Abstract
Intracerebral hemorrhage (ICH) is a devastating disease with the high mortality. The poor outcome of ICH is partially due to a combination of various secondary insults, including in the ischemic area. Xuemaitong capsule (XMT), a kind of traditional Chinese medicine, has been applied to clinic practice. The purpose of this study is to explore the mechanism of XMT in alleviating secondary damage in the ischemic area after ICH. We screened XMT target, compound components, and ICH-related targets using network pharmacology, cluster analysis, and enrichment analysis. We found that the tumor necrosis factor (TNF) signaling pathway might be the key signaling pathway for XMT treatment of ICH. An ICH rat model was established, as demonstrated by poor neurologic score. In the ICH rats, Western blot analysis and immunofluorescence indicated the upregulated expression of TNF receptor 1 (TNFR1), mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and caspase-3 (CASP3). Importantly, administration of XMT alleviated inflammation, edema, and increased perfusion in the ischemic area, whereas the expression of TNFR1, MAPK, NF-κB, and CASP3 was decreased. Furthermore, Fluoro-Jade B and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining revealed that XMT application also inhibited apoptosis and degradation of ischemic area neurons. In conclusion, this evidence elucidates that XMT alleviates neuron apoptosis, ischemic area inflammation, edema, and perfusion through the TNFR1-mediated CASP3/NF-κB/MAPK axis.
SIGNIFICANCE STATEMENT Tumor necrosis factor (TNF) is the key signaling pathway of Xuemaitong (XMT) to intervention during intracerebral hemorrhage. Fourteen key targets [intercellular adhesion molecule 1, interleukin (IL) 6, TNF, C-C motif chemokine ligand 2, prostaglandin-endoperoxide synthase 2, v-rel reticuloendotheliosis viral oncogene homolog A, matrix metalloproteinase 9, endothelin-1 (EDN1), mitogen-activated protein kinase (MAPK) 1, fos proto-oncogene protein, caspase-3 (CASP3), jun proto-oncogene, IL1B, MAPK8] are retrieved from the data base. XMT can inhibit neuron apoptosis in the ischemic area via regulating TNF receptor 1 (TNFR1)/CASP3. XMT alleviates inflammation and edema through regulating TNFR1/nuclear factor-κB and TNFR1/MAPK signaling pathways. XMT alleviates hypoperfusion in the cerebral ischemic area through mediating TNFR1/MAPK/EDN1.
Introduction
Intracerebral hemorrhage (ICH), one of the common stroke with high morbidity and mortality globally, is characterized by the pathologic aggregation of blood in the patient’s brain (Giakoumettis et al., 2017; Zeng et al., 2018). Studies indicate that hypertension and cerebral amyloid angiopathy are the main risk factors for ICH (Passos et al., 2016). Not only structural damage but also secondary injury leads to poor prognosis in the perihemorrhagic region (Mittal and LacKamp, 2016). It is known that clinical recovery after stroke relies on the salvage of the ischemic area (Carrera et al., 2013) and the viable tissue near the injured ischemic core (Liu et al., 2010). Notably, application of traditional Chinese medicine (TCM) has been suggested for treatment of ICH (Chen, 2015). Therefore, there is potential to develop TCM for preventing secondary damage in the ischemic area after ICH.
Recent years have witnessed the improvement and advancement of multitarget drugs and multicomponent therapy. TCM has been suggested as a crucial resource for discovering multitarget drugs (Wang et al., 2012). Network pharmacology, a novel discipline following the systems biology theory, is used to analyze the biologic network. Also, it helps to select the nodes of interest and promote drug development in a more cost-effective way, which can be instructive for analysis of TCM (Hao and Xiao, 2014). In TCM, Xuemaitong (XMT) is a known Miao medicine and has been widely applied to clinical practice in China. It is composed of Cinnamomum cassia ramulus, Salvia miltiorrhiza radix, Ligusticum chuanxiong rhizoma, Pueraria lobata radix, Gardenia jasminoides fructus, and Alisma orientale (Sam.) Juz. According to previous reports, XMT can attenuate carotid atherosclerosis (Zhang et al., 2014). XMT granules have also been reported to improve blood rheology in rat atherosclerosis (Qi et al., 2005). In addition, many previous studies have indicated that components of XMT are beneficial for stroke therapy. For instance, Shuan-Tong-Ling, a fermented Chinese formula that contains P. lobata radix and S. miltiorrhiza radix, has been shown to ameliorate ischemic stroke through suppression of inflammation and apoptosis (Mei et al., 2017). Moreover, combined use of P. lobata radix and L. chuanxiong rhizoma is widely applied for treatment of cerebrovascular diseases and has been found beneficial for alleviating cerebral ischemic stroke (Chen et al., 2019b). Intriguingly, our data base–based network analysis identified the tumor necrosis factor (TNF) signaling pathway as the key pathway for XMT treatment of ICH. TNF, a proinflammatory cytokine, contributes to mammalian immunity and cellular homeostasis, and its aberrant expression is associated with many inflammatory disorders, including stroke (Brenner et al., 2015). The progression of stroke is affected by the intricate relationship between the blood-brain barrier (BBB) and TNF (Pan and Kastin, 2007). A previous protein-protein interaction (PPI) network-based analysis also revealed the involvement of the TNF signaling pathway in stroke (Lv et al., 2020). Furthermore, a TCM prescription, Huang-Lian-Jie-Du decoction, has 28 target proteins in the stroke network, and it alleviates stroke via a pharmacological mechanism in part by mediating the TNF signaling pathway (Wang et al., 2019). However, little evidence elucidates the role of XMT in ICH. Therefore, in this study we performed network pharmacology to investigate whether XMT can be applied for ICH treatment. Based on the results of bioinformatic analysis, we established a rat model of ICH and administered rats with XMT, and target gene expression and signaling pathway were detected.
Materials and Methods
Ethics Statement.
The present study was approved by the Ethics Committee of the Animal of Guizhou Medical University. All animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Composition Analysis of XMT Compounds.
Through the TCM Systems Pharmacology data base (http://tcmspw.com/tcmsp.php) (screening conditions: oral bioavailability ≥30%, drug-likeness ≥0.18), we analyzed the compound components of C. cassia ramulus, S. miltiorrhiza radix, L. chuanxiong rhizoma, P. lobata radix, G. jasminoides fructus, and A. orientale (Sam.) Juz in XMT. The compound components of Euscaphis japonica (score cutoff ≥20, P ≥ 0.05) were screened through the BATMAN data base (http://bionet.ncpsb.org/batman-tcm/). A total of 65 compounds were retrieved, including two in E. japonica, six in C. cassia ramulus, 34 in S. miltiorrhiza radix, six in L. chuanxiong rhizoma, three in P. lobata radix, 12 in G. jasminoides fructus, and seven in A. orientale (Sam.) Juz.
Target Analysis of XMT Compounds.
TCM Systems Pharmacology and BATMAN data bases were used to retrieve the targets of compounds in XMT. The official gene symbols corresponding to the target protein were retrieved from the UniProt data base (http://uniprot/) (the species condition was limited to “HOMO sapiens”). As a result, 189 targets were obtained, including six corresponding compounds in E. japonica, 37 corresponding compounds in C. cassia ramulus, 68 corresponding compounds in S. miltiorrhiza radix, 22 corresponding compounds in L. chuanxiong rhizoma, 46 corresponding compounds in P. lobata radix, 160 corresponding compounds in G. jasminoides fructus, and 11 corresponding compounds in A. orientale (Sam.) Juz.
Analysis of ICH-Related Targets.
Through the Comparative Toxicogenomics Database (CTD; http://ctdbase.org/) and the GeneCards data base (https://www.genecards.org/), the targets of ICH were retrieved, and the screening condition was set as follows: CTD: influence score ≥30. In total, 3046 targets were retrieved from CTD, and 2489 were retrieved from the GeneCards data base. Moreover, 971 ICH-related targets were obtained by screening the intersecting targets retrieved from the two data bases.
PPI Network.
Based on the STRING data base (https://string-db.org), the XMT compound targets and ICH-related target interaction network (the species condition was limited to “HOMO sapiens”) were obtained.
Construction of Target Interaction Networks.
Major networks were as follows: 1) an ICH-related target network; 2) a TCM-compound-target network; 3) a key target of XMT treatment of ICH–compound-TCM network; and 4) a TCM-compound–TNF signal pathway target–TNF signal pathway network. All networks were drawn using Cytoscape (http://cytoscape.org/) version 3.7.1. At the same time, we analyzed the results of the networks using the Cytoscape website and selected the key targets for XMT treatment of ICH with the screening conditions set at BC ≥ Avg (BC), CC ≥ Avg (CC), and De ≥ Avg (De).
Cluster Analysis.
In large PPI networks, the tightly connected regions that may represent molecular complexes are defined as topological modules or clusters, which have pure network properties. The aggregation of nodes with similar or related functions in the same network is called a functional module. A disease module is a set of network components that together destroy cellular function and then cause a specific disease phenotype. Because a topological module, a functional module, and a disease module have the same meaning in the network, a functional module is equivalent to a topological module, and a disease module can be regarded as interference and destruction of a functional module. Finally, we can perform cluster analysis to obtain the topological modules through the Cytoscape plug-in MCODE.
Enrichment Analysis.
The functional enrichment webpage on Database for Annotation, Visualization and Integrated Discovery (DAVID) online (https://david.ncifcrf.gov/) was used for analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and the signal pathway of key target of XMT in the treatment of ICH. DAVID version 6.8 comprises a full Knowledgebase update to the sixth version of its original web-accessible programs and a comprehensive set of functional annotation tools. KEGG (https://www.kegg.jp/ or https://www.genome.jp/kegg/) is used to interpret genome sequences and other high-throughput data with three generic categories of systems information, genomic information, and chemical information, as well as the human-specific category of health information.
Animal Grouping and Administration Regimen.
A total of 126 adult male Sprague-Dawley rats weighing 200 ± 10 g were purchased from Guizhou Laboratory Animal Engineering Technology Center. The specific-pathogen–free animals underwent adaptive feeding at 20–25°C under 45%–50% relative humidity for 3 to 4 days (12-hour light/dark cycles). The rats were randomly and equally divided into seven groups in a blinded manner, including a sham-operated group, an ICH group, an ICH + vehicle group, three ICH + XMT groups (21, 42, 84 mg/kg), and an ICH + edaravone group (2 mg/kg). Edaravone, a drug known to alleviate ICH with neuroprotective effect was taken as a positive control in this study (Miao et al., 2020). XMT dose conversion between human and rats was as follows: the dose given to experimental animals (D2) = human dose (D1) × 6.3 mg/kg. The adult dose was 2.34 g/70 kg per day; therefore, D2 = 2.34 g ÷ 70 kg × 6.3 × 0.2 kg = 0.04 g per day). After ICH modeling, each rat was gavaged twice a day in the XMT treatment groups. In each gavage, each rat was treated with 0.04 g per day. ICH rats in the edaravone group were injected intraperitoneally once a day, whereas those in the vehicle group were given an equal volume of saline. XMT, edaravone, and normal saline were administrated at 10 minutes after modeling. At 24 hours after ICH, 16 brain tissues in each group were selected for subsequent a series of experiments. The last eight rats in each group were tested for brain edema and neurologic function scoring 72 hours after ICH.
ICH Modeling.
As previously described (Meng et al., 2018), an ICH model was established in rats by injecting 100 μl of autogenous blood. The rats were anesthetized and placed in a stereotactic frame (ZH-Blue Star B brain stereotactic apparatus; Huaibei Zhenghua Biologic Instrument Equipment Co., Ltd., Anhui, China). Next, the whole blood was collected via femoral artery puncture. The microinjector (Hamilton Company, Reno, NV) was inserted into bone hole (3 mm lateral to midline and 1 mm front to bregma, ventral side of cortex surface: 5.5 mm). First, 20 μl blood was injected and maintained for 5 minutes, and then 30 μl blood was injected and maintained for 5 minutes. After the injection, the microinjector was slowly withdrawn.
Neurologic Scoring.
After XMT treatment, the ICH model rats were monitored for appetite, activity, and neural defects, and their behavioral disorders were assessed according to the scoring system (Table 1) as mentioned previously (Li et al., 2018).
Behavior and activity scores
Determination of Brain Water Content.
As previously described (Wang et al., 2017), after 72 hours of ICH treatment, the content of brain water was determined by means of dry and wet method. After collecting brain tissues, the samples were divided into five parts: ipsilateral basal ganglia, ipsilateral cortex, contralateral basal ganglia, contralateral cortex, and cerebellum. After that, the above samples were immediately weighed to obtain wet weight and then reweighed for dry weight after drying at 100°C for 24 hours. The percentage of moisture content was calculated by the following formula: (wet weight − dry weight)/wet weight × 100%.
Terminal Deoxynucleotidyl Transferase–Mediated Digoxigenin-Deoxyuridine Nick-End Labeling Staining.
As described previously (Wu et al., 2017), terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) and neuronal nuclear antigen double staining were used to quantify the apoptotic neurons according to the instructions of the in situ cell death detection kit (Roche, Madison, WI). Three sections of each rat were examined and photographed in parallel for TUNEL-positive cell counting.
Fluoro-Jade B Staining.
The brain slices of Sprague-Dawley rats were dewaxed with xylene and a series of graded ethanol solutions. After incubation with 0.06% KMnO4 at room temperature for 15 minutes, the slices were washed with PBS, dyed with Fluoro-Jade B (FJB) working solution at room temperature for 1 hour, and then dehydrated and air-dried. Finally, the brain slices were observed and photographed under a fluorescence microscope (BX50/BX-FLA/DP70; Olympus, Tokyo, Japan). FJB-positive cells were counted.
Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction.
Trizol reagent (15596026; Invitrogen, Carlsbad, CA) was used to extract the total RNA. According to the instructions of the Primescript RT Regent Kit (RR047A; Takara Bio Inc., Otsu, Shiga, Japan), the RNA was reverse-transcribed into cDNA. The synthesized cDNA was detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) with a Fast SYBR Green PCR kit (Applied Biosystems, Carlsbad, CA) on the ABI PRISM 7300 RT-PCR system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal parameter to analyze the relative expression of genes by 2−ΔΔCt method. The primer design is shown in Table 2.
Primers used for real-time polymerase chain reaction
H&E Staining.
After deep anesthesia, rats were injected rapidly with 0.9% normal saline through the left ventricular aorta until the color of the liver became lighter and the clear fluid flowed out of the right atrial appendage incision. Subsequently, 250 ml of 4% polyoxymethylene (Sigma-Aldrich Chemical Company, St. Louis, MO) was used for perfusion until the limbs of rats became stiff. The brain tissues were fixed in 4% paraformaldehyde at 4°C for 24 hours, followed by embedding. The paraffin sections of rat brains were baked in an oven at 60°C for 30 minutes, stained with H&E, then sealed with neutral gum and finally observed under a microscope (Olympus).
Immunofluorescence.
The tissues were cut into 12 μm slices, and the apoptotic neurons were stained with Nissl staining solution. Next, the sections were incubated with the primary antibody cleaved caspase-3 (CASP3; 1:100; Cell Signaling Technologies, Beverly, MA) overnight at 4°C. After three washes in PBS, goat anti-rabbit Alex594 was applied at room temperature for 1 hour. Afterward, the sections were washed with PBS and observed under a Nikon-A1RS confocal microscope.
Western Blot Assay.
After ICH, the tissues on the marginal area of lesions were lysed with the enhanced radioimmunoprecipitation assay lysate containing protease inhibitor (Boster Biologic Technology Co., Ltd., Wuhan, China), and then the protein concentration was determined with a bicinchoninic acid protein quantitative Kit (Boster Biologic Technology Co., Ltd.). The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated protein was transferred to a polyvinylidene fluoride membrane. After blockade with 5% bovine serum albumin sealed at room temperature for 2 hours to block the nonspecific binding, the membrane was incubated overnight at 4°C with diluted rabbit primary antibodies: TNF receptor 1 (TNFR1) (13377S), TNFRSF1A associated via death domain (TRADD) (3694S), TNF receptor–associated factor (TRAF) 2 (4712S), and cleaved receptor-interacting protein 1 (RIP1)/RIP1 (3493S) from Cell Signaling Technologies; Fas-associated death domain (FADD) (ab24533), CASP3 (ab4051), cleaved CASP3 (ab49822), extracellular signal-regulated kinase (ERK) (ab17942), phosphorylated (p)-ERK (ab201015), p38 (ab170099), p-p38 (ab47363), c-Jun amino-terminal kinase (JNK) (ab179461), p-JNK (ab124956), and β-actin (ab8227) from Abcam Inc. (Cambridge, MA). The following day, the horseradish peroxidase–labeled goat anti-rabbit secondary antibody (ab205719; 1: 2000; Abcam) was used to incubate the membrane at room temperature for 1 hour. Enhanced chemiluminescence working solution (Millipore, Billerica, MA) was used to visualize protein bands. ImageJ analysis software was used to quantify the gray level of each band in Western blot images with β-actin as internal reference.
Statistical Analysis.
Statistical analysis of the data was performed using the statistical software SPSS 21.0 (IBM Corp., Armonk, NY). Measurement data were expressed as means ± S.D. Initially, a test of normality and homogeneity of variance was conducted. Data comparison among multiple groups obeying normal distribution and homogeneous variance was conducted using one-way ANOVA or repeated measures of ANOVA, followed by Tukey’s test. P < 0.05 demonstrated that the difference was statistically significant.
Results
ICH-Related Target Network.
ICH-related target genes were screened from CTD and the GeneCards data base, and 971 genes at the intersection of two data bases were obtained. These 971 overlapping genes were displayed in a PPI network. The network consists of 971 nodes and 34920 edges, where each node refers to a gene and each edge connects to two genes (Fig. 1). Among them, 20 nodes in red [GAPDH, RAC-alpha serine/threonine-protein kinase (AKT1), albumin, interleukin (IL) 6, insulin, vascular endothelial growth factor A (VEGFA), tumor protein 53 (TP53), TNF, endothelial growth factor receptor (EGFR), endothelial growth factor (EGF), fibronectin 1, mitogen-activated protein kinase (MAPK) 3, V‑myc avian myelocytomatosis viral oncogene homolog (Myc), signal transducer and activator of transcription (STAT) 3, CASP3, MAPK8, MAPK1, chemokine ligand 18, jun proto-oncogene (JUN), matrix metalloproteinase (MMP) 9] had high degree with more edges: 501 for GAPDH, 489 for AKT1, 488 for ALB, 471 for IL6, 462 for INS, 433 for VEGFA, 426 for TP53, 412 for TNF, 389 for EGFR, 375 for EGF, 367 for FN1, 361 for MAPK3, 337 for Myc, 337 for STAT3, 329 for CASP3, 309 for MAPK8, 308 for MAPK1, 307 for CXCL8, 305 for JUN, and 294 for MMP9. These indicated that these 20 genes are at the core of the gene network, and thus they may be the key genes in the cause and development of ICH.
Network of ICH-related targets. Each circle in the figure represents a gene, and each edge connects to two genes that bind to each other. The increasing number of edge responses to higher degree. Core genes (red) have higher degrees.
TCM-Compound-Target Network.
To further screen TCM-related branches, we performed analysis on the TCM-compound-target network and established another network. The TCM-compound-target network consists of 261 nodes and 4139 edges (Fig. 2), including seven kinds of TCM, 65 compounds, and 189 targets. In this network, we found that many targets are regulated by various compounds at the same time, such as prostaglandin-endoperoxide synthase 2 (PTGS2), AKT1, TP53, ESR1, CASP3, Jun, and TNF, but there are also targets, such as IL6, ECE1, CLDN4, CTRB1, NPEPPS, CACNA2D1, PCOLCE, ACPP, EIF6, and PKIA, only regulated by one compound. For example, PTGS2 was regulated by all compounds in TCM except E. japonica and A. orientale (Sam.) Juz, whereas IL6 was only regulated by quercetin in G. jasminoides fructus. These results suggest that the compounds in XMT may act synergistically on these targets, thereby exerting a pharmacological effect on the treatment of ICH. In addition, it also suggests the multicomponent, multitarget, and multidisease therapeutic characteristics of TCM.
TCM-compound-target network. This network contains seven kinds of TCMs, 65 compounds, and 189 targets. The red V-shaped nodes represent TCM, the pink hexagons represent compounds, and the purple circles represent targets.
Cluster Analysis for the TCM-Compound-Target Network.
The TCM-compound-target network was next assessed by cluster analysis. Using the Cytoscape plug-in MCODE, we clustered the relation on the TCM-compound-target network for analysis and obtained eight topological modules (Fig. 3): cluster 1: score = 43.36, nodes = 51, edges = 1085; cluster 2: score = 6.933, nodes = 16, edges = 52; cluster 3: score = 5.474, nodes = 20, edges = 52; cluster 4: score = 5.2, nodes = 6, edges = 13; cluster 5: score = 5.077, nodes = 14, edges = 33; cluster 7: score = 3, nodes = 5, edges = 6; cluster 8: score = 3, nodes = 3, edges = 3.
Cluster analysis for the TCM-compound-target network. The pink hexagons represent compounds, and the purple circles represent targets. Panels (A) through (H) represent clusters 1 through 8, respectively.
Key Target–Compound-TCM-XMT Network.
Subsequently, we retrieved XMT potential targets from the ICH-related target network and the TCM-compound-target network, where the genes at the intersections were selected. A total of 121 potential targets for XMT treatment of ICH were obtained (Fig. 4A). Further screening conditions for these targets were set at BC ≥ Avg (BC), CC ≥ Avg (CC), and De ≥ Avg (DE). As a result, we obtained 34 key targets [MAPK8, JUN, TP53, CASP3, TNF, EGFR, EGF, Fos, SERPINE1, endothelin-1 (EDN1), AKT1, CCND1, VEGFA, STAT3, RELA, IL1B, IL6, IL10, MYC, MAPK1, ERBB2, IL4, C-C motif chemokine ligand 2 (CCL2), ESR1, MMP9, intercellular adhesion molecule 1 (ICAM1), STAT1, AR, NR3C1, PPARG, PTGS2, MMP2, CAT, MPO] (Fig. 4B). These 34 key targets correspond to 55 compounds and seven kinds of TCM in XMT (Fig. 4C).
Key target–compound-TCM-XMT network. (A) One hundred twenty-one putative targets for XMT treatment of ICH. (B) Thirty-four key targets for XMT treatment of ICH. (C) Key target–compound-TCM-XMT network. The red V-shaped nodes represent TCM, the pink hexagons represent compounds, and the purple circles represent targets.
KEGG Enrichment Analysis and Key Signal Pathway Target–Compound-Drug Network.
Through DAVID, the 121 candidate genes for XMT treatment of ICH obtained above were analyzed by KEGG analysis (Fig. 5A). XMT mainly affected 91 pathways, including pathways in cancer (hsa05200), the TNF signaling pathway (hsa04668), hepatitis B (hsa05161), pancreatic cancer (hsa05212), bladder cancer (hsa05219), Chagas disease (American trypanosomiasis) (hsa05142), proteoglycans in cancer (hsa05205), the hypoxia-inducible factor 1 signaling pathway (hsa04066), leishmaniasis (hsa05140), pertussis (hsa05133), colorectal cancer (hsa05210), inflammatory bowel disease (hsa05321), the MAPK signaling pathway (hsa04010), the prolactin signaling pathway (hsa04917), the Toll-like receptor signaling pathway (hsa04620), endometrial cancer (hsa05213), influenza A (hsa05164), prostate cancer (hsa05215), osteoplast differentiation (hsa04380), and hepatitis C (hsa05160) (P < 0.05). The TNF signaling pathway is the main signaling pathway in the treatment of ICH with XMT. There are 14 key targets enriched in the TNF signaling pathway. These 14 key targets correspond to 49 compounds and five kinds of TCM in XMT (Fig. 5B).
KEGG enrichment analysis through DAVID and key signal pathway target–compound-drug network. (A) KEGG enrichment analysis. (B) Key signal pathway target–compound-drug network. The red V-shaped nodes represent TCM, the pink hexagons represent compounds, and the purple circles represent targets.
XMT Regulated the TNF Signaling Pathway in the Cerebral Ischemic Area Upon ICH.
We then explored the potential regulatory role of XMT on the TNF signaling pathway in the cerebral ischemic area. As illustrated in Fig. 6A, regarding the TNF signaling pathway, TNFR1 was indicated to regulate the expression of CASP3 through the death-inducing signaling complex, including TRADD, TRAF2, FADD, and RIP1. Meanwhile, TNFR1 regulated the expression of CASP3 through signalosome (complex 1), including TRADD, TRAF2, and RIP1, which regulated MAPK and nuclear factor-κB (NF-κB) signaling pathways and their downstream genes (CCL2, IL1B, IL6, TNF, Fos, Jun, MMP9, EDN1, ICAM1, PTGS2). Figure 6B shows the ischemic core area and ischemic area at the site of cerebral hemorrhage. The neurologic scoring (Fig. 6C) showed that compared with sham-operated rats, the ICH rats had notably impaired neurobehavioral ability (P < 0.05), whereas ICH rats could partially alleviate the injury and restore neurobehavioral ability under the XMT treatment at a dose of 42 or 84 mg/kg (P < 0.05). The effect of XMT was similar to that of edaravone. Compared with sham-operated rats, Western blot assay revealed that the protein expression of TNFR1, TRADD, TRAF2, FADD, and RIP1 and the extent of RIP1 phosphorylation were significantly increased in ICH rats (P < 0.05), whereas those in the XMT group were obviously decreased (P < 0.05; Fig. 6, D and E). Meanwhile, the results of immunofluorescence assay for TNFR1 were consistent with those of Western blot assay. XMT markedly inhibited the abnormal increase of TNFR1 protein expression in the cerebral ischemic area induced by ICH (P < 0.05; Fig. 6F; Supplemental Fig. 1). Collectively, XMT significantly alleviated aberrant expression of TNRP1 after ICH and cerebral ischemia.
XMT inhibited TNFR1 aberrant expression and cerebral ischemia upon ICH. (A) TNF signaling pathway. (B) Ischemic area and core area. (C) Neurologic function scoring. (D and E) Western blot assay for detection of the protein expression of TNFR1, TRADD, TRAF2, FADD, and RIP1 and the extent of RIP1 phosphorylation in the ischemic area. (F) Quantification of the protein expression and location of TNFR1 in the ischemic area. *P < 0.05 vs. sham-operated rats. These data were measurement data, expressed as means ± S.D. Data comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s test. All experiments were repeated three times.
XMT Inhibited the Apoptosis of Neurons in the Ischemic Area by Regulating TNFR1/CASP3.
In addition, we investigated the effect of XMT on TNFR1/CASP3 in the ischemic area through network pharmacology. It was shown that XMT mainly regulated the expression of the death-inducing signaling complex and its downstream CASP3 through the TNF signaling pathway to inhibit the apoptosis of neurons (Fig. 7A). To verify the mechanism revealed by the network, we established a rat model and administered rats with XMT to observe alteration of CASP3. Compared with that in sham-operated rats, ICH rats exhibited elevated expression of cleaved CASP3/CASP3 in ICH rats (P < 0.05), whereas administration of 42 or 84 mg/kg of XMT notably decreased cleaved CASP3/CASP3 level (P < 0.05) (Fig. 7B). At the same time, the results of immunofluorescence assay for cleaved CASP3 were consistent with Western blot assay revealing increased cleaved CASP3/CASP3 level in the ICH rats. Upon XMT administration, the abnormal increase of cleaved CASP3 protein expression in the ischemic area secondary to ICH was decreased (P < 0.05; Fig. 7C; Supplemental Fig. 2). These results suggest that XMT may inhibit the apoptosis of neurons by regulating the protein expression of cleaved CASP3.
XMT decreased the apoptosis of neurons in the ischemic area by regulating the TNFR1/CASP3 signaling pathway. (A) Diagrammatic sketch of the TNFR1/CASP3 signaling pathway. (B) Western blot analysis of cleaved caspase-3 and caspase-3 in the ischemic area. (C) Quantification of cleaved caspase-3 in the ischemic area. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. ICH rats. These data were measurement data, expressed as means ± S.D. Data comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s test. All experiments were repeated three times.
XMT Inhibited the Degeneration and Apoptosis of Neurons in the Ischemic Area by Regulating Cleaved CASP3.
To explore how XMT regulates neurons progression upon ICH through cleaved CASP3, we stained the neurons with FJB and TUNEL double staining to further observe the effect of XMT on the degeneration and apoptosis. The results revealed that compared with sham-operated rats, the number of TUNEL-positive cells (P < 0.05; Fig. 8A) and FJB-positive cells (P < 0.05; Fig. 8B) was significantly increased in the ICH rats, whereas ICH rats treated with XMT or edaravone had decreased TUNEL- and FJB-positive cells (Supplemental Fig. 3). The effect of 84 mg/kg XMT was similar to 2 mg/kg edaravone, but 84 mg/kg XMT was more effective. The above experimental results demonstrated that XMT has a certain rescue effect on the lesion of ICH, more effective than edaravone at the same dose. Of note, XMT inhibited apoptosis and degradation of ischemic nerve cells.
XMT inhibited the degeneration and apoptosis of neurons in the cerebral ischemic area by regulating cleaved CASP3. (A) Quantification of the degeneration of neurons in the ischemic area. (B) Quantification of the apoptosis of neurons in the ischemic area. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. ICH rats. These data were measurement data, expressed as means ± S.D. Data comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s test. All experiments were repeated three times.
XMT Inhibited the TNFR1/NF-κB Pathway Upon ICH.
Next, we analyzed the regulatory effect of XMT on the TNFR1/MAPK pathway in the ischemic area. Network pharmacology revealed that XMT can regulate the abnormal activation of the NF-κB signaling pathway mediated by signalosome through the TNF signaling pathway (Fig. 9A). We therefore determined the expression of related proteins and downstream genes in the NF-κB signaling pathway. Western blot assay showed that compared with sham-operated rats, ICH rats exhibited increased protein expression of p-p65/p65 and p-IκBα/IκBα (P < 0.05). In addition, treatment with XMT had notably decreased protein expression of p-p65/p65 and p-IκBα/IκBα (P < 0.05; Fig. 9B). Moreover, ICH rats also displayed increased expression of inflammatory cytokines IL6, IL1B, TNF (Fig. 9C), adhesion molecule ICAM1 (Fig. 9D), and PTGS2 (Fig. 9E) in comparison with that of sham-operated rats (P < 0.05), whereas the expression of all these genes was notably decreased in ICH rats treated with XMT relative to that in ICH rats (P < 0.05). The aforementioned results validated our network pharmacology results that XMT could block the TNFR1/NF-κB pathway in the ischemic area.
XMT suppressed the TNFR1/NF-κB pathway in the cerebral ischemic area. (A) Schematic diagram of the TNFR1/NF-κB signaling pathway. (B) The protein expression of p65 and IκBα as well as the extent of p65 and IκBα phosphorylation in the ischemic area as detected by Western blot assay. (C–E) The mRNA expression of IL6, IL1B, TNF, ICAM1, and PTGS2 as detected by RT-qPCR. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. ICH rats. These data were measurement data, expressed as means ± S.D. Data comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s test. All experiments were repeated three times.
XMT Suppressed the TNFR1/MAPK Pathway in the Ischemic Area.
Next, we analyzed the interaction between XMT and the TNFR1/MAPK pathway in the ischemic area. The network pharmacology showed that XMT alleviated the abnormal activation of the signalosome-mediated MAPK signaling pathway through the TNF signaling pathway (Fig. 10A). To verify the result of the network, the expression of related proteins and downstream genes in the MAPK signaling pathway in rats was determined by Western blot assay. p-ERK/ERK, p-p38/p38, and p-JNK/JNK were highly expressed in ICH rats relative to sham-operated rats (P < 0.05). Addition of XMT reduced protein expression of p-ERK/ERK, p-p38/p38, and p-JNK/JNK (P < 0.05; Fig. 10B). Compared with that in sham-operated rats, the expression of Fos and Jun (P < 0.05; Fig. 10C), CCL2 (P < 0.05; Fig. 10D), MMP9 (P < 0.05; Fig. 10E), and EDN1 in ICH rats was significantly higher (P < 0.05; Fig. 10F), whereas XMT administration decreased the expression of the above factors as well (P < 0.05). All these results verified our network pharmacology results that XMT could inhibit the TNFR1/MAPK pathway in the ischemic area.
XMT blocked the TNFR1/MAPK pathway in the cerebral ischemic area. (A) Schematic diagram of the TNFR1/MAPK signaling pathway. (B) The protein expression of ERK, p38, and JNK as well as the extent of ERK, p38, and JNK phosphorylation in the ischemic area as detected by Western blot assay. (C–F) The mRNA expression of Fos and Jun, CCL2, MMP9, and EDN1 as detected by RT-qPCR. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. ICH rats. These data were measurement data, expressed as means ± S.D. Data comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s test. All experiments were repeated three times.
XMT Alleviated Inflammation and Edema and Increased Perfusion in the Cerebral Ischemic Area.
Proinflammatory factors, chemokines, and MMPs are all related to the destruction of the BBB (Zhang et al., 2018; Lasek-Bal et al., 2019). At the same time, the destruction of the BBB can further induce inflammation by promoting leukocyte infiltration. In addition to promoting inflammation, the destruction of the BBB can also lead to the formation of angiogenic edema after ICH. Whether XMT alleviates the formation of angiogenic edema after ICH by inhibiting inflammation and MMPs remained elusive and aroused our interest. We applied H&E staining and tissue water content measurement to assess the effect of XMT. In sham-operated rat, brain tissues contained rich neurons, the neurons were arranged orderly with normal tissue gaps, and the staining of cell tissue structure was uniform with clear nucleus (blue). In ICH rats and ICH rats treated with vehicle, glial cells and neurons were swollen, decreased, and unevenly arranged, along with widened extracellular space and weakened eosinophilic cells of the cytoplasm; some cells were pyknotic and necrotic, and the staining was deepened, accompanied by infiltration of a large number of neutrophils and microglial cells. Upon treatment with XMT or edaravone, all symptoms were alleviated: the swelling was attenuated, the widening shrank, the the eosinophilic cells of the cytoplasm were enhanced, and the number of pyknosis necrosis cells and inflammatory cells was reduced (Fig. 11A). Meanwhile, the water content of brain tissues in the ischemic area was notably higher in ICH rats than in sham-operated rats (P < 0.05). Relative to the ICH rats, rats treated with XMT or edaravone displayed a decline in the water content of brain tissues in the ischemic area (P < 0.05; Fig. 11B).
XMT alleviated inflammation and edema and increased perfusion in the cerebral ischemic area. (A) H&E staining for the cerebral ischemic area. (B) Water content measurement in ipsilateral basal ganglia, ipsilateral cortex, contralateral basal ganglia, contralateral cortex, and cerebellum. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. ICH rats. These data were measurement data, expressed as means ± S.D. Data comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s test. All experiments were repeated three times.
Discussion
ICH is a fatal neurologic disorder with poor prognosis (Kim et al., 2014). In spite of modern treatment effectively realizing rapid stabilization, management of patients with ICH requires long-term anticoagulation, and patients are prone to other neurodegenerative diseases. Novel medical and surgical approaches are urgently required (Schrag and Kirshner, 2020). Notably, network pharmacology in combination with TCM research have been highlighted as novel insights in the development of modern drugs (Wu and Wu, 2015). In this study, we elucidated that XMT ameliorates the secondary damage after ICH through modulation of TNFR1/CASP3, TNFR1/NF-κB, and TNFR1/MAPK.
Our study identified the TNF signaling pathway as the main signal pathway in the treatment of ICH with XMT. We also predicted 14 TNF signaling pathway–enriched genes (ICAM1, IL6, TNF, CCL2, PTGS2, v-rel reticuloendotheliosis viral oncogene homolog A, MMP9, EDN1, MAPK1, Fos, CASP3, JUN, IL1B, MAPK8) as key targets for the intervention efficacy on ICH. Furthermore, we found that XMT-mediated TNFR1/CASP3, TNFR1/NF-κB, and TNFR1/MAPK were involved in neuron apoptosis, inflammation/edema, and the ischemic area after ICH. TNFR1, one of the TNF signaling receptors, is accountable for the pathogenesis of Alzheimer’s disease through facilitating neuron death (Steeland et al., 2018). CASP3 is a well known proapoptotic gene (Yamabe et al., 1999), and its activation has been implicated in ischemic stroke (Li et al., 2020). Moreover, upregulated plasma level of TNFR1 was correlated with incident ICH, suggesting the underlying association between TNF-mediated inflammation and vascular changes prior to ICH (Svensson et al., 2017). Consistently, downregulated expression of TNF-α, TNFR1, and NF-кB by cannabidiol in the total, core, and ischemic areas could play a cerebroprotective role in ischemic injury (Khaksar and Bigdeli, 2017). Additionally, inactivation of the AMP-activated protein kinase/JNK/p38 MAPK pathway by melanocortin receptor 4 with RO27-3225 was found to reduce neuroinflammation after ICH in a mouse model (Chen et al., 2018). The inactivation of the Toll-like receptor 4/TRAF6/NF-κB pathway by luteolin also ameliorates neuroinflammation after ICH (Yang et al., 2020). Furthermore, downregulation of TNF-α and TNFR1 was found to attenuate endothelial necroptosis and improve stroke outcomes (Chen et al., 2019a). EDN1 is well known for its vasoconstrictor function in controlling brain microcirculation (Petrov et al., 2002). Interference with EDN1 could also improve cerebral hypoperfusion and restore CBF in multiple sclerosis (D’Haeseleer et al., 2013). Overall, these previous reports are supportive of our finding regarding the involvement of TNFR1/CASP3, TNFR1/NF-κB, and TNFR1/MAPK in ICH.
Mechanistically, our study revealed that XMT could mediate the TNF signaling pathway, mainly by regulating TNFR1/CASP3, TNFR1/NF-κB, and TNFR1/MAPK in the ischemic area after ICH. In fact, another TCM prescription, Danggui-Honghua, was also detected through network analysis. It was considered a promising therapy for blood stasis syndrome by targeting genes mainly enriched in the TNF signaling pathway (Yue et al., 2017). Additionally, Longxuetongluo capsule, a prescription for ischemic stroke treatment, could attenuate neuro-inflammation in BV2 microglial cells partially by diminishing IL1β, IL6, and TNF-α production and NF-κB translocation (Hong et al., 2020). The regulatory effect of XMT on TNF signaling pathway–related genes has been reported but not extensively elucidated. Zhang et al. (2014) found that XMT granules could diminish IL6, TNF-α, and high-sensitivity C-reactive protein expression, which ameliorated carotid atherosclerosis. A previous study has found that Xuemaitong granules could decrease the expression of MMP1, MMP9, and endothelin in a rabbit model of atherosclerosis, thereby improving blood rheology (Qi et al., 2005). In addition, many of the compounds in XMT have been revealed as involved in the regulation of TNF signaling pathway–related genes. For instance, AA-24-a extracted from A. orientale (Sam.) Juz. was found to be capable of elevating glucose uptake via the calmodulin-dependent protein kinase kinase β–AMP-activated protein kinase–p38 MAPK/AS160 pathway (Chen et al., 2020). Similarly, P. lobata radix–derived puerarin conferred renal protection against cisplatin nephrotoxicity by inactivating the Toll-like receptor 4/NF-κB pathway (Ma et al., 2017). Collectively, it was concluded that XMT yields therapeutic efficacy on ICH via regulation of TNFR1/CASP3, TNFR1/NF-κB, and TNFR1/MAPK.
In conclusion, the results obtained from this study demonstrated that XMT is able to alleviate secondary damage in the ischemic area after ICH through mediating TNFR1/CASP3, TNFR1/NF-κB, and TNFR1/MAPK. This finding also suggests the role of network pharmacology in analyzing the molecular mechanism of TCM in treatment of stroke. However, the clinical efficacy of the therapeutic targets still needs further validation in clinical research.
Acknowledgments
We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study.
Authorship Contributions
Participated in research design: Zhang.
Conducted experiments: Zeng.
Contributed new reagents or analytic tools: Zhang, Zeng, Wu.
Performed data analysis: Wu.
Wrote or contributed to the writing of the manuscript: Zhang, Zeng, Wu.
Footnotes
- Received May 1, 2020.
- Accepted October 5, 2020.
↵1 B.Z., Z.Z., and H.W. are co-first authors.
This work was supported by the Science and Technology Foundation of Guizhou Province (No. [2017]1015) and the Project of Science and Technology of Guizhou Educational Committee (No. KY[2018]053).
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This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- BBB
- blood-brain barrier
- CASP3
- caspase-3
- CCL2
- C-C motif chemokine ligand 2
- CTD
- Comparative Toxicogenomics Database
- DAVID
- Database for Annotation, Visualization and Integrated Discovery
- EDN1
- endothelin-1
- EGF
- endothelial growth factor
- EGFR
- endothelial growth factor receptor
- ERK
- extracellular signal-regulated kinase
- FADD
- Fas-associated death domain
- FJB
- Fluoro-Jade B
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- ICAM1
- intercellular adhesion molecule 1
- ICH
- intracerebral hemorrhage
- IL
- interleukin
- JNK
- Jun amino-terminal kinase
- JUN
- jun proto-oncogene
- KEGG
- Kyoto Encyclopedia of Genes and Genomes
- MAPK
- mitogen-activated protein kinase
- MMP
- matrix metalloproteinase
- NF-κB
- nuclear factor-κB
- p-
- phosphorylated
- PPI
- protein-protein interaction
- PTGS2
- prostaglandin-endoperoxide synthase 2
- RT-qPCR
- reverse transcription quantitative real-time polymerase chain reaction
- RIP1
- receptor-interacting protein 1
- STAT
- signal transducer and activator of transcription
- TCM
- traditional Chinese medicine
- TNF
- tumor necrosis factor
- TNFR1
- TNF receptor 1
- TRADD
- TNFRSF1A associated via death domain
- TRAF
- TNF receptor–associated factor
- TUNEL
- terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling
- VEGFA
- vascular endothelial growth factor A
- XMT
- Xuemaitong
- Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics
