Protection of flavonoids from Smilax china L. rhizome on phenol mucilage-induced pelvic inflammation in rats by attenuating inflammation and fibrosis

Highlights

• The chemical compositions of FSCR were analysed by UPLC-PDA-MS.

• A PID model induced by phenol mucilage was successfully established in rats.

• The effects of FSCR on rats with PID were studied.

• FSCR attenuated pelvic inflammation and prevented fibrosis.

• The ERK1/2 and Smad2/3 signalling pathways may be involved in the effects of FSCR.

Abstract

Pelvic inflammatory disease (PID), an infection and inflammation disorder of the upper genital tract, results in infertility in approximately 20% of cases. No antibiotic that can alleviate the sequelae of PID has been identified. Smilax china L. rhizome (SCR), which are widely used in functional food and folk medicine, exhibit multiple pharmacological activities, such as anti-inflammatory, antioxidant and antitumour activities. In this study, the flavonoids of SCR (FSCR) were purified and analysed by UPLC-PDA-MS. The therapeutic effects of FSCR in a rat PID model were evaluated. FSCR-treated PID rats exhibited significantly decreased inflammation, ameliorated fibrosis, and enhanced production of matrix metalloproteinase (MMP)-2 and MMP-9 in uteri. Moreover, the phosphorylation of ERK1/2 and Smad2/3 was markedly inhibited by FSCR. These results indicate that FSCR exhibits robust anti-inflammatory and anti-fibrosis effects, possibly via inhibition of the ERK1/2 and TGF-β-Smad2/3 signalling pathways, and may be an effective candidate natural medicine for PID treatment.

Introduction

Pelvic inflammatory disease (PID) involves infection and inflammation of the uterus, ovaries, and other female reproductive organs (Mitchell & Prabhu, 2013). In the United States, there are more than 800,000 patients with PID, and approximately 20% of cases develop infertility (Blanchard et al., 1998; Ness, Trautmann et al., 2005). PID is often elicited by sexually transmitted infections but is occasionally induced by postsurgical and post-delivery infections or intrauterine devices. Neisseria gonorrhoeae and Chlamydia trachomatis have been implicated as the most common causes (Bevan et al., 1995, Haggerty, 2008, Ness et al., 2005b); however, in up to 70% of cases, no pathogen is identified (Burnett et al., 2012, Oakeshott et al., 2010). A considerable proportion of cases result in long-term sequelae, including ectopic pregnancy, infertility, intrauterine adhesion and chronic pelvic pain (Sweet, 2009).

Inflammation and hyperplasia of fibrous tissue are the main pathological processes responsible for PID (Bulun et al., 2005). Pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are increased significantly in endometritis (Zhang et al., 2015) and other female genital tract infections (Agrawal et al., 2011, Takenaka et al., 2010) as well as in many other inflammatory diseases (Chen et al., 2014). Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play an important role in regulating these pro-inflammatory mediators, and enhanced phosphorylation of ERK1/2 has been observed in LPS-induced mice with endometritis (Zhang et al., 2015). In the development of PID, inflammation is often accompanied by fibrosis due to the recurring infection and excessive tissue remodelling. Transforming growth factor-β1 (TGF-β1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) aggravate the deposition of extracellular matrix (ECM) in the process of fibrosis, and increases in these proteins have been observed in many diseases, such as left ventricular fibrosis (Lauer et al., 2014), fibrogenesis (Zhao et al., 2014), and chronic spinal cord wounds (Ahmed et al., 2014). Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) play critical roles in the degradation of ECM by hydrolysing gelatines, collagens, and fibronectin; however, their activity is inhibited by TIMP-1 (Shimokawa Ki et al., 2002). In normal uteri, TIMPs and MMPs maintain the homeostasis of the ECM, but this balance is disrupted in the development of PID (Wang et al., 2009).

PID is usually treated with antibiotics to provide broad-spectrum treatment of likely pathogens. Antibiotics are effective in reducing short-term morbidity but have no effects on long-term complications, owing to the disease’s complex mechanism and long-term process (Mitchell & Prabhu, 2013). Although the incidence of PID has declined because of screening for gonorrhoea and chlamydia and the early use of broad-spectrum antibiotics, damage to the reproductive system caused by infection has not improved (Ross, 2013). Thus, the therapeutic goal for the treatment of PID should include both short-term microbiological effects and long-term prevention of sequelae (Mitchell & Prabhu, 2013). In addition, the use of antibiotics is limited by the emergence of antibiotic resistance and PID without an identified pathogen. Thus, there is an unmet need for new drugs that inhibit the progression and alleviate the long-term sequelae of PID.

Smilax china L. (SCR) is commonly known as “Ba Qia” and “Jin Gang Teng” in China and has a long history of use as a traditional Chinese medicine. Its tender stems are also eaten as a vegetable in China, and in Korea, the leaves are traditionally used to wrap rice cakes to enhance flavour and impart antimicrobial activity (Wu et al., 2010). The rhizome of SCR possess anti-inflammatory (Shu, Gao, & Yang, 2006), antioxidant (Lee, Ju, & Kim, 2001), antitumour (Li et al., 2007, Nho et al., 2015) and neuroprotective (Ban et al., 2008) activities. SCR rhizomes have been extensively used in the treatment of various inflammatory diseases, particularly for pelvic inflammation (Shu et al., 2006). Jin Gang Teng capsules, a crude extract of SCR, have been used to treat PID through traditional Chinese medicine (TCM) for years in clinics in China. We have previously demonstrated that the ethyl acetate extract of SCR is therapeutic in rats with PID, and the bioactive fraction inhibits the expression of TNF-α in rat uteri (Luo et al., 2014, Ma et al., 2013). However, the mechanism and active ingredients underlying the effects of SCR on PID have not been identified. In this study, flavonoids of SCR (FSCR) were prepared, and the main ingredients and contents were identified and detected with Ultra Performance Liquid Chromatography-Photo Diode Array-Mass Spectrometer (UPLC-PDA-MS). After identification of the effective substance, we explored the therapeutic effects and possible mechanisms of FSCR by assessing inflammation and fibrosis in rats with PID induced by phenol mucilage.