Abstract

   Rhus chinensis Mill. fruits are a kind of widely distributed edible
   seasoning, which have been documented to possess a variety of
   biological activities. However, its inhibitory effect on osteoclast
   formation has not been determined. The objective of this study was to
   evaluate the effect of the fruits on osteoclast differentiation of
   RAW264.7 cells, induced by receptor activator of nuclear factor-κB
   ligand (RANKL) and to illuminate the potential mechanisms using network
   pharmacology and western blots. Results showed that the extract
   containing two organic acids and twelve phenolic substances could
   effectively inhibit osteoclast differentiation in RANKL-induced
   RAW264.7 cells. Network pharmacology examination and western blot
   investigation showed that the concentrate essentially decreased the
   expression levels of osteoclast-specific proteins, chiefly through
   nuclear factor kappa-B, protein kinase B, and mitogen-activated protein
   kinase signaling pathways, particularly protein kinase B α and
   mitogen-activated protein kinase 1 targets. Moreover, the extract
   likewise directly down regulated the expression of cellular oncogene
   Fos and nuclear factor of activated T-cells cytoplasmic 1 proteins.
   Citric acid, quercetin, myricetin-3-O-galactoside, and
   quercetin-3-O-rhamnoside were considered as the predominant bioactive
   ingredients. Results of this work may provide a scientific basis for
   the development and utilization of R. chinensis fruits as a natural
   edible material to prevent and/or alleviate osteoporosis-related
   diseases.

   Keywords: Chinese sumac, osteoclast, osteoporosis, network
   pharmacology, phenolic compounds

1. Introduction

   Human bones are constantly modified, and the dynamic balance of
   osteoblasts and osteoclasts is an important factor to maintain human
   bone health [[30]1]. However, when this homeostasis becomes imbalanced,
   it can induce the growth of bone-solubilizing diseases, such as
   osteoporosis (OP) [[31]2,[32]3], osteosclerosis [[33]4], etc. The main
   manifestations of OP are exacerbation of bone organization
   microarchitecture, reduction of bone density and an increase in
   susceptibility to fragile fractures [[34]5]. OP can be mainly divided
   into primary OP and secondary OP, and the underlying pathogenesis is
   that bone loss is faster than bone formation [[35]6,[36]7,[37]8]. With
   the development of medical technology and the standard of living, the
   average life expectancy of people is increasing which is accelerating
   the aging of society, resulting in a higher prevalence of OP [[38]9].
   The excessive activation of osteoclasts assumes a critical part in the
   pathology of OP [[39]10]. Therefore, the inhibition of osteoclast
   differentiation has been considered as a potential therapy for the
   treatment and/or prevention of OP with few side effects.

   Osteoclasts are a kind of novel multinucleated cell with bone resorbing
   capacity that originates from the bone marrow monocyte-macrophage
   lineage [[40]11]. A major cascade of signals regulating osteoclast
   maturation and activation is the receptor activator of nuclear
   factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL)
   pathway. RANKL facilitates osteoclast activation by combination with
   RANK on the cell membrane of osteoclast lineage cells [[41]12]. After
   activation, the expression of many key transcription activators and
   enzymes increases, which in turn promote transdifferentiation,
   multiplication, and multi-nucleation of the osteoclast [[42]4]. RANKL
   recruits tumor necrosis factor receptor-associated factor 6 (TRAF6) to
   activate a series of downstream cascades, including NF-κB, Akt, and
   MAPKs signaling pathways, and then further activates nuclear factor of
   activated T-cells cytoplasmic 1 (NFATc1) and cellular oncogene Fos
   (c-Fos), which are the final step for osteoclast formation [[43]13].
   Currently, the representative drugs for OP treatment are
   bisphosphonates and estrogens [[44]14], but these substances inevitably
   cause varying degrees of side effects and complications in humans, such
   as osteonecrosis of the jaw and unrepresentative femur fractures
   [[45]15]. Therefore, it is essential to develop some alternative
   dietary therapy with fewer side effects to prevent and/or improve OP.

   Polyphenols are abundant in common edible fruits, vegetables and herbs.
   They have good antioxidant and anti-inflammatory activities and can
   maintain the health of the body [[46]16]. At present, many studies have
   found that plant polyphenols have inhibitory effects on the formation
   of osteoclasts [[47]17,[48]18]. The study by Thomas et al. [[49]17]
   showed that TRAP activity, an indicator of osteoclast differentiation,
   exhibited a downward trend with treatment using tart cherry
   polyphenols; moreover, result also showed that high doses of tart
   cherry polyphenols (300 µg/mL) could reduce the production of
   inflammatory markers, including nitric oxide content, cyclooxygenase 2,
   in RANKL-induced RAW264.7 cells, thereby reducing the differentiation
   and resorption activity of osteoclasts. Aronia melanocarpa ‘Viking’
   (AM) was rich in phenolic compounds, and the study of Ghosh et al.
   [[50]18], showed that the water and alcohol extracts of AM could
   inhibit the excessive accumulation of ROS, and the expression of
   osteoclast-related genes, including integrin β[3], TRAP, cathepsin K
   and calcitonin receptor; in addition, both extracts inhibited
   osteoclastogenesis by acting on the MAPKs pathway and blocking the
   signaling of c-Fos and NFATc1. Rhus chinensis pertains to the genus
   Rhus of the Anacardiaceae family, and is extensively distributed in
   Asia, including China, and Japan [[51]19]. The fruits are commonly used
   as a kind of appetizer, beverage or natural vinegar by local people
   [[52]19]. In addition, the fruits are recorded as a traditional herb
   with a wide range of bioactivities, which can be used to preclude
   and/or cure some diseases such as jaundice, alcoholism, hepatitis, and
   inflammatory diseases [[53]20,[54]21]. Rhus chinensis Mill. fruits have
   high nutritional value and are rich in a variety of polyphenols,
   polyunsaturated fatty acids and other phytochemicals [[55]19]. For
   example, quercetin, myricetin-3-O-galactoside, and
   quercetin-3-O-rhamnoside are polyphenols that are abundant in R.
   chinensis fruits [[56]21,[57]22]. Some studies have reported that
   quercetin has related activities such as inhibiting osteoclastogenesis
   [[58]23,[59]24]. Kim et al. [[60]23], found that quercetin could play
   an immunomodulatory role in interleukin-17 (IL-17) produced
   osteoclastogenesis. The results of Guo et al. [[61]24], showed that
   quercetin could inhibit lipopolysaccharide-induced osteoclast bone
   resorption. Based on the above related findings, we speculated that R.
   chinensis fruits may have a bioactivity that inhibits the
   differentiation and formation of osteoclasts. However, no study has
   been carried out to investigate the preventive effects and the
   underlying mechanisms involved in the differentiation and formation of
   osteoclasts. The aim of this work was to investigate the preventive
   effects and potential mechanisms of the ethanolic extract from R.
   chinensis fruits against the differentiation and formation of
   osteoclasts by using network pharmacology and validation of results
   using cellular experiments ([62]Figure 1).

Figure 1.

   [63]Figure 1
   [64]Open in a new tab

   Workflow of the research. OP, osteoporosis; MTT,
   3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TRAP,
   tartrate-resistant acid phosphatase; RANKL, nuclear factor-κB ligand;
   NF-κB, nuclear factor kappa-B; c-Fos, cellular oncogene Fos; NFATc1,
   nuclear factor of activated T-cells cytoplasmic 1.

2. Materials and Methods

2.1. Reagents and Chemicals

   RANKL was obtained from R&D Systems (Minneapolis, MN, USA). BCA protein
   assay kit, 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
   (MTT) and tartrate-resistant acid phosphatase (TRAP) activity kits were
   supplied by Beyotime Biotechnology (Shanghai, China). Penicillin,
   streptomycin, Dulbecco’s modified Eagle’s medium (DMEM) and fetal
   bovine serum (FBS) were purchased from Gibco-Invitrogen (Karlsruhe,
   Baden-Württemberg, Germany). Cell lysis buffer with inhibitors of
   protease and phosphatase were provided by Nanjing Jiancheng
   Bioengineering Institute (Nanjing, China). Antibodies used in the
   current work, including NF-κB, p-IκBα, IκBα, p-JNK
   (phospho-JNK1-T183/Y185 + JNK2-T183/Y185 + JNK3-T221/Y223), JNK, p-ERK
   (phospho-ERK1-T202/Y204 + ERK2-T185/Y187), ERK, p38, p-p38 (phospho-p38
   MAPK-T180/Y182 Rabbit pAb), Akt, p-Akt (Ser 473) and β-Actin were
   supplied by Wuhan Abclonal, China, and p-NF-κB, c-Fos, and NFATc1 were
   obtained from Affinity (Carlsbad, CA, USA).

2.2. Sample Preparation

   R. chinensis fruits were collected by Kunming Plant Classification
   Biotechnology Co., Ltd. (Kunming, China) from Tengchong County, Yunnan
   Province, in November 2019. All fruit materials were washed with tap
   water to remove impurities, dried naturally, and stored at −20 °C. The
   fruit was then freeze–dried, crushed to pass through a 40 mesh sieve.
   The 80% ethanolic extract was prepared in accordance with a former
   report [[65]22] and the extract concentrated by a Heidolph Hei-VAP
   rotary evaporator (Hei-VAP Advantage, Heidolph, Germany) and
   freeze-dried.

2.3. Characterization of Phytochemical Composition with UHPLC-ESI-HRMS/MS

   Phytochemical components in the extract were characterized by using the
   Ultimate 3000 UHPLC System of Thermo Fisher coupled with a Thermo
   Fisher Scientific Q-Exactive Orbitrap mass (Bremen, Germany).
   Phytochemical substances of the ethanolic extract from the fruits were
   first separated with a Zorbax SB-C18 column (Agilent, 2.1 × 100 mm, 1.7
   μm). The injection volume and flow rate were 3 µL and 0.2 mL/min. The
   column temperature was set at 35 °C. Ultrapure water acidified by 0.1%
   formic acid (phase A) and acetonitrile (phase B) were applied as mobile
   phases. The following program was used as gradient elution conditions:
   0–2 min, 5% B; 2–10 min, 5–15% B; 10–25 min, 15–30% B; 25–30 min,
   30–50% B; 30–32 min, 50–5% B. The mass spectrum conditions were set
   according to our previous study [[66]25]. The compounds were
   preliminarily or positively characterized by comparing the mass data of
   the compounds with the data in the corresponding standards, databases
   (mass bank) or references. Identified compounds were (semi-) quantified