Pharmacodynamic Evaluation Of The In-hospital Formulation Huangxiong Formula For The Treatment Of Ischemic Stroke

Huangxiong Formula（HXF;Huangxiong Kangshuan Capsules,Tongnaojing Capsules）is a hospital preparation from the First Affiliated Hospital of Anhui University of Chinese Medicine,consisting of Rheum palmatum L.,Ligusticum striatum DC.,Curcuma aromatica Salisb.,and Acorus gramineus Aiton,which has the effects of regulating the flow of Qi and expelling phlegm,promoting blood circulation,and removing toxins,which may improve neurological impairment and inflammatory damage caused by ischaemia and hypoxia,thus better protecting the brain.Ischemic stroke（IS）was defined on the basis of a narrowing or occlusion of the blood supplying arteries to the brain,resulting in brain tissue necrosis.Although thrombolysis is one of the main strategies for the clinical management of IS,it has limitations due to the narrow time window and risk of bleeding.Due to the complexity of IS pathology,it is important to find an aggressive and effective treatment approach.HXF has therapeutic properties for IS,however,its mechanism of action is unclear.Traditional Chinese medicine is multi-component,multi-target and multi-pathway,and has a long history of application in the prevention and treatment of stroke and post-stroke recovery.Network pharmacology is an effective approach to predict the active ingredients and disease targets of a compound from the systemic level,establish a multi-level network of drug-compound-target-disease,target the key targets of drug-disease interconnection,and use bioinformatics analysis to elucidate the mechanism of Chinese medicine and the compound to exert therapeutic effects.The objective is to explore the mechanism of action in the treatment for IS with HXF by means of network pharmacology and in vitro experimental validation,and to provide a basis for further development of the formulation.ObjectiveNetwork pharmacology,molecular docking,drug affinity responsive target stability（DARTS）,cellular thermal shift assay（CETSA）and in vivo and in vitro assays were used to investigate the mechanism of action of HXF for the treatment of IS and to provide a scientific basis for clinical application.Method1.Network pharmacology study of the mechanism for HXF treatment for IS（1）The main active ingredients of HXF were obtained using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）and literature,and their corresponding component targets were retrieved through the Swiss Target Prediction data platform.（2）IS targets were obtained through Genecards,Online Mendelian Inheritance in Man（OMIM）,Therapeutic Target Database（TTD）and Dis Ge NET.（3）HXF and IS intersection target analysis by using Venny 2.1.0.（4）The active herbal-component-target network was constructed by Cytoscape 3.9.0software.（5）The protein-protein interaction（PPI）network of potential targets was constructed by STRING database,and the key targets were screened by topology analysis using Cytoscape 3.9.0 software.（6）GO function and KEGG pathway enrichment analysis of key targets were performed using the DAVID database.（7）Component-target-pathway network construction using Cytoscape 3.9.0 software.2.Protective effect of HXF on I/R rats and validation of targets and pathways（1）A rat cerebral ischemia/reperfusion（I/R）model was established by using a modified wire embolization method,and neurobehavioural scores were performed.（2）TTC staining was used to detect the volume of cerebral infarction in each group of rats.（3）Cerebral edema was observed in the rats by brain index.（4）Histomorphological changes in the brain of each group of rats were detected by H&E staining and Nissl staining.（5）The expression of m RNA in the core genes in brain tissue was detected by Real-Time PCR.（6）The expression of PI3K/AKT pathway proteins in the brain tissues of each group was detected by Western blot.3.Protective effect of HXF-containing formula-cerebrospinal fluid（HXF-CSF）on OGD/R-injured HT22 cells（1）A model of I/R was established in vitro by Oxygen-Glucose Deprivation and Reperfusion（OGD/R）.（2）The safe concentration of HXF-CSF on HT22 cells and the survival rate of HT22cells after OGD/R injury were screened by CCK8 method.（3）Colorimetric assay to detect the amount of LDH release from HT22 cells after OGD/R injury.（4）Morphological changes of HT22 cells were observed by inverted microscopy.（5）DCFH-DA fluorescent probe staining and flow cytometry were used to detect the effect of HXF-CSF on the changes in ROS production after OGD/R injury in HT22 cells.（6）Intracellular SOD activity was detected by WST-8 assay.（7）Intracellular MDA levels were measured by TBA assay.（8）The effect of HXF-CSF on Ca²⁺after OGD/R injury in HT22 cells was detected byflow cytometry.（9）Hoechst 33258 staining was used to observe the apoptosis of HT22 cells in each group.（10）Flow cytometry to detect the apoptosis rate of HT22 cells.（11）The expression of PI3K/AKT pathway protein was detected by Western blot.4.The protective effect ofα-Asarone components of HXF on OGD/R-damaged HT22cells validated（1）Ultra Performance Liquid Chromatography（UPLC）was used for detecting the components in HXF.（2）OGD/R was used to establish an in vitro model of I/R.（3）The CCK8 method was used to screen the safe concentration ofα-Asarone on HT22cells and the survival rate of HT22 cells after OGD/R injury.（4）The LDH release from HT22 cells after OGD/R injury was measured by colorimetric assay.（5）Morphological changes of HT22 cells were observed by inverted microscopy.（6）DCFH-DA fluorescent probe staining was used to detect the effect ofα-Asarone onROS production in OGD/R-injured HT22 cells.（7）The WST-8 assay was used to detect intracellular SOD activity.（8）TBA assay for intracellular MDA levels.（9）Hoechst 33258 staining to observe the apoptosis of HT22 cells in each group.（10）Flow cytometry to detect the apoptosis rate of HT22 cells.（11）Western blot to detect the expression of PI3K/AKT pathway protein.（12）RT-PCR was performed to detect the m RNA expression of Bax and Bcl-2 in each group of cells.（13）Molecular docking,DARTS and CETSA were used to verify whetherα-pentaerythrin binds to PI3K.Results1.Network pharmacology study of the mechanism for HXF treatment for IS（1）A total of 44 active components and 795 component targets of HXF,2244 IS targets and 317 intersecting targets of HXF and IS were screened.（2）PPI network topology analysis identified 72 key targets based on Degree,Closeness Centrality and Betweenness Centrality.（3）GO enrichment analysis showed that HXF therapeutic IS was associated with biological processes including negative regulation of apoptosis and positive regulation of cell proliferation and apoptosis.（4）KEGG pathway analysis showed that the PI3K/AKT signalling pathway,thyroxine signalling pathway and HIF-1 signalling pathway were mainly involved.（5）The component-target-pathway network suggested that naringenin,kaempferol andα-Asarone may be potential active components of HXF.2.Protective effect of HXF on I/R rats and validation of targets and pathways（1）HXF significantly reduced neurobehavioral scores and brain infarct volume in I/R rats.（2）HXF significantly reduced brain index.（3）HXF significantly improved histomorphological brain injury in I/R rats.（4）RT-PCR results showed that HXF significantly downregulated the m RNAexpression of SRC,TP53,STAT3 and CTNNB1 and upregulated the expression of AKT1,MAPK3,HRAS,EGFR,VEGFA and PIK3R1 compared with the model group.（5）HXF increased the expression of p-PI3K,p-AKT and Bcl-2,and decreased the expression of Bax and cleaved-caspase9 in each dose of HXF compared with the model group.3.Protective effect of HXF-CSF on OGD/R-injured HT22 cells（1）According to the results of CCK8,the HXF-CSF 20%concentration group was selected as the most suitable.concentration for the subsequent experiment.（2）When observed by inverted microscopy,HT22 cells in the OGD/R group werewrinkled,had shortened cell synapses,poorly adhered to the wall and floated easily,while the HXF-CSF administration group had uniform cell morphology,less cell shedding and more adherent cells.（3）Compared with OGD/R group,MDA,ROS and Ca²⁺in HXF-CSF weresignificantly decreased,while SOD activity was increased.（4）The results of Hoechst staining and AV/PI flow showed that HXF-CSF attenuated the apoptosis induced by OGD/R.（5）Western Blot results showed that compared with the model group,HXF-CSFincreased I/R injury-induced p-PI3K,p-AKT and Bcl-2 expression and decreased Bax and cleaved-caspase9 expression compared to the model group.4.The protective effect ofα-Asarone components of HXF on OGD/R-damaged HT22cells validated（1）Six active ingredients of HXF were detected by UPLC,which wereα-Asarone,ferulic acid（FER）,senkyunolide I,rhubarb acid（rhein）,β-Asarone and physcion.（2）Combining with the results of the network pharmacological analysis,α-Asarone may be an important component in HXF.（3）Based on the CCK8 results,theα-Asarone（5n M,10n M,20n M）concentration groups were selected as the low,medium and high doses for subsequent experiments.（4）When the cells were observed by inverted microscope,the HT22 cells in the OGD/R group were wrinkled,had shortened cell synapses,poorly adhered to the wall and floated easily,while theα-Asarone group had uniform cell morphology,less cell shedding and more adherent cells.（5）The MDA and ROS were significantly lower and SOD activity was higher inα-Asarone compared to OGD/R.（6）Hoechst fluorescence results and AV/PI flow results showed thatα-Asarone reducedOGD/R-induced apoptosis.（7）Molecular docking showed thatα-Asarone can bind to PI3K,and the results of DARTS and CETSA experiments indicated thatα-Asarone can bind directly to PI3K,which may be the target ofα-Asarone.ConclusionTo visualize the relationship between components and disease targets and pathways through network pharmacology.HXF has a therapeutic effect on IS,which is related to the activation of the PI3K/AKT pathway,andα-Asarone is one of the potential active components of HXF.