Potency of Tokishakuyakusan in treating preeclampsia: Drug repositioning method by in vitro screening of the Kampo library

Abstract

Introduction: Preeclampsia therapy has not been established, except for the termination of pregnancy. The aim of this study was to identify a potential therapeutic agent from traditional Japanese medicine (Kampo) using the drug repositioning method.

Materials and methods: We screened a library of 74 Kampo to identify potential drugs for the treatment of preeclampsia. We investigated the angiogenic effects of these drugs using human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were performed to measure the levels of placental growth factor (PlGF) in conditioned media treated with 100 μg/mL of each drug. We assessed whether the screened drugs affected cell viability. We performed tube formation assays to evaluate the angiogenic effects of PlGF-inducing drugs. PlGF was measured after administering 10, 50, 100, and 200 μg/mL of the candidate drug in the dose correlation experiment, and at 1, 2, 3, 6, 12, and 24 h in the time course experiment. We also performed tube formation assays with the candidate drug and 100 ng/mL of soluble fms-like tyrosine kinase 1 (sFlt1). PlGF production by the candidate drug was measured in trophoblastic cells (BeWo and HTR-8/SVneo). The Mann-Whitney U test or one-way analyses of variance followed by the Newman-Keuls post-hoc test were performed. P-values < 0.05 were considered significant.

Results: Of the 7 drugs that induced PlGF, Tokishakuyakusan (TS), Shoseiryuto, and Shofusan did not reduce cell viability. TS significantly facilitated tube formation (P = 0.017). TS administration increased PlGF expression in a dose- and time-dependent manner. TS significantly improved tube formation, which was inhibited by sFlt1 (P = 0.033). TS also increased PlGF production in BeWo (P = 0.001) but not HTR-8/SVneo cells (P = 0.33).

Conclusions: By using the drug repositioning method in the in vitro screening of the Kampo library, we identified that TS may have a therapeutic potential for preeclampsia. Its newly found mechanisms involve the increase in PlGF production, and improvement of the antiangiogenic state.

Fig 1. Production of PlGF from HUVECs treated with a library of Kampo drugs.

A: First screening. PlGF concentrations produced by 74 drugs. B: Second screening. PlGF concentrations produced by 15 drugs increased PlGF by more than 120 above the control in the first screening. 1: Kakkonto; 3: Otsujito; 4: Anchusan; 5: Hachimijiogan; 6: Daisaikoto; 7: Saikokeishito; 8: Saikokeishikankyoto; 9: Saikokaryukotsuboreito; 12: Shoseiryuto; 13: Shofusan 14: Tokishakuyakusan 23: Shichimotsukokato; 25: Juzentaihoto; 36: Bofutsushosan; 58: Sansoninto. PlGF, placental growth factor; HUVECs, human umbilical vein endothelial cells.

Fig 2. Effect of selected Kampo drugs on the viability of HUVECs.

A: The MTS assay with one of 7 selected drugs. B: The CCK-8 assay with one of 7 selected drugs. * indicates P < 0.05. 1: Kakkonto; 4: Anchusan; 5: Hachimijiogan; 9: Saikokaryukotsuboreito; 12: Shoseiryuto; 13: Shofusan 14: Tokishakuyakusan. HUVECs, human umbilical vein endothelial cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium inner salt; CCK, Cell Counting Kit.

Fig 3. Effect of selected Kampo drugs on microvascular tube formation in HUVECs.

A: Quantification of total tube length. B: Representative images. * indicates P < 0.05. 12: Shoseiryuto; 13: Shofusan; 14: Tokishakuyakusan. HUVECs, human umbilical vein endothelial cells.

Fig 4. Effect of TS on PlGF expression in HUVECs.

A: Dose correlation experiment. B: Time course experiment. * indicates P <0.05; ** indicates P <0.01. TS, Tokishakuyakusan; PlGF, placental growth factor; HUVECs, human umbilical vein endothelial cells.

Fig 5. Effect of TS on tube formation of HUVECs with antiangiogenic factors.

A: Quantification of total tube length. B: Representative images. * indicates P <0.05. TS, Tokishakuyakusan; HUVECs, human umbilical vein endothelial cells; sFlt1, soluble fms-like tyrosine kinase 1.

Fig 6. Effect of TS on PlGF expression and the cell viability of trophoblastic cells.

A: PlGF concentrations produced by TS from HTR-8/SVneo and BeWo cells. B: The CCK-8 assay with TS in HTR-8/SVneo and BeWo cells. * indicates P <0.05. TS, Tokishakuyakusan; PlGF, placental growth factor; HTR-8, HTR-8/SVneo cells; BeWo, BeWo cells.