Dihydrocelastrol induces antitumor activity and enhances the sensitivity of bortezomib in resistant multiple myeloma by inhibiting STAT3-dependent PSMB5 regulation.

Multiple myeloma (MM) is characterized by excessive aggregation of B-cell-derived malignant plasma cells in the hematopoietic system of bone marrow. Previously, we synthesized an innovative molecule named dihydrocelastrol (DHCE) from celastrol, a triterpene purified from medicinal plant Tripterygium wilfordii. Herein, we explore the therapeutic properties and latent signal transduction mechanism of DHCE action in bortezomib (BTZ)-resistant (BTZ-R) MM cells. In this study, we first report that DHCE shows antitumor activities in vitro and in vivo and exerts stronger inhibitory effects than celastrol on BTZ-R cells. We find that DHCE inhibits BTZ-R cell viability by promoting apoptosis via extrinsic and intrinsic pathways and suppresses BTZ-R MM cell proliferation by inducing G0/G1 phase cell cycle arrest. In addition, inactivation of JAK2/STAT3 and PI3K/Akt pathways are involved in the DHCE-mediated antitumor effect. Simultaneously, DHCE acts synergistically with BTZ on BTZ-R cells. PSMB5, a molecular target of BTZ, is overexpressed in BTZ-R MM cells compared with BTZ-S MM cells and is demonstrated to be a target of STAT3. Moreover, DHCE downregulates PSMB5 overexpression in BTZ-R MM cells, which illustrates that DHCE overcomes BTZ resistance through increasing the sensitivity of BTZ in resistant MM via inhibiting STAT3-dependent PSMB5 regulation. Overall, our findings imply that DHCE may become a potential therapeutic option that warrants clinical evaluation for BTZ-R MM.

ETCM v2.0: An update with comprehensive resource and rich annotations for traditional Chinese medicine.

Existing traditional Chinese medicine (TCM)-related databases are still insufficient in data standardization, integrity and precision, and need to be updated urgently. Herein, an Encyclopedia of Traditional Chinese Medicine version 2.0 (ETCM v2.0, http://www.tcmip.cn/ETCM2/front/#/) was constructed as the latest curated database hosting 48,442 TCM formulas recorded by ancient Chinese medical books, 9872 Chinese patent drugs, 2079 Chinese medicinal materials and 38,298 ingredients. To facilitate the mechanistic research and new drug discovery, we improved the target identification method based on a two-dimensional ligand similarity search module, which provides the confirmed and/or potential targets of each ingredient, as well as their binding activities. Importantly, five TCM formulas/Chinese patent drugs/herbs/ingredients with the highest Jaccard similarity scores to the submitted drugs are offered in ETCM v2.0, which may be of significance to identify prescriptions/herbs/ingredients with similar clinical efficacy, to summarize the rules of prescription use, and to find alternative drugs for endangered Chinese medicinal materials. Moreover, ETCM v2.0 provides an enhanced JavaScript-based network visualization tool for creating, modifying and exploring multi-scale biological networks. ETCM v2.0 may be a major data warehouse for the quality marker identification of TCMs, the TCM-derived drug discovery and repurposing, and the pharmacological mechanism investigation of TCMs against various human diseases.

Pharmacological characterization of the small molecule 03A10 as an inhibitor of α-synuclein aggregation for Parkinson's disease treatment.

Aggregation of α-synuclein, a component of Lewy bodies (LBs) or Lewy neurites in Parkinson's disease (PD), is strongly linked with disease development, making it an attractive therapeutic target. Inhibiting aggregation can slow or prevent the neurodegenerative process. However, the bottleneck towards achieving this goal is the lack of such inhibitors. In the current study, we established a high-throughput screening platform to identify candidate compounds for preventing the aggregation of α-synuclein among the natural products in our in-house compound library. We found that a small molecule, 03A10, i.e., (+)-desdimethylpinoresinol, which is present in the fruits of Vernicia fordii (Euphorbiaceae), modulated aggregated α-synuclein, but not monomeric α-synuclein, to prevent further elongation of α-synuclein fibrils. In α-synuclein-overexpressing cell lines, 03A10 (10 µM) efficiently prevented α-synuclein aggregation and markedly ameliorated the cellular toxicity of α-synuclein fibril seeds. In the MPTP/probenecid (MPTP/p) mouse model, oral administration of 03A10 (0.3 mg· kg-1 ·d-1, 1 mg ·kg-1 ·d-1, for 35 days) significantly alleviated behavioral deficits, tyrosine hydroxylase (TH) neuron degeneration and p-α-synuclein aggregation in the substantia nigra (SN). As the Braak hypothesis postulates that the prevailing site of early PD pathology is the gastrointestinal tract, we inoculated α-synuclein preformed fibrils (PFFs) into the mouse colon. We demonstrated that α-synuclein PFF inoculation promoted α-synuclein pathology and neuroinflammation in the gut and brain; oral administration of 03A10 (5 mg· kg-1 ·d-1, for 4 months) significantly attenuated olfactory deficits, α-synuclein accumulation and neuroinflammation in the olfactory bulb and SN. We conclude that 03A10 might be a promising drug candidate for the treatment of PD. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, in an MPTP/p mouse model, and PFF-inoculated mice.

Glycosides from Buddleja officinalis with their protective effects on photoreceptor cells in light-damaged mouse retinas.

A new phenylethanoid, hebitol IV (1), along with fifteen known glycosides (2-16), were isolated from water extract of the flower buds of Buddleja officinalis. Their structures were elucidated on the basis of 1 D-NMR, 2 D-NMR and MS data. Molecular docking showed the potential activities of the natural products against VEGFR-2. Bioassay results revealed that the compounds 10 and 14 exhibited strong inhibitory activity against VEGFR-2 with IC50 values of 0.51 and 0.32 µM, respectively. Moreover, the potential retinal protective effects of 10 and 14 were then investigated in the mouse model featuring bright light-induced retinal degeneration. The results demonstrated remarkable photoreceptor protective activities of 10 and 14 in vivo.

DCZ0014, a novel compound in the therapy of diffuse large B-cell lymphoma via the B cell receptor signaling pathway.

Diffuse large B cell lymphoma (DLBCL) is a clinical and genetically heterogeneous lymphoid malignancy. Although R-CHOP (rituximab plus cyclophosphamide, vincristine, doxorubicin, and prednisone) treatment can improve the survival rate of patients with DLBCL, more than 30% of patients exhibit treatment failure, relapse, or refractory disease. Therefore, novel drugs or targeted therapies are needed to improve the survival of patients with DLBCL. The compound DCZ0014 is a novel chemical similar to berberine. In this study, we found that DCZ0014 significantly inhibited the proliferation and activity of DLBCL cells, and induced cell apoptosis. Following treatment with DCZ0014, DLBCL cells accumulated in G0/G1-phase of the cell cycle and showed decreased mitochondrial membrane potential. Additionally, DCZ0014 inhibited DNA synthesis, enhanced DNA damage in DLBCL cells, as well as inhibited Lyn/Syk in B cell receptor signaling pathway. Further experiments demonstrated that DCZ0014 did not significantly affect peripheral blood mononuclear cells. Tumor xenograft model showed that DCZ0014 not only inhibited tumor growth but also extended the survival time of mice. Thus, DCZ0014 showed potential for clinical application in the treatment of patients with DLBCL.

Competitive CatSper Activators of Progesterone from Rhynchosia volubilis.

The root Rhynchosia volubilis was widely used for contraception in folk medicine, although its molecular mechanism on antifertility has not yet been revealed. In human sperm, it was reported that the cation channel of sperm, an indispensable cation channel for the fertilization process, could be regulated by various steroid-like compounds in plants. Interestingly, these nonphysiological ligands would also disturb the activation of the cation channel of sperm induced by progesterone. Therefore, this study aimed to explore whether the compounds in R. volubilis affect the physiological regulation of the cation channel of sperm. The bioguided isolation of the whole herb of R. volubilis has resulted in the novel discovery of five new prenylated isoflavonoids, rhynchones A - E (1:  - 5: ), a new natural product, 5'-O-methylphaseolinisoflavan (6: ) (1H and 13C NMR data, Supporting Information), together with twelve known compounds (7:  - 18: ). Their structures were established by extensive spectroscopic analyses and drawing a comparison with literature data, while their absolute configurations were determined by electronic circular dichroism calculations. The experiments of intracellular Ca2+ signals and patch clamping recordings showed that rhynchone A (1: ) significantly reduced cation channel of sperm activation by competing with progesterone. In conclusion, our findings indicat that rhynchone A might act as a contraceptive compound by impairing the activation of the cation channel of sperm and thus prevent fertilization.

[A new hexenol glycoside from Buddleja officinalis].

The chemical constituents of the flower buds of Buddleja officinalis were investigated in this study. Eight compounds were isolated from the water extract of B. officinalis by column chromatography, and their structures were elucidated on the basis of physicochemical properties and spectral data. These compounds were identified as(Z)-hex-3-en-1-ol-1-O-ß-D-glucopyranosyl-(1→2)-[ß-D-xylcopyranosyl-(1→6)]-ß-D-glucopyranoside(1), ebracteatoside B(2), jasmonic acid-11-O-ß-D-glucopyranoside(3), 6-hydroxyluteolin-7-O-ß-D-glucopyranoside(4), luteolin-7-O-galacturonide(5), vicenin-2(6), decaffeoylverbascoside(7), and 6-O-(E)-feruloyl-D-glucopyranoside(8). Compound 1 is a new 3-hexenol glycoside. Compounds 2, 3, and 6 were isolated from Buddleja genus for the first time, and compounds 4 and 5 were isolated from this plant for the first time.

Phenols and γ-Lactams from the Aerial Part of Pseudolysimachion linariifolium subsp. dilatatum.

Linariifolioside II (1) and (2S)-2-hydroxy-5-oxoproline methyl ester (2), two new compounds along with 13 known compounds were obtained from the aerial part of Pseudolysimachion linariifolium Holub subsp. dilatatum (Nakai & Kitag.) D.Y. Hong. Their chemical structures were revealed mainly through NMR and MS data. The absolute configuration of 2 was deduced by comparing its experimental CD with the calculated ECD spectra. At a concentration of 1 mm, total antioxidant capacities of compounds 1-15 were measured using a rapid ABTS method in vitro. Compounds 1, 3-5, and 11-14 exhibited approximately equal antioxidant capacity to that of vitamin C (Vc).

Bisindole alkaloids with nitric oxide inhibitory activities from an alcohol extract of the Isatis indigotica roots.

Eight bisindole alkaloids including six undescribed ones (1a/1b-5) were isolated from an alcohol extract of the Isatis indigotica roots. Their structures and absolute configurations were supported by extensive spectroscopic data analysis, including 1D, 2D NMR, HRESIMS data, specific rotation data, and comparison of the experimental and calculated ECD data. Compounds 1a and 1b were determined to be a pair of enantiomers with a ratio of approximately 1:1 by chiral-phase chromatography analysis while compound 4 was elucidated as a new type of bisindole alkaloid with the aglycone categorized as bis(indole-1'/3″-yl)methane. All the isolated compounds were tested for their nitric oxide (NO) inhibitory effects and 1-4 and 6 exhibited inhibitory effects with IC50 values ranging from 11.0 to 37.6 µM.

Alopecines A-E, five chloro-containing matrine-type alkaloids with immunosuppressive activities from the seeds of Sophora alopecuroides.

Alopecines A-E (1-5), five unusual matrine-type alkaloids featuring with an additional dichlorocyclopropane (1-3) or a di/tri-chloromethyl (4/5) attached on the D ring, were isolated from the seeds of Sophora alopecuroides. Their structures and absolute configurations were elucidated by extensive spectroscopic techniques, and X-ray diffraction analyses or time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations. Alkaloid 4 exhibited potent inhibitory effects on the proliferation of ConA-induced T lymphocytes or LPS-induced B cells with IC50 value of 3.98 or 3.74 µM, respectively.

Lignans from Isatis indigotica roots and their inhibitory effects on nitric oxide production.

Seventeen lignans (1-17) were obtained from the roots of Isatis indigotica (I. indigotica). Among them, isatindigosesquilignans A-C (1-3) were deduced as three undescribed sesquilignans, which possess unusual carbon skeleton of aryltetralin unit connected with a C6-C3 moiety via a five-membered ring of C-3-C-8″-C-7″-O-C-4. Isatindigosesquilignans B and C were determined as the first examples of its glycosides from a natural source and a plausible biosynthetic pathway was proposed. Moreover, all of the isolated lignans were assayed regarding their inhibitory effects on nitric oxide (NO) production in RAW 264.7 cells and compounds 1, 2 and 7 showed inhibitory effects with IC50 values ranging from 19.46 µM to 64.82 µM.

Efficacy and safety of selective internal radiotherapy versus sorafenib for intermediate-locally advanced hepatocellular carcinoma: a systematic review and meta-analysis.

BACKGROUND: Sorafenib (SOR) is recommended for locally advanced and metastatic hepatocellular carcinoma (HCC), but the tolerability of SOR is unsatisfactory. Selective internal radiotherapy (SIRT) has shown efficacy in intermediate-locally advanced HCC patients. This meta-analysis aimed to compare the efficacy and safety of SIRT and SOR in the treatment of intermediate-locally advanced HCC. METHODS: We systematically searched the PubMed, Embase, Cochrane Library and Web of Science databases for eligible studies. The endpoints evaluated included the overall survival (OS), disease control rate (DCR), objective response rate (ORR) and grade≥3 adverse events (AEs). RESULTS: Six studies were included in this analysis. The OS was similar between the two groups (HR 1.06, 95%CI 0.93-1.20; P = 0.40). There was no difference in the DCR between the two groups (RR 1.13, 95%CI 0.87-1.46; P = 0.35). However, the ORR in the SIRT group was significantly higher than that in the SOR group (RR 4.10, 95%CI 1.92-8.76; P = 0.0003). The incidence rate of grade≥3 AEs was higher in the SOR group. CONCLUSIONS: In patients with intermediate-locally advanced HCC, SIRT and SOR result in similar survival rates. The improved toxicity profile of SIRT may help when choosing between the two treatments.

Flueggenoids A - E, new dinorditerpenoids from Flueggea virosa.

A phytochemical investigation on the twigs and leaves of Flueggea virosa (Euphorbiaceae) led to the isolation of flueggenoids A - E (1-5), five new 13-methyl-ent-podocarpanes, together with eleven known compounds (6-16). Their structures and absolute configurations were elucidated on the basis of extensive MS and NMR data analysis, and/or single-crystal X-ray diffraction, time-dependent density functional theory (TDDFT)-based electronic circular dichroism (ECD) calculations, and chemical transformation. All isolates were evaluated for anti-HCV activity, the results showed that terpenoids of F. virosa had nonnegligible contribution for the anti-HCV activity.

Baicalin relaxes vascular smooth muscle and lowers blood pressure in spontaneously hypertensive rats.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin is the most abundant flavone compound present in Scutellaria baicalensis Georgi and endothelium-dependent vascular activities of baicalin have been suggested. However, the pharmacological implications and mechanisms of baicalin under hypertensive conditions remain to be investigated. The current study examined the blood pressure-lowering effect of baicalin in a spontaneously hypertensive rat (SHR) model. Moreover, vascular activities and mechanisms of baicalin were investigated under hypertensive conditions. The results demonstrate that baicalin treatment lowers the blood pressure in SHRs in vivo. Ex vivo vascular reactivity assay reveals that baicalin relaxes phenylephrine (PE)-constricted SHR aortas in an endothelium-independent manner. Baicalin attenuates Angiotensin II (Ang II) or potassium chloride (KCl)-induced vasoconstriction in SHR aortas as well. Baicalin also relaxes SHR aortas in the presence of different Ca2+ channel blockers such as nifedipine and SKF96365 in response to PE-induced constriction. Most importantly, ATP-sensitive potassium channel (KATP) blockade partially abrogated the vasorelaxant effect of baicalin. In summary, the current study demonstrates for the first time that intracellular Ca2+ regulation in vascular smooth muscle is mechanistically implicated in the vasorelaxant effect of baicalin under hypertensive conditions. Furthermore, activated KATP channels are in part required for the vasorelaxant effect of baicalin under hypertensive conditions. Thus, the work here sheds novel pharmacological and mechanistic insights into the blood pressure-lowering effect of baicalin, which may help better understand the therapeutic application of Scutellaria baicalensis Georgi in the treatment of hypertension.

Dihydrocelastrol exerts potent antitumor activity in mantle cell lymphoma cells via dual inhibition of mTORC1 and mTORC2.

Mantle cell lymphoma (MCL) is a distinct and highly aggressive subtype of B-cell non-Hodgkin lymphoma. Dihydrocelastrol (DHCE) is a dihydro-analog of celastrol, which is isolated from the traditional Chinese medicinal plant Tripterygium wilfordii. The present study aimed to investigate the effects of DHCE treatment on MCL cells, and to determine the mechanism underlying its potent antitumor activity in vitro and in vivo using the Cell Counting kit-8 assay, clonogenic assay, apoptosis assay, cell cycle analysis, immunofluorescence staining, western blotting and tumor xenograft models. The results demonstrated that DHCE treatment exerted minimal cytotoxic effects on normal cells, but markedly suppressed MCL cell proliferation by inducing G0/G1 phase cell cycle arrest, and inhibited MCL cell viability by stimulating apoptosis via extrinsic and intrinsic pathways. In addition, the results revealed that DHCE suppressed cell growth and proliferation by inhibiting mammalian target of rapamycin complex (mTORC)1-mediated phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E binding protein. Simultaneously, DHCE induced apoptosis and inhibited cell survival by suppressing mTORC2-mediated phosphorylation of protein kinase B and nuclear factor-κB activity. In addition to in vitro findings, DHCE treatment reduced the MCL tumor burden in a xenograft mouse model, without indications of toxicity. Furthermore, combined treatment with DHCE and bortezomib, a proteasome inhibitor, induced a synergistic cytotoxic effect on MCL cells. These findings indicated that DHCE may have the potential to serve as a novel therapeutic agent for the treatment of MCL through dually inhibiting mTORC1 and mTORC2.

Combined Virtual Screening and Substructure Search for Discovery of Novel FABP4 Inhibitors.

Fatty acid-binding protein 4 (FABP4, AFABP) is a potential drug target for diabetes and atherosclerosis. In this study, a series of novel FABP4 inhibitors were discovered through combining virtual screening and substructure search. Seventeen compounds exhibited FABP4 inhibitory activities with IC50 < 10 µM, among which 11 compounds showed high selectivity against FABP3. The best compound 36b displayed an IC50 value of 1.5 µM. Molecular docking and point mutation studies revealed that Gln95, Arg126, and Tyr128 play key roles for these compounds binding with FABP4. Interestingly, Gln95 seems to be essential for conformation stability of FABP4. The new scaffolds of these compounds and their interaction mechanisms binding with FABP4 should provide an important clue for the further development of novel FABP4 inhibitors.

Apigenin-7-diglucuronide protects retinas against bright light-induced photoreceptor degeneration through the inhibition of retinal oxidative stress and inflammation.

Vision impairment in retinal degenerative diseases such as age-related macular degeneration is primarily associated with photoreceptor degeneration, in which oxidative stress and inflammatory responses are mechanistically involved as central players. Therapies with photoreceptor protective properties remain to be developed. Apigenin-7-diglucuronide (A7DG), a flavonoid glycoside, is present in an assortment of medicinal plants with anti-inflammatory or ant-oxidant activities. However, the pharmacological significance of A7DG remains unknown in vivo. The current study isolated A7DG from Glechoma longituba (Nakai) Kuprian and investigated the retinal protective effect A7DG in mice characterized by bright light-induced photoreceptor degeneration. The results showed that A7DG treatment led to remarkable photoreceptor protection in bright light-exposed BALB/c mice. Moreover, A7DG treatment alleviated photoreceptor apoptosis, mitigated oxidative stress, suppressed reactive gliosis and microglial activation and attenuated the expression of proinflammatory genes in bright light-exposed retinas. The results demonstrated for the first time remarkable photoreceptor protective activities of A7DG in vivo. Inhibition of bright light-induced retinal oxidative stress and retinal inflammatory responses was associated with the retinal protection conferred by A7DG. The work here warrants further evaluation of A7DG as a pharmacological candidate for the treatment of vision-threatening retinal degenerative disorders. Moreover, given the general implication of oxidative stress and inflammation in the pathogenesis of neurodegeneration, A7DG could be further tested for the treatment of other neurodegenerative disorders.

Dihydrocelastrol inhibits multiple myeloma cell proliferation and promotes apoptosis through ERK1/2 and IL-6/STAT3 pathways in vitro and in vivo.

Multiple myeloma (MM) is the second most frequent malignant hematological disease. Dihydrocelastrol (DHCE) is synthesized by hydrogenated celastrol, a treterpene isolated from Chinese medicinal plant Tripterygium regelii. In this study, we first reported the anti-tumor activity of DHCE on MM cells. We found that DHCE could inhibit cell proliferation and promote apoptosis through caspase-dependent way in vitro. In addition, DHCE could inactivate the expression of interleukin (IL)-6 and downregulate the phosphorylation of extracellular regulated protein kinases (ERK1/2) and the signal transducer and activator of transcription 3 (STAT3) in MM. It also retained its activity against MM cell lines in the presence of IL-6. Furthermore, treatment of MM cells with DHCE resulted in an accumulation of cells in G0/G1 phase of the cell cycle. Notably, DHCE reduced the expression of cyclin D1 and cyclin-dependent kinases 4 and 6 in MM cell lines. Additionally, its efficacy toward the MM cell lines could be enhanced in combination with the histone deacetylase inhibitor panobinostat (LBH589), which implied the possibility of the combination treatment of DHCE and LBH589 as a potential therapeutic strategy in MM. In addition, treatment of NCI-H929 tumor-bearing nude mice with DHCE (10 mg/kg/d, i.p., 1-14 days) resulted in 73% inhibition of the tumor growth in vivo. Taken together, the results of our present study indicated that DHCE could inhibit cellular proliferation and induce cell apoptosis in myeloma cells mediated through different mechanisms, possibly through inhibiting the IL-6/STAT3 and ERK1/2 pathways. And it may provide a new therapeutic option for MM patients.

Repositioning organohalogen drugs: a case study for identification of potent B-Raf V600E inhibitors via docking and bioassay.

Drug repositioning has been attracting increasingly attention for its advantages of reducing costs and risks. Statistics showed that around one quarter of the marketed drugs are organohalogens. However, no study has been reported, to the best of our knowledge, to aim at efficiently repositioning organohalogen drugs, which may be attributed to the lack of accurate halogen bonding scoring function. Here, we present a study to show that two organohalogen drugs were successfully repositioned as potent B-Raf V600E inhibitors via molecular docking with halogen bonding scoring function, namely D(3)DOCKxb developed in our lab, and bioassay. After virtual screening by D(3)DOCKxb against the database CMC (Comprehensive Medicinal Chemistry), 3 organohalogen drugs that were predicted to form strong halogen bonding with B-Raf V600E were purchased and tested with ELISA-based assay. In the end, 2 of them, rafoxanide and closantel, were identified as potent inhibitors with IC50 values of 0.07 µM and 1.90 µM, respectively, which are comparable to that of vemurafenib (IC50: 0.17 µM), a marketed drug targeting B-Raf V600E. Single point mutagenesis experiments confirmed the conformations predicted by D(3)DOCKxb. And comparison experiment revealed that halogen bonding scoring function is essential for repositioning those drugs with heavy halogen atoms in their molecular structures.

Deciphering the pharmacological mechanism of the Chinese formula huanglian-jie-du decoction in the treatment of ischemic stroke using a systems biology-based strategy.

AIM: Huanglian-jie-du decoction (HLJDD) is an important multiherb remedy in TCM, which is recently demonstrated to be effective to treat ischemic stroke. Here, we aimed to investigate the pharmacological mechanisms of HLJDD in the treatment of ischemic stroke using systems biology approaches. METHODS: Putative targets of HLJDD were predicted using MetaDrug. An interaction network of putative HLJDD targets and known therapeutic targets for the treatment of ischemic stroke was then constructed, and candidate HLJDD targets were identified by calculating topological features, including 'Degree', 'Node-betweenness', 'Closeness', and 'K-coreness'. The binding efficiencies of the candidate HLJDD targets with the corresponding compositive compounds were further validated by a molecular docking simulation. RESULTS: A total of 809 putative targets were obtained for 168 compositive compounds in HLJDD. Additionally, 39 putative targets were common to all four herbs of HLJDD. Next, 49 major nodes were identified as candidate HLJDD targets due to their network topological importance. The enrichment analysis based on the Gene Ontology (GO) annotation system and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway demonstrated that candidate HLJDD targets were more frequently involved in G-protein-coupled receptor signaling pathways, neuroactive ligand-receptor interactions and gap junctions, which all played important roles in the progression of ischemic stroke. Finally, the molecular docking simulation showed that 170 pairs of chemical components and candidate HLJDD targets had strong binding efficiencies. CONCLUSION: This study has developed for the first time a comprehensive systems approach integrating drug target prediction, network analysis and molecular docking simulation to reveal the relationships between the herbs contained in HLJDD and their putative targets and ischemic stroke-related pathways.