Abstract
Shaogan Fuzi Decoction (SGFD), one of the classical prescriptions of
Chinese Medicine, has a long history in the treatment of rheumatoid
arthritis (RA), but definitive studies on its efficacy and mechanism of
action are lacking. This study aims to elucidate the pharmacodynamic
role of SGFD against RA and the potential mechanisms based on a
combination of network pharmacology and experimental verification. The
RA model in rats was induced by intradermal injection of bovine type Ⅱ
collagen and incomplete Freund’s adjuvant at the tail root. SGFD was
administered once a day by oral gavage for 4 weeks. After SGFD
administration, rat’s arthritis index (AI) score and paw swelling
decreased to some extent, and synovial inflammation, vascular
hyperplasia, and cartilage destruction of the ankle joint were
improved. Simultaneously, thymus and spleen index and serum levels of
C-reactive protein (CRP) were lowered. Network pharmacology revealed
that quercetin, kaempferol, naringenin, formononetin isorhamnetin and
licochalcone A were the potentialiy active components, and IL6, TP53,
TNF, PTGS2, MAPK3 and IL-1β were potential key targets for SGFD in the
treatment of RA. Ingredients-targets molecular docking showed that the
components had the high binding activity to these target proteins. The
mechanism of SGFD for RA involves various biological functions and is
closely correlated with TNF signaling pathway, Osteoclast
differentiation, T cell receptor signaling pathway, mitogen-activated
protein kinase (MAPK) signaling pathway, NF-κB signaling pathway,
toll-like receptor signaling pathway, and so on. Western blot and ELISA
showed that the expression of toll-like receptor 4 (TLR4), nuclear
factor kappa-B (NF-κB) p65, phosphorylated c-Jun N-terminal kinase
(p-JNK), p-p38, phosphorylated extracellular regulated kinase (p-ERK)
and TNF-α was significantly upregulated in the synovium of RA rats, and
the levels of serum inflammatory factors were significantly increased.
SGFD inhibits the activation of the TLR4/NF-κB/MAPK pathway and the
expression/production of pro-inflammatory cytokines. In summary, SGFD
could improve the symptoms and inflammatory response in
collagen-induced arthritis (CIA) rat model. The mechanism might be
related to the regulation of TLR4/MAPKs/NF-κB signaling pathway and the
reduction of inflammatory factor release, which partially confirms the
results predicted by network pharmacology.
Keywords: shaogan fuzi Decoction, rheumatoid arthritis, network
pharmacology, TLR4/MAPKs/NF-κB signaling pathway, inflammatory
Introduction
As a chronic progressive autoimmune disease, the major pathological
features of rheumatoid arthritis (RA) are infiltration of inflammatory
cells, the aberrant proliferation of synovial cells, formation of
pannus, erosion of cartilage and bone, and ultimately leading to
destruction, deformation and loss of function of the affected joints.
It is associated with a high prevalence of concomitant diseases such as
cardiovascular disease, lung disease and other extra-articular and
systemic diseases ([49]Mcinnes and Schett, 2011). RA affects 0.5–1% of
the global population and represents a significant economic and social
burden due to high rates of morbidity, disability, and mortality
([50]Cross et al., 2014). The pathogenesis and aetiology of RA remain
unclear; however, immunological, genetic, infectious, environmental,
and hormonal factors have been shown to be involved in its development
([51]Guo et al., 2018). Currently, nonsteroidal anti-inflammatory drugs
(NSAIDs), disease-modifying antirheumatic drugs (DMARDs),
glucocorticoids, and biologics are commonly used in clinics as regular
and primary candidates for the treatment of RA ([52]Smolen and Aletaha,
2015; [53]Aletaha and Josef, 2018). Although these agents play a role
in relieving symptoms and delaying relapses, they have side effects
such as gastrointestinal abnormalities, liver and kidney damage, and
bone marrow toxicity ([54]Wang et al., 2018). So it is necessary to
explore new therapeutic agents with low toxicity and high efficacy.
According to the theory of traditional Chinese medicine (TCM), RA is
classified as a “Bi syndrome” associated with an insufficiency of the
body’s vital energy (known as qi), and the intrusion of “wind (feng)”,
“cold (han)”, “dampness (shi)”, “heat (re)” and other evil qi from
outside, resulting in a blockage of qi and blood in the meridians
([55]Lü et al., 2015; [56]Li and Zhang, 2020). Chinese Medicine has
accumulated abundant clinical experience and effective formulas for
treating RA. Shaogan Fuzi Decoction (SGFD) was first described in the
Treatise on Febrile Diseases (Shang Han Lun). It is consists of
Paeoniae Radix Alba (Paeonia lactiflora Pall., Bai-Shao), Glycyrrhizae
Radix et Rhizoma (G. uralensis Fisch., G. inflata Bat., or G. glabra
L., Gan-Cao), and Aconiti Lateralis Radix Praeparata (Aconitum
carmichaelii Debx., Fu-Zi) with the functions of reinforcing deficiency
and reducing excess, warming and activating meridians, nourishing blood
and extending sinew, relieving spasm and pain ([57]Zhang et al., 2015).
In the clinical treatment of RA, SGFD and its modification have shown
good efficacy and safety. However, the efficacy and mechanism of action
of SGFD have not yet been systematically studied.
Network pharmacology is a strategy and technology of bioinformatics
network construction and network topology analysis based on a network
database query, high-throughput histological data analysis, and
computer virtual computing ([58]Hopkins, 2007). It adopts a holistic
perspective to systematically elucidate drug mechanisms of action by
revealing the relationships between drugs, targets, and diseases.
Network pharmacology is consistent with TCM’s multi-component and
multi-target features and the ideas of dialectical theory and
systematic regulation. It provides a novel idea for studying herbal
compounds and has been extensively used to predict the possible active
ingredients and targets of TCM and explore its mechanism of action
([59]Liu and Sun, 2012; [60]Yuan et al., 2017). In this study, we used
a network pharmacology approach to predict the molecular targets and
signaling pathways of SGFD at RA, validate its pharmacological efficacy
using the rat model of collagen-induced arthritis (CIA), and
preliminarily explore the underlying mechanism. The system flowchart of
this work is shown in [61]Figure 1.
FIGURE 1.
[62]FIGURE 1
[63]Open in a new tab
The flowchart of this study based on an integration strategy of network
pharmacology and experimental verification.
Methods
Establishment of the collagen-induced arthritis model and drug administration
Wistar rats (SPF grade, male, 170–200 g) were purchased from Beijing
Weitong Lihua Experimental Animal Technology Co., Ltd. The license
number for experimental animal production is SCXK (Jing) 2016–0011. All
rats were housed in a barrier-free environment at the Beijing
University of Chinese Medicine, and the license number for experimental
animal use is SYXK (Jing) 2020–0033. The animals were housed in a
temperature of 22–24°C, relative humidity of 50–70%, and a light-dark
cycle of 12 h. All rats were acclimatized to the environment for 1 week
before the experiment. The animal study was reviewed and approved by
the Medical and Experimental Animal Ethics Committee of the Beijing
University of Chinese Medicine (No. BUCM-4-2019100501-4122).
Bovine type II collagen (2 mg/ml in 0.05 M acetic acid; Chondrex, Inc.,
Redmond, WA, United States) was emulsified in an equal volume of
incomplete Freunds adjuvant (Sigma-Aldrich, St. Louis, MO, United
States). Each rat was immunized on day 0 with 0.2 ml of the cold
emulsion by multiple intradermal injections on the back and tail base.
On day 7, after the first immunization, 0.1 ml of the emulsified
collagen II was injected intradermally at the base of the tail for
booster immunization ([64]Trentham et al., 1977; [65]Wang et al.,
2019). Normal control rats were injected with an equal volume of saline
at the same site and time point. The progression of arthritis was
monitored daily. At 17 days after the first immunization, the CIA rats
were scored by the arthritis index (AI). The AI was graded on a scale
of 0–4 ([66]Brand et al., 2007): 0 = no evidence of hyperemia and/or
inflammation; 1 = slight redness and swelling at the toe joints; 2 =
moderate swelling of the toe and ankle joints; 3 = swelling and
hyperemia in all areas below the ankle; and 4 = all joints, including
the ankle, were swollen ([67]Figure 2). The sum of the four paw scores
is the AI of each rat, with a maximum score of 16, and rats with an AI
score greater than two were selected as successful rats for the CIA
model.
FIGURE 2.
[68]FIGURE 2
[69]Open in a new tab
Example of arthritis index score in rats. (A) score 0. (B) score 1. (C)
score 2. (D) score 3. (E) score 4.
A total of 65 CIA model rats were successfully duplicated and randomly
divided into five groups as follows (n = 13/group): the model group,
the positive drug tripterygium glycosides tablet group (9 mg/kg) (TG),
the SGFD group with low-dose (1.05 crude drug g/kg) (SGFD-L),
medium-dose (2.1 crude drug g/kg) (SGFD-M) and high-dose (4.2 crude
drug g/kg) (SGFD-H). Meanwhile, the normal group was established.
The formula of SGFD was purchased from the Pharmacy Department of
Dongfang Hospital, Beijing University of Chinese Medicine (Beijing,
China). Each packet contains Bai-Shao 9g, Gan-Cao 9g, and Fu-Zi 3g (in
crude drug). TG was purchased from Zhejiang Deende Pharmaceutical Co.,
Ltd. The tablet and Chinese granules were ground and mixed with
distilled water before use. Animals were treated with 10 ml/kg by oral
gavage, once a day for 4 weeks (D18 to D45). In addition, rats in the
normal and model groups received the same amount of distilled water.
The rat’s spontaneous activity, mental status, and weight were
observed.
Evaluation of collagen-induced arthritis
The swelling of the hind paws of rats was measured with a paw volume
meter (YLS-7B; Jinan Yiyan Science and Technology Development Co.,
Ltd.) and the AI of each rat was assessed at days 0, 7, 17, 24, 31, 38,
and 45 as described previously. The paw swelling rate was calculated as
follows:
[MATH: Paw swelling rate(%)<
mo>=(paw volume at the time point − initial paw
volume)/initial paw volume×100% :MATH]
Histopathological examination
At the end of the experiment, the rats were anesthetized and then
sacrificed. The tissues of the right ankle joint were collected and
fixed with 4% paraformaldehyde for 24 h and decalcified with 10% EDTA.
After processing by conventional histological procedures, including
dehydration, transparency, wax immersion, and embedding, the
paraffin-embedded tissues were cut into 5-μm-thick sections.
Histopathological examination (HE) with staining (G1003, Servicebio,
China) was used to assess the pathological and morphological changes in
the ankle joint.
Index of spleen and thymus assay
Thymus and spleen were collected after sacrificed rats, washed in
normal saline, blotted with filter paper, and weighed. The thymus and
spleen index were calculated as follows:
[MATH: spleen (thymus)
index (‰)=the spleen (thymus) mass (g)/the body weight (g)×1000 :MATH]
ELISA
The blood of rats was collected by the abdominal aortic method, and the
serum was separated by centrifugation. The serum levels of rheumatoid
factor (RF), C-reactive protein (CRP), interleukin-1β (IL-1β), IL-6,
tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and IL-17 were
determined using the respective ELISA kits. The specific operation was
performed according to the instructions of the kit.
Network pharmacology analysis
The ingredients of Bai-Shao (BS), Gan-Cao (GC), and Fu-Zi (FZ) in SGFD
were obtained from the Traditional Chinese Medicine systems
pharmacology (TCMSP: [70]http://tcmspw.com/tcmsp.php) database ([71]Ru
et al., 2014). Oral bioavailability (OB) and drug similarity (DL), as
essential pharmacokinetic parameters in the ADME process and drug
design, can be used to estimate the druggability of ingredients
([72]Wang and Craik, 2016). Therefore, components with OB ≥ 30% and DL
≥ 0.18 were chosen as active ingredients of SGFD. The SMILE numbers of
the active ingredients were acquired from the Pubchem database
([73]Sunghwan et al., 2020) ([74]https://pubchem.ncbi.nlm.nih.gov/) and
then sequentially imported into the SwissTargetPrediction database
([75]Daina et al., 2019) ([76]http://www.swisstargetprediction.ch/) for
target prediction. Targets with a probability ≥0.5 were selected as
potential targets for active ingredients. The TCMSP database was also
used to obtain the target information for each active ingredient.
RA-related targets were obtained from the Drugbank database
([77]Wishart et al., 2017) ([78]https://www.drugbank.ca/), Therapeutic
Target Database ([79]Zhou et al., 2021)
([80]http://db.idrblab.net/ttd/), and DisGeNET database ([81]Janet et
al., 2020) ([82]https://www.disgenet.org/) by searching the keyword
“rheumatoid arthritis”. The gene names and Uniprot ID of all targets
were obtained from the Uniprot database ([83]UP Consortium, 2020)
([84]https://www.uniprot.org/), and the species was restricted to “Homo
sapiens”.
The overlapping targets between active compound targets and RA-related
targets were used to construct a protein-protein interaction (PPI)
network by STRING database ([85]Damian et al., 2020)
([86]http://string-db.org/). The species studied was “Homo sapiens”
with a required minimum interaction score of 0.4. The overlapping
targets were imported into the DAVID database ([87]Huang et al., 2009;
[88]Wei et al., 2009) ([89]https://david.ncifcrf.gov/tools.jsp) to
perform a gene ontology (GO) analysis and a Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathway enrichment analysis, where the GO analysis
consists of three modules: biological process (BP), Molecular Function
(MF) and Cellular Component (CC). Then, the PPI network and the
herb-compound-target-pathway interaction network were visualized using
Cytoscape 3.7.2 software ([90]Shannon et al., 2003).
Molecular docking
The top six targets from the PPI network, and the main active compounds
of SGFD were selected for the molecular docking analysis. The files of
active ingredients in Mol2 format were downloaded from the TCMSP
database, and the files of the protein targets in PBD format were
downloaded from the PDB database ([91]http://www1.rcsb.org/). AutoDock
4.2 was used to further modify the protein by incorporating new polar
hydrogen atoms and calculating the Gasteiger charges. Hydrogen atoms,
Gasteiger charges, and rotatable bonds were also assigned to the
ligands. Molecular docking was then performed using AutoDock 4.2 and
visualized using Pymol software.
Western bolting
Protein expressions of toll-like receptor 4 (TLR4), nuclear factor
kappa-B (NF-κB) p65, c-Jun N-terminal kinase (JNK), p-JNK,
extracellular regulated kinase (ERK), p-ERK, p38, p-p38, and TNF-α were
measured by Western blotting. RIPA Lysis Buffer containing protease and
phosphatase inhibitors was used to lysis synovial tissue from knee
joints. Protein concentration was determined using a BCA protein
detection kit (P0010S, Beyotime, China). The total protein (10 μg) of
each sample was added to SDS-PAGE gel, separated by electrophoresis,
and then transferred to the PVDF membrane. The PVDF membranes
containing the proteins were placed in a closed solution for 1.5 h and
subsequently incubated overnight at 4°C with the required primary
antibodies, including TLR4 antibody (1:500, ab13556, Abcam, United
Kingdom), NF-κB p65 antibody (1:2000, ab16502, Abcam, United Kingdom),
JNK antibody (1:5000, 66210-1-Ig, Proteintech, United States), p-JNK
antibody (1:2000, 9255, Cell Signaling Technology, United States), ERK
antibody (1:5000, 67170-1-Ig, Proteintech, United States), p-ERK
antibody (1:2000, 4370, Cell Signaling Technology, United States), p38
antibody (1:1000, 14064-1-AP, Proteintech, United States), p-p38
antibody (1:1000, 4511, Cell Signaling Technology, United States) and
TNF-α antibody (1:1000, 17590-1-AP, Proteintech, United States) and
β-tubulin antibody (1:2000, 10094-1-AP, Proteintech, United States)
overnight at 4°C. After incubation with HRP-conjugated Affinipure Goat
Anti-Mouse IgG (H + L) (1:5000, SA00001-1, Proteintech, United States)
or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:5000,
SA00001-2, Proteintech, United States) for 1 h, the PVDF membranes were
washed with TBST solution and then processed with chemiluminescent
reagents for exposure and photography. Western blot bands were
quantified using ImageJ 1.52V and their relative expression levels were
quantified as the ratio of the corresponding protein to β-tubulin.
Statistical analysis
Experimental data were statistically analyzed using SPSS 22.0 and
GraphPad Prism 8 software and expressed as mean ± standard deviation
(SD). One-Way ANOVA was used when each group’s data were normally
distributed and the variances were equal. The Fisher’s LSD post-hoc
test was used for comparison between groups. When the data were not
normally distributed or the variance was not homogeneous, a
nonparametric test was used. The difference was statistically
significant when p-value < 0.05.
Results
Shaogan fuzi decoction treatment alleviated collagen-induced
arthritis-related symptoms
After booster immunization, redness or swelling of the hind paws and
ankle joints was gradually observed in CIA rats. On day 17, 65 rats
showed arthritic symptoms, with a success rate of 65%. Moreover, they
were randomly divided into five groups and received appropriate
treatment. The rats in the normal groups moved freely, showed normal
appetite and water consumption and shiny hair, while their weight
gradually increased. The rats in the model group exhibited yellowish
and lusterless hair, poor appetite, slow weight gain, low mood and
activity, and severe paw swelling. After administration of SGFD or TG,
the AI score and paw swelling rate decreased compared with the model
group. TG and SGFD at high and low doses exhibited the favorable
effects of suppressing paw swelling from day 38 (p < 0.05 or p < 0.01).
Similar to the trend of paw swelling results, TG and the high dose of
SGFD significantly decreased AI from day 38 (p < 0.05 or p < 0.01). At
the end of the study (day 45), the paw swelling rate and AI of rats in
each administration group were significantly lower than those in the
model group, and the hair and mental status of rats in the treatment
group gradually recovered ([92]Figure 3).
FIGURE 3.
[93]FIGURE 3
[94]Open in a new tab
Effect of SGFD on joint swelling in RA rats. (A). Images of rats’ paw
swelling in each group (a1-f1: before treatment; a2-f2: after
treatment). (B). The results of right hind paw swelling rate. (C). The
results of left hind paw swelling rate. (D). The results of AI scores.
Data were shown as mean ± SD (Normal, n = 13; Model, TG, SGFD-L,
SGFD-M, n = 12; SGFD-H, n = 10). ^* p < 0.05, ^** p < 0.01 vs. normal
group; ^# p < 0.05, ^## p < 0.01 vs. model group.
Shaogan fuzi decoction treatment alleviated the histopathological changes in
the joints
After treatment, histological analysis of the right ankle joint tissues
was performed to further evaluate the therapeutic effect of SGFD. The
normal rats showed normal articular cartilage structures with smooth
articular surfaces, the chondrocytes were well arranged, with a clear
hierarchical structure and uniform distribution, and the nucleus was
oval in the center of the cell; the synovial membrane had a regular
cellular arrangement, without synoviocyte hyperplasia or inflammatory
cell infiltration. In the model group, the articular cartilage surface
of the rats was rough, with localized fissure defects, the chondrocytes
were disorganized, and the hierarchical structure was blurred; the
synovial tissue was hyperplastic and disorganized, accompanied by
neovascularization and pannus formation and inflammatory cell
infiltration. The degree of ankle joint destruction was significantly
improved in all treated rat groups compared with the model group, with
mild synovial hyperplasia, destruction of articular cartilage, and less
infiltration of inflammatory cells ([95]Figure 4).
FIGURE 4.
[96]FIGURE 4
[97]Open in a new tab
Effect of SGFD on pathological changes of the ankle joint in RA rats.
Shaogan fuzi decoction treatment decreased the spleen and thymus index and
the levels of rheumatoid factor and C-reactive protein
The spleen and thymus index of rats in the model group were
significantly increased compared with those in the normal group.
Compared with the model group, the spleen index in the SGFD-M and
SGFD-H groups and the thymus index in the SGFD-H group showed a
significant decrease (p < 0.05) ([98]Figures 5A,B). As shown in
[99]Figures 5C,D, the serum CRP level was significantly increased in
the model group compared with the normal group (p < 0.05). Compared
with the model group, treatment with SGFD at a high dose significantly
decreased the CRP level of rats (p < 0.05). Meanwhile, there was no
significant difference in RF levels between groups.
FIGURE 5.
[100]FIGURE 5
[101]Open in a new tab
Effects of SGFD on the spleen and thymus index and the serum levels of
RF and CRP. (A). Spleen index. (B). Thymus index. (C). Serum RF
concentration. (D). Serum CRP concentration. Data were shown as mean ±
SD (Normal, n = 13; Model, TG, SGFD-L, SGFD-M, n = 12; SGFD-H, n = 10).
^* p < 0.05, ^** p < 0.01 vs. normal group; ^# p < 0.05, ^## p < 0.01
vs. model group.
Prediction of targets and pathways by network pharmacology technology
Firstly, after ADME screening by OB ≥ 30% and DL ≥ 0.18, 127 active
ingredients were obtained for SGFD ([102]Figure 6A), and 334 potential
human targets corresponded to these compounds. In the corresponding
database, 794 targets associated with RA were collected. Through Venn
analysis, a total of 75 overlapping targets were screened as key
targets for SGFD treatment of RA ([103]Figure 6B). PPI analysis of the
above 75 targets was performed using the STRING database, and the
network was further constructed using Cytoscape software ([104]Figure
6C). The PPI network consisted of 71 nodes and 619 edges (4 targets
were no protein-protein interaction, so they were not shown in the PPI
network). There were 30 targets higher than the average degree value,
including IL6, TP53, TNF, PTGS2, MAPK3, IL1β, JUN, IL10, CASP3, MYC,
RELA, MAPK1, MAPK14, PPARG, MPO ([105]Supplementary Table S1). These
proteins might play an essential pharmacological function in the RA
process.
FIGURE 6.
[106]FIGURE 6
[107]Open in a new tab
Network pharmacology analysis for screening targets and pathways of
SGFD. Venn diagram of (A). Active compounds in SGFD and (B). Common
target of SGFD and RA. (C). PPI network of common targets. The target
is represented by a circle node. The color of node changed from green
to red corresponds to a degree from small to bigger. The thickness of
the edge and the combined score value between the protein have a
positive correlation. (D). GO enrichment analysis for common targets.
(E). KEGG pathway analysis for common targets. (F).
Herb-Compound-Target-Pathway network. The red triangles represent the
herbal medicines; the yellow squares represent the active chemical
compounds of SGFD; the blue dots represent the key targets in the
treatment of RA with SGFD; the green dots represent the pathways based
on enrichment analysis of key targets.
To further explore the mechanism of SGFD action on RA at the systemic
level, we uploaded 75 overlapping targets to DAVID. GO analysis
revealed that these targets were enriched for 114 BP, 10 CC and 52 MF
(p < 0.01, FDR < 0.01). The top 10 terms in BP, CC and MF according to
the number of genes are shown in [108]Figure 6D. Enriched BPs included
positive regulation of transcription from RNA polymerase II promoter,
cytokine-mediated signaling pathway, and positive regulation of
transcription. Enriched CCs included cytosol, cytoplasm, and
nucleoplasm. Enriched MFs included protein binding, enzyme binding, and
protein homodimerization activity. The results of KEGG pathway
enrichment analysis showed that 75 key targets were significantly
enriched in 29 signaling pathways (p < 0.01, FDR < 0.01), including TNF
signaling pathway, Osteoclast differentiation, T cell receptor
signaling pathway, MAPK signaling pathway, NF-κB signaling pathway,
Toll-like receptor signaling pathway, etc. ([109]Figure 6E).
An Herb-Compound-Target-Pathway network was constructed to further
explore the association between the active ingredients of SGFD and the
efficacy of treatment on RA ([110]Figure 6F), consisting of 201 nodes
(3 herbal medicines, 94 active chemical compounds, and 75 key targets
and 29 pathways). The mean degree value of the active ingredients was
6.8, and 21 compounds had a degree value >7, indicating that most
compounds modulate multiple targets to exert different therapeutic
effects. Quercetin, kaempferol, naringenin, formononetin, isorhamnetin
and licochalcone A, acted on 57, 55, 16, 13, 12 and 11 targets,
respectively, indicating that compounds might be crucial for the
treatment of RA. The mean degree value of the targets was 11.3, and 20
targets had a degree value >12. The association of multiple targets
with multiple compounds suggests that the various components of SGFD
act synergistically in the therapeutic process. Moreover, these targets
are involved in different pathways and coordinate with each other,
suggesting that SGFD may treat RA through multiple pathways and
multiple targets. The detailed information is shown in
[111]Supplementary Table S2.
Ingredients-targets molecular docking
Molecular docking was used to further evaluate the interaction between
components and targets and validate the accuracy of the network
analysis. The binding strength of ligand and receptor depends on the
binding energy. The lower the binding energy between ligand and
receptor, the stronger the stability and the higher the possibility of
interaction. The top six core active ingredients quercetin, kaempferol,
naringenin, formononetin, isorhamnetin and licochalcone A, and six
targets IL6, tumor protein p53 (TP53), TNF, prostaglandin-endoperoxide
synthase 2 (PTGS2), MAPK3 and IL-1β were used as ligands and receptors,
respectively. Molecular docking results showed that each component had
good binding activity to each target (Binding Energy <0 kcal/mol)
([112]Wu et al., 2021) ([113]Figure 7A, [114]Supplementary Table S3).
Quercetin, formononetin, kaempferol, isorhamnetin and naringenin showed
the best binding activity to TNF with the binding energy of −8.77,
−8.70, −8.54, −8.27 and −7.30 kcal/mol, respectively. Licochalcone A
showed the best binding activity to IL-1β with a binding energy of
−8.57 kcal/mol. The compounds and targets displayed diverse binding
patterns to the active sites, including hydrogen bonding, H-π and π-π
interactions. Moreover, these compounds bind to the targets through
interactions with various amino acid residues, such as LYS-63, GLN-62,
GLU-64, ASN-60, SER-65, VAL-41, SER-65, GLN-62. The binding
interactions and the binding sites between the compounds and the
targets are shown in [115]Figure 7B.
FIGURE 7.
[116]FIGURE 7
[117]Open in a new tab
Ingredients-Targets Molecular Docking (A). The binding energy of the
main active components of SGFD and the key targets. (B). The binding
site of the main active components of SGFD and the key targets. The
molecular docking poses of Quercetin—TNF (a), Formononetin—TNF (b),
Licochalcone A—IL1β (c), Kaempferol—TNF (d), Licochalcone A—TNF (e),
Isorhamnetin—TNF (f), Quercetin—IL1β (g), Isorhamnetin—IL1β (h),
Formononetin—PTGS2 (i), Naringenin—TNF (j), Kaempferol—IL1β (k),
Formononetin—IL1β (l), Naringenin—IL1β(m), Kaempferol—PTGS2 (n),
Isorhamnetin—PTGS2 (o), Kaempferol—MAPK3 (p), Formononetin—MAPK3 (q),
Quercetin—PTGS2 (r), Naringenin—IL6 (s), Quercetin—IL6 (t),
Licochalcone A—PTGS2 (u), Kaempferol—IL6 (v), Isorhamnetin—IL6 (w),
Naringenin—PTGS2 (x), Formononetin—IL6 (y), Naringenin—TP53 (z),
Isorhamnetin—MAPK3 (aa), Licochalcone A—MAPK3 (ab), Quercetin—MAPK3
(ac), Licochalcone A—IL6 (ad), Naringenin—MAPK3 (ae), Formononetin—TP53
(af), Licochalcone A—TP53 (ag), Kaempferol—TP53 (ah), Quercetin—TP53
(ai), Isorhamnetin—TP53 (aj).
Shaogan fuzi decoction treatment decreased the levels of inflammatory
cytokines
As shown in [118]Figures 8A–E, compared with the normal group, serum
levels of IFN-γ, IL-17, TNF-α, IL-1β and IL-6 were significantly
increased in the model group (p < 0.05 or p < 0.01). Compared with the
model group, SGFD treatment at low and medium doses significantly
decreased the levels of IL-17 (p < 0.01); SGFD treatment at low and
high doses significantly decreased TNF-α level (p < 0.05 or p < 0.01),
and SGFD at all doses notably decreased the serum levels of IFN-γ,
IL-1β and IL-6 (p < 0.01).
FIGURE 8.
[119]FIGURE 8
[120]Open in a new tab
Effects of SGFD on the serum levels of inflammatory cytokines. (A).
Serum IFN-γ concentration. (B). Serum IL-17 concentration. (C). Serum
TNF-α concentration. (D). Serum IL-1β concentration. (E). Serum IL-6
concentration. Data were shown as mean ± SD (n = 8). ^* p < 0.05, ^** p
< 0.01 vs. normal group; ^# p < 0.05, ^## p < 0.01 vs. model group.
Shaogan fuzi decoction treatment downregulated the toll-like receptor
4/nuclear factor kappa-B/mitogen-activated protein kinases pathway
As shown in [121]Figure 9, protein expressions of TLR4, NF-κB p65,
p-JNK, p-ERK, p-p38 and TNF-α and the ratio of p-JNK/JNK, p-ERK/ERK and
p-p38/p38 in the synovial tissues of the knee joints were increased in
the model groups compared with the normal group (p < 0.01). Compared
with the model group, a high dose of SGFD treatment decreased the
expression of TLR4, NF-κB p65, and TNF-α (p < 0.01), and inhibited the
increase in phosphorylation of p38 and JNK (p < 0.01). The p-ERK1/2 was
slightly suppressed but not statistically significant.
FIGURE 9.
[122]FIGURE 9
[123]Open in a new tab
SGFD treatment suppressed TLR4/NF-κB/MAPKs pathway. (A). Western
blotting assay was performed to measure the expression of TLR4, NF-κB
p65, JNK, p-JNK, ERK, p-ERK, p38, p-p38 and TNF-α in synovial tissues
of knee joints (B–G). Relative expression of TLR4, NF-κB p65,
p-p38/p38, p-ERK/ERK, p-JNK/JNK and TNF-α were quantified. Data were
shown as mean ± SD (n = 3). ^* p < 0.05, ^** p < 0.01 vs. normal group;
^# p < 0.05, ^## p < 0.01 vs. model group.
Discussion
RA is a complex, chronic systemic autoimmune disease characterized by
joint or synovium inflammation, angiogenesis, cartilage, and bone
destruction, leading to joint swelling, pain and morning stiffness,
eventually deformity and functional disability ([124]Mcinnes and
Schett, 2017). Current RA treatment tends to focus more on improving
disease activity. The ultimate goal is to relieve pain and
inflammation, maintain joint mobility, and limit the loss of functional
capacity ([125]Smolen et al., 2010). SGFD is a representative
prescription for the treatment of RA, has a tonic effect on
deficiencies, expels wind and dampness, warms the meridians and
nourishes the blood to dredge the collaterals, and relieves pain.
Clinical studies have shown that treatment with SGFD effectively
alleviates clinical symptoms and signs in RA patients and has fewer
side effects ([126]Zhang, 2017; [127]Zheng et al., 2018; [128]Zhang,
2021). However, the exact pharmacological effects of SGFD and the major
active compounds and molecular mechanisms exerting these effects, have
not yet been elucidated. In this study, we demonstrated that SGFD could
effectively alleviate the symptoms of RA. The underlying mechanism is
related to the suppression of inflammation through the regulation of
the TLR4/NF-κB/MAPK signaling pathway.
Currently, the CIA model is the most frequently used animal model to
study RA, because it has similar immunological and histological
characteristics to human RA patients ([129]Mcnamee et al., 2015;
[130]Miyoshi and Liu, 2018). This study successfully established a CIA
model in rats, which showed weight loss, joint swelling, pain,
inflammation, synovial hyperplasia, and bone and cartilage damage.
Tripterygium glycosides tablet is a Chinese patent medicine made from
extracts of the traditional Chinese medicinal herb Triptergii Radix
(Tripterygium wilfordii Hook. f.) ([131]Zhang et al., 2021). It mainly
contains diterpenoids, triterpenoids and alkaloids ([132]Zhang et al.,
2019). Tripterygium glycosides tablet has anti-inflammatory and
suppressive effects on cellular and humoral immunity, and has been
approved by the China Food and Drug Administration for the treatment of
autoimmune and inflammatory diseases, including RA (State medical
license No. Z32021007) ([133]Na et al., 2020). Therefore, tripterygium
glycosides tablet was selected as a positive control drug in this
study. After treatment with SGFD and tripterygium glycosides tablet,
the AI score and paw swelling of rats decreased to a certain extent,
and the histopathological lesions in the joints also improved,
suggesting that SGFD shows benefit in alleviating the symptoms in RA.
RF is an autoantibody that reacts against the Fc portion of IgG
antibodies, and is produced locally by B cells in lymphoid follicles
([134]Moura et al., 2012). The immune complex formed by RF is deposited
on the vascular wall and articular cartilage, which can enhance the
inflammatory and destructive response ([135]Fang et al., 2019). The
titer of RF correlates positively with the severity and active stage of
RA. Therefore, RF can be used to determine the severity of RA and the
efficacy of treatment ([136]Song and Kang, 2010). In our present study,
it was found that the serum RF level slightly decreased in RA rats
after SGFD treatment, but it was not statistically significant, which
may be due to the lower specificity of RF. CRP is an acute-phase
protein, and usually increases rapidly during the acute phase of the
host inflammatory response. CRP is a reliable and accurate marker of
the inflammatory response in vivo, and when combined with RF, can help
avoid misdiagnosis ([137]Machold et al., 2007). The serum CRP level was
significantly increased in the model group, whereas it was decreased
after SGFD treatment. The results of the present study show that SGFD
has an effect on reducing RA disease activity.
The pharmacological components and complex molecular mechanism of SGFD
on RA were investigated using network pharmacology. By screening the
active ingredients and analyzing the Herb-Compound-Target-Pathway
network, quercetin, kaempferol, naringenin, formononetin isorhamnetin
and licochalcone A were considered as the potential active components
of SGFD exerting pharmacological effects on RA. Quercetin ([138]Yuan et
al., 2020; [139]Zhao et al., 2020; [140]Costa et al., 2021) has been
attributed with a variety of biological activities, such as
anti-inflammatory and antioxidant, immunomodulatory, inhibition of MMPs
activity and synovial hyperplasia, which can inhibit the pathological
process of RA. Kaempferol suppresses proliferation, migration and
invasion of fibroblast-like synoviocytes, and alleviates inflammation
([141]Yoon et al., 2013; [142]Lee et al., 2018; [143]Pan et al., 2018).
Naringenin ([144]Hajizadeh et al., 2021; [145]Wang et al., 2021),
formononetin ([146]Machado Dutra et al., 2021), isorhamnetin ([147]Gong
et al., 2020), and licochalcone A ([148]de Freitas et al., 2020;
[149]Li et al., 2022) have been reported to have antioxidant,
anti-inflammatory, anti-cancer and osteogenic effects, showing great
potential as agents for the treatment of RA.
Combined with network pharmacology analysis and related literature,
IL6, TP53, TNF, PTGS2, MAPK3 and IL-1β could be potential key targets
for SGFD in treating RA. TNF-α, IL-1β and IL-6, as typical
pro-inflammatory factors, play important roles in the pathogenesis and
development of RA. Currently, TNF-α inhibitory drugs such as the
monoclonal antibodies infliximab and adalimumab ([150]Richmond et al.,
2015), and IL-6 receptor inhibitors such as the monoclonal antibody
tocilizumab, and a monoclonal antibody against IL-1β anakinra are
widely used in the clinical treatment of RA ([151]Prieto-Peña and
Dasgupta, 2021). PTGS2, also known as cyclooxygenase (COX)-2, is a
proinflammatory enzyme that mediates the conversion of arachidonic acid
to prostaglandin (PG) E2. This in turn stimulates the production of
MMPs, promotes angiogenesis, destruction of cartilage and bone, and
inhibits apoptosis of T-cells ([152]Smith et al., 2000). In joint
tissue, abnormal expression of the protein COX-2 is an important marker
for RA ([153]Kang et al., 1996). A previous study by our group showed
that SGFD significantly reduced the expression of PTGS2 protein in the
synovial tissues of the ankle of RA rats ([154]Dong et al., 2022). TP53
is a transcription factor that modulates cell cycle initiation.
Mutations in the p53 gene (named TP53) may contribute to the tumor-like
growth and pro-inflammatory properties of fibroblast-like synoviocytes
(FLS), such as aggressive growth of RA-FLS, invasion and destruction of
cartilage ([155]Yu et al., 2020). MAPK3, also known as ERK, acts in a
signaling cascade that regulates various cellular processes such as
proliferation, differentiation and cell cycle progression, and plays an
important role in inflammation, proliferation and bone destruction in
RA ([156]Ohori, 2008). Molecular docking showed that the active
components of SGFD have the high binding activity to these target
proteins. This evidence suggests that the pharmacological activity of
SGFD against RA is due to the synergistic effect between multiple
components and targets.
Among the 29 signaling pathways predicted by network pharmacology, the
regulation of inflammatory mediators is an interesting pathway
described. RA is a chronic autoinflammatory disease with abnormal
innate and adaptive immune responses. The markedly increased thymus and
spleen indices in RA rats indicate immune system activation
([157]Steiniger and Meide, 1993). As modulators and effectors of the
immune system, cytokines are closely associated with the initiation and
development of RA ([158]Hazekawa et al., 2017). The pro-inflammatory
cytokines are directly or indirectly involved in the activation and
differentiation of pathogenic cells (e.g., Th17), the transcriptional
regulation of inflammatory cytokines and chemokines, the processes of
angiogenesis, the production of matrix metalloproteinases (MMPs) by
synovial cells and chondrocytes, and the development and activation of
osteoclasts, leading to synovial inflammation and the destruction of
bone and cartilage ([159]Venkatesha et al., 2014). Among the enriched
signaling pathways, inflammation-related signaling pathways include the
TNF signaling pathway, T cell receptor signaling pathway, MAPK
signaling pathway, Toll-like receptor signaling pathway, NOD-like
receptor signaling pathway and B cell receptor signaling pathway. T
lymphocytes and B lymphocytes are the main cells involved in
antigen-specific defense. Activation of the T-cell receptor and B-cell
receptor signaling pathways regulates T-cell and B-cell activation,
proliferation, differentiation, and death. Abnormal expression of these
signaling pathways can lead to defects in the body’s immune response or
autoimmune diseases. Pattern recognition receptors (PRRs), represented
by TLRs distributed on the membrane surface and Nod-like receptors
(NLRs) present in the cytoplasm, play an important role in innate
immunity. They recognize pathogen-associated molecular patterns (PAMPs)
and damage-associated molecular patterns (DAMPs) and regulates immune
responses ([160]Marshak-Rothstein, 2006). Members of NLRs are involved
in the control of inflammasome activation, antigen-presentation, signal
transduction, transcriptional regulation, and autophagy. NLRs also act
as a key regulator of apoptosis and early development. Thus, NLRs are
closely associated with various of infection- and immune-related
diseases ([161]Kim et al., 2016). TLRs, expressed in a variety of cells
such as macrophages, endothelial cells and epithelial cells, are among
the most well-studied and well-characterized PRRs. They can detect a
variety of PAMPs such as lipids, proteins, lipoproteins and nucleic
acids. Activation of TLRs leads to the engagement of diverse
intracellular signaling pathways that determine the host inflammatory
response. As a member of the toll family, TLR4 activates NF-κB and MAPK
signaling pathways through MyD88 dependent or independent pathways and
induces the expression of inflammatory cytokines and chemokines
([162]Pålsson-McDermott and O'Neill, 2004). The MAPK signaling pathway
can transduce extracellular stimulatory signals into cells to mediate
proliferation, differentiation, transformation, apoptosis, and
autophagy. MAPK signal transduction pathways associated with RA mainly
include the ERK, JNK, and p38 pathways. The abnormally activated MAPK
signaling pathway may be involved in the activation, proliferation and
differentiation of T cells and induce the expression of inflammatory
cytokines, chemokines and MMPs, thereby promoting the inflammatory
response of synovitis cells, participating in the excessive
proliferation and apoptosis inhibition of synovitis cells, and causing
long-lasting chronic synovitis. Moreover, the MAPK signaling pathway
induces the expression of MMPs in chondrocytes, regulates chondrocyte
apoptosis and osteoclast differentiation, and plays an important role
in the process of cartilage and bone destruction in RA joints
([163]Thalhamer et al., 2008). NF-κB is a nuclear transcription
activating factor that can initiate or enhance the transcription
process of genes and release downstream cytokines such as TNF-α, IL-1β,
IL-6, etc ([164]Hayden and Ghosh, 2008). The latter activates NF-κB
through a positive feedback loop, resulting in a cascade response that
amplifies and sustains inflammation. TLR4, NF-κB and MAPK pathways play
important roles in the inflammatory response, and a complex
co-regulatory relationship exists between them, that jointly influences
the type, magnitude, and duration of the inflammatory response. Given
the multi-component, multi-target, and multi-pathway effects of TCM, we
used TLR4/NF-κB/MAPK signaling pathway as an entry point to investigate
the comprehensive regulatory effect of SGFD on the inflammatory
response in RA rats. Our study confirmed that the expression of TLR4,
NF-κB p65, p-JNK, p-p38, p-ERK, and TNF-α in the synovium of RA rats
was significantly upregulated and suppressed after SGFD administration.
Moreover, SGFD treatment significantly reduced the serum levels of
TNF-α, IL-1β, IL-6, IFN-γ and IL-17, as well as thymus and spleen
indexes in rats, demonstrating that SGFD inhibited the activation of
the TLR4/NF-κB/MAPK pathway and the expression/production of
pro-inflammatory cytokines at RA.
Conclusion
In summary, this study explored the therapeutic effects and underlying
mechanism of SGFD against RA based on a combination of network
pharmacology and experimental verification. The results showed that
SGFD could improve the symptoms and inflammatory response in the CIA
rat model. Its mechanism might be related to the regulation of the
TLR4/MAPKs/NF-κB signaling pathway, and the reduction of inflammatory
factor release ([165]Figure 10), which partially confirms the results
predicted by network pharmacology. This study provided the experimental
basis for the clinical application and further research of SGFD in
treating of RA.
FIGURE 10.
[166]FIGURE 10
[167]Open in a new tab
A model representing SGFD molecular targets in TLR4/MAPK/NF-κB pathway.
SGFD exerts its anti-inflammatory effect by inhibiting TLR4 and NF-κB
expression, the phosphorylation of JNK and p38 in synovial tissues. The
action of SGFD is indicated by red lines.
Data availability statement
The original contributions presented in the study are included in the
article/[168]Supplementary Materials, further inquiries can be directed
to the corresponding authors.
Ethics statement
The animal study was reviewed and approved by the Medical and
Experimental Animal Ethics Committee of Beijing University of Chinese
Medicine.
Author contributions
WS and JW contributed to the design concepts of this whole study. LS,
FM, and LD carried out the animal experiment and collected important
background information. AS and JZ carried out network pharmacologic
analysis. CF and YZ drafted the manuscript. LS carried out data
acquisition and analysis. TW, JT, and KL revised the article. All
authors have read and approved the content of the manuscript.
Funding
This work was supported by the National Key Basic Research Program of
China (973 Program, grant no. 2009CB522803) and the Beijing University
of Chinese Medicine Research Development Fund (no. 2020072120047).
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:
[169]https://www.frontiersin.org/articles/10.3389/fphar.2022.967164/ful
l#supplementary-material
[170]Click here for additional data file.^ (26.7KB, docx)
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