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. 2019 Jun 14:10:688.
doi: 10.3389/fphar.2019.00688. eCollection 2019.

A Novel Antiplatelet Aggregation Target of Justicidin B Obtained From Rostellularia Procumbens (L.) Nees

Yan-Fang Yang  1   2   3 , Song-Tao Wu  1 , Bo Liu  1   2   3 , Zhou-Tao Xie  1 , Wei-Chen Xiong  1 , Peng-Fei Hao  1 , Wen-Ping Xiao  1 , Yuan Sun  1 , Zhong-Zhu Ai  1   2   3 , Peng-Tao You  1   2   3 , He-Zhen Wu  1   2   3
Affiliations

A Novel Antiplatelet Aggregation Target of Justicidin B Obtained From Rostellularia Procumbens (L.) Nees

Yan-Fang Yang et al. Front Pharmacol. .

Abstract

The present study explored the possible bioactive ingredients and target protein of Rostellularia procumbens (L.) Nees. Firstly, we found that the ethyl acetate extraction obtained from R. procumbens could inhibit platelet aggregation. Then, gene chip was used to investigate differentially expressed genes and blood absorption compounds were investigated using high performance liquid chromatography-mass spectrometry characterization (LC-MS). Depending on the results of gene chip and LC-MS, the targets of blood absorption compounds were predicted according to the reverse pharmacophore matching model. The platelet aggregation-related genes were discovered in databases, and antiplatelet aggregation-related gene targets were selected through comparison. The functions of target genes and related pathways were analyzed and screened using the DAVID database, and the network was constructed using Cytoscape software. We found that integrin αIIbβ3 had a highest degree, and it was almost the intersection of all pathways. Then, blood absorption compounds were screened by optical turbidimetry. Western blot (WB) revealed that justicidin B separated from the ethyl acetate fraction may inhibit the expression of integrin αIIbβ3 protein. For the first time, we used Prometheus NT.48 and MST to detect the stability of this membrane protein to optimize the buffer and studied the interaction of justicidin B with its target protein. To our best knowledge, this is the first report to state that justicidin B targets the integrin αIIbβ3 protein. We believe that our findings can provide a novel target protein for the further understanding of the mechanism of R. procumbens on platelet aggregation.

Keywords: LC-MS; Prometheus NT.48; gene chip; integrin αIIbβ3; justicidin B; microscale thermophoresis; network pharmacology; platelet aggregation.

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Figures

Figure 1
Figure 1
Mass spectrometry.
Figure 2
Figure 2
Top 10 components of the KEGG pathway and GO enrichment analyses. (A) KEGG pathways. (B) Biological process. (C) Cell components. (D) Molecular function.
Figure 3
Figure 3
Network diagram constructed by Cytoscape. (A) Network diagram of active components/target genes/enrichment pathways. (B) Network diagram of active components/platelet aggregation-related target genes.
Figure 4
Figure 4
MIR (%) of different compounds.
Figure 5
Figure 5
The influence of justicidin B on integrin αIIbβ3 protein. (A) Western blot. (B) Grayscale value.
Figure 6
Figure 6
Molecular interaction of integrin αIIbβ3 using NT. LabelFree analysis. (A) SD-TEST capillary shap. (B) SD-TEST capillary scan. (C) MST time traces of 16 different justicin B concentrations (ranging from 0.00748 to 245 µM). (D) Dependence of the MST signal on the justicin B concentration (measured 30 s after turning on heating; data from C).
Figure 7
Figure 7
Molecular interaction of integrin αIIbβ3 using NT. 115 analysis. (A) SD-TEST capillary shap. (B) SD-TEST capillary scan. (C) MST time traces of 16 different justicin B concentrations (ranging from 0.0032 to 13.1 mM). (D) Dependence of the MST signal on the justicin B concentration (measured 30 s after turning on heating; data from C).
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