| Objective:Through the collection of clinical cases of cerebrovascular diseases,this paper identifies the population of Vascularmild cognitive impairment(VaMCI),and objectively analyzes the correlation between the risk factors of this disease and the characteristics of TCM syndrome and cognitive function.Clinical observation was made on VaMCI patients by Bushen Yisui Jiedu method,which provided visual and quantitative basis for clinical application of the method.Meanwhile,based on the molecular level of network pharmacology,the potential active ingredients and effective molecular mechanism of Bushen Yisui Jiedu in VaMCI were explored,and the mechanism of kidney-tonifying medulla detoxification therapy in regulating cAMP/CREB/BDNF signaling pathway in VaMCI rat model was further explored.Methods:(1)Collected the general clinical data and relevant medical history from outpatients and inpatients in the Department of Encephalopathy of the Affiliated Hospital of Changchun University of Chinese Medicine and The Third Affiliated Hospital of Changchun University of Chinese Medicine.Take part the patients into two groups by the VaMCI diagnostic criteria:one is the Vascular disease normal cognition group(Control group),the other is the Vascular mild cognitive impairment group(Case group).The influencing factors were described and compared between groups.Statistical analysis of TCM syndromes in VaMCI patients was conducted to determine the distribution pattern and its correlation with cognitive function.(2)72 patients were randomly divided into two groups according to the principle of randomized controlled clinical design.There were 36 cases in TCM treatment group and 36 cases in Nimodipine contrast group.The treatment group was given oral prescription(Cistanches Herba15g、Alpiniae oxyphyllae Fructus15g、Salvia miltiorrhiza15g、Radix Paeoniae Rubral5g、Safflower15g、Earthworms15g、Corni Fructus15g、Red Yeast Rice12g、Spatholobi Caulis30g、Wolfberry fruitl5g、Cuscuta chinensis15g、Lotus Leaf20g),150ml each time,twice a day.The contrast group was given oral prescription(Nimodipine),30mg each time,three times a day.The observation course lasted for 6 months.Main outcome measures:ADAS-cog,TCM syndrome scale and IADL.(3)TCMSP database was used to search the active ingredients and corresponding target proteins of various Traditional Chinese medicines of tonifying kidney,enriching marrow and detoxifying method,Genecards database was searched,VaMCI-related targets were screened,and the intersection targets of kidney-nourishing marrow and detoxifying method compound and VaMCI were extracted by drawing Venn diagram.PPI network of intersecting targets was established with STRING database,and network diagram of"Traditional Chinese medicine-active component-intersecting target" was established with Cytoscape3.9.0 software.GO enrichment analysis and KEGG analysis of intersecting targets were conducted with Matescape database.(4)Healthy aged rats(10-20 months of age)were selected as subjects and bilateral common carotid artery stenosis was performed to establish the VaMCI rat model.The protective effects of kidney tonifying,marrow nourishing and detoxification on VaMCI were studied by behavioral,morphological and molecular biological methods.Morris water maze was used to test the ability of rats to navigate and explore space,and to detect the behavioral changes of rats.HE staining was used to observe the pathological changes of hippocampal tissues in each group.The protein expression levels of PKA,CREB1,p-CREB1 and BDNF were detected by Western-blot method.The gene expression levels of PKA,CREB1,p-CREB1 and BDNF were determined by fluorescence quantitative RT-PCR.Results:(1)After analysis,age(>60 years),excessive alcohol consumption,irritability and depression,and a history of cerebrovascular disease were independent risk factors for VaMCI.High education,proper diet and regular exercise are protective factors.According to the neuropsychological assessment,VaMCI patients have more impairment in four cognitive domains,such as memory,language,behavior,and visual space,and less impairment in overall cognitive ability and daily living ability.The score of TCM syndromes had an impact on VaMCI.Statistical analysis of TCM syndromes in the case group showed that the deficiency of kidney essence,blood stasis and obstruction of collatals,and phlegm and turbidity and obstruction of body organs accounted for a large proportion.The TCM syndromes of VaMCI were correlated with a variety of cognitive domain impairment to a certain extent.Kidney essence deficiency syndrome was significantly correlated with CDT,MMSE and IADL.Phlegm and turbidity obstruction affect the function of CDT,TMT-A and HVLT.Blood stasis and collateral obstruction affect BNT-2 function.(2)72 patients who met VaMCI diagnostic criteria were selected from the screening cases and assigned to the treatment group and the control group in a 1:1 ratio using the randomized control number method.There were no significant differences in the baseline of basic information,vascular risk factors and pre-treatment evaluation index scores between the two groups(p>0.05).After 6 months of treatment and 6 months after drug withdrawal,the overall cognitive score ADAS-cog score of 2 groups decreased significantly(p<0.05,p<0.001),with statistical difference.IADL scale decreased 6 months after treatment and 6 months after drug withdrawal in 2 groups(p<0.05,p<0.001),showing statistical difference.After 6 months of treatment,the scores of TCM syndrome efficacy decreased in both groups(p<0.05).The total effective rate of Bushen Yisui Jiedu method was 80.556%,indicating that Bushen Yisui Jiedu method had certain effect on VaMCI after 6 months of treatment,which was significantly improved compared with the control group.After 6 months of treatment,there was 1 case of dementia in the treatment group and 2 cases of dementia in the control group,and the difference in dementia conversion rate between the two groups was statistically significant(p<0.05).After 6 months of treatment,there were 2 cases of dementia in the treatment group and 6 cases of dementia in the control group,and the difference in dementia conversion rate between the two groups was statistically significant(p<0.05).(3)Network Pharmacology Studies:There were 116 active components of Bushen Yisui Jiedu method,corresponding to 410 target genes,and 198 intersecting targets were extracted by Venn diagram drawing with VaMCI target genes.Sytoscape3.9.0 was used to construct the network construction diagram of "TCMactive component-target"of VaMCI treated by Bushen Yisui Jiedu method.It was known that the Degree values of the main active components were quercetin,β-sitosterol,kaempferol,stigmasterol,etc.GO enrichment analysis showed that the biological process of Bushen Yisui Jiedu method treatment of VaMCI included the same protein binding,enzyme binding,signal receptor binding,transcription factor binding,phosphatase binding,and transmembrane signal receptor activity.Biological processes may include reactions of oxygen-containing compounds,organics,cells to chemical stimuli,endogenous stimuli,cell proliferation and apoptosis,etc.Cell components may mainly include membrane rafts,receptor complexes,plasma membranes,vesicles,synapses,axons and so on.According to KEGG pathway enrichment analysis,the Bushen Yisui Jiedu method treatment of VaMCI pathway mainly involves cancer pathway,age-rage pathway,PI3K-Akt pathway,lipid and atherosclerosis pathway,fluid shear stress and atherosclerosis pathway,prostate cancer signaling pathway,proteoglycan signaling pathway,and cAMP Signal pathway and other aspects.(4)Behavioral monitoring results:In the results of the navigation experiment,the escape latency of rats in each group was shortened to varying degrees,which proved that the ability of rats to remember the location of quadrant platform was gradually improved with training.Compared with sham operation group,the escape latency of rats in model group,Chinese medicine group and positive control group was significantly prolonged,with statistical significance(p<0.01,p<0.05,p<0.01).Compared with model group,the escape latency of rats in positive control group and Chinese medicine group was significantly shortened(p<0.05,p<0.01).In the experiment of space exploration,the learning ability and memory ability of VaMCI rats in each group were improved to varying degrees after training.Compared with sham operation group,the times of crossing platform and the percentage of time in target quadrant in model group were significantly decreased(p<0.05,p<0.01),and the learning and memory ability was significantly decreased.Compared with model group.The Times of crossing platform in treatment groups increased,and the percentage of time in target quadrant increased,with significant differences(p<0.05,p<0.01).HE staining results:In model group,the neurons in hippocampal CA1 region were incomplete,irregular in shape and loose in hierarchy,with obvious intercellular edema,deep staining of nucleus pyknosis,widening of the gap between neurons and surrounding tissues,and obvious neuron loss.Compared with the model group,the hippocampal CA1 neurons in the TCM group and the positive control group were relatively intact,with regular morphology,clear layers and uniform arrangement.Interstitial edema was less obvious than that in the model group,and the nucleus solid shrinkage was less,the gap between neurons and surrounding tissues was smaller,and a small number of neurons were lost.Western-blot results:Compared with sham operation group,the protein expressions of PKA,CREB1,pCREB1 and BDNF in VaMCI model group were significantly decreased(p<0.05,p<0.001,p<0.0001,p<0.0001),and the differences between the two groups were statistically significant.The expression of PKA,CREB1,p-CREB1 and BDNF protein was up-regulated in TCM group.RT-PCR results:PKA,CREB1,p-CREB1 and BDNF levels in the model group were significantly lower than those in the sham group(p<0.0001,p<0.0001,p<0.0001,p<0.0001).The expression of PKA,CREB1,p-CREB1 and BDNF in TCM group was significantly up-regulated.Conclusion:(1)General data Statistical analysis Age(>60 years old),excessive alcohol consumption,emotional irritability and depression,and history of cerebrovascular disease were independent risk factors for VaMCI.The protective factors were high education level,reasonable diet,and regular exercise.Among VaMCI patients,the TCM syndrome types with statistical significance were 84.78%phlegm obstruction,82.79%kidney essence deficiency,and 63.93%blood stasis obstruction,respectively,which were correlated with different cognitive functions.(2)Bushen Yisui Jiedu self-prescribed prescription and nimodipine control group had therapeutic effects on VaMCI without obvious adverse reactions.(3)Based on network pharmacology,the Bushen Yisui Jiedu method treatment of VaMCI was preliminatively explored and verified to have a multi-component,multi-target and multi-pathway overall direction regulation,and it was predicted that the cAMP/CREB/BDNF signaling pathway could regulate the synaptic regulation process,which might be the potential mechanism of Bushen Yisui Jiedu method treatment of VaMCI.(4)Bushen Yisui Jiedu method can significantly up-regulate the expression of PKA,CREB1,p-CREB1,BDNF protein and PKA,CREB1,p-CREB1,BDNF gene in rat hippocampus.This may be achieved by regulating the expression of factors related to cAMP/CREB/BDNF signaling pathway to repair synaptic plasticity and improve neuronal damage,because the effect of Bushen Yisui can only protect the brain,because the"neurotoxic" effect is anti-apoptosis,thus improving VaMCI learning and cognitive function.Implement a protective mechanism against VaMCI disease. |
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