Abstract

Background

   Gouty arthritis is a common metabolic disease caused by long-term
   purine metabolic disorder and elevated serum uric acid.
   Jiang-Suan-Chu-Bi recipe (JSCBR), a traditional Chinese herbal formula
   prescribed according to utilization frequency and cluster analysis, has
   been clinically validated remedy for gouty arthritis. However, its
   therapeutic composition and mechanism remains unclear.

Methods

   In the present study, a simple, rapid, and sensitive ultraperformance
   liquid chromatography coupled with quadrupole time-of-flight mass
   spectrometry (UHPLC-QTOF-MS)-based chemical profiling was firstly
   established for comprehensively identifying the major constituents in
   JSCBR. A phytochemistry-based network pharmacology analysis was further
   performed to explore the potential therapeutic targets and pathways
   involved in JSCBR bioactivity. Finally, THP-1 cell model was used to
   verify the prediction results of network pharmacology by western blot
   analysis.

Results

   A total of 139 compounds containing phenolic acids, flavonoids,
   triterpenoid saponins, alkaloids, amino acids, fatty acids,
   anthraquinones, terpenes, coumarins, and other miscellaneous compounds
   were identified, respectively. 175 disease genes, 51 potential target
   nodes, 80 compounds, and 11 related pathways based on network
   pharmacology analysis were achieved. Among these pathways and genes,
   NOD-like receptor signaling pathway may play an important role in the
   curative effect of JSCBR on gouty arthritis by regulation of
   NRLP3/ASC/CASP1/IL1B. The results of cellular and molecular experiments
   showed that JSCBR can effectively reduce the protein expression of ASC,
   caspase-1, IL-1β, and NRLP3 in monosodium urate-induced THP-1 cells,
   which indicated that JSCBR mediated inflammation in gouty arthritis by
   inhibiting the activation of NOD-like receptor signaling pathway.

Conclusion

   Thus, the integrated approaches adopted in the present study could
   contribute to simplifying the complex system and providing directions
   for further research of JSCBR.

   Keywords: Jiang-Suan-Chu-Bi recipe (JSCBR), gouty arthritis, chemical
   profile, network pharmacology, NOD-like receptor signaling pathway

Introduction

   Gouty arthritis is a metabolic disease caused by the deposition of
   monosodium urate crystals in joints and soft tissues ([39]Pascart
   et al., 2018) and closely associated with chronic hyperuricemia, which
   seriously affects quality of life of patients due to severe pain
   ([40]Nielsen et al., 2018). Across the globe, the age of onset is
   getting younger and younger ([41]Zhou et al., 2014). It is estimated
   that the number of gout patients in China will exceed 100 million in
   2020. Now, gout has become the second largest metabolic disease in
   China ([42]Wilson and Saseen, 2016).

   At present, western medicines including colchicine, allopurinol,
   benzbromarone, and febuxostat have been used as the conventional
   therapy method for gouty arthritis. Although their short-term effect on
   inhibiting uric acid production, promoting uric acid excretion, or
   analgesic effect is optimistic, the side effects such as skin mucosal
   injury, kidney and liver injury, digestive tract injury, as well as
   some potential neurotoxicity and muscular toxicity caused by long-term
   use could not be ignored. Meanwhile, as the main form of traditional
   Chinese medicine (TCM) that based on the compatibility theory,
   traditional Chinese herbal formula has been applied for the treatment
   of gouty arthritis for thousands of years in view of remarkable
   therapeutic effect and little adverse reactions ([43]Zhou et al.,
   2014).

   Jiang-Suan-Chu-Bi recipe (JSCBR) is a formula prescribed according to
   utilization frequency and cluster analysis by retrieving gout-related
   data obtained from multiple databases ([44]Xiao et al., 2019), which
   has been clinically validated remedy for gouty arthritis under the
   guidance of TCM doctors. It consists of 12 herbs, namely, Smilax glabra
   Roxb. (tu-fu-ling in Chinese, TFL, batch No. 20160925), Paeonia
   lactiflora Pall (chi-shao in Chinese, CS, batch No. 20160814),
   Reynoutria japonica Houtt. (hu-zhang in Chinese, HZ, batch No.
   20160728), Cremastra appendiculata (D.Don) Makino (shan-ci-gu in
   Chinese, SCG, batch No. 20160723), Rheum officinale Baill. (da-huang in
   Chinese, DH, batch No. 20160719), Clematis chinensis Osbeck
   (wei-ling-xian in Chinese, WLX, batch No. 20160915), Angelica pubescens
   Maxim. (du-huo in Chinese, Dh, batch No. 20160822), Phellodendron
   chinense C.K.Schneid. (huang-bai in Chinese, HB, batch No. 20160918),
   Atractylodes lancea (Thunb.) DC. (cang-zhu in Chinese, CZ, batch No.
   20160814), Glycyrrhiza glabra L. (gan-cao in Chinese, GC, batch No.
   20160816), Codonopsis pilosula (Franch.) Nannf. (dang-shen in Chinese,
   DS, batch No. 20160723), and Angelica sinensis (Oliv.) Diels (dang-gui
   in Chinese, DG, batch No. 20160801). Although its prescription is
   scientific and the efficacy is definite. To date, no reports on the
   chemical composition and functional mechanisms of JSCBR contribute to
   its bioactivity has been published, which restrict its further
   application and development.

   In the present study, a systematic dissection of JSCBR was employed by
   integrating chemical profile, network pharmacology, and experimental
   support using molecular cell biology. The schematic diagram of the
   present study was depicted in [45]Figure 1 .

Figure 1.

   [46]Figure 1
   [47]Open in a new tab

   The schematic diagram of the present study.

Materials and Methods

Chemicals, Reagents, and Materials

   UHPLC-MS grade acetonitrile and methanol were supplied by Merck Company
   Inc., (Darmstadt, Germany). MS grade formic acid was purchased from
   Fisher Scientific Company (Inc., Fairlawn, NJ). Ultrapure water (18.2
   MΩ) was prepared with a Milli-Q water purification system (Millipore,
   Milford, MA, USA). All other reagents were of analytical grade and
   purchased from Tianjin Concord Technology Co. Ltd. (Tianjin, China).

   The reference compounds ferulic acid (56), polydatin (57), paeoniflorin
   (79), atractylodin (81), liquiritin (84), aloe-emodin (94), rhein
   (115), glycyrrhizic acid (119), osthole (126), columbianadin (127), and
   oleanolic acid (134) were purchased from Chengdu Pufei De Biotech
   (Chengdu, Sichuan, China). The reference compounds astilbin (60),
   chysophanol (93), physcion (100), and emodin (122) were purchased from
   Baoji Herbest Biotech (Baoji, Shanxi, China). The purity of each
   reference standard was determined to be over 98% by UHPLC analysis. All
   the 12 herbs of JSCBR were purchased from Beijing Tong-Ren-Tang
   Technologies Co., Ltd. (Taiyuan, Shanxi Province, China), and
   authenticated by Professor Yunpeng Diao (College of Pharmacy, Dalian
   Medical University). Voucher specimens were deposited at the authors’
   laboratory.

   THP-1 cell was purchased from Yipu Biological Technology Co., Ltd.
   (Wuhan, Hubei Province, China). RPMI 1640 cell culture mediums and
   pencillin-streptomycin were purchased from Gibco BRL, Invitrogen
   Corporation (Grand Island, NY). Fetal bovine serum was purchased from
   Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, Zhejiang Province,
   China). Cell Counting Kit -8 (CCK-8) was purchased from Yiyuan
   Biotechnology Co., Ltd. (Guangzhou, Guangdong Province, China).
   Phorbol-12-myristate-13-acetate (PMA) was purchased from Abcam Ltd.,
   (Cambridge, UK). Monosodium urate (MSU) was purchased from
   Sigma-Aldrich (St Louis, MO, USA). Colchicine was supplied by Shengshi
   Kangpu Chemical Technology Research Institute (Beijing, China).
   Antibodies against caspase-1, β-actin, apoptosis-associated speck-like
   protein (ASC), IL-1β, and antirabbit IgG-HRP were purchased from Bioss
   (Beijing, China). The BCA Protein Quantification Kit was purchased from
   KeyGen Biotech Co., Ltd. (Nanjing, Jiangsu Province, China). RIPA lysis
   buffer and PMSF protease inhibitor were purchased from Beyotime
   Institute of Technology (Shanghai, China). ECL-plus chemiluminescence
   reagent was purchased from Bio-rad (Richmond, CA, USA).

Preparation of JSCBR Extract

   The usage of each crude herb were accurately weighed as follows:
   Smilacis Glabrae Rhizome (30 g), Paeoniae Radix Rubra (10 g), Polygoni
   Cuspidati Rhizoma et Radix (15 g), Cremastrae Pseudobulbus (8 g), Rhei
   Radix et Rhizoma (10 g), Clematidis Radix et Rhizome (15 g), Angelicae
   Pubescentis Radix (10 g), Phellodendri Chinensis Cortex (10 g),
   Atractylodis Rhizome (10 g), Glycyrrhizae Radix et Rhizome (10 g),
   Codonopsis Radix (15 g), and Angelicae Sinensis Radix (10 g). They were
   mixed and prepared by the decocting method as described below: a total
   of 153 g mixture was immersed in sevenfold mass of water for 2 h,
   heated and refluxed for 2 h and then filtered with six-layer absorbent
   gauze. A fivefold mass of water was subsequently added to the residues
   and boiled for 2 h. After being filtered with six-layer absorbent
   gauze, the two filters were combined and concentrated to 150 ml (equal
   to 1 g crude herb/ml). The liquid was finally transformed into powder
   by vacuum freeze drying technology.

   A 1.0 g of the freeze-dried powder was accurately weighted and
   extracted with 50 ml of methanol/water (1:1, v/v) for 30 min under
   ultrasound. The extract solution was centrifuged at 13,000 rpm for 10
   min at 4°C and the supernatant was filtered through a 0.22 μm membrane
   filters. 1.0 μl of filtrate was injected to UHPLC-QTOF-MS for analysis.

UHPLC-QTOF-MS Analysis

   The chromatography and MS conditions were almost the same as those
   reported in literature ([48]Huang et al., 2019). The only difference is
   the change of elution gradient, which was listed as follows: 0–25 min,
   5%–45% B; 25–35 min, 45%–99% B; 35–38 min, 99%–5% B; 38–40 min, 5% B.

Establishment of In-House Library for JSCBR and Generation of Empirical
Molecular Formula

   The in-house library that covers all previous reported compounds from
   the 12 formulated herbs was established in a Microsoft office excel
   table, which includes compound name, molecular formula, molecular
   weight, chemical structures, natural source, and related references