The effects of tokishakuyakusan, a traditional Japanese medicine (kampo), ferulic acid and paeoniflorin, on human endometriotic stromal cells and peritoneal macrophages

Highlights

• TSS and FA reduce IL-8 and VEGF secretion by ESC.

• FA reduces IL-8 secretion by peritoneal macrophages.

• FA distributes to plasma and the uterus after the oral administration of TSS.

Abstract

Objectives

To evaluate the anti-inflammatory and anti-angiogenetic effects of Tokishakuyakusan (TSS), a traditional Japanese medicine (Kampo), and its ingredients, ferulic acid (FA) and paeoniflorin (PA) on endometriotic stromal cells (ESC) and peritoneal macrophages.

Study design

Endometriotic tissues were obtained from 16 patients and peritoneal macrophages were obtained from 11 patients that had undergone laparoscopic surgery for ovarian endometriosis. ESC isolated from endometriotic tissues and peritoneal macrophages were cultured, and pre-treated with 300 μg/mL of TSS, 500 μM FA or 50 μM PA. ESC and peritoneal macrophages were then stimulated with IL-1β. Concentrations of IL-8 and VEGF protein in supernatants were then detected and measured using specific ELISAs. TSS (4 g/kg body weight) was orally administered to female Sprague-Dawley rats. The concentration of FA in plasma and uteri was measured using liquid chromatography–mass spectrometry with tandem mass spectrometry (LC–MS/MS).　

Results

TSS and FA but not PA decreased the secretion of inflammatory cytokine (IL-8) and angiogenic factor (VEGF) in ESC. TSS and FA also suppressed the secretion of inflammatory cytokine (IL-8) from peritoneal macrophages. FA was detected in plasma and in uterine tissues after the oral administration of TSS to rats.

Conclusions

Our study demonstrates that TSS has anti-inflammatory and anti-angiogenic effects on endometriosis related cells by controlling inflammatory cytokine and growth factor secretion from cells, and these effects, at least partially, may be due to the direct effects of the TSS ingredient FA.

Introduction

Traditional Japanese medicines (Kampo) are increasingly recognized to have useful clinical applications in Japan. Tokishakuyakusan (TSS), a pharmaceutical-grade Kampo, is currently manufactured under strict quality controls and according to Japan’s good manufacturing practice (GMP). This medicine has been approved as a prescription drug by the Japanese Ministry of Health, Labour and Welfare, and has been prescribed for various gynecologic disorders (Terauchi et al., 2014). TSS is especially well known for managing dysmenorrhea (Kotani et al., 1997; Yoshino et al., 2016) in patients with endometriosis and adenomyosis (Tanaka, 2003), although its size reductive effects on these lesions have not been shown. Until today, the mechanism by which TSS reduces dysmenorrhea associated with endometriosis has not been determined.

Ferulic acid (FA) and paeoniflorin (PA) are the main ingredients of TSS. FA has been shown to exert anti-inflammatory effects in various cell types (Navarrete et al., 2015) (Lampiasi and Montana, 2016). PA is also known to induce various pharmacological responses such as anti-oxidative, anti-inflammatory and immuno-regulatory effects (Liu et al., 2006) (Zhao et al., 2018).

Endometriosis and endometriosis-related pain is associated with a local inflammatory and angiogenic microenvironment in the pelvis (Zondervan et al., 2018). In this study, we examined: i) the expression of interleukin-8 (IL-8) as a cytokine that enhances inflammation in the pathogenesis of endometriosis (Harada et al., 2001); ii) IL-8 and vascular endothelial growth factor (VEGF) as factors responsible for angiogenesis in endometriosis (Ricci et al., 2011) (Hsiao et al., 2014), and tested the effect of TSS, FA and PA on the expression of IL-8 and VEGF. In addition, a pharmacokinetic study in rats was performed to investigate whether FA distributes to plasma and the uterus.