Abstract
Rheumatoid arthritis (RA) is a complex autoimmune condition primarily
affecting synovial joints, which targeted synthetic drugs have damaging
safety issues. Saussurea laniceps, a reputed anti-rheumatic medicinal
herb, is an excellent place to start looking for natural products as
safe, effective, targeted therapeutics for RA. Via biomimetic
ultrafiltration, umbelliferone and scopoletin were screened as two
anti-rheumatic candidates with the highest specific affinities towards
the membrane proteomes of rheumatic fibroblast-like synoviocytes (FLS),
the pivotal effector cells in RA. In vitro assays confirmed that the
two compounds, to varying extents, inhibited RA-FLS proliferation,
migration, invasion, and NF-κB signaling. Network pharmacology analysis
and molecular docking analysis jointly revealed that umbelliferone and
scopoletin act on multiple targets, mostly tyrosine kinases, in
combating RA. Taken together, our present study identified
umbelliferone and scopoletin as two major anti-rheumatic components
from SL that may bind and inhibit tyrosine kinases and subsequently
inactivate NF-κB in RA-FLSs. Our integrated drug discovery strategy
could be valuable in finding other multi-target bioactive compounds
from complex matrices for treating multifactorial diseases.
Keywords: rheumatoid arthritis, Saussurea laniceps, ultrafiltration,
network pharmacology, ErbB network, drug discovery
Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that
causes progressive articular destruction and deformity, and affects
approximately 1% of the global population ([42]Almutairi et al., 2021).
Despite advances in RA management, the disease is still not well
controlled in up to 30% of patients due to the individualized
pathogenic network ([43]McInnes and Schett, 2017). Current first-line
RA medications include non-steroidal inflammatory drugs (NSAIDs) and
disease-modifying anti-rheumatic drugs (DMARDs). These drugs can only
relieve symptoms but fail to control disease progression ([44]McInnes
and Schett, 2011). Recently, highly targeted small-molecule agents,
such as tyrosine kinase inhibitors (TKI), open a new avenue for RA
therapy with improved clinical responses ([45]Koenders and Van DenBerg,
2015). However, such synthetic molecules bring inevitable safety
issues. For example, tofacitinib and baricitinib, two TKIs, have black
box warnings from the US Food and Drug Administration (FDA) for severe
infections and malignancies ([46]Petronelli, 2021). The adverse events
may not be correlated to specific protein inhibition but the synthetic
structures ([47]Schwartz et al., 2019). Therefore, novel scaffolds with
safe, effective, targeted biomarker inhibition are desired for
precision medicine in RA.
Natural products from herbal medicines have long been valuable sources
for drug discovery due to their enormous scaffold diversity and
structural complexity ([48]Wang et al., 2021). The therapeutic
rationale of herbal medicines is a paradigm of the emerging drug
discovery concept of “polypharmacology”, i.e., multiple components
hitting multiple targets ([49]Shen, 2015). Multi-targeted chemicals
provide a better balance of efficacy and safety than single-targeted
drugs in multifactorial diseases ([50]Fang et al., 2019). “Snow lotus”
has been reputed as an effective anti-rheumatic herbal medicine in Asia
for centuries ([51]Committee of Chinese Pharmacopoeia, 1995). We
previously discovered that, among official “snow lotus” species
([52]Chik et al., 2015; [53]Fan et al., 2015; [54]Chen et al., 2016),
Saussurea laniceps Hand.-Mazz (SL) exerts the most outstanding chemical
composition ([55]Chen et al., 2017) and most potent anti-rheumatic
efficacies ([56]Yi et al., 2010, [57]2012), and SL significantly
ameliorates RA symptoms by targeted inhibition of multiple therapeutic
biomarkers while maintaining good safety profiles ([58]Chen et al.,
2021). Therefore, it is believed that SL is a promising source of safe,
effective, targeted anti-rheumatic agents. However, therapeutic
components in SL and corresponding action mechanisms have not been
thoroughly investigated.
Isolation and activity screening of active compounds from herbal
medicines, which are structurally diverse and sometimes in trace
amounts, has long been challenging with existing conventional methods
([59]Cannell, 1998). An advanced approach, bio-affinity
ultrafiltration, based on receptor-ligand interaction, is a powerful
tool for compound (ligand) fishing in complicated matrices due to its
excellent high-throughput online screening ability, sensitivity, and
selectivity ([60]Chen et al., 2018). Generally, bio-affinity
ultrafiltration uses one or a few recombinant cytosolic proteins as
receptors ([61]Wang et al., 2018). As one step further, a “biomimetic
ultrafiltration” approach can be designed by employing natural
disease-specific proteomes as receptors, which is more reasonable for
screening active agents from complex matrices to treat multifactorial
diseases, where most of the therapeutic targets remain highly
underexplored ([62]Fang et al., 2019).
Fibroblast-like synoviocytes (FLS) are the most common cell type at the
pannus–cartilage junction and are critical effector cells in RA
([63]Bustamante et al., 2017). A significant hallmark of FLS activation
attributes to increased expression of surface proteins under
inflammatory conditions ([64]Wu Z et al., 2021). Therapies that target
FLS, especially against their surface markers, are emerging as
promising therapeutic tools for RA ([65]Nygaard and Firestein, 2020).
Therefore, proteins extracted from FLS of RA patients (HFLS-RA),
including membrane and cytosolic proteomes, can be used as receptors in
a tailored biomimetic ultrafiltration, to discover anti-rheumatic
compounds from SL. The screened compounds are expected to hit multiple
and even possibly unknown targets with minimized unspecific bindings.
Such parallel study of compounds targeting natural proteomes from
membrane and cytosol has not been reported.
In the present study, biomimetic ultrafiltration with FLS proteome
fractions was conducted, followed by ultra-performance liquid
chromatography coupled to a quadrupole/time-of-flight-mass spectrometer
(UPLC-QTOF-MS) analysis, to fish out anti-rheumatic candidate compounds
from SL. In vitro pharmacological performances of the screened
compounds were verified, in terms of inhibiting RA-FLS proliferation,
migration, invasion, and NF-κB activation. Protein targets and action
pathways of the screened compounds were identified by network
pharmacology analysis. Interaction modes between the compounds and key
protein targets were investigated by molecular docking analysis. Our
work provides valuable insights into the pharmacodynamic material basis
of SL. It also serves as a case study to develop new drugs from natural
resources for RA and other complex diseases.
Materials and methods
Chemicals and reagents
The standard compounds of umbelliferone, scopoletin, and celecoxib were
purchased from Biomart Biotechnology Ltd. (Beijing, China).
Acetonitrile (ACN) and methanol of chromatography grade were purchased
from Lab-scan (Bangkok, Thailand). All aqueous solutions were prepared
using ultra-pure water with a Milli-Q water purification system from
Millipore (MA, United States). All chemicals not otherwise mentioned
were purchased from Sigma-Aldrich (MO, United States) and were used
without further purification.
Plant material and extract preparation
SL was collected from Lhasa, Tibet, in 2008. The plant was
authenticated by Prof. Hubiao Chen. Voucher specimens were deposited in
the Hong Kong Baptist University. The aerial parts of the plant were
powdered with a Fargo RT-04 grinder (Century Equipment Ltd., Kowloon,
HK) and passed through a 20 mesh (0.9 mm) sieve. The dried and powdered
sample (0.5 g) was reflux extracted with 25 ml water at 100°C for 1 h,
twice. Total extracts were combined into a 100-ml volumetric flask and
added up to the calibration mark with water. The extract was
centrifuged at 14,000 g for 10 min before passed through a 0.2-µm
syringe membrane (Alltech, IL, United States of America).
Cell culture
Normal HFLS (HFLS-N) and HFLS-RA were purchased from Otwo Biotech Co.,
Ltd (Shenzhen, China) and Guandao Biological Engineering Co., Ltd
(Shanghai, China), respectively. The cells were separately cultured in
Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, MD, United States)
containing 10% (v/v) heat-inactivated fetal bovine serum (Gibco), 100
U/mL penicillin and 100 mg/ml streptomycin (Gibco). Cells were grown in
a humidified atmosphere with 5% CO[2] at 37°C.
Biomimetic ultrafiltration coupled with UPLC-QTOF-MS analysis
HFLS-N and HFLS-RA cells were collected and washed three times with PBS
(pH 7.4) with 1 mM phenylmethylsulfonyl fluoride (PMSF). The isolation
of cell membrane and cytosol proteins was conducted using the Membrane
and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China)
following the manufacturer’s instructions. Protein contents were
determined by the Bradford method.
The screening procedure was conducted based on previous reports with
modifications ([66]Chen et al., 2018). Extracted proteins (2 mg/ml) and
SL extract (5 mg/ml) were mixed in PBS buffer (pH 7.4) and rotated
under 4°C for 30 min. To intercept the ligand–protein complexes, the
mixtures were ultracentrifuged through filters (Molecular weight
cut-off: 3 kDa; Amicon, Darmstadt, Germany) at 14,000 g under 4°C for
10 min, then washed with PBS four times. To release ligands, the
obtained complexes were incubated with 500 μl MeOH for 10 min, then
ultracentrifuged at room temperature for 15 min thrice. The combined
filtrates were collected for chemical analysis. Heat-inactivated
proteins were used as negative controls.
A UPLC-QTOF-MS system (Agilent Technologies, United States) was used
for chemical analyses of SL extract and the ligand filtrates. The
chromatographic and spectrometric parameters were set as previously
published ([67]Chen et al., 2017, [68]2021), with minor modifications
regarding the elution gradient (solvent A: 0.1% formic acid in water;
solvent B: 0.1% formic acid in ACN): 0–2 min with 2% B; 2–10 min with
2–10% B; 16–20 min with 18–25% B; 20–25 min with 25–55% B.
Affinity degree (AD) of a ligand towards an extracted proteome =
(A[1]—A[2])/A[0] × 100%, where A[1], A[2], and A[0] represented the
peak areas of selected compounds obtained from the incubations of the
SL extract with active, inactive, and without respective proteins,
respectively.
RA- and membrane-specificity index = (AD[RM]/AD[NM])/(AD[RC]/AD[NC]),
where AD[RM], AD[NM], AD[RC], and AD[NC] represented the AD towards
proteome from HFLS-RA membrane, HFLS-N membrane, HFLS-RA cytosol, and
HFLS-N cytosol, respectively.
MTT assay
Proliferation rate of HFLS-RA cells was monitored by an MTT assay as
previously described ([69]Chen et al., 2021). Briefly, cells (3 ×
10^4/well) in 100 μl complete culture medium were seeded in 96-well
microtiter plates for 24 h. They were then exposed to varying doses of
celecoxib (positive control), umbelliferone, and scopoletin in
serum-free medium. After incubation for 24 and 48 h, MTT (5 mg/ml,
10 μl) was added to each well, and the plates were incubated for 2 h.
Formazan crystals were dissolved with 100 μl DMSO, and absorbance at
570 nm was measured by a Benchmark microplate reader (Bio-Rad,
Hercules, CA, United States) with 630 nm as reference filter.
Wound healing assay
Collective migration of HFLS-RA cells was assessed by a scratch wound
healing assay as reported ([70]Li et al., 2019). Cells (3 ×
10^5/well) in 2 ml complete culture medium were seeded in 12-well
microtiter plates for 24 h to reach 80% confluence. A linear wound was
made in the cellular monolayer with a sterile 200 µl pipette tip. After
removing the cell debris, the cells were incubated with 20 µM
celecoxib, umbelliferone, and scopoletin in serum-free medium. Wound
closures were observed and photographed at 0, 24, and 48 h under a
light microscope (Leica DMI3000B, Wetzlar, Germany). The wound area was
determined using ImageJ software.
Transwell migration and invasion assay
24-well Transwell chambers (8 μm pore size; Corning, NY, United States)
were used as published ([71]Huang H et al., 2021). For single-cell
migration assessment, HFLS-RA cells (4 × 10^4/well) in 200 μl
serum-free medium were seeded in the upper chambers and treated with
20 µM celecoxib, umbelliferone, and scopoletin, respectively. 600 μl
complete culture medium was added in the lower chambers. After
incubation at 37°C for 24 h, non-migrating cells on the upper surface
of the Transwell membrane were removed; cells migrated to the lower
surface of the membrane were fixed with methanol, stained with 0.5%
crystal violet, photographed under the microscope, and counted. The
invasion assay was performed similarly, except that the membranes were
pre-coated with Matrigel.
Western blot analysis
HFLS-RA cells treated with 20 µM celecoxib, umbelliferone, and
scopoletin, respectively, for 24 h, were collected and lysed using RIPA
buffer (containing phosphatase inhibitor and PMSF) to obtain protein
samples. An equal amount of protein was separated by 10% SDS-PAGE and
transferred to PVDF membranes (Millipore). The membranes were blocked
with 5% non-fat milk for 1 h at room temperature, and then incubated
overnight at 4°C with the following primary antibodies: anti-NF-κB p65
(#8242; CST, MA, United States), phospho-NF-κB p65 (#3033; CST), IκBα
(#4814; CST), phospho-IκBα (#2859; CST), and anti-GAPDH (#AF7021;
Affinity, OH, United States). Then, the membranes were washed with 3
[MATH: × :MATH]
TBST and incubated with corresponding HRP-conjugated secondary
antibodies for 1 h at room temperature. The blots were developed using
the enhanced chemiluminescence solution and imaged by ChemiDoc™ XRS +
system.
Network pharmacology analysis
To predict protein targets of umbelliferone and scopoletin, the two
compounds were input in the form of Simplified Molecular Input Line
System (SMILES) into SwissTargetPrediction database
([72]http://www.swisstargetprediction.ch) and Search Tool for
Interactions of Chemical (STITCH; [73]http://stitch.embl.de); targets
with literature support after checking at Uniprot
([74]https://www.uniprot.org) were retained for subsequent analysis.
RA-related genes were collected using GeneCards database
([75]https://www.genecards.org) and Therapeutic Target Database (TTD,
[76]http://db.idrblab.net/ttd). Candidate targets related to both
umbelliferone/scopoletin and RA were selected using a Venn diagram.
To conduct gene ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway enrichment analysis, the selected targets were
imported into the Database for Annotation, Visualization and Integrated
Discovery (DAVID; [77]https://david.ncifcrf.gov). The annotations with
adjusted p < 0.05 were considered significantly enriched.
For each compound, compound–target–pathway (C-T-P) networks were
constructed using Cytoscape ([78]https://cytoscape.org) to examine the
relationships between the compound, corresponding protein targets, and
related pathways; protein–protein interaction (PPI) networks were
generated using STRING ([79]https://string-db.org) and visualized by
Cytoscape to present the relationships between major protein targets of
the compound.
Molecular docking
Discovery Studio Biovia 2019 (Dassault Syst`emes, OH, United States),
AutoDock Vina (The Scripps Research Institute, CA, United States), and
PyMol (DeLano Scientific LLC, CA, United States) were employed for
receptor/ligand structure modification, docking, 3D visualization,
respectively. The crystal structures of the receptors were obtained
from the Protein Data Bank (PDB; [80]https://www.rcsb.org). All water
molecules were removed, hydrogen polarities were assigned, and
Gasteiger charges were computed. For each receptor, a grid was created
to ensure the binding site covers the active pocket.
Statistical analysis
Data are presented as the mean ± SD. Comparisons were performed using
ANOVA tests unless specified. p < 0.05 was considered significant.
Results
Umbelliferone and scopoletin are ligands of RA-FLS membrane proteome
Ligand fishing from the anti-rheumatic SL extract was based on
biomimetic ultrafiltration coupled with UPLC-QTOF-MS analysis. Membrane
and cytosolic protein fractions were employed as receptors in the
ultrafiltration. To eliminate false-positive ligand fishing results,
inactivated HFLS-RA-derived proteins were used as activity-negative
control, and HFLS-N derived proteins as disease-negative control
([81]Figure 1).
FIGURE 1.
[82]FIGURE 1
[83]Open in a new tab
Schematic diagram of biomimetic ultrafiltration coupled with LC/MS.
Based on our previous chemical profiling of SL, ten major components
were detected from the herbal extract ([84]Figure 2) ([85]Chen et al.,
2017). Among the extract components, three compounds showed a AD
towards rheumatic membrane (RM) proteins than that towards normal
membrane (NM) proteins, namely, umbelliferone (peak 6; AD[RM] = 25.82%;
AD[NM] = 10.27%), scopoletin (peak 7; AD[RM] = 1.55%; AD[NM] = 1.42%),
and involucratolactone-β-D-glucoside (peak 10; AD[RM] = 1.08%; AD[NM] =
1.05%) ([86]Table 1). Four compounds exerted higher AD towards
rheumatic cytosol (RC) than normal cytosol (NC) fraction, namely,
skimmin (peak 1; AD[RC] = 3.40%; AD[NC] = 1.88%), chlorogenic acid
(peak 2; AD[RC] = 0.10%; AD[NC] = 0%), piceol (peak 5; AD[RC] = 2.76%;
AD[NC] = 0.88%), and 3,5-dicaffeoylquinic acid (peak 8; AD[RC] = 0.63%;
AD[NC] = 0%).
FIGURE 2.
[87]FIGURE 2
[88]Open in a new tab
Proteome ligands separated from SL extract by biomimetic
ultrafiltration coupled to UPLC-QTOF-MS. RM, RC: membrane and cytosolic
proteomes of HFLS-RA, respectively. NM, NC: membrane and cytosolic
proteomes of HFLS-N, respectively. IRM, IRC, INM, INC: inactivated
corresponding proteomes, respectively.
TABLE 1.
UPLC-QTOF-MS data and proteome affinity degrees of SL extract
components.
Peak t[R] (min) Compound Formula Major fragment AD[RM] (%)[89] ^a
AD[NM](%) AD[RC] (%) AD[NC] (%) RA- and membrane-specific index
m/z Adduct ion
1 8.43 Skimmin C[15]H[16]O[8] 369.0893 [M + HCOO]^- 0.31 ± 0.62 0.43 ±
0.58 3.40 ± 1.46 1.88 ± 0.13 0.40
2 9.59 Chlorogenic acid C[16]H[18]O[9] 399.1004 [M-H]^- 0.15 ± 0.01
0.26 ± 0.25 0.10 ± 0.13 0 /[90] ^b
3 10.11 Scopolin C[16]H[18]O[9] 399.0933 [M + HCOO]^- 0.04 ± 0.06 0.51
± 0.24 2.13 ± 1.33 2.78 ± 1.57 0.10
4 10.35 Syringin C[17]H[24]O[9] 417.1402 [M + HCOO]^- 0.34 ± 0.28 0.60
± 0.32 1.99 ± 1.21 2.83 ± 0.21 0.81
5 11.84 Piceol C[8]H[8]O[2] 135.0481 [M-H]^- 1.03 ± 1.51 1.06 ± 0.93
2.76 ± 1.54 0.88 ± 0.13 0.31
6 13.56 Umbelliferone C[9]H[6]O[3] 161.0244 [M-H]^- 25.82 ± 12.03 10.27
± 9.45 10.64 ± 6.17 18.09 ± 2.33 4.27
7 14.38 Scopoletin C[10]H[8]O[4] 191.0372 [M-H]^- 1.55 ± 1.99 1.42 ±
1.13 2.00 ± 2.08 6.54 ± 1.02 3.57
8 18.22 3,5-Dicaffeoylquinic acid C[25]H[24]O[12] 515.1195 [M-H]^- 0.38
± 0.46 0.98 ± 0.60 0.63 ± 0.62 0 /[91] ^b
9 18.73 Apigenin 7-O-β-D-glucoside C[21]H[20]O[10] 577.1550 [M-H]^-
1.60 ± 0.27 3.60 ± 2.43 8.03 ± 4.28 10.72 ± 2.77 0.59
10 22.31 Involucratolactone-β-D-glucoside C[21]H[30]O[8] 455.2004 [M +
HCOO]^- 1.08 ± 1.10 1.05 ± 2.04 0 16.66 ± 2.12 /[92] ^b
[93]Open in a new tab
Data as mean ± SD (n = 3).
^a
AD[RM], AD[NM], AD[RC], and AD[NC], represented affinity degrees (AD)
towards proteome from HFLS-RA, membrane; HFLS-N, membrane; HFLS-RA,
cytosol, and HFLS-N, cytosol, respectively.
^b
Not applicable.
Cell surface proteins play crucial physiological roles in vivo and are
currently the most successful class of drug targets for pharmaceuticals
([94]Huang Y et al., 2021). In this regard, specific RA- and
membrane-targeting compounds will be selected as high potential
anti-rheumatic agents. Two coumarins from SL extract exhibited
outstanding RA- and membrane-specificity index, i.e. umbelliferone
(peak 6; index = 4.27) and scopoletin (peak 7; index = 3.57) ([95]Table
1). Therefore, umbelliferone and scopoletin were screened from SL
extract as key ligands of RA-FLS membrane proteome for subsequent
analyses.
Umbelliferone and scopoletin differentially inhibited RA-FLS activities
Inhibitory effects of the two compounds on RA-FLS activities were
assessed regarding proliferation, migration, and invasion. An MTT study
showed that umbelliferone and scopoletin shared similar inhibitory
profiles on HFLS-RA proliferation with celecoxib, a first-line RA drug
yet associated with increased cardiovascular risks ([96]Figure 3A)
([97]Fidahic et al., 2017). Specifically, scopoletin significantly
inhibited HFLS-RA proliferation at 30 μM and onwards. In our previous
research, umbelliferone and scopoletin were proved with evidently lower
cardiomyocyte toxicity and less cardiac remodeling in rats than
celecoxib did ([98]Chen et al., 2021). Our previous and current
findings collectively indicate that the two screened natural compounds
are safer for the cardiovascular system yet match competitiveness in
inhibiting RA-FLS proliferation compared to celecoxib.
FIGURE 3.
[99]FIGURE 3
[100]Open in a new tab
Umbelliferone and scopoletin differentially inhibited RA-FLS
activities. (A) Proliferation, (B) wound healing, (C) Transwell
migration, (D) Transwell invasion, and (E) western blot assays on
HFLS-RA. Data are means ± SD of independent experiments performed in
triplicate. ns, not significant at p > 0.05, *p < 0.05, ***p < 0.001,
and ****p < 0.0001 vs blank control group; ns, not significant vs
celecoxib group.
For inhibition of HFLS-RA migration, umbelliferone showed similarly
potent efficacies with celecoxib and was more effective than
scopoletin. At 20 μM, where the dosage was proved with no significant
impact on cell proliferation, umbelliferone and celecoxib significantly
reduced both single-cell and collective migration of HFLS-RA cells with
24 and 48 h incubation (p < 0.0026) ([101]Figures 3B,C). Scopoletin
exhibited significant reduction of single-cell migration (p < 0.0001)
but only slight inhibition of collective migration (p
[MATH: ≤ :MATH]
0.8919) ([102]Figures 3B,C). Since cell-extracellular matrix (ECM)
adhesion and cell-cell adhesion are central mechanisms in the process
of collective migration ([103]Pijuan et al., 2019), our results
indicated that umbelliferone is more potent in inhibiting such
adhesions than scopoletin.
The compounds’ inhibitory effects on HFLS-RA invasion were also
evaluated ([104]Figure 3D). Umbelliferone exerted significant (71.82%
inhibition; p < 0.0001) and even more evident reduction of invasive
cells than celecoxib (69.42% inhibition; p < 0.0001). Scopoletin also
reduced the number of invasive cells, yet the inhibitory rate was
approximately half that of umbelliferone. The inhibitory pattern of
invasion being similar to that of collective migration can be explained
that the two processes share similar cell behaviors: they both involve
cells with mesenchymal characteristics to degrade the ECM for directed
cell movements ([105]Friedl and Gilmour, 2009; [106]Yang et al., 2019).
The transcription factor NF-κB is a pivotal mediator of inflammatory
responses. NF-κB activation is associated with RA-FLS
hyperproliferation, migration, and invasiveness, leading to hyperplasia
in the rheumatic synovium ([107]Nejatbakhsh Samimi et al., 2020). We
hereby investigated whether the tested compounds inhibit the NF-κB
signaling pathway. The western blot results indicated that the
compounds decreased the phosphorylation of NF-κB p65 and IκB-α,
paralleling their inhibitory levels ([108]Figure 3E). Such suppressed
NF-κB canonical signaling can decrease the expression of inflammatory
cytokines, adhesion molecules, and other promoters in the inflammatory
cascade ([109]Liu et al., 2017). The conclusion of the two compounds
suppressing NF-κB signaling can be supported by other studies showing
that umbelliferone ([110]Ouyang et al., 2019; [111]Wu G et al., 2021)
and scopoletin ([112]Li et al., 2009; [113]Chen et al., 2021) can
effectively suppress phosphorylation of specific NF-κB subunits (e.g.
p65 and IĸBα) and expression of downstream genes (e.g. IL-1β, TNF-α,
MMP-3, MMP-9, COX-2, Bcl-2) in RA synovial tissues and FLSs. Since both
umbeliferone and scopoletin had high binding affinities towards RA-FLS
membrane proteins ([114]Table 1) and inhibited RA-FLS activities
([115]Figure 3), it is believed that NF-κB is a primary node of the two
compounds in treating RA.
Umbelliferone and scopoletin target key pathways and proteins against RA
A network pharmacology analysis was conducted to identify direct
biological targets of umbelliferone and scopoletin in treating RA. From
various databases, 5104 RA-related proteins, 116 umbelliferone protein
targets, and 110 scopoletin protein targets were collected with
literature support. It is worth noticing that over half of the protein
targets of the two screened compounds are linked with RA ([116]Figure
4A and [117]Figure 5A), i.e., 70 (60.34%) proteins and 62 (56.36%)
proteins for umbelliferone and scopoletin, respectively, corresponding
to their abovementioned promising anti-rheumatic efficacy. The
overlapping proteins were recognized as candidate anti-rheumatic
targets of the two respective compounds ([118]Supplementary Tables S1,
S2).
FIGURE 4.
[119]FIGURE 4
[120]Open in a new tab
Network pharmacology analysis of umbelliferone treating RA. (A) Venn
diagram of umbelliferone and RA-related targets. (B) The top ten GO
enrichment. (C) The top ten KEGG enrichment. (D) PPI network showing
top ten protein targets. (E) C-T-P network based on top ten protein
targets.
FIGURE 5.
[121]FIGURE 5
[122]Open in a new tab
Network pharmacology analysis of scopoletin treating RA. (A) Venn
diagram of scopoletin and RA-related targets. (B) The top ten GO
enrichment analysis. (C) The top ten KEGG enrichment analysis. (D) PPI
network showing top ten protein targets. (E) C-T-P network based on top
ten protein targets.
C-T-P networks were constructed for the two compounds. The C-T-P
network of umbelliferone consisted of 70 targets and 117 pathways, and
that of scopoletin included 62 targets and 109 pathways
([123]Supplementary Figures S1, S2). On such basis, Gene Ontology (GO)
and KEGG pathway enrichment analysis were employed to characterize the
functional annotations of the proteins ([124]Supplementary Figures S3,
S4). Interestingly, umbelliferone and scopoletin shared highly similar
GO and KEGG profiles. According to the GO enrichment results, the two
compounds are both heavily involved with transmembrane receptor
tyrosine kinases (RTKs), especially with their activities (GO: 0004714,
0004173, 0004672), phosphorylation (GO: 0038083, 0046777, 0018108), and
signaling pathways (GO: 0007169) ([125]Figure 4B and [126]Figure 5B).
As for the KEGG results, adherens junction (KEGG: hsa04520), focal
adhesion (KEGG: hsa04510), and Rap1 signaling pathway (KEGG: hsa04015)
are among the ten most significant pathways for both compounds
([127]Figure 4C and [128]Figure 5C). Apart from the shared KEGG
enrichments, umbelliferone is particularly enriched in
phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling
pathway (KEGG: hsa04151), while scopoletin particularly in signaling
pathways of ErbB (ErbB; KEGG: hsa04012) and chemokines (KEGG:
hsa04062). Therefore, it is predicted that both umbelliferone and
scopoletin can act on TKs to regulate biological processes, including
cell adhesion, cell-cell junction formation, and cell polarization,
which are all critical during RA progression ([129]Weyand and Goronzy,
2021).
PPI networks for umbelliferone and scopoletin were also analyzed
([130]Supplementary Figures S5, S6). There were seven common proteins
among the top ten targets of the two compounds, namely, epidermal
growth factor receptor (EGFR/ErbB1), proto-oncogene tyrosine-protein
kinase Src (Src), Akt serine/threonine kinase 1 (Akt1), receptor
tyrosine-protein kinase ErbB-2 (ErbB2/HER2), protein tyrosine kinase 2
(PTK2/FAK), prostaglandin-endoperoxide synthase 2 (PTGS2/COX-2), and
estrogen receptor 1 (ESR1/ERα) ([131]Figure 4D and [132]Figure 5D).
Most of the listed proteins can be located on cell membrane; many of
them can be categorized as RTKs (e.g., EGFR and ErbB2), non-receptor
TKs (e.g. Src and FAK), or proteins closely crosstalking with TKs (e.g.
Akt1 and ERα). Simplified C-T-P networks were constructed based on
umbelliferone and scopoletin’s top ten protein targets, respectively
([133]Figure 4E and [134]Figure 5E). Specifically, the present network
pharmacology analysis results correspond with our previous study in
which the two compounds exerted significant and selective COX-2
inhibition in rheumatic rat synovium tissues ([135]Chen et al., 2021).
Molecular docking analyses of umbelliferone and scopoletin against the
abovementioned seven common top protein targets were performed to
investigate the ligand-receptor interactions ([136]Figures 6, [137]7).
The docking studies were all validated by redocking the co-crystalized
ligand for each protein, where the root mean square deviation (RMSD)
value below 2Å was considered good solutions ([138]Supplementary Figure
S7) ([139]Ramírez and Caballero, 2018). Generally, the analyzed
proteins all showed satisfactory affinities towards the two compounds
([140]Supplementary Table S3). Among the analyzed TKs, HER2 is the
protein exhibiting the lowest binding energies towards umbelliferone
(-6.9 kcal/mol) and scopoletin (-7.2 kcal/mol), respectively. All
interacting residues of HER2 with both compounds lie in the active
pocket. The interactions are mainly via conventional H-bonds
(umbelliferone: Met[801], Thr[862]; scopoletin: Asn[520], Thr[862]),
π-alkyl bonds (umbelliferone: Leu[726], Val[734], Ala[751], Leu[852];
scopoletin: same except without Leu[726]), and π-σ bonds
(umbelliferone: Val[734], Leu[852]; scopoletin: Val[734]).
Collectively, the molecular docking results indicate favorable
interactions and binding mechanisms between the two compounds and their
top target proteins.
FIGURE 6.
[141]FIGURE 6
[142]Open in a new tab
Docking patterns of umbelliferone with selected top target proteins. 3D
residues in cyan indicate representative ligand–receptor interactions.
Numbers indicate bond distance (Å).
FIGURE 7.
[143]FIGURE 7
[144]Open in a new tab
Docking patterns of scopoletin with selected top target proteins. 3D
residues in cyan indicate representative ligand–enzyme interactions.
Numbers indicate bond distance (Å).
Discussion
Umbelliferone and scopoletin act on FLSs by targeting TKs and blocking NF-κB
signaling
According to our present study, in treating RA, umbelliferone and
scopoletin 1) exert high binding affinities towards membrane proteins
of RA-FLSs, 2) directly target TKs (mostly membrane-bound) and proteins
with close interaction with TKs, 3) inhibit NF-κB signaling in RA-FLSs,
and 4) attenuate RA-FLS activities ([145]Figures 2–[146]5). A schematic
overview of the signaling network regarding the two compounds
inhibiting FLS activation is drawn, featuring main protein targets
shared by the two compounds, such as EGFR, ErbB2, Src, FAK, ERα, and
Akt ([147]Figure 8). In the ErbB signaling network, ErbB family
members, including EGFR, ErbB2, and their heterodimers, signal through
Src and FAK to activate a myriad of downstream signaling pathways
([148]Yarden and Pines, 2012). Src-induced tyrosine phosphorylation of
FAK is a central mediator of focal adhesion turnover and cell migration
([149]Wu et al., 2015). ErbB RTKs, Src, and FAK activate the PI3K/Akt
cascade to affect diverse cellular functions, including chemokine
signaling and endocrine resistance. Akt also regulates NF-κB signaling
to induce target gene expression ([150]Liu et al., 2020). In the
estrogen signaling network, stimulated ERα not only forms a complex
with Src and PI3K, leading to Akt activation, but also activates Src,
which in turn enhances matrix metalloproteinase (MMP) expression to
facilitate ECM destruction ([151]Pan et al., 2020). In summary,
umbelliferone and scopoletin inhibit NF-κB activation in RA FLSs mainly
via the ErbB/PI3K/Akt signaling axis.
FIGURE 8.
[152]FIGURE 8
[153]Open in a new tab
Schematic overview of umbelliferone and scopoletin targeting membrane
and cytosolic proteins in RA-FLS in attenuating RA development via
NF-κB blockade.
NF-κB activation in RA-FLSs can lead to a series of cancerous and
inflammatory features of the synoviocytes ([154]Figure 8). The
activation of NF-κB induces the expression of cyclin D1 and c-Myc,
which are cell growth promoters, subsequently boosting cell
proliferation. Various anti-apoptotic signals are delivered, including
inhibition of pro-apoptotic genes (e.g., p53 activity loss due to p65
Ser536 phosphorylation) and increased expression of anti-apoptotic
genes (e.g. FLIP in the Fas/FasL pathway). Since gene promoters of most
MMPs have canonical sites for NF-κB, activated RA-FLSs secret elevated
levels of MMPs, resulting in increased invasiveness and cartilage
erosion. IκB kinase (IKK) can be stimulated by inflammatory cytokines
(e.g. IL-2 and TNF-α) and further mediate the inflammatory signaling
cascade ([155]Ghosh and Karin, 2002; [156]Liu et al., 2017). In
addition, FLS-induced RA pathogenesis switches on inflammatory
responses from various immune cells in the RA synovium, such as
dendritic cells, lymphocytes, and monocytes, which further maintain FLS
activation and perpetuate the disease progression ([157]Nejatbakhsh
Samimi et al., 2020). Therefore, it is believed that the anti-rheumatic
effects of umbelliferone and scopoletin can be at least partly
attributed to the blockage of NF-κB signaling via targeting TKs on
activated FLSs.
Umbelliferone and scopoletin targeting RA-FLS membrane raises hope to combat
RA
Membrane proteins perform a myriad of biological functions, including
cell communication, substance transportation, and catalytic reactions.
Such proteins on the cell surface account for over 60% of the targets
of all FDA-approved small-molecule drugs ([158]Huang Y et al., 2021).
As FLS are the critical effector cells in RA, FLS membrane proteins,
such as transmembrane TKs, play pivotal roles in RA progression through
orchestrating cellular signaling among different cell types
([159]Kovács et al., 2014). However, despite the significance, drug
discovery against membrane proteins is notoriously challenging, mainly
because 1) membrane proteins are difficult to be isolated with
maintained structure and functions, and 2) this category of proteins is
still largely under-investigated. Our present study demonstrates an
integrated drug discovery strategy, using SL herb as an example,
featuring biomimetic ultrafiltration to screen compounds targeting
RA-specific membrane proteins. As a result, our strategy enables the
discovery of safe, effective compounds hitting intact, multiple
membrane targets with minimized unspecific bindings to combat RA.
Therapeutic agents discovered by this strategy are promising advantages
compared with current first-line drugs for RA, e.g. NSAIDs and DMARDs,
that lack clinical responses because 1) selectively inhibiting multiple
and possibly novel targets, e.g., TKs, helps to control RA as a
heterogeneous disease, 2) minimizing unspecific bindings can prevent
adverse events of some current synthetic targeted drugs ([160]Koenders
and Van DenBerg, 2015).
There have been reports on anti-rheumatic performances of umbelliferone
and scopoletin. On a rat model with Freund’s complete adjuvant
(FCA)-induced arthritis, umbelliferone was found to reduce
pro-inflammatory cytokines, such as TNF-α and IL-1β ([161]Ouyang et
al., 2019; [162]Wu G et al., 2021), and osteoclastogenesis biomarkers,
such as MMP-3 and MMP-9 ([163]Wu G et al., 2021); according to the
authors, such reduced gene expressions were due to suppressed NF-κB
signaling upon umbelliferone administration. On the same rat model,
scopoletin significantly alleviated clinical symptoms, immune
responses, and joint pathological conditions ([164]Chen et al., 2021);
scopoletin was also proved to induce RA FLS apoptosis by inhibiting
NF-κB activation ([165]Li et al., 2009). Despite such findings, neither
umbelliferone nor scopoletin has reached routine clinical therapy for
RA. One possible reason might be a lack of confirmation on their direct
target proteins and subsequent pivotal signaling axis. Our current
study fills this gap by elucidating that membrane proteins on FLSs,
especially TKs, are among the direct targets of umbelliferone and
scopoletin; the two compounds may function via the RTK/PI3K/Akt axis to
reach inhibition of NF-κB and downstream cascades.
Drug discovery for natural resource preservation
Drug discovery and development from herbal medicines that are derived
from rare herbal species can significantly preserve natural resources.
There is little cultivation of SL; almost all SL material in commerce
has been collected from the wild. In the Himalayan region, due to the
reputed anti-rheumatic potency of SL, the population vitality and
survival of the species are threatened by heavy and illegal harvesting
of the plant ([166]Chen et al., 2016). Since umbelliferone and
scopoletin are the two most abundant and therapeutically potent
components in SL, precise elucidation of action mechanisms of the two
compounds enables the development of synthetic analog drugs, which can,
in turn, effectively save the endangered situation of SL.
Conclusion
Taken together, our present study demonstrates three main steps in
elucidating therapeutic components and corresponding action mechanisms
in the anti-rheumatic herb SL. Firstly, umbelliferone and scopoletin
were screened from SL as two compounds with the highest specific
binding affinities towards RA-FLS membrane proteins. Secondly, reduced
activities of RA-FLSs and decreased NF-κB activation were confirmed
under the administration of either umbelliferone or scopoletin.
Thirdly, the two compounds are identified with crucial target proteins
(including TKs) and pathways (including RTK/PI3K/Akt/NF-κB) against RA.
Therefore, we can conclude that umbelliferone and scopoletin, as major
active ingredients from SL, can target tyrosine kinases on FLSs to
block NF-κB signaling in attenuating progression of RA. Our study not
only contributes to elucidating the multi-component and multi-target
anti-rheumatic mechanisms of the endangered species of SL but also
helps develop safe, effective anti-rheumatic drugs based on chemical
scaffolds of umbelliferone and scopoletin.
Data availability statement
The original contributions presented in the study are included in the
article/[167]Supplementary Materials, further inquiries can be directed
to the corresponding authors.
Author contributions
Conceptualization, HC and JZ; Funding acquisition, HC and JZ;
Investigation, QC, WZ, SW, WL, and KY; methodology, QC, WZ, YH;
Supervision, HC and JZ; writing—original draft, QC, SW; writing—review
and editing, WZ, QC, YT. All authors have read and agreed to the
published version of the manuscript.
Funding
This work was supported by the National Natural Science Foundation of
China (82074123, U1903126), the Health and Medical Research Fund in
Hong Kong (16170251), and the Innovation and Technology Fund in Hong
Kong (PRP/036/20FX; MHP/023/20).
Conflict of interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by
the publisher.
Supplementary material
The Supplementary Material for this article can be found online at:
[168]https://www.frontiersin.org/articles/10.3389/fphar.2022.946210/ful
l#supplementary-material
[169]Click here for additional data file.^ (10.2MB, docx)
References