.(3)Morris water maze and Y maze were used to test the learning and memory ability of rats.HE staining and Nissl staining were used to observe the histomorphological changes of hippocampal CA1 region and cortical tissues of rats.(4)Immunohistochemical staining was used to detect the expression of endothelial cell specific antigens CD31 and CD34,microvessel density in the hippocampal CA1 region and cortex tissues of rats was observed.The protein expression and mRNA transcription of HIF-la and VEGFA in hippocampal and cortical tissues of rats were detected by real-time quantitative PCR and Western Blot.(5)Network pharmacology was employed to predict pharmacological action and mechanism of Qinzhi Zhudan prescription on angiogenesis of VD.(6)The expression levels of PI3K,P-PI3K,Akt and P-Akt in hippocampal and cortical tissues of rats were detected by Western Blot,verifying the reliability of prediction results of network pharmacology.Results:(1)Data mining of VD TCM syndrome retrieved 18 literatures on syndrome research,4 literatures on syndrome differentiation treatment and 167 literatures on basic prescription treatment.In syndrome research literatures,the basic syndromes with frequency>10%were deficiency of kidney essence syndrome,phlegm stasis syndrome,blood stasis syndrome,liver-yang hyperactivity syndrome and syndrome for deficiency of both qi and blood.In syndrome differentiation treatment literatures,the syndromes with higher frequency were liver-kidney yin deficiency syndrome>phlegm stasis syndrome>kidney essence deficiency,phlegm stasis syndrome>kidney essence deficiency blood stasis syndrome>kidney essence deficiency syndrome.In basic prescription treatment literatures,the syndromes with higher frequency were kidney essence deficiency syndrome>kidney essence deficiency,phlegm stasis syndrome>blood stasis syndrome>kidney essence deficiency,blood stasis syndrome.(2)In the navigation experiment,the escape latency of model group was significantly longer than that of blank group(P<0.05).Compared with model group,the escape latency of Qinzhi Zhudan group and nimodipine group were both shorter(P<0.05).There was no significant difference in escape latency between Qinzhi Zhudan group,nimodipine group and blank group(P>0.05).In the space exploration experiment,times of crossing platform and target quadrant and time of staying in platform area in model group were reduced compared with blank group(P<0.05).Compared with model group,times of crossing platform and target quadrant and time of staying in platform area of Qinzhi Zhudan group and nimodipine group were increased(P<0.01).There were no significant differences in the number of crossing platform and target quadrant and the stay time in platform area among Qinzhi Zhudan group,nimodipine group and blank group(P>0.05).In the Y maze,when there was no significant difference in the total number of arm entries,the rate of spontaneous alternation of model group was significantly lower than that in blank group(P<0.01).The rate of spontaneous alternation in Qinzhi Zhudan group and nimodipine group was significantly higher than that in model group(P<0.01).There was no significant difference in the rate of spontaneous alternation between Qinzhi Zhudan group,nimodipine group and blank group(P>0.05).(3)HE staining results showed that cells in the hippocampal CA1 area and cortex of model group were disordered,and the numbers of cells were decreased.There were more denatured and necrotic cells,and nuclear pyknosis,rupture and dissolution occurred in pyramidal cells of neurons in model group.The cell structure of hippocampal CA1 area and cortex in Qinzhi Zhudan group and nimodipine group was clear,the numbers of cells were increased by varying degrees,and the apoptosis and necrosis of neurons were significantly reduced compared with the model group.Nissl staining results showed that the numbers and volume of Nissl corpuscle in hippocampal CA1 area and cortex of the model group decreased,cells were hyper-stained,wrinkled and necrotic,and nerve processes were not obvious.Compared with model group,the degeneration and necrosis of neurons in hippocampal CA1 area and cortex tissue of Qinzhi Zhudan group and nimodipine group were significantly reduced,number of Nissl corpuscle increased,the cells were arranged neatly,stained darker,and the nerve processes were obvious.(4)The expression numbers of CD31 and CD34 positive cells in hippocampal CA1 area and cortex of model group was higher than that of blank group(P<0.05).Compared with model group,the expression numbers of CD31 and CD34 positive cells in hippocampal CA1 area and cortex of Qinzhi Zhudan group and nimodipine group were increased significantly(P<0.01).Compared with nimodipine group,there was no significant difference in the expression numbers of CD31 and CD34 positive cells in hippocampal CA1 area and cortex of Qinzhi Zhudan group(P>0.05).The relative expression levels of VEGFA and HIF-1α proteins in hippocampus and cortex of model group were higher than that of blank group(P<0.05).Compared with model group,the relative expression levels of VEGFA and HIF-la in hippocampus and cortex of Qinzhi Zhudan group and nimodipine group significantly increased(P<0.01).Compared with nimodipine group,the relative expression levels of VEGFA and HIF-la proteins in hippocampus and cortex of Qinzhi Zhudan group were not significantly different(P>0.05).The mRNA levels of VEGFA and HIF-la in hippocampus of rats in model group were not significantly different from those in blank group(P>0.05),but showed an upward trend.Compared with blank group,mRNA levels of VEGFA and HIF-1α in the cortex of model group rats increased(P<0.05).Compared with model group,mRNA levels of VEGFA and HIF-la in hippocampus and cortex of Qinzhi Zhudan group and nimodipine group increased(P<0.05).Compared with nimodipine group,.there was no significant difference in mRNA levels of VEGFA and HIF-1α in hippocampus and cortex of Qinzhi Zhudan group(P>0.05).(5)The results of network pharmacology showed that Qinzhi Zhudan prescription,VD and angiogenesis jointly acted on 75 target proteins,including IL-6,Aktl,TNF,MAPK3,etc.Qinzhi Zhudan prescription mainly interfered with VD angiogenesis by affecting oxidative stress,lipopolysaccharide response and growth factor activities,and regulates MAPK signaling pathway,PI3K/Akt signaling pathway and HIF-1α signaling pathway.(6)The relative expression levels of p-PI3K and p-Akt in hippocampus and cortex tissues of model group were higher than that of blank group(P<0.05).Compared with model group,the relative expression levels of p-PI3K and p-Akt in hippocampus and cortex of Qinzhi Zhudan group and nimodipine group increased(P<0.05).Compared with nimodipine group,the relative expression levels of p-PI3K and p-Akt in hippocampus and cortex of Qinzhi Zhudan group had no significant difference(P>0.05).Conclusion:After usage of Qinzhi Zhudan prescription,for VD model rates,microvascular densities in hippocampal CA1 area and cortex increased,levels of VEGFA and HIF-la protein and gene transcription in hippocampus and cortex were promoted,angiogenesis was promoted,ischemic and hypoxia injury were improved,ability of learning memory and working memory was improved.Qinzhi Zhudan prescription could promotes the expression of p-PI3K and p-Akt in hippocampus and cortex,suggesting that the prescription may interfere with VD angiogenesis by promoting the expression of PI3K/Akt signaling pathway. |
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