| Ischemic stroke(IS)is the main cause of cerebrovascular disease,with high morbidity,high mortality and high recurrence rate,and IS the second leading cause of death.Buyang Huanwu decoction was first published in"Yilingaicuo" by Qingren Wang,and its clinical effective rate for ischemic stroke was as high as 80%.Studies have shown that glycosides and alkaloids are the effect parts of Buyang Huanwu decoction,which have the activities of inhibiting neuron apoptosis,anti-oxidation and anti-inflammation.On the basis of literature reports,the elution parts of 40%and 70%ethanol of Buyang Huanwu decoction were enriched by macroporous resin column,explore its efficacy and mechanism at the animal level,and then screen the components specifically combined with hippocampal neurons in the effective parts by cell membrane solid phase chromatography,the efficacy and mechanism of Calycosin-7-0-β-D-glucoside were discussed at the cell level,so as to provide scientific basis for the clarification of the pharmacodynamic basis and mechanism of Buyang Huanwu decoction.ObjectiveTo explore the pharmacodynamic basis of Buyang Huanwu decoction in promoting the rehabilitation of ischemic stroke.Methods1.Establishment of fingerprint of Buyang Huanwu decoctionThe fingerprint of Buyang Huanwu decoction was established by High performance liquid chromatography(HPLC)to control its quality.2.Study on the protective effect and mechanism of the fraction of Buyang Huanwu decoction on cerebral ischemia reperfusion model ratsFirstly,the lyophilized powder of the fraction of Buyang Huanwu decoction and the water extract of Buyang Huanwu decoction were prepared.The rat model of middle cerebral artery occlusion was reproduced by the method of thread bolt.After the Zea longa 5-level system score,the animals were divided into Sham group(0.4%CMC Na),Model group(0.4%CMC Na),Fraction of Buyang Huanwu decoction group(300 mg/kg),Buyang Huanwu decoction group(27.5 g/kg),Nimodipine group(20 mg/kg).The neurological function score was used to evaluate the recovery of neurological function;the water content of brain tissue was used to evaluate the brain edema;the volume of cerebral infarction was detected by TTC staining;the levels of LDH,SOD and MDA in serum were detected by chemical method;the ultrastructural changes of hippocampal neurons were observed by transmission electron microscope;the expression of SIRT1,FOXO1,Bax,Bcl2 and PGC-1α protein in brain tissue of rats was detected by Western Blot and ELISA.3.Establishment of membrane solid phase chromatography of hippocampal neurons and identification of specific binding componentsSolid phase extraction(SPE)was used to pretreat the samples,determine the appropriate concentration of drug,elution procedure and washing times of non-specific binding components,and establish the membrane solid phase chromatography of hippocampal neurons.The components specifically bound to hippocampal neurons in the fraction of Buyang Huanwu decoction were qualitatively analyzed by solid-phase chromatography of cell membrane combined with high resolution liquid mass.The molecular formula of the compatible components was determined by matching the secondary spectral information with the literature.4.Network pharmacology was used to screen the possible mechanism of action of specific binding componentsThe 3D structure of each specific binding component was obtained through PubChem database,and then the 3D structure of specific binding component was imported into PharmMapper database to predict the target of each specific binding component;the related target of ischemic stroke was obtained through OMIM and Gene cards database.Affinity ingredients targets and targets of ischemic stroke disease import STRING 11.0 database,construction of protein-protein interaction network,and then by DAVID online database of network analysis of core targets to GO analysis and KEGG biological pathway analysis,so as to explore the possible mechanism of specific binding components on ischemic stroke rehabilitation.5.Study on the protective effect and mechanism of Calycosin-7-O-β-Dglucoside on the model of oxygen-glucose deprivation/reperfusion in hippocampal neuronsUnder different cell inoculation density and reoxygenation conditions,CCK-8 method was used to detect cell viability,chemical method was used to detect LDH,SOD and MDA levels in cell supernatant to determine the best modeling method.Then the cells were divided into Control group,Model group,Calycosin-7-O-β-D-glucoside group(33.6 μM),Nimodipine group(5.9μM).CCK-8 method was used to detect cell viability,chemical method was used to detect LDH,SOD and MDA levels in supernatant,immunofluorescence method was used to detect ROS content in cells and mitochondrial membrane potential,flow cytometry was used to detect apoptosis rate,Western Blot and ELISA were used to detect SIRT1,FOXO1,Bax,Bcl-2 and PGC-1α protein expression in hippocampal neurons.6.Effect of Calycosin-7-O-β-D-glucoside on the model of oxygenglucose deprivation/reperfusion in hippocampal neurons after EX527 inhibited SIRT1The cells were divided into Control group,Model group,EX527 group and EX527+Calycosin-7-O-β-D-glucoside group.CCK-8 method was used to detect cell viability,chemical method was used to detect LDH,SOD and MDA levels in supernatant,immunofluorescence method was used to detect ROS content in cells and mitochondrial membrane potential,flow cytometry was used to detect apoptosis rate,Western Blot and ELISA were used to detect SIRT1,FOXO1,Bax,Bcl-2 and PGC-1α protein expression in hippocampal neurons.Results1.Establishment of fingerprint of Buyang Huanwu decoctionA fingerprint construction method with good specificity,precision and stability was established.Through the similarity evaluation system,similarity fitting was carried out for the 10 batches of Buyang Huanwu decoction test solution,and 9 common peaks were determined.The similarity of the 10 batches of Buyang Huanwu decoction test solution was above 0.9,which could be used for quality control.2.Study on the protective effect and mechanism of the fraction of Buyang Huanwu decoction on middle cerebral artery occlusion model ratsThe rat model of middle cerebral artery occlusion with low mortality was successfully reproduced,and the neurological function score showed that the fraction of Buyang Huanwu decoction could significantly improve the neurological damage in the model rats.The results of brain water content and TTC staining showed that the fraction of Buyang Huanwu decoction could alleviate the brain tissue edema and brain tissue infarction of the model rats.The results showed that the fraction of Buyang Huanwu decoction could significantly reduce the content of LDH and MDA,and increase the activity of SOD.In addition,through the observation of the ultrastructure of rat hippocampal tissue,we found that the fraction of Buyang Huanwu decoction could also reduce the damage of hippocampal nerve cells.At the same time,the fraction of Buyang Huanwu decoction significantly increased the expression of SIRT1,FOXO1,Bcl-2 and PGC-1α protein in rat brain,and decreased the expression of Bax protein.3.Establishment of membrane solid phase chromatography of hippocampal neurons and identification of specific binding componentsThe best elution method of SPE colum was established.The elution steps were as follows:chromatographic methanol,deionized water activation and equilibrium SPE colum,standing at room temperature for 1 min after uniform sample loading,washing SPE colum with deionized water,drying with nitrogen after elution with 50%acetonitrile aqueous solution,and storing at low temperature after constant volume.The optimal concentration and elution times of non-specific components were determined.Finally,the test solution of Buyang Huanwu decoction(55 mg/ml)was added to the cells for combination,and the elution times of non-specific components were 5 times.Combined with literature to parse of secondary spectra were collected,16 components specifically bound to hippocampal neurons were identified,namely,Calycosin-7-O-β-D-glucoside,Ononin,7,2’-Dihydroxy-3’,4’dimethoxyisoflavan,Phenylalanine,Calycosin,Formononetin,3-Hydroxy9,10-dimethoxyptercarpan,Calycosin 7-(6’-acetylglucoside),Formononetin 7-O-(6"-acetylglcoside),Leucine,Tryptophan,Isomucronulatol-7-Oglucoside,6-Hydroxykaempferol 3,6-diglucoside,Paeoniflorin,Galloylpaeoniflorin,Catechin.4.Network pharmacology was used to screen the possible mechanism of specific binding componentsThe interaction network of specific binding component target proteins was constructed,which has 23 nodes and 29 edges;the interaction network of target proteins of ischemic stroke disease was constructed,which has 68 nodes and 249 edges;the interaction network of specific binding component target proteins and ischemic stroke disease target proteins was combined,the combined network consisted of 99 nodes and 395 edges,and a total of 26 key genes were screened through "degree>10".By using DAVID for GO annotation and KEGG biological pathway analysis of key genes,84 biological process information was obtained through GO annotation analysis,among which 21 biological process information was of significant significance.These biological processes were related to cell proliferation regulation,protein phosphorylation,hypoxia response and angiogenesis.A total of 60 gene pathways were enriched by KEGG biological pathway analysis,among which 20 were of significant significance,mainly involving FOXO signaling pathway,AMPK signaling pathway,NF-κB signaling pathway and apoptosis pathway.5.The protective effect and mechanism of Calycosin-7-O-β-D-glucoside on oxygen-glucose deprivation/reperfusion model of hippocampal neuronsA reliable and stable oxygen-glucose deprivation/reperfusion model of hippocampal neurons was established,that is,the density of 9000 cells/well was inoculated to 96 well plates,and other well plates could be converted to the appropriate cell planting density according to the base area.The hypoxia compartment was hypoxic for 8 h and reoxygenated for 6 h.The results showed that the Calycosin-7-O-β-D-glucoside could significantly improve the survival rate of hippocampal neurons.The detection results of LDH,SOD and MDA levels in cell supernatant showed that the Calycosin-7-Oβ-D-glucoside could significantly reduce the content of LDH and MDA,increase the activity of SOD,and reduce the content of ROS in cells.In addition,the detection results of apoptosis rate and mitochondrial membrane potential of apoptotic cells showed that the Calycosin-7-O-β-Dglucoside could significantly reduce the apoptosis rate of hippocampal neurons and mitochondrial membrane potential of apoptotic cells.At the same time,the expression of SIRT1,FOXO1,Bcl-2 and PGC-1α proteins in hippocampal neurons was significantly increased and Bax protein was decreased.6.Effect of Calycosin-7-O-β-D-glucoside on the model of oxygenglucose deprivation/reperfusion in hippocampal neurons after EX527 inhibited SIRT1The results of cell viability test showed that the decreased number of hippocampal neurons induced by EX527 inhibitor could be significantly alleviated by the intervention of Calycosin-7-O-β-D-glucoside.The results of LDH,SOD and MDA in the supernatant of cells showed that after treatment,the content of LDH and MDA increased,the activity of SOD decreased,and the content of ROS increased.In addition,the apoptotic rate and mitochondrial membrane potential of apoptosis were detected.The results showed that the apoptotic rate of hippocampal neurons and mitochondrial membrane potential of apoptosis induced by EX527 inhibitor were significantly decreased.At the same time,it can significantly alleviate the decrease of SIRT1,FOX01,Bcl-2 and PGC-1α protein expression and the increase of Bax protein expression in hippocampal neurons induced by EX527 inhibitor.ConclusionThe fraction of Buyang Huanwu decoction could alleviate the damage caused by cerebral ischemia-reperfusion by alleviating oxidative stress and neuron apoptosis.Through the method of membrane solid phase chromatography of hippocampal neurons combined with high-resolution liquid-mass spectrometry,16 components specifically combined with hippocampal neurons were screened out,among which the Calycosin-7-O-β-D-glucoside had significant anti-oxidative stress and anti-apoptotic activity.The effective components of Buyang Huanwu decoction in alleviating cerebral ischemia-reperfusion injury may be some affinitive components represented by Calycosin-7-O-β-D-glucoside. |
PDF Full Text Request