Abstract

Background

   Evodia rutaecarpa is a traditional Chinese herbal medicine known for
   its potential benefits in the treatment of cardiovascular and
   cerebrovascular diseases. Despite its recognized effects, the effects
   of Evodia rutaecarpa on ischemic stroke (IS), along with the primary
   active compounds and precise mechanisms of action, require elucidation.

Methods

   Network pharmacology analyses and molecular docking were performed to
   integrate information related to Evodia rutaecarpa and IS. Cell
   oxygen–glucose deprivation (OGD) and rat middle cerebral artery
   occlusion (MCAO) models were established to simulate cerebral ischemic
   injury. The effects of rutaecarpine on these models were evaluated to
   assess its effect on IS.

Results

   Network pharmacological analysis indicated that rutaecarpine from
   Evodia rutaecarpa showed therapeutic effects against IS. The mechanism
   underlying these effects mainly involved the mitogen-activated protein
   kinase (MAPK), and targets such as matrix metalloproteinase (MMP)-9,
   caspase 3 and MMP-2 may be activated to exert these effects. In vitro
   studies showed that rutaecarpine significantly improved the
   mitochondrial membrane potential of HT22 cells, reduced the production
   of reactive oxygen species, and reversed OGD-induced cytotoxicity. In
   the MCAO rat model, pretreatment with rutaecarpine significantly
   reduced neuronal death, decreased infarct volume, and improved
   neurological functional deficits. In addition, rutaecarpine alleviated
   damage to the blood–brain barrier in the brain tissue. These effects
   may be related to the regulation of the MAPK-mediated MMPs pathway.

Conclusion

   This study revealed the neuroprotective effects and molecular
   mechanisms of rutaecarpine on IS, providing a new theoretical basis for
   the clinical application of Evodia rutaecarpa.

1. Introduction

   Stroke, including ischemic stroke (IS) and hemorrhagic stroke, is one
   of the main causes of death and severe disability worldwide. IS
   accounts for more than 80% of all stroke cases [[34]1]. The main
   pathological feature of IS is the blockage or severe stenosis of blood
   vessels in functional areas of the brain, resulting in a decrease in
   cerebral blood perfusion. This leads to ischemia and hypoxia, causing
   death of brain tissue and defects in brain function in the supply area
   of the cerebrovascular system. Drug thrombolysis and mechanical
   thrombectomy are the most commonly used treatment strategies for
   restoring blood flow. However, their clinical efficacy is poor, and
   they can also lead to cerebral ischemia-reperfusion (I/R) injury
   [[35]2]. I/R injury, which involves various pathophysiological events
   such as neural apoptosis, neuroinflammation, blood–brain barrier (BBB)
   destruction, and mitochondrial dysfunction, is a common
   pathophysiological condition in IS that leads to more severe neural
   injury and functional impairment [[36]3]. Thus, it is necessary to
   conduct additional research to determine safer and more effective
   therapeutic drugs and methods for the prevention and treatment of IS.

   Chinese herbal medicines have recently shown notable developmental
   potential in the treatment of neurological diseases. These medicines
   have the advantages of being natural, safe, and effective, with few
   side effects and a lower probability of drug resistance [[37]4]. Evodia
   rutaecarpa is a well-known herbal medicine with multiple biological
   activities and therapeutic applications. It contains many chemical
   components with pharmacological activities, including analgesic,
   anti-inflammatory, anti-ulcer, anti-emetic, anti-diarrhea,
   vasodilatory, blood pressure-reducing, and cardiotonic effects
   [[38]5,[39]6]. However, the effects of Evodia rutaecarpa on IS are
   still unclear. Evodiamine and rutaecarpine (Rut) are the two main
   bioactive indole alkaloids found in Evodia rutaecarpa. Modern
   pharmacological research has shown that Rut has notable
   anti-inflammatory, antioxidant, antithrombotic, and cardioprotective
   effects [[40]7]. Evodiamine has also been shown to reduce myocardial
   ischemic injury and protect the nerves [[41]8]. Therefore, these active
   ingredients of Evodia rutaecarpa may show therapeutic effects against
   ischemic cerebrovascular diseases.

   Owing to its systematization and integrity, network pharmacology
   represents a new method for evaluating the complex components of
   traditional Chinese medicines and exploring the mechanisms underlying
   drug treatment for diseases [[42]9]. Therefore, we applied network
   pharmacology and molecular docking analyses to predict the effective
   components and corresponding targets in IS, and selected active
   components with strong correlations and significant influences.
   Subsequently, we performed step-by-step experiments using in vitro to
   in vivo models to verify the protective effects and potential
   mechanisms of IS ([43]Fig 1).

Fig 1. Schematic diagram of the relationship between Evodia rutaecarpa and
IS.

   [44]Fig 1
   [45]Open in a new tab

   This study aimed to explore the protective effect of Evodia rutaecarpa
   on IS on the basis of network pharmacology and molecular docking
   analyses and verify the mechanisms of action of Rut against IS both in
   vitro and in vivo.

2. Materials and methods

2.1. Cell culture

   HT22 cells were obtained from ProCell Life Science & Technology Co.,
   Ltd. The cells were routinely grown in Dulbecco’s modified Eagle’s
   medium (Solarbio, Beijing, China) containing 10% fetal bovine serum
   (Gibco, USA) and 1% penicillin-streptomycin solution (HyClone, USA) in
   an incubator maintained at 37°C with a humidified environment of 5%
   CO[2].

2.2. Animals

   Male Sprague–Dawley rats (250–280 g, n = 75) were provided by Hunan
   Slaike Jingda Experimental Animal Co., Ltd. (License No. SCXK [Xiang]
   20210002). The animal experiments and methods performed in this study
   were in accordance with the ethical guidelines for animal studies and
   were approved by the Laboratory Animal Ethics Committee of the Jiangxi
   Health Industry Institute of Traditional Chinese Medicine (Jiangxi,
   Nanchang, China; animal ethics number: 2024020), and followed the
   National Guidelines for Animal Protection. The rats used in this study
   were raised under standard laboratory conditions (temperature:
   22°C ± 2°C, relative humidity: 55% ± 10%, light/dark cycle: 12 h/12 h)
   and had free access to water and food.

2.3. Network pharmacology analysis

2.3.1. Establishment of the ingredient–target network between Evodia
rutaecarpa and IS.

   The bioactive ingredients of Evodia rutaecarpa were identified using
   the Traditional Chinese Medicine Systems Pharmacology Database and
   Analysis Platform (TCMSP; [46]https://tcmsp-e.com/). To retrieve the
   active ingredient information of Evodia rutaecarpa, the conditions for
   screening were set as oral bioavailability (OB) ≥ 30% and drug-likeness
   (DL) ≥ 0.18. The relevant effective targets of Evodia rutaecarpa were
   predicted using the Pharm Mapper database
   ([47]https://lilab.ecust.edu.cn/pharmmapper), and the targets were
   converted into corresponding gene names using the Universal Protein
   Resource database (Uniprot, [48]https://www.uniprot.org) and the STRING
   database ([49]https://cn.string-db.org/). Disease targets were
   identified using the search term “ischemic stroke” in the DisGeNET
   database ([50]https://disgenet.org/). After removing duplicates, a Venn
   diagram was drawn to obtain the intersection of the potential active
   ingredients of Evodia rutaecarpa and the disease targets. Data
   visualization and detailed analysis were performed using Cytoscape
   3.9.1 to draw the “Evodia rutaecarpa-active ingredients-IS targets”
   network relationship diagram.

2.3.2. Protein–protein interaction (PPI) network construction and pathway
enrichment analysis.

   To study the relationships among Evodia rutaecarpa, targets, and
   diseases, the intersection targets were imported into the STRING
   database, with the species source as “Homo sapiens,” to construct a PPI
   network. Network topology analysis was performed using Cytoscape 3.9.1
   software with the following criteria: degree > 2 times median,
   closeness centrality > 1 times median, and betweenness centrality > 1
   times median. The core targets of Evodia rutaecarpa in IS treatment
   were imported into the DAVID database ([51]https://david.ncifcrf.gov/)
   for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) enrichment analyses, and the results were further visualized
   using a bioinformatics platform
   ([52]https://www.bioinformatics.com.cn/). The relationship between the
   potential targets of active ingredients in Evodia rutaecarpa and the
   intersection of KEGG pathways was input into Cytoscape 3.9.1 to
   generate a network analysis graph of targets acting on pathways.

2.4. Molecular docking

   The structures of the drug molecule and target macromolecules were
   downloaded from PubChem ([53]https://pubchem.ncbi.nlm.nih.gov/) and
   Protein Data Bank ([54]https://www.rcsb.org) databases. The protein
   structures were dehydrated using PyMOL 2.5.2, and modified ligands were
   removed. AutoDockTools 1.5.6 was used for format conversion and
   location of the active pocket. Molecular docking was performed using
   AutoDock Vina 1.1.2, and the binding sites of the small molecules to
   the core proteins were visualized and analyzed using PyMOL 2.5.2.

2.5. Establishment of the oxygen–glucose deprivation (OGD) model

   HT22 cells were subjected to OGD to induce stroke-like injury in
   neurons. The detailed protocol for OGD was as follows: the cultured
   HT22 cells were washed three times with phosphate-buffered saline
   (PBS). Then, the cells were transferred into a glucose-free medium and
   placed in a hypoxia chamber containing 5% CO[2] and 95% N[2] to create
   an anoxic environment. After hypoxia and glucose deficiency at 37°C for
   2 h, the cells were collected; the medium was switched back to normal
   medium; and the cells were cultured in a 5% CO[2] incubator at 37°C.

2.5.1. Assessment of cell viability.

   Rut was purchased from Dalian Meilun Biotechnology Co. Ltd. (Dalian,
   China). HT22 cells in the logarithmic growth phase were seeded into a
   96-well cell culture plate. After a 24-hour treatment with different
   concentrations of Rut (0, 0.5, 0.75, 1, 2, 4, 6 μM), 5 mg/mL 3-[4,
   5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT)
   solution was added to each well. After incubation for 4 h, the culture
   medium was removed, and 150 μL dimethyl sulfoxide (DMSO) was added and
   shaken. The absorbance value of each well was measured at 570 nm using
   a microplate reader. Cell viability (%) was calculated as the optical
   density (OD) value of drug-treated group/OD value of control
   group × 100%.

2.5.2. Detection of reactive oxygen species in cells.

   HT22 cells were treated with different concentrations of Rut (0, 0.1,
   0.25, and 0.5 μM). The cells were then subjected to OGD for 2 h. After
   3, 6, and 12 h of reoxygenation and re-glucose supplementation, the
   culture medium was discarded. A pre-diluted 2′-7′-
   dichlorodihydrofluorescein diacetate fluorescent probe was added until
   the cells were fully covered. The cells were incubated in a 37°C
   incubator in the dark for 20 min. After washing the cells with
   serum-free medium, the reactive oxygen species (ROS) content was
   detected using a fluorescence microplate reader.

2.5.3. Detection of mitochondrial membrane potential.

   Changes in mitochondrial membrane potential were detected by treating
   HT22 cells with JC-1 (Wanleibio, Shenyang, China). Fluorescence was
   analyzed and observed using an inverted fluorescence microscope (Leica,
   Germany), transitioning from red (excitation 525 nm/emission 590 nm) to
   green (excitation 490 nm/emission 530 nm).

2.5.4. Detection of apoptosis in cells.

   Apoptosis in HT22 cells was detected using the acridine orange
   (AO)/ethidium bromide (EB) double-fluorescence staining kit (Yuanye,
   Shanghai, China). Transformation of the fluorescence color was analyzed
   and observed using an inverted fluorescence microscope.

2.6. Middle cerebral artery occlusion (MCAO) rat model preparation and drug
administration

   The rats were anesthetized with 2% isoflurane, and MCAO was induced by
   inserting a nylon monofilament suture (diameter, 0.36 mm) into the
   right internal carotid artery. After occluding the right middle
   cerebral artery for 2 h, the filament was carefully withdrawn to allow
   blood reperfusion. The sham group underwent the same procedure without
   nylon monofilament insertion. Low-dose (L-Rut) and high-dose (H-Rut)
   Rut groups were established by intraperitoneally injecting low-dose Rut
   (0.5 mg/ [kg·d]) or high-dose Rut (2.5 mg/ [kg·d]) into model rats. The
   edaravone group (Eda) was established by intraperitoneal injection of
   edaravone (3 mg/ [kg·d]). Rats in the model and sham operation groups
   were intraperitoneally injected with equal volumes of normal saline
   containing 5% DMSO. After continuous administration for 6 days in all
   groups, MCAO surgery was performed and the administration was repeated
   the following day (a total of 7 days of administration). After 24 hours
   of reperfusion, rats were deeply anesthetized using isoflurane and then
   rapidly decapitated to ensure humane euthanasia and minimize animal
   distress. Brain tissues were immediately collected for subsequent
   analysis.

2.6.1. 2, 3, 5-Triphenyl tetrazolium chloride (TTC) staining and neurological
evaluation.

   The rat brain tissue was isolated and coronally sliced at a thickness
   of 2 mm/slice. The brain slices were placed in 2% TTC solution and
   stained at 37°C in the dark for 15 min. The images were analyzed using
   ImageJ software. The cerebral infarct rate was calculated as follows:
   (infarct area/total brain area) × 100%. After reperfusion for 24 h, the
   neurological function scores of the rats in each group were evaluated
   using Zea-Longa’s 5-point and Ludmila-Belayev 12-point scores.

2.6.2. Measurement of BBB permeability.

   Rats were injected with 2% Evans blue solution through the tail vein.
   After 2 h, cardiac perfusion was performed using PBS. The brains were
   removed by decapitation, weighed, and cut into pieces. Then, a 50%
   trichloroacetic acid solution was added for homogenization.
   Centrifugation was performed at 14000 × g and 4°C for 20 min. The
   supernatants were collected, and three times the volume of absolute
   ethanol was added and mixed thoroughly. Evans blue content in brain
   tissue was determined at 620 nm using a fluorescence microplate reader.

2.6.3. Nissl staining.

   The rat brain tissue was fixed with 4% paraformaldehyde, dehydrated,
   and made transparent using a gradient of ethanol and xylene. Then, it
   was embedded in paraffin and sectioned into 4-µm-thick slices. The
   tissues were dewaxed in xylene and rehydrated using an ethanol gradient
   prior to Nissl staining with 0.1% methyl violet. After routine
   dehydration and transparency, tissue samples were mounted with neutral
   resin. Cells were observed under a microscope to calculate the number
   of Nissl-positive neurons in the penumbra.

2.6.4. Immunohistochemistry.

   Paraffin sections of brain tissue were dewaxed with water, subjected to
   antigen retrieval, blocked for endogenous peroxidase activity, and
   blocked at room temperature. Appropriate amounts of occludin and zonula
   occludens-1 (ZO-1) antibodies (both purchased from Proteintech Company)
   were added as primary antibodies at a concentration of 1: 2000 and
   incubated overnight at 4°C. Horseradish peroxidase-labeled biotin was
   used as the secondary antibody. Color development was terminated when
   the cells showed a brownish-yellow color under a microscope. The
   sections were mounted and observed under a microscope after dehydration
   and transparency to check for the positive expression of tight junction
   proteins (TJPs).

2.6.5. Western blot (WB) assay.

   In accordance with a previously reported scheme [[55]10], protein
   samples were extracted from the cerebral ischemic penumbra and cerebral
   microvessels and transferred to a polyvinylidene fluoride membrane
   (Millipore, USA). After blocking with 5% BSA for 1 h at room
   temperature, the membrane was incubated overnight at 4°C with primary
   antibodies against p38MAPK (1: 1000) (Servicebio, Wuhan, China),
   p-p38MAPK (1: 500), MMP-9 (1: 1000), cleaved caspase 3 (1: 500)
   (Wanleibio, Shenyang, China), Bax (1: 1000) (Bioss, Beijing, China),
   MMP-2 (1: 500), ZO-1 (1: 10000), Occludin (1: 10000), Bcl-2 (1: 1000),
   and the internal reference β-actin (1: 20000) (Proteintech, Rosemont,
   USA). The membrane was then incubated with a horseradish
   peroxidase-conjugated secondary antibody (Proteintech, Rosemont, USA)
   for 1 h. After development, the levels of the target proteins were
   photographed and analyzed using a chemiluminescence imaging system
   (Bio-Rad, USA).

2.7. Statistical analysis

   The data were expressed as mean ± standard deviation and analyzed using
   GraphPad Prism 9.5 software. The mean values were analyzed using
   one-way analysis of variance and Student’s t-test. All P values < 0.05
   indicated a statistically significant difference. The probability
   values were noted as follows: *P < 0.05; **P < 0.01; ***P < 0.001;
   ****P < 0.0001.

3. Results

3.1. Network pharmacology analysis

   A total of 31 active ingredients of Evodia rutaecarpa were identified
   by screening using the TCMSP database ([56]Table 1). The PharmMapper
   database was used to identify potential targets of these active
   ingredients. After removing the duplicates, 172 potential targets were
   identified. Using “ischemic stroke” as the keyword, a total of 1,159
   disease-related targets were retrieved and summarized in the DisGeNET
   database. A Venn diagram was used to map the active ingredient and
   disease targets, and 54 intersection targets of the active ingredients
   of Evodia rutaecarpa and IS were obtained ([57]Fig 2A). The active
   ingredient and disease targets were matched and visualized to construct
   a network relationship diagram of Evodia rutaecarpa active ingredients
   with IS ([58]Fig 2B). To explore the mechanisms underlying the effects
   of Evodia rutaecarpa on IS, the core targets of diseases and active
   ingredients were imported into the STRING database, and Cytoscape 3.9.1
   was used to construct a PPI network. As shown in [59]Fig 2C, target
   proteins such as albumin (ALB), matrix metalloproteinase (MMP)-9,
   caspase 3 (CASP3), MMP-2, and estrogen receptor 1 (ESR1), were located
   in the center of the network and had high degree values, suggesting
   that Evodia rutaecarpa plays a therapeutic role by acting on these key
   targets. The DAVID database was used for functional and pathway
   enrichment analyses. The top 10 entries were selected to draw a GO
   functional analysis diagram ([60]Fig 2D). The GO analysis revealed that
   the enriched biological process terms were primarily related to signal
   transduction, proteolysis, and apoptotic processes. The enriched
   cellular component terms were primarily associated with the nucleus,
   extracellular region, and extracellular space. The enriched molecular
   function terms included identical proteins, zinc ions, and
   sequence-specific DNA binding. KEGG pathway analysis assigned
   significance to gene functions at the molecular level and provided a
   systematic and comprehensive analysis of specific target genes that
   were significantly enriched in the biological pathways determined
   above. Using the MicroBioInfo platform for KEGG pathway enrichment
   analysis, we selected the top 20 signaling pathways sorted by p-value.
   [61]Fig 2E showed that the key gene targets of Evodia rutaecarpa in
   treating IS were mainly enriched in signaling pathways, such as the
   mitogen-activated protein kinase (MAPK) and
   phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathways.

Table 1. Active ingredients of Evodia rutaecarpa.

   MOLID Molecule Name OB% DL
   MOL003958 Evodiamine 86.02 0.64
   MOL004018 Goshuyuamide I 83.19 0.39
   MOL003988 2-Hydroxy-3-formyl-7-methoxycarbazole 83.08 0.18
   MOL004014 Evodiamide 73.77 0.28
   MOL003963 hydroxyevodiamine 72.11 0.71
   MOL004019 GoshuyuamideII 69.11 0.43
   MOL003942 Rutaevine 66.05 0.58
   MOL004021 Gravacridoneshlirine 63.73 0.54
   MOL004025 N-(2-Methylaminobenzoyl)tryptamine 56.96 0.26
   MOL004017 Fordimine 55.11 0.26
   MOL000354 isorhamnetin 49.6 0.31
   MOL003974 Evocarpine 48.66 0.36
   MOL003950 1-methyl-2-[(Z)-undec-6-enyl]-4-quinolone 48.48 0.27
   MOL003947 1-methyl-2-[(Z)-pentadec-10-enyl]-4-quinolone 48.45 0.46
   MOL003972 1-methyl-2-nonyl-4-quinolone 48.42 0.2
   MOL003964 1-methyl-2-undecyl-4-quinolone 47.59 0.27
   MOL004004 6-OH-Luteolin 46.93 0.28
   MOL000098 quercetin 46.43 0.28
   MOL003975 icosa-11,14,17-trienoic acid methyl ester 44.81 0.23
   MOL003957 1-methyl-2-pentadecyl-4-quinolone 44.52 0.46
   MOL013352 Obacunone 43.29 0.77
   MOL003956 dihydrorutaecarpine 42.27 0.6
   MOL003943 Rutalinidine 40.89 0.22
   MOL002662 rutaecarpine 40.3 0.6
   MOL003994 24-methyl-31-norlanost-9(11)-enol 38 0.75
   MOL000358 beta-sitosterol 36.91 0.75
   MOL000359 sitosterol 36.91 0.75
   MOL001454 berberine 36.86 0.78
   MOL004020 gossypetin 35 0.31
   MOL004002 5alpha-O-(3’-Methylamino-3’-phenylpropionyl)nicotaxine 30.86
   0.49
   MOL003960 1-(5,7,8-trimethoxy-2,2-dimethylchromen-6-yl)ethanone 30.39
   0.18
   [62]Open in a new tab

Fig 2. Network pharmacology analysis.

   [63]Fig 2
   [64]Open in a new tab

   (A) Venn diagram of component target–disease target. (B) Evodia
   rutaecarpa–active component–IS target network. (C) PPI network of
   targets of Evodia rutaecarpa in treating IS. (D) GO functional
   enrichment analysis. (E) KEGG pathway enrichment analysis.

3.2. Molecular docking

   The main active ingredient, Rut, was screened and subjected to
   molecular docking verification using ALB, MMP-9, CASP3, MMP-2, and ESR1
   to evaluate the affinity between receptors and ligands ([65]Fig
   3A–[66]3E). The results were visualized using PyMOL software. The
   results showed that the binding energies of Rut with ALB, MMP-9, CASP3,
   MMP-2, and ESR1 were all < −5.0 kcal/mol, indicating that the docking
   results were relatively stable.

Fig 3. Molecular docking diagram of rut and core targets.

   [67]Fig 3
   [68]Open in a new tab

   (A) Molecular docking model of ALB with Rut. (B) Molecular docking
   model of MMP-9 with Rut. (C) Molecular docking model of CASP3 with Rut.
   (D) Molecular docking model of MMP-2 with Rut. (E) Molecular docking
   model of ESR1 with Rut.

3.3. Rut reversed OGD-induced neuronal injury

   The effect of Rut on the viability of HT22 cells was evaluated by the
   MTT assay. As shown in [69]Fig 4A, at Rut concentrations up to 1 μM,
   the survival rate of HT22 cells was above 90%, indicating that Rut was
   non-toxic to HT22 cells within this concentration range. To further
   explore whether the protective mechanism of Rut is closely related to
   the mitochondria, we measured the production of intracellular ROS and
   changes in mitochondrial membrane potential. HT22 cells were subjected
   to OGD to induce cell damage. The cells were treated with Rut at three
   different concentration levels (low, medium, and high). To study the
   antioxidant effect, changes in ROS levels in OGD-exposed cells were
   detected after 3, 6, and 12 h of reoxygenation. As shown in [70]Fig 4C,
   the concentration of ROS in OGD-exposed cells was higher than that in
   normal cells, indicating that OGD induced cell damage by increasing ROS
   production. Rut treatment reduced ROS levels, and the antioxidant
   effect of Rut was the most significant after 12 h of reoxygenation. The
   JC-1 assay was used to study the effect of Rut on the mitochondrial
   membrane potential of HT22 cells. In comparison with normal HT22 cells,
   the red fluorescence in the mitochondria of the OGD group was weaker,
   and the green fluorescence was stronger. This result indicated that OGD
   caused a loss of mitochondrial membrane potential and induced
   mitochondrial damage. In contrast, after treatment with Rut, enhanced
   red fluorescence and weakened green fluorescence were observed,
   indicating that Rut improved mitochondrial function by increasing the
   mitochondrial membrane potential ([71]Fig 4D–[72]4E). The inhibitory
   effect of Rut on OGD-induced neuronal injury was studied using the MTT
   assay and the AO/EB double-staining method. As shown in [73]Fig 4B, the
   survival rate of OGD-exposed HT22 cells significantly decreased, while
   the survival rate of Rut-treated cells significantly increased at 24 h.
   The results of the AO/EB double-staining method showed that the number
   of non-apoptotic cells showing green fluorescence reduced and the
   number of apoptotic cells showing red fluorescence increased in the OGD
   group. Treatment with Rut weakened red fluorescence and enhanced green
   fluorescence, suggesting that Rut inhibited OGD – induced apoptosis
   ([74]Fig 4F–[75]4G).

Fig 4. Effect of Rut on neuronal injury caused by OGD.

   [76]Fig 4
   [77]Open in a new tab

   Normal HT22 cells served as the control group. (A) Cell viability of
   HT22 cells treated with different concentration gradients of Rut. (B)
   Cell viability of OGD cells treated with Rut. (C) Relative cellular ROS
   levels of OGD cells treated with Rut at 3 h, 6 h, and 12 h of
   reoxygenation. The relative ROS level (%) was determined by calculating
   the ratio of the fluorescence intensity of Dichlorofluorescein in each
   group to the fluorescence intensity of Dichlorofluorescein in the OGD
   group. (D) Detection of changes in mitochondrial membrane potential in
   OGD cells treated with Rut by the JC-1 method. The scale bar is 50 μm.
   (E) Quantitative analysis of relative fluorescence in OGD cells treated
   with Rut. (F) Detection of apoptosis in OGD cells treated with Rut by
   AO/EB double-staining. The scale bar is 100 μm. (G) Quantitative
   analysis of relative fluorescence in OGD cells treated with Rut. All
   data are presented as mean ± SD (n = 3 per group). *P < 0.05, **
   P < 0.01, ***P < 0.001, ****P < 0.0001.

3.4. Rut reduced neuronal injury in the ischemic penumbra and improved BBB
permeability in rats

   Since Rut showed the potential to reduce excessive ROS generation,
   restore mitochondrial membrane potential, and alleviate OGD-induced
   neuronal injury, we further studied Rut-mediated neurological
   improvement in vivo. As shown in [78]Fig 5A and [79]5B, in comparison
   with the sham group, the MCAO group showed a significantly higher
   cerebral infarction volume. After treatment with Rut and Eda, the
   ischemia-induced infarct volumes significantly reduced. The
   neurological function of the rats was evaluated using the Zea-Longa and
   Ludmila-Belayev neurological scores. Consistent with the results of TTC
   staining, the neurological function scores in the MCAO group were
   higher than those in the sham group. In contrast, the administration
   and positive drug groups showed reduced neurological function scores
   compared with those seen in the MCAO group ([80]Fig 5C and [81]5D). To
   determine whether Rut regulates BBB integrity and permeability after
   cerebral I/R injury, we used Evans blue staining to evaluate the
   effects of Rut on BBB damage caused by MCAO. In comparison with the
   sham group, the MCAO rats showed significantly higher Evans blue
   content on the cerebral ischemic side. After administration of Rut, the
   amount of Evans blue on the cerebral ischemic side of MCAO rats
   decreased significantly, indicating that Rut alleviated the
   MCAO-mediated BBB damage in the rat brain tissue ([82]Fig 5E and
   [83]5F). Nissl staining of the sham group showed that the neuronal cell
   structure was completely and neatly arranged; the cytoplasm was dark
   blue; Nissl bodies were abundant; and the dendritic structure was
   normal. In the MCAO group, neuronal cells were sparsely distributed,
   the number of Nissl bodies was reduced, and dendritic structures
   disappeared. After treatment with Rut and Eda, a large number of
   surviving neurons were observed, and the extent of degeneration and
   necrosis was relatively small, significantly improving the integrity of
   the basic neuronal structure ([84]Fig 5G and [85]5H). These results
   indicated that Rut treatment reversed neuronal death caused by MCAO,
   effectively reduced neuronal damage in the ischemic area, and improved
   BBB damage.

Fig 5. Rut alleviated BBB damage in MCAO rats and exerted neuroprotective
effects.

   [86]Fig 5
   [87]Open in a new tab

   (A) Representative infarcted brain sections in the L-Rut, H-Rut, and
   Eda groups were stained with TTC after ischemia for 2 h and reperfusion
   for 24 h. (B) The percentages of infarct volumes in the MCAO models
   treated with L-Rut, H-Rut, and Eda. (C) Zea–Longa neurological score.
   (D) Ludmila–Belayev neurological score. (E) Quantification of Evans
   blue leakage from the brains of rats. (F) Representative images of
   Evans blue exudation in the L-Rut, H-Rut, and Eda groups after ischemia
   for 2 h and reperfusion for 24 h. (G) Nissl staining images of the
   ischemic penumbra in MCAO rats treated with L-Rut, H-Rut, or Eda. (H)
   The relative number of Nissl bodies was determined by calculating the
   ratio of the number of Nissl bodies in the L-Rut group, H-Rut group,
   and Eda group to the number of Nissl bodies in the sham operation
   group. All data are presented as mean ± SD (n = 6 per group).
   *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.5. Rut weakened BBB disruption and inhibited brain tissue apoptosis by
regulating the MAPK pathway

   Studies have shown that the MAPK pathway involved in the pathological
   process of cerebral I/R injury and mediated the activation of MMPs and
   regulated the expression of TJPs, thereby affecting BBB injury
   [[88]11]. Immunohistochemical staining was used to compare the
   distribution and expression of ZO-1 and occludin in the brain tissues
   of each group. As shown in [89]Fig 6A, in comparison with the sham
   group, the model group showed reduced expression of ZO-1 and occludin
   in the brain tissues, while the expression and distribution of these
   proteins increased in the L-Rut, H-Rut, and Eda groups. The WB results
   were consistent with the immunohistochemical findings. In comparison
   with the model group, the ratio of p-p38MAPK/p38MAPK in the L-Rut,
   H-Rut, and Eda groups was significantly decreased. The expression
   levels of MMP-2 and MMP-9 were also significantly reduced, while the
   expression levels of ZO-1 and occludin were significantly increased in
   the model group. The results indicated that Rut and Eda inhibited the
   activation of MAPK in the brain tissue of MCAO rats, further inhibited
   the activation of MMP-2 and MMP-9, slowed down the degradation of ZO-1
   and occludin ([90]Fig 6B–[91]6I). All these evidences suggested that
   Rut plays an important role in protecting the integrity of the BBB.
   Furthermore, the effect of Rut on the expression of apoptotic proteins
   in the ischemic penumbra of rat brains was investigated. In comparison
   with the sham group, the model group showed a significantly lower
   Bcl-2/Bax ratio and a significantly higher expression level of cleaved
   caspase 3. In comparison with the model group, the H-Rut and Eda groups
   showed a significantly higher Bcl-2/Bax ratio and a significantly lower
   expression level of cleaved caspase 3, confirming that Rut inhibited
   brain tissue apoptosis ([92]Fig 6J–[93]6L).

Fig 6. Rut inhibited the degradation of TJPs to reduce BBB breakdown and also
suppressed apoptosis of brain tissue cells.

   [94]Fig 6
   [95]Open in a new tab

   (A) The representative immunohistochemical images of ZO-1 and occludin
   in rat brain tissue. (B) The expression levels of p38MAPK and p-p38MAPK
   proteins. (C) The expression levels of MMP-2 and MMP-9 proteins. (D)
   The expression levels of ZO-1 and occludin proteins. (E) The
   statistical analysis of the p-p38MAPK/p38MAPK ratio. (F) The
   statistical analysis of the MMP-2 protein level. (G) The statistical
   analysis of the MMP-9 protein level. (H) The statistical analysis of
   the ZO-1 protein level. (I) The statistical analysis of the occludin
   protein level. (J) The expression levels of cleaved caspase 3, Bcl-2,
   and Bax proteins. (K) The statistical analysis of cleaved caspase 3
   protein level. (L) The statistical analysis of the Bcl-2/Bax ratio. All
   data are presented as mean ± SD (n = 3 per group). *P < 0.05,
   **P < 0.01, ***P < 0.001.

4. Discussion

   IS is a serious cerebrovascular disease with a high incidence and high
   rates of disability and mortality. The main risks associate with IS
   include inflammation, oxidative stress, cell death, BBB damage, and
   cerebral infarction, all of which are caused by cerebral I/R injury
   [[96]12]. The existing primary treatment methods for IS do not yield
   satisfactory results. Therefore, safe and effective new drugs to
   prevent stroke and improve prognosis are essential. With the
   modernization of traditional Chinese medicine, the protective effects
   of natural, safe, and effective traditional Chinese medicines and their
   active ingredients on cerebrovascular diseases have attracted
   widespread attention [[97]13,[98]14]. Network pharmacology is a novel
   method for drug discovery. It constructs a “drug-gene-target-disease”
   map to help identify the active ingredients of drugs and analyze their
   action mechanisms in preventing and treating diseases. It offers a
   brand-new systematic perspective and research approach for drug
   research and development as well as disease research, and holds broad
   application prospects in the biomedical field [[99]15].

   In this study, we used network pharmacology to analyze the potential
   active ingredients, targets, and mechanisms of action of Evodia
   rutaecarpa in the treatment of IS. Based on the network analysis of
   active ingredients and potential targets of Evodia rutaecarpa, Rut, an
   active ingredient of Evodia rutaecarpa with a high degree of value, was
   screened. Studies have confirmed that Rut regulates oxidative stress
   and apoptosis in PC12 cells and prevents neuronal oxidative damage
   [[100]16]. Xu et al. [[101]14] found that Rut reduced the neurological
   damage induced by cerebral I/R by improving oxidative stress. In
   addition, Han et al. [[102]17] found that Rut reduced the inflammatory
   response, improved oxidative stress, and inhibited neuronal apoptosis
   by activating the ERK1/2-mediated Nrf2/HO-1 signaling pathway, thus
   providing a pharmacological basis for the development and utilization
   of Rut as a drug for stroke treatment. The findings of these research
   reports are consistent with the predicted results, suggesting that Rut
   has a therapeutic effect on IS. In a PPI network analysis, five core
   targets, ALB, MMP-9, CASP3, MMP-2, and ESR1, were identified. GO
   analysis showed that Evodia rutaecarpa had multiple functions. KEGG
   analysis indicated that the therapeutic effects of Evodia rutaecarpa on
   IS were mainly related to the MAPK and PI3K/AKT signaling pathways.
   Molecular docking was used to verify the connections between the main
   active ingredients determined by network pharmacology analyses and the
   key protein targets. The results showed that Rut had good binding
   affinity for ALB, MMP-9, CASP3, MMP-2, and ESR1, which was suitable for
   further experimental verification.

   The BBB is primarily composed of capillary endothelial cells and is a
   key barrier in the central nervous system. It prevents neurotoxic
   substances, immune-related factors, and other toxic substances from
   entering the central nervous system and maintains the homeostasis
   between the brain parenchyma and peripheral circulation [[103]18].
   After cerebral ischemia and reperfusion, multiple pathological
   processes, such as oxidative stress and inflammation-induced apoptosis,
   collaborate to damage the brain and destroy the structure and location
   of TJPs between the brain capillary endothelial cells, thereby
   increasing the permeability of the BBB. This results in severe
   complications and worsen brain damage [[104]19]. Therefore, protecting
   the structural integrity of the BBB may be an effective treatment
   strategy for reducing the progression of ischemic brain injury. The
   MAPK signaling pathway is a key signaling pathway that regulates cell
   growth, reproduction, differentiation, apoptosis, and stress responses
   under normal and pathological conditions, and is particularly important
   in the pathogenesis of cerebral ischemia [[105]20]. It has been widely
   demonstrated that during cerebral infarction, MAPK promotes the
   expression of apoptotic proteins and enhances neuronal cell death.
   Inhibiting the MAPK signaling pathway reduces the apoptosis of neuronal
   cells and exert a neuroprotective effect [[106]21]. MMPs are a group of
   enzymes in brain tissue that participate in the dynamic remodulation of
   the extracellular matrix. Their overexpression can degrade various
   components of extracellular matrix and TJPs of BBB, destroy the BBB,
   and increase the risk of hemorrhagic transformation after vascular
   recanalization treatment [[107]22]. Under pathological conditions, the
   p38MAPK sub-pathway is activated by hypoxia and ischemia, mediating the
   degradation of extracellular endothelial TJPs by MMP-2/9 and destroying
   the integrity of the BBB [[108]23]. In addition, after a stroke,
   necrosis and apoptosis increase significantly, which may be related to
   the abnormal expression of p38MAPK and oxidative stress [[109]24]. Hou
   et al. [[110]25] confirmed that endogenous inhibition of p38MAPK
   reduced neurological deficits, neuronal apoptosis, and infarct volume
   in a mouse model of cerebral IR injury. Zou et al. [[111]23] found that
   by regulating the MAPK-mediated MMPs pathway and upregulating the
   expression of claudin 3, occludin, and ZO-1, the BBB was protected from
   I/R injury. In conclusion, the MAPK signaling pathway, MMPs, and TJPs
   interact in the neuroprotection of IS, exerting a comprehensive
   protective effect by regulating cell apoptosis and the integrity of the
   BBB. Previous studies have shown that Rut improves osteoarthritis in
   mice by inhibiting the PI3K/AKT/NF-κB and MAPK signaling pathways
   [[112]26]. Wang et al. [[113]27]found that Rut inhibits the JNK/p38
   MAPK signaling pathway, interferes with the oxidative stress response,
   and reduces renal I/R injury in rats. Huang et al. [[114]28]confirmed
   that Rut inhibits the MAPK and NF-κB signaling pathways, thereby
   alleviating acute pancreatitis. These findings also suggest that Rut
   may exert its therapeutic effects by inhibiting the MAPK signaling
   pathway.

   Based on the results of network pharmacology analysis, Rut was screened
   for further studies. Rut is one of the main bioactive components of the
   traditional Chinese medicine Evodia rutaecarpa. Although a small number
   of studies have reported that Rut has some protective effects against
   cerebral ischemic diseases, many gaps exist in the existing
   understanding of the biological mechanisms by which Rut exerts its
   therapeutic effects. Therefore, we evaluated the effectiveness of Rut
   in treating IS in vivo and in vitro, explored its mechanisms of action,
   and verified the reliability of the predicted targets. In this study,
   HT22 cells underwent OGD to simulate neuronal stroke-like injury. The
   results showed that Rut improved the survival rate of neurons by
   inhibiting the excessive generation of ROS and improving mitochondrial
   membrane potential. We also verified the protective effects of Rut
   against IS injury in vivo. Eda is an effective hydroxyl radical
   scavenger and is widely used in the treatment within 24 hours after the
   onset of acute IS [[115]29]. Therefore, Eda was selected as the
   positive drug to help evaluate the therapeutic effect and clinical
   application value of Rut. The results showed that Rut inhibited the
   activation of p38MAPK in the brain tissue of MCAO rats, reduced the
   expression levels of MMP-2 and MMP-9, inhibited the degradation of the
   TJPs (ZO-1 and occludin), and reduced damage to the BBB. In addition,
   cerebral I/R injury is closely associated with the expression of
   apoptosis-related proteins [[116]30]. Rut reduced the expression level
   of cleaved caspase 3 protein, increased the Bcl-2/Bax ratio, reduced
   neuronal death in the rat brain tissue, reduced infarct volume,
   improved neurological function scores, and alleviated cerebral I/R
   injury in rats.

   Although this study has yielded positive results, it is still confined
   to the stages of cell and animal experiments, and lacks sufficient
   clinical data. It remains unknown whether it can be smoothly applied to
   the treatment of patients with cerebral IS. In addition, the mechanisms
   of cerebral ischemia and its reperfusion are complex. In this study,
   Rut alleviated I/R injury by regulating the MAPK signaling pathway to
   inhibit MMPs expression, reducing TJPs degradation, scavenging
   oxidative stress, inhibiting neuronal apoptosis, and protecting the
   BBB. Interestingly, Huang et al. [[117]31]found that Rut inhibits
   platelet activation and reduces microvascular thrombosis through the
   PLCγ2/PKC and PI3K/Akt/GSK3β pathways. Rut may act through these
   mechanisms throughout the entire process from microvascular thrombosis
   to post-thrombolytic rehabilitation. There is also evidence indicating
   that the composition of thrombi affects the thrombectomy strategies in
   endovascular treatment [[118]32]. it is unknown whether it also affects
   the therapeutic effect of Rut on anti-thrombosis. Therefore, it is
   necessary to further explore and clarify the mechanism of Rut in
   treating Cerebral I/R Injury, strengthen the systematic research on the
   multi-pathway and multi-target mechanisms of the active ingredient Rut
   in Evodia rutaecarpa, focus on the combination of experimental research
   and clinical practice, expand the sample size and the sources of
   samples, so as to enhance the potential for the translational
   application of Rut in the treatment of IS.

5. Conclusion

   In conclusion, we utilized network pharmacology and molecular docking
   analyses and in vitro and in vivo experiments to investigate the
   effects and mechanisms of Rut in IS treatment. The results confirmed
   that Rut alleviated BBB damage and antioxidative stress and reduced
   neuronal apoptosis by regulating the MAPK pathway after cerebral
   ischemia, thereby exerting neuroprotective effects in MCAO rats. Our
   results suggest a new mechanism for Rut in the treatment of IS and
   provide a new theoretical basis for the clinical application of Evodia
   rutaecarpa.

Supporting information

   S1 File. XXX.

   (ZIP)
   [119]pone.0327133.s001.zip^ (1.4MB, zip)

Abbreviations

   ALB
          albumin

   AO
          acridine orange

   BBB
          blood-brain barrier

   DMSO
          dimethyl sulfoxide

   EB
          ethidium bromide

   ESR1
          estrogen receptor 1

   GO
          Gene Ontology

   I/R
          ischemia-reperfusion

   IS
          ischemic stroke

   KEGG
          Kyoto Encyclopedia of Genes and Genomes

   MAPK
          mitogen-activated protein kinase

   MCAO
          middle cerebral artery occlusion

   MMP
          matrix metalloproteinase

   MTT
          3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide

   OD
          optical density

   OGD
          oxygen-glucose deprivation

   PBS
          phosphate-buffered saline

   PPI
          protein-protein interaction

   ROS
          reactive oxygen species

   TCMSP
          Traditional Chinese Medicine Systems Pharmacology

   TJPs
          tight junction Proteins

   TTC
          2,3,5-Triphenyl tetrazolium chloride

   ZO-1
          zonula occludens-1

Data Availability

   All relevant data are within the manuscript and its [120]Supporting
   Information files.

Funding Statement

   the Fundamental Research Funds for the Central Public Welfare Research
   Institutes (ZZ16-ND-12, ZZ16-YQ-050, ZZ17-ND-12).

References