In Silico-based Screen Synergistic Drug Combinations From Herb Medicines: A Case Using Cistanche Tubulosa-Ⅱ

Neuroprotection Module

Neuroprotection is the mechanisms and strategies used to protect against neuronal injury or degeneration in the CNS following acute disorders (e.g. stroke or nervous system injury/trauma) or as a result of chronic neurodegenerative diseases (e.g. Parkinson's, Alzheimer's, Multiple Sclerosis). The goal of neuroprotection is to limit neuronal dysfunction/death after CNS injury and attempt to maintain the highest 

possible integrity of cellular interactions in the brain resulting in an undisturbed neural function. As can be seen from Fig. 4, some targets on the Serotonergic synapse pathway are involved in the function of neuroprotection. For example, the production of prostaglandins through PTGS1 and PTGS2 (also known as COX-1 and COX-2) is an essential mediator in evoking anti-inflammatory and novel pro-resolving mechanisms56. A recent study has shown that gene expression of ADCY5, an enzyme that catalyzes the generation of cAMP57, is reduced by promoter methylation in COX-2-induced human HCC cell lines58. Based on the above analysis, we speculate that COX-2 accumulation may influence the secretion of sAPPα, the α cleavage of APP cleaved by α-secretase that is modulated by cAMP and further exerts the neuroprotective efect59,60. Our results show that neuroprotection plays an important role in the treatment of neuroinflammation.

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Experimental Validation 

The Viability Of BV2 Microglia Cells Treated By The Compounds

BV2 microglia cells (8×104 cells/ ml) are fed with concentrations of 37.5 to 300 μM per milliliter culture media with no serum of the four compounds for 24 h. We regard the cell viability of the control group cultured in the absence of serum with less than 0.1% DMSO as 100% (Fig. 5(a–d)). No significant cellular cytotoxicity is observed at the prescribed dosages of the groups. 

Validation Of Drug Synergy And Potential Anti-Inflammatory Effect In Vitro

To further assess the obtained results in silico, four compounds covering three synergistic pairs, namely isoacteoside, 2′-acetylacteoside, echinacoside, and verbascoside, are selected to examine their drug synergistic effects and potential anti-inflammatory effect using BV2 cells treated with LPS. In particular, we conduct western blot analysis for iNOS and COX-2 protein expression to confirm the synergy and anti-inflammatory effects of the predicted drug combinations.

As shown in Fig. 5(e–g), the levels of iNOS and COX-2 proteins in the panel of BV2 cell lines tested are reported. We observe that in either isoacteoside or 2′-acetylacteoside treatment, the protein expressions of iNOS and COX-2 in BV2 cells both declined significantly at different dose levels. However, treatment with the combination of isoacteoside and 2′-acetylacteoside induces a significant increase in the inflammatory factors iNOS and COX-2 (Fig. 5(e)). Figure 5(f) illustrates that echinacoside or verbascoside treatment, as a single agent, causes a decrease in the iNOS and COX-2 expression. Moreover, as expected, treatment with echinacoside in combination with verbascoside at the concentration of 150 μM resulted in a more pronounced decrease in the 

levels of protein expression (iNOS and COX-2), indicating the synergistic anti-inflammatory effects of this drug combination. Similarly, as indicated in Fig. 5(g), the combination of echinacoside and 2′-acetylacteoside shows a significant synergistic effect on the inhibition of COX-2 at the concentration of 75 μM or 300 μM. However, for iNOS, at the dosage of 300 μM, the combination represents marked suppression of the protein. In contrast, we find that there are no obvious inhibition effects on both iNOS and COX-2 about the combination of isoacteoside and 2′-acetylacteoside or echinacoside and verbascoside at the concentration of 75μM (Supplementary Figure 2), besides, treatment with other pairs shows a much weaker effect compared with the single agents at the dosage of 150 or 300μM that can be seen in Fig. 5 (e–g).

To sum up, the in vitro study provides additional information for screening drug combinations with potentially anti-inflammatory effects and demonstrates the reliability of the in silico screen strategy.

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Discussion 

Neuroinflammation is implicated in the majority of neurological, psychiatric, and neurodevelopmental diseases to that is not only a consequence but could be a trigger of the pathology. However, current treatments for neuroinflammatory are monotherapies mostly, limited by well-known side effects as we know, COX-2 inhibitors may lead to cardiovascular defects in response to long-term treatment, and TNF-targeted treatment could cause infection through immunosuppression. Combinatorial therapeutic approaches may be imperative to improve the treatment of complex diseases with the following advantages: the countered network robustness and bypass compensation, the increased clinical efficacy while maintaining minimal human toxicity, and the reduced dosage of each compound63. However, exploration of the synergistic drug combinations among compounds derived from herb medicines based on system pharmacology is restricted by the possible main reason for large amounts of compounds.

In the work, we first gain 63 potential bioactive compounds from the herb Cistanche tubulosa, fulfilling the criteria (DL≥0.18) for further analysis with the aid of the prediction which is indispensable to screen out more promising molecules with desirable properties. After mapping the 133 targets of the 63 potential bioactive compounds to the database, we get 43 targets related to neuroinflammation, and then GOBP clustering analysis of the predicted targets can probably contribute to the treatment of neuroinflammation. The analytical result of the C-T network displayed an average degree per compound of 11.209 and 7.651 per target, respectively, and 38 of them adjusted more than 7 targets (larger than the average degree). For example, echinacoside (mol41) predicted with 7 targets, verbascoside (mol33), with 9 targets, or tableside B (mol57), with 8 targets could play key roles in neuroprotection in line with the increasing literature.

We achieve direct therapeutic targets such as APP, MAPT (also known as Tau), PPARG70, MMP9, MMP2, and HTR2A (also known as 5-HT2A), GRIN2B (glutamate ionotropic receptor NMDA type subunit 2B), and GRIA1 (glutamate ionotropic receptor AMPA type subunit 1)or downstream potential targets such as PTGS274 or NOS275 that are associated with neuroinflammation or various diseases of nervous system.

The analysis of the Compound-Target-Pathway network displays 12 compounds from the top 10 drug pairs through the PEA algorithm, connected with the 43 potential targets and the pathways linked with neuroinflammation, for example, Calcium signaling pathway, Neuroactive ligand-receptor interaction or TNF signaling pathway and so on. In the system, these predicted compounds could act on not only the proteins of the upstream but also downstream pathways associated with neuroinflammation and inflammatory biomarkers, in particular. Moreover, additional information for screening drug combinations with potentially anti-inflammatory effects is provided and the reliability of the in silico screen strategy is verified by experimental validation. The neuroinflammation pathway is comprised of the Alzheimer's disease pathway, Calcium signaling pathway, GnRH signaling pathway, VEGF signaling pathway, and the Serotonergic synapse. The analytical results distinctly explained to us that cell death, inflammation, and neuroprotection modules are exemplified to decipher the mechanism of Cistanche tubulosa for the treatment of neuroinflammation.

Neuroinflammation accompanies various neurodegenerative diseases which could be not only a consequence but a trigger of pathology, thus, anti-inflammatory therapies are suggested to be a promising treatment approach. To our disappointment, though we have realized the limitations of the monotherapies, the evaluation and the underlying mechanisms of combination therapies are still the major challenges in the development of the novel alternative strategy. This work therefore could offer new therapeutic opportunities for neuroinflammation and may open up a new avenue for discovering drug combinations from natural products.

Materials And Methods 

Compounds Collection

A total of 66 chemical ingredients of Cistanche tubulosa are manually gathered from TCMSP (http://lsp.nwu.edu.cn/)76, including 26 phenylethanoid glycosides, 22 iridoids, 4 lignans, 7 monoterpene glycosides, 2 nitrogenous substances, 3 benzene acryloyl sugars, 1 sterol, 1 ketol. Given that glycosides in Cistanche tubulosa are usually hydrolyzed to liberate aglycone which is then absorbed in the intestinal mucosa, thus, we take the molecules without glycolic and into consideration, which are tagged as _qt. This led to the generation of the 103 compounds. These molecules are provided in Supplementary Table S1. 

Drug-Likeness Evaluation

To obtain the potential bioactive compounds from Cistanche tubulosa, we evaluate the drug-likeness of these ingredients by calculating the Tanimoto similarity between herbal compounds and the average molecular properties of all chemicals in the Drugbank database. And, the DL prediction model has been applied successfully in many studies, to select bioactive compounds. In the work, the DL index≥0.18 of the candidates is defined as the threshold value to better suit subsequent analysis. 

Drug Target Prediction

The identification of the efficacy targets for leading compounds remains a key step to progressing compounds into drug development. Here, two in-house tools: SysDT and WES are carried out to derive the molecular target information for drug fshing. SysDT is an in-solo model that is performed with the combination of chemical, genomic, and pharmacological information based on two powerful mathematical tools: Random Forest (RF) and Support Vector Machine (SVM) to tackle the issue of target identification effectively. The obtained model served as a valuable platform for the prediction of drug-target interactions with an overall accuracy of 97.3%, an activated prediction accuracy of 87.7%, and an inhibited prediction accuracy of 99.8%. To capture more promising components, the filtering criteria are defined as RF value≥0.7 or SVM≥0.8 in this study.

Weighted ensemble similarity (WES) is a new powerful computational model to pinpoint the drug's direct targets of the actual bioactive ingredients. As a novel tool, the obtained model performs well in predicting the binding with an average sensitivity of 85% (SEN) and the non-binding patterns with 71% (SPE) with the average areas under the receiver operating curves (ROC, AUC) of 85.2% and an average concordance of 77.5%. The obtained targets are further mapped to Uniprot to normalize their names and organisms subsequently. Here, we only choose the targets of Homo sapiens for further analysis. Candidate targets of the selected compounds are mapped to the CTD database to get their related diseases and we screen out potential targets related to neuroinflammation finally.

GO Enrichment And Analysis For Targets

To probe the involved biological processes of the obtained targets, we map the targets to DAVID and the terms with P-value less than 0.05 are chosen in this section. 

Drug Combination Analysis

In our previous work, a system pharmacology framework was exploited to predict drug combinations on a newly designed model, termed the Probability Ensemble Approach (PEA) to analyze the clinical efficacy and adverse effects of drug combinations. In detail, a Bayesian network integrating with a similarity algorithm was developed to model the combinations from compound molecular and pharmacological effects. The combined evaluation that covered the clinical efficacy and adverse effects for the predicted pairs was presented then. Briefly, it shows that PEA could predict the efficacy of the pairs with high specificity and sensitivity (AUC=0.90) in our work. In this work, we select the top ten drug combinations based on their synergy probabilities, which represent the possibility of inducing synergy between two compounds.

Network/Pathway Building And Analysis

To investigate relationships between the active ingredients and the inflammatory diseases, compound-target (C-T) network and compound-target-pathway network (C-T-P) are generated by Cytoscape 2.8.1, a popular bioinformatics package for biological network visualization and data integration. The quantitative properties of the network are analyzed by the two following plugins Network Analyzer and CentiScaPe 1.2. In the graphic network, nodes indicate either compounds, targets, or pathways while edges encode the drug-target interaction. To further explore the biological effects of how cellular targets work through modulating multiple metabolism pathways, an incorporated "pathway" is assembled by the up-to-date information on neuroinflammation pathology. Firstly, using mapping them onto the KEGG database, the achieved target profiles are aggregated into several pathways. After abandoning the indirect sections, then, a relatively synthesized pathway is manually integrated on account of the pathological and clinical data.

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Experimental Validation 

Samples Preparation

Echinacoside, verbascoside, isoacteoside and 2′-acetylacteoside are purchased from Nanjing Zelang Biological Technology Co., Ltd. (Nanjing, Jiangsu, China). Test samples are dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA) to get 100 mM, as a stock solution, and then stored at 4°C. The final dilutions of DMSO added to the culture medium never exceeded 0.1% which ensured there was no effect on cell viability.

Cell Culture

BV2 mouse microglia cells are originally developed by Chinese Academy of Sciences Shanghai cell bank and cultured in 25 or 75 cm2 tasks with Dulbecco's modified Eagle's medium (DMEM/25mM HEPES) (Gibco BRL, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, USA), penicillin G (100 units/ mL) and streptomycin (100 mg/mL) in a humidified incubator with 5% CO2/95% O2 at 37 °C.

Cell Viability Assay

BV2 microglia cells are seeded into a 96-well plate at a density of 1×105 cells/ml, after incubated for 18 h, cells are treated with 100 μl of fresh medium with or without various indicated concentrations of test samples for an additional 24 h. CCK-8 assay (BestBio, Shanghai, China) is a convenient, reliable method to determine the viability of the cells. To eliminate the background of test samples, we discard the whole culture medium, after which 100 μl/well fresh media containing 10% CCK-8 solution is added then. The OD values at 450 nm are read on a microplate reader (Molecular Devices, California, USA) after a 3 h incubation at 37 °C and 5% CO2.

Western Blot Analysis

The cellular protein is extracted from cell lines using a Qproteome™ Mammalian Protein Prep Kit (Qiagen, Germany) after the indicated procedures by the manufacturer's protocol. Quick Stari Bradford Protein Assay Kit (Bio-Rad, USA) is applied to protein quantification. Equivalent amounts of protein (50μg) are denatured by boiling at 100 °C for 10 min with 2*laemmli sample loading buffer (Bio-Rad, USA) plus 5% β-mercaptoethanol in a ratio of 1:1 and loaded per lane onto 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide minigels), electrotransferred onto 0.45 μm polyvinylidene fuoride membranes (PDVF) (Millipore, Bedford, MA, USA) for 150 min at 200 mA. Subsequently, the membranes are blocked in 3% bovine serum albumin (BSA) at room temperature and incubated with the primary antibodies iNOS and COX-2 (Abcam) at 4 °C overnight. Following three thorough washes in Tris-buffered Saline-Tween (TBST) each for 5 min, the membranes are probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000 dilutions; Abcam) for 1.5 h at room temperature. The immunoreactive bands are then visualized by using an ECL chemiluminescence detection kit (Bio-Rad Laboratories, Richmond, California, USA) after washing twice in TBST and once in TBS, each time for 5 min. Densitometric values are normalized using β-actin as loading internal control.

Statistical Analysis

Data are presented as means±standard error, and Western blot analysis is repeated in three independent experiments with the same result. One-way analysis of variance is used to compare the differences of means for three or more groups, statistical significance is analyzed with the Student's t-test between two groups.

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