Abstract

   Kangfuxiaoyan suppository (KFXYS) is a commonly used traditional
   Chinese medicine (TCM) preparation for the treatment of chronic pelvic
   inflammatory disease (CPID) clinically, and its safety and
   effectiveness have been well verified. However, the potential mechanism
   remains unclear. The integrated strategy of metabolomics and network
   pharmacology was employed in the study to reveal the potential
   mechanism of KFXYS in the treatment of CPID. Our research consists of
   five steps. First, the effect of KFXYS in reversing uterine
   inflammation indexes was verified. Second, based on the comprehensive
   characterization of 123 chemical ingredients of KFXYS, the ingredients
   of KFXYS absorbed into blood were identified by UPLC-Q-TOF/MS, then
   ADME research was carried out on the main ingredients. Third, the
   differential metabolites with significant correlation to inflammatory
   indexes were discovered by metabolomics and correlation analysis.
   Fourth, the potential targets and pathways of KFXYS in treating CPID
   were predicted by network pharmacology based on the ingredients which
   had good ADME behavior. Fifth, the proteins in common pathways of
   metabolomics and network pharmacology were used to screen the key
   targets from the potential targets of network pharmacology, and the
   potential mechanism of KFXYS in treating CPID was clarified. As a
   result, KFXYS significantly reversed the uterine inflammation indexes,
   including IL-1 and IL-6. The ingredients absorbed into blood including
   matrine, sophocarpine, aloin, esculetin-O-glucuronide,
   7,4′-dihydroxyisoflavone-O-glucuronide, and
   4′-methoxyisoflavone-7-O-glucuronide had good ADME behavior in vivo.
   Among the differential metabolites, Leukotriene A4,
   5-Hydroxyindoleacetic acid, Ornithine, Arginine, and PC (20:1
   (11Z)/20:4 (8Z,11Z,14Z,17Z)) were significant correlation to
   inflammation indexes. The integration analysis of metabolomics and
   network pharmacology shows that KFXYS may regulate the key targets
   including ARG1, NOS2, NOS3, etc. We speculate that ingredients of
   KFXYS, such as matrine, sophocarpine, aloin etc. act on the key
   proteins including ARG1, NOS2, and NOS3, to exert anti-inflammatory
   effect.

   Keywords: kangfuxiaoyan suppository, chronic pelvic inflammatory
   disease, UPLC-Q-TOF/MS, network pharmacology, metabolomics

Introduction

   Chronic pelvic inflammatory disease (CPID) is a chronic inflammation of
   female pelvic reproductive organs, surrounding connective tissue and
   pelvic peritoneum ([40]Cao, 2008), which can lead to infertility,
   ectopic pregnancy, or chronic pelvic pain ([41]Soper, 2010).
   Antibiotics such as levofloxacin or metronidazole are mostly used in
   treating CPID ([42]Duarte et al., 2015), but the therapeutic effect is
   not ideal due to drug resistance and side effects. Therefore, it is
   necessary to develop new alternative drugs to treat CPID.

   Kangfuxiaoyan suppository (KFXYS) is a Chinese patent medicine commonly
   used for the treatment of CPID ([43]Standardized project group of
   "Clinical Application Guidelines of the Chinese Medicine for Treatment
   of Common Disease, 2020). The formula of KFXYS is composed of Sophorae
   Flavescentis Radix (SFR, derived from Sophora flavescens Aiton.),
   Andrographis Herba [AH, derived from Andrographis paniculata (Burm.f.)
   Nees], Arnebiae Radix [AR, derived from Arnebia euchroma (Royle ex
   Benth.) I.M.Johnst.], Herba Patriniae (HP, Patrinia villosa (Thunb.)
   Dufr. or Patrinia scabiosifolia Link), Taraxaci Herba (TH, derived from
   Taraxacum mongolicum Hand.-Mazz. or Taraxacum sinicum Kitag.), Violsse
   Herba (VH, derived from Viola philippica Cav.), Aloe [Aloe, derived
   from Aloe vera (L.) Burm. f. or Aloe ferox Mill.], and Suis Fellis
   Pulvis (SFP, derived from Sus scrofa domestica Brisson.) ([44]Chinese
   Pharmacopoeia Commission, [CPC], 2020). Our previous study has shown
   that KFXYS could alleviate the degree of uterine swelling in CPID,
   inhibit the secretion of inflammatory factors, and increase the immune
   organ index, the efficacy of KFXYS was verified in rat with CPID
   ([45]Xie et al., 2018), but the underlying mechanism is still unknown.

   In order to reveal the mechanism of the synergistic effect of
   multi-components of TCM, integrated metabolomics and network
   pharmacology strategy has been successfully used ([46]Li et al., 2021).
   In recent years, metabolomics has been successfully applied to the
   study of the mechanism of TCM in treating diseases ([47]Geiger et al.,
   2016). However, due to the lack of specificity of non-targeted
   metabolomics research, the discovery of differential metabolites
   correlation with pharmacodynamic indicators contribute to the following
   mechanism research. Network pharmacology can explore the changes of
   targets and pathways in vivo caused by diseases, and the disturbance of
   one or more nodes in the biological network after TCM preparation
   acting on the body ([48]Hopkins, 2008), so as to effectively establish
   a “compound-protein/gene-disease” network, reveal the regulation
   principles of small molecules in a high-throughput manner ([49]Zhang et
   al., 2019). However, we believe that the results of network
   pharmacology based on the components absorbed into blood which had good
   ADME behavior is more accurate and reliable.

   In this study, the reversal effect of KFXYS on inflammatory indexes was
   verified in CPID model rats. Based on clarifying the chemical
   ingredients of KFXYS, the ingredient of KFXYS absorbed into blood were
   further characterized by UPLC-Q-TOF/MS technology, then ADME research
   was carried out. Among the differential metabolites discovered by
   metabolomics, the metabolites with significant correlation to
   inflammatory indexes were discovered by correlation analysis. Then
   based on the ingredients absorbed into blood which had good ADME
   behavior, network pharmacology was used to predict the potential
   targets and pathways of KFXYS in the treatment of CPID. The
   metabolomics and network pharmacology common pathways were considered
   the key metabolic pathways, and the key targets were screened from the
   potential targets obtained by network pharmacology through the proteins
   in the key pathways. Finally, the mechanism of KFXYS in the treatment
   of CPID was explored through literature research ([50]Figure 1).

FIGURE 1.

   [51]FIGURE 1
   [52]Open in a new tab

   Schematic diagram of the action mechanism of KFXYS in treating CPID.

Materials and Methods

Chemicals and Reagents

   Kangfuxiaoyan suppository (batch No. 20171225) was obtained from the
   Sunflower Pharmaceutical Group Co., Ltd. Sodium pentobarbital (batch
   No. 11011) was obtained from Merck. Mass spectrometry-grade
   acetonitrile and methanol, and HPLC-grade formic acid were obtained
   from Thermo Fisher Scientific. The water used for LC/MS analysis was
   purchased from Watsons.

Animal Model Establishment and Treatment

   Animal Sprague Dawley (SD) rats, female, weight 190–210 g, SPF grade,
   approval number: SCXK (Beijing) 2016-0011, raised in the Animal
   Experimental Center of Beijing University of Chinese Medicine, the
   temperature was controlled at 21–25°C, the relative humidity was
   55-65%, and the cycle of day and night was 12 h alternately. The rats
   adapted to the environment for 7 days, during which they drank and ate
   freely.

   Animal Model Establishment The rat model of CPID was established by
   implanting extraneou materials. The method of establishing CPID model
   is shown in [53]Supplementary Material. The rats are grouped as
   follows: 20 rats were randomly divided into the normal group and the
   sham operation group, the normal group did not do any treatment, the
   sham operation rats received only a 2 cm incision wound in the middle
   of the lower abdomen, and then sutured without any treatment, and the
   other rats were used to establish the rat model of CPID by implanting
   extraneous materials. Finally, the rats were divided into normal group
   (n = 10), sham operation group (n = 8), model group (n = 10),
   KFXYS-dosed group (n = 11), levofloxacin-dosed group (n = 10).

   Appropriate amounts of KFXYS and normal saline were added into the
   clean evaporation dish, heat in a water bath at 50°C until melting, and
   finally, obtain 0.4 g mL^−1 KFXYS solution. KFXYS was given by rectal
   administration according to the clinical equivalent dose of
   408 mg kg^−1 for 21 days, and the rats in each group ate normal diet
   during the treatment.

Sample Collection and Preparation

   Sample Collection For the identification of ingredients absorbed into
   blood of KFXYS and the study of pharmacokinetic, the blood was
   collected from orbital vein at different time points (15, 30 min, 1, 2,
   3, 4, 6, 8, 12, and 24 h) after the last administration of KFXYS in
   CPID rats, and placed in a centrifuge tube covered with heparin sodium,
   centrifuged at 1,100 ×g for 10 min at 4°C, and the plasma samples were
   collected.

   For the study of metabolomics, the blood samples were collected from
   the abdominal aorta in each group rats, and these samples were placed
   at room temperature for half an hour, centrifuged at 1,100 ×g for
   10 min at 4°C, then the serum samples were collected, including eight
   sham operation group serum samples, 10 model group serum samples, and
   11 KFXYS-dosed group serum samples.

   Sample Preparation For the identification of ingredients absorbed into
   blood of KFXYS, mixed the plasma sample from each time point evenly,
   took 1 ml mixed plasma sample, three times the amount of methanol was
   added to precipitate the protein, and vortex for 30 s, centrifuged at
   13,600 ×g for 15 min at 4°C, and the supernatant was dried with
   nitrogen. Then the residue was redissolved with 100 μL 70% methanol,
   centrifuged at 13,600 ×g for 10 min at 4°C, and the supernatant was
   taken as the plasma sample to be tested in each group. As to the study
   of pharmacokinetic, the plasma samples at each time point were prepared
   according to the above method.

   For the study of metabolomics, in order to obtain comprehensive
   information of serum metabolites, the polar part and weak polar part of
   serum samples were treated respectively. The polar samples were
   processed as follows: taken 120 μL of the serum sample, added 360 μL
   methanol to precipitate protein, and vortex for 30 s, static 10 min,
   centrifuged at 13,600 ×g for 10 min at 4°C. After the 300 μL
   supernatant was dried with nitrogen, redissolved with 100 μL
   acetonitrile-water (1:1, v/v), vortex for 30 s, centrifuged at 13,600
   ×g for 10 min at 4 °C, and the supernatant was collected. The weak
   polar samples were processed as follows: taken the serum sample 120 μL,
   added 300 μL chloroform-methanol (2:1, v/v), vortex for 2 min, static
   10 min, centrifuged at 13,600 ×g for 10 min at 4°C, then taken 200 μL
   lower liquid (chloroform layer), dried with nitrogen, redissolved with
   100 μL isopropanol-acetonitrile (1:1, v/v), vortex for 30 s,
   centrifuged at 13,600 ×g for 10 min at 4°C, and the supernatant was
   collected.

UPLC-Q-TOF/MS Analysis

   For the identification of ingredients absorbed into blood of KFXYS, the
   UPLC-Q-TOF/MS analysis was performed on Waters ACQUITY UPLC I-CLASS
   liquid phase system equipped with SYNAPT G2-SI high-resolution mass
   spectrometer. Chromatographic separations were performed at 40°C on
   ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters, UK). The
   mobile phase composed of 0.1% formic acid-water (A) and acetonitrile
   (B). The elution program was as follows: 0–2 min, 2%–5% B; 2–3.5 min,
   5%–8% B; 3.5–6 min, 8%–11% B; 6–8.5 min, 11%–15% B; 8.5–9.5 min,
   15%–17% B; 9.5–11 min, 17% B; 11–13.5 min, 17%–20% B; 13.5–15 min,
   20%–23% B; 15–17 min, 23%–30% B; 17–20.5 min, 30%–40% B; 20.5–25 min,
   40%–60% B; 25–29 min, 60% B; 29–32 min, 60%–100% B; 32–32.1 min,
   100%–2% B; 32.1–35 min, 2% B. The flow rate was set to 0.25 ml min^−1
   and the injection volume was 2 μL.

   An electrospray ionization source (ESI) was used both in positive and
   negative mode. The MS conditions were as follows: capillary voltage, +3
   KV/-2.5 KV; cone voltage, 40 kV; ion source temperature, 100°C; cone
   gas flow, 50 L h^−1; desolvation gas temperature, 400°C; desolvation
   gas flow, 600 L h^−1. The Lockmass solution was Leucine encephalin. The
   collision energy was set as 6 eV (trap) for low-energy scan, and
   10–65 eV ramp (trap) for high-energy scan. The mass range was
   100-1,200 m/z and the data acquisition mode was 3D data acquisition in
   Continuum mode.

   As to the study of pharmacokinetics of main ingredients, the
   UPLC-Q-TOF/MS analysis was performed according to the above method, but
   the data acquisition mode was MRM mode.

   For the study of metabolomics, all serum samples were performed on
   Waters Acquity^TM UPLC analysis system equipped with Xevo^TM G2 Q/TOF
   tandem quadrupole time-of-flight mass spectrometer, and chromatographic
   separations were performed at 45°C on ACQUITY UPLC BEH C18 column (2.1
   × 100 mm, 1.7 μm, Waters, UK). The UPLC-Q-TOF/MS analysis conditions
   are shown in [54]Supplementary Material. Quality control (QC) samples
   were prepared by mixing an equal amount of each sample, and the
   stability and repeatability of sample analysis were monitored by
   analyzing QC samples after every 10 samples.

Identification of Ingredients Absorbed Into Blood of KFXYS

   Based on the previous research, the identification steps of the
   ingredients absorbed into blood of KFXYS were as follows: 1)
   Summarizing the possible metabolic pathways of various chemical
   components in vivo by comparing with some representative ingredients
   and tracking the literature. 2) The prototype components and
   metabolites in the KFXYS-dosed plasma were discovered by comparing with
   the KFXYS-treated and KFXYS sample. Then the ingredients absorbed into
   blood of KFXYS were comprehensively identified based on the mass
   fragmentation pattern, characteristic fragments and chromatographic
   retention behavior.

Study on the Pharmacokinetics of Main Components Absorbed into Blood of KFXYS

   The optimized ion pairs of the main components and parameters of cone
   voltage and capillary voltage are shown in [55]Table 1.

TABLE 1.

   Mass spectrum parameters of the main components in MRM mode.
   References substance Parent ions Ion pairs Cone voltage (V) Capillary