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Anti-inflammatory activity of Radix Angelicae biseratae in the treatment of osteoarthritis determined by systematic pharmacology and in vitro experiments

Radix Angelicae biseratae is a widely used Chinese traditional herbal medicine for osteoarthritis (OA). Its therapeutic efficacy has been confirmed in clinical practice. However, its mechanisms of action in treating OA have remained elusive. The purpose of the present study was to identify active components with good oral bioavailability and drug-like properties from Radix Angelicae biseratae through systematic pharmacology and in vitro experiments to determine targets of Radix Angelicae biseratae in the treatment of OA. The functional components of Radix Angelicae biseratae were screened from the Traditional Chinese Medicine Systems Pharmacology database based on oral bioavailability and drug-like properties. Subsequently, the databases STITCH, Open Targets Platform and DrugBank were searched and microarray analysis was performed to screen the active components of Radix Angelicae biseratae to treat OA and predict its potential target proteins. The interaction network and protein interaction network were then generated and examined, molecular docking between active components and targets was performed and the enrichment of potential target proteins was analyzed. Finally, reverse transcription-quantitative (RT-q)PCR and western blot analyses were used to verify the therapeutic effect of Radix Angelicae biseratae extract on the expression of OA-associated target proteins. The results provided eight active components in Radix Angelicae biseratae, which were firmly linked to 20 targets of OA. In combination with molecular docking and the analysis of the interaction network between components and targets, it was suggested that sitosterol was a major active component of Radix Angelicae biseratae in the treatment of OA. Protein interaction network analysis suggested that prostaglandin-endoperoxide synthase 2 (PTGS2), nitric oxide synthase 3 and cytochrome P450 2B6 may be critical targets for Radix Angelicae biseratae in the treatment of OA. In addition, RT-qPCR and western blot analyses suggested that Radix Angelicae biseratae extract inhibited the mRNA and protein expression of PTGS2 in degenerative articular cartilage cells in vitro, whilst other targets remain to be verified. Functional enrichment analysis indicated that Radix Angelicae biseratae confers pharmacological efficacy towards OA through exerting anti-inflammatory effects and immune regulation.


Introduction
Osteoarthritis (OA) is a complex chronic, severe degenerative arthropathy involving the synovium of the joint. It has a significant economic impact on the public health sector and as the population ages, the number of patients is expected to increase (1). OA is widely recognized as an 'arthralgia syndrome' in Traditional Chinese Medicine (TCM). Inflammation is a critical process in the occurrence and development of OA, as it is able to stimulate and enhance the catabolism of chondrocytes and lead to joint injury. One of the primary symptoms of OA is pain, which may be caused and worsened by inflammation. Inhibition of inflammation is able to provide an effective treatment for OA by relieving pain and reducing joint injury (2)(3)(4).
Radix Angelicae biseratae is the root of the plant Angelica biserrata, known as 'Duhuo' in China. As a natural herb, Radix Angelicae biseratae has long been used to treat 'arthralgia syndrome' by eliminating dampness and relieving pain according to the concepts of TCM (5). Modern pharmacological studies have demonstrated that Radix Angelicae

Anti-inflammatory activity of Radix Angelicae biseratae in the treatment of osteoarthritis determined by systematic pharmacology and in vitro experiments
biseratae has a significant anti-inflammatory effect for the treatment of OA (6). However, the chemical composition of Radix Angelicae biseratae is complex and the mechanism for treating OA at the molecular level has remained elusive. Systematic pharmacology is a subject where the knowledge of systems biology is applied to pharmacology. The purpose of this discipline is to clarify how drugs act on the body through biological systems. Instead of evaluating the effect of a drug by analyzing a particular protein-drug interaction, systematic pharmacology suggests that drugs act by forming a network of interactions (7). Interaction systems may include drug-protein and protein-protein interactions, as well as genetic, signaling and physiological interactions (7). Systematic pharmacology uses bioinformatics and statistics to integrate and explain the network of drug interactions, which provides an effective method to investigate the underlying mechanisms of TCM and other naturally occurring compounds for the treatment of a number of diseases (8,9).
Therefore, in the present study, systematic pharmacology was used to screen the potential active components of Radix Angelicae biseratae for the treatment of OA, to predict and verify its targets and provide a theoretical basis for drug development and disease treatment. An experimental flowchart for the present study is presented in Fig. 1.

Materials and methods
Screening for active components. The TCM systems pharmacology (TCMSP) database (http://lsp.nwu.edu.cn/tcmsp. php) is a systematic pharmacological database for evaluating the pharmacokinetic properties of TCM or associated components (10). It provides data on the absorption, distribution, metabolism and systemic excretion (ADME) of drugs with potential biological effects, such as oral bioavailability (OB) and druglikeness (DL), as well as Caco-2 and blood-brain barrier permeability. OB is the most critical feature of oral drugs because it is important in evaluating the effectiveness of drug distribution through the systemic circulation (11). DL refers to the similarity between components and known drugs. In drug development, DL evaluation helps to identify qualified components and improve the success rate of candidate drugs (12). Based on the published literature and preliminary information in the TCMSP database (13), the drug name 'Radix Angelicae biseratae' was entered into the search window in the TCMSP database. The ingredients with OB ≥30% and DL ≥18% were screened for further study.
Identification of drug targets. The STITCH (http://stitch.embl.de/) database was used to identify the potential targets of active components in Radix Angelicae biseratae (14). STITCH is a database used to explore and predict the interaction between components and proteins. Through experiments, databases and literature evidence, chemicals associated with other chemicals and proteins were retrieved. In the present study, the active components were inputted into the STITCH database and human species were set up to screen the target proteins of Radix Angelicae biseratae with a confidence score >0.4 for further analysis.
Identification of disease targets. OA targets were collected from the Open Targets platform (https://www.targetvalidation. org) (15,16) and the DrugBank database (https://www. drugbank.ca) (17)(18)(19). The Open Targets platform is a comprehensive and robust data integration platform for accessing and visualizing potential drug targets related to diseases. It brings together a variety of data types designed to help users identify and prioritize targets for further research (20). The disease name 'osteoarthritis' was entered into the search window in the Open Targets platform database and target proteins were collected for further analysis. DrugBank database is a comprehensive online database that contains information concerning drugs and drug targets. The DrugBank database was searched with the key word 'osteoarthritis' to screen and collect targets for known drugs in the treatment of OA. Subsequently, the proteins overlapping the Radix Angelicae biseratae targets were selected for further study.

Construction of component-target network.
A 'component-target network' diagram was constructed using Cytoscape 3.7.2 (https://cytoscape.org/) software in order to reveal the major active components and regulatory mechanisms of Radix Angelicae biseratae in the treatment of OA (21).

Construction of target-target interaction network.
In order to identify the critical proteins among the target proteins identified as specified above, a 'target-target interaction network' was established using the Search Tool for the Retrieval of Interacting Genes and proteins (STRING; v11.0; http://string-db.org/) and visualized with Cytoscape 3.7.2 (22).
Gene functional enrichment analysis. ClusterProfiler was used to analyze the enrichment of gene function and signaling pathways. ClusterProfiler is an R package for gene cluster enrichment analysis, which may be used to understand the function of genes and the enrichment of pathways (23). When provided with the overlapping target gene list, ClusterProfiler uses the Gene Ontology (GO) database to analyze the biological function and signaling pathway enrichment of these genes (24,25).

Verification of target proteins by OA gene expression array.
A set of microarray data of gene expression in cartilage tissue of patients with OA was obtained from the gene expression omnibus (GEO) database (https://www.ncbi.nlm. nih.gov/geo/; dataset no. GSE75181). The dataset contained 48 samples, of which 12 non-control samples and 12 control samples treated with IL-1β were selected. The gene matrix file of the GSE75181 dataset was downloaded and it was standardized with the limma package (26), including background correction, standardization and normalization. The impute package was used to fill in the missing values (27). The platform annotation file was used to annotate the probes and delete those probes (genesymbols) that did not match the gene. For multiple probes targeting the same gene, the average value of different probes was used as the expression value of the gene. Depending on whether the samples were treated with IL-1β or not, the samples were divided into two groups: The non-control group and the control group. Regarding the difference in expression, the P-value and fold change (FC) in expression of the non-control vs. control samples were calculated using the limma package. Adjusted P-value (adj.P.Val) <0.05 and |log2FC|>1 were chosen as the threshold for filtering the significant differentially expressed mRNA and display the results in the form of volcano plot. Critical proteins on the volcano plot were then marked.

Prediction of hub target protein-component docking.
The Blind Dock ing ser ver (https:// bio-hpc.ucam. edu/achilles/entry) was used for molecular docking experiments (28). The crystal structure of active components was downloaded from the PubChem database (https://pubchem. ncbi.nlm.nih.gov) and the structure of the hub target proteins was obtained from the protein databank (PDB; https://rscb.org) (29). BIOVIA DiscoveryStudio ® 2016 software (Dassault Systemes) was used to modify the protein structure for molecular docking. All water molecules and ligands were removed and the necessary hydrogen atoms were added. The obtained receptor and ligand structures were uploaded to the Blind Docking server for docking calculation and the best structures with the lowest binding energy were used.  Preparation and analysis of Radix Angelicae biseratae extract. Radix Angelicae biseratae was purchased from the local Guo Yi Tang Chinese herbal medicine store. The original herb was identified as Radix Angelicae biseratae by Dr Wen Xu at the Department of Pharmacology, Fujian University of Traditional Chinese Medicine (Fujian, China). Radix Angelicae biseratae (500 g) was crushed into a fine powder and boiled twice with 4 l of 80% ethanol for 1 h. The ethanolic extract was collected and filtered. The filtrate was concentrated to 500 ml at 50˚C under reduced pressure and the concentration was 1 g/ml. The content of sitosterol in the extract was determined by high-performance liquid chromatography (HPLC) method using an Agilent 1200 HPLC system (Agilent Technologies, Inc.) and the Thermo Scientific™ BETASIL™ C18 column (4.6x150 mm, 5 µm; Thermo Fisher Scientific, Inc.). The column temperature was set at 35˚C and the injection volume was 10 µl. The mobile phase was 100% methanol and the flow rate was 1.0 ml/min. Ultraviolet detection wavelengths were set at 210 nm at 0-30 min.
Chondrocyte observation and identification. Chondrocytes were isolated from the knee articular cartilage of 4-week-old Sprague Dawley rats. After the rats were anesthetized by intraperitoneal injection of 30 mg/kg pentobarbital, they were sacrificed via rapid decapitation. The chondrocytes were isolated, cultured and verified, as previously described (31,32).
Degenerative chondrocyte model. The degenerative chondrocyte model was established as previously described (33,34). In brief, the chondrocytes at passage three were exposed to 10 ng/ml IL-1β for 24 h.
Experimental grouping. Cells were divided into the following groups: i) control group without treatment; ii) degenerative chondrocyte group; iii) degenerative chondrocytes with 1 µM Radix Angelicae biseratae extract group; iv) degenerative chondrocytes with 5 µM Radix Angelicae biseratae extract group; and v) degenerative chondrocytes with 25 µM Radix Angelicae biseratae extract group. The intervention time for all groups was 24 h.

Reverse transcription-quantitative PCR (RT-qPCR).
After 24 h of intervention, total RNA was extracted from chondrocytes of each group using a TRIzol ® kit (Invitrogen; Thermo Fisher Scientific, Inc.). RNA(1 µg) was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (cat. no. RR0047A; Takara Bio, Inc.) in the PCR amplifier (S1000 Thermal Cycler; Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. The following primer sequences were used: Prostaglandin-endoperoxide synthase 2 (PTGS2) forward, 5'-AGC ACA ATA GAC GCC CAA GA-3' and reverse, 5'-GGA GTC AAA GCA TAG GTC TTC A-3'; β-actin forward, 5'-ACC ACT GGC ATT GTG ATG GA-3' and reverse, 5'-CGC TCG GTC AGG ATC TTC T-3'. β-actin was used to normalize the expression of mRNA. AceQ qPCR SYBR ® Green Master Mix (cat. no. Q111-02; Vazyme Biotech Co., Ltd.) was used to detect the respective mRNA expression levels. qPCR was conducted using the 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following reaction conditions: Initial denaturation at 95˚C for 3 min, followed by 40 cycles at 95˚C for 10 sec and 60˚C for 30 sec; dissolution curve at 95˚C for 15 sec, 60˚C for 60 sec and 95˚C for 15 sec. The experiment was performed three times and the gene expression ratios were expressed in the form of average value and standard deviation according to the independent measurement results of the three experiments. The 2 -ΔΔCq method (35) was used to evaluate the experimental results.
Statistical analysis. The experimental data were processed and analyzed by Graph Pad Prism 8.02 software (GraphPad Software, Inc.). The Shapiro-Wilk test was used to determine the normality of all groups of data. If the data exhibited a normal distribution, they were compared by one-way analysis of variance followed by a least significant difference or Games Howell post-hoc test; if not, the Kruskal-Wallis test was used and the Mann-Whitney U test with Bonferroni's correction was applied as a post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results
Components and potential targets for Radix Angelicae biseratae. The TCMSP database was searched with the key words 'Radix Angelicae biseratae' for 99 herbal ingredients. When applying the threshold specified in the methods section, nine herbal ingredients were obtained.
These nine components screened from the TCMSP database were searched in the STITCH database, of which eight components had 30 target proteins. Among them, ammidin had five targets, isoimperatorin had seven targets, sitosterol had 14 targets, O-acetyl columbianetin had nine targets, angelol D had two targets, angelol G had one target, angelicone had five targets and nodakenin had eight targets. The ADME properties of these eight components are presented in Table I.
Identification of OA-associated proteins. By searching the Open Targets Platform and DrugBank database, 20 targets linked to the treatment of OA were screened ( Fig. 2A). As presented in Fig. 2B, a network of pharmacologically active components and targets was established. Of the eight components, seven had OA-related targets. Among these seven components, sitosterol (degree=11) may have an essential role in the treatment of OA, followed by O-acetylcolumbianetin (degree=7) and nodakenin (degree=6). All components were linked to PTGS2, which suggests that Radix Angelicae biseratae may be a potential inhibitor of PTGS2.
Target-target interaction analysis. Using Cytoscape 3.7.1, a diagram of the 'target-target network' was constructed based on the query results of the STRING database (Fig. 3A). In addition, the bar plot was constructed according to the degrees of connection between the targets (Fig. 3B). The results indicated that nitric oxide synthase 3 (NOS3), PTGS2 and cytochrome P450 2B6 (CYP2B6) were the hub targets in this network with high degree values.
Pathway analysis. The ClusterProfiler package in R was used to perform GO analysis of the functional characteristics of targets and the threshold was set as P≤0.01. The results of the GO analysis suggested that 75 GO terms were enriched in 'Biological Process'. The first 20 items with the most significant P-values are provided in Fig. 4. The neurotransmitter metabolic process, response to nutrient levels and nitric oxide metabolic process were among the functional roles of Radix Angelicae biseratae and this may explain its anti-inflammatory action.
Verification of the target protein in GSE75181. A total of 665 differentially expressed genes were identified from two groups of cartilage samples. As presented in Fig. 5, compared with the hub target of Radix Angelicae biseratae, PTGS2 was significantly upregulated in the IL-1β-treated group (adj.P.Val <0.05; |log2FC|>1).

Docking of hub target proteins and major components.
Docking studies were performed using the Blind Docking server in the active sites of the two critical targets to investigate  the possible interactions between the active components and potential targets, namely PTGS2 (PDB ID: 5F19) and NOS3 (PDB ID: 3NOS). In general, a lower binding energy indicates a stronger binding between the molecules (37). The conformation with the lowest binding energy was selected in the component-target complex between each component and the target and their binding energy is listed in Table II. As presented in Table II, among the active components, a certain conformation of the complex of sitosterol and hub targets had the highest binding energy. Discovery Studio was used to map the binding pattern of sitosterol to hub target proteins. The binding pattern of sitosterol in the active site of PTGS2 is presented as a three-dimensional model in Fig. 6A and a two-dimensional diagram is shown in Fig. 6B. Sitosterol was indicated to interact with the ASN537 residue in the B chain and also, the GLY533 residue in the B chain has two hydrogen bonds, and sitosterol has two hydrophobic interactions with the PHE142 and LEU145 amino acid residues. The binding pattern of sitosterol at the active site of NOS3 is presented in its three-dimensional model in Fig. 6C and a  two-dimensional diagram is provided in Fig. 6D. Sitosterol exhibited an electrostatic interaction in the TRP356 residue on the B chain, and also involved in the alkyl-alkyl interaction with ALA423, MET358, VAL185, VAL418, CYS184, PHE353, ARG183, MET339, PHE473, ALA181, TRP178 and PRO334 in the A chain. These interactions increased the binding affinity between the two molecules.
Analysis of active components of Radix Angelicae biseratae extract. HPLC analysis indicated that the Radix Angelicae biseratae extract contained sitosterol ( Fig. 7A and B), which was demonstrated to be a significant active compound in the treatment of OA that may be responsible for its anti-inflammatory activity, as determined by network analysis and molecular docking.
Radix Angelicae biseratae extract regulates gene and protein expression in the degenerative chondrocyte model. In order to verify the effect of Radix Angelicae biseratae extract on inflammatory factors in degenerative chondrocytes, the rat chondrocytes were treated with 10 ng/ml IL-1β for 24 h, following which they were treated with Radix Angelicae biseratae extract (1, 5 or 25 µM) for 24 h. As presented in Fig. 8, the expression of PTGS2 in the IL-1β group was significantly higher compared with that in the regular group, which was consistent with the results of the gene expression array. At the same time, Radix Angelicae biseratae extract significantly decreased the expression of PTGS2 in degenerative chondrocytes treated with IL-1β (P<0.05), which indicated that certain active components in Radix Angelicae biseratae extract were able to inhibit the expression of PTGS2 in degenerative chondrocytes.
The protein expression of PTGS2 was determined to verify the effect of Radix Angelicae biseratae on degenerative chondrocytes (Fig. 9A and B). Compared with the regular group, the expression of PTGS2 protein in IL-1β-induced degenerative chondrocytes was significantly increased (P<0.01). Compared with that in the degenerative chondrocyte group, the expression of PTGS2 protein in the Radix Angelicae biseratae groups (1, 5 and 25 µM) was significantly inhibited (P<0.05).

Discussion
The systematic pharmacology method has been developed for determining molecular biological networks and may be used to discover novel therapeutic effects of drugs from medicinal plants (8). Therefore, it provides a systematic method to expand the application of available drug components in TCM in a variety of complex diseases (9). OA, which is a common disease of chronic arthritis, is associated with painful symptoms and significantly affects the patients' quality of life (1,2). Although Radix Angelicae biseratae has been used as an effective Chinese herbal medicine for the treatment of OA for several centuries, its pharmacological mechanism has remained to be elucidated (5).
In the present study, the active components and potential targets of Radix Angelicae biseratae in the treatment of OA were evaluated based on systematic pharmacology, including ADME system evaluation, network analysis, pathway analysis and molecular docking. A total of eight active components were screened and were indicated to interact with 30 different targets related to OA. According to the analysis of the components-targets network model, sitosterol had the most significant number of target connections (degree=11), followed by O-acetyl columbianetin (degree=7) and nodakenin (degree=6). Sitosterol has a wide range of biological functions in vivo, including antitumor, antioxidant, antidiabetic and anti-inflammatory activity and roles in reducing gallstone activity (38). Combined with the results of molecular docking  in the present study, it is proposed that sitosterol is a critical potential active component of Radix Angelicae biseratae in the treatment of OA. PTGS2 is the coding gene of cyclooxygenase-2, which is an essential target of OA anti-inflammatory therapy and one of the major inflammatory factors involved in articular cartilage degradation (39). NOS encoded by the NOS3 gene is also a crucial inflammatory mediator in the occurrence and development of OA. It can promote the production of NO in the microenvironment, increase the production of reactive oxygen species and induce chondrocyte apoptosis (40). CYP2B6 is a member of the cytochrome P450 superfamily of enzymes that is closely related to drug metabolism (41). A recent study demonstrated that CYP2B6 may affect the metabolism of NO under inflammatory stimulation (42). The present study indicated that PTGS2, NOS3 and CYP2B6 are potential targets for Radix Angelicae biseratae in the treatment of OA, with high degree values in the 'target-target' network. Microarray  analysis demonstrated that compared with the non-control group, PTGS2 was highly expressed in the degenerative chondrocyte group. RT-qPCR and western blot analysis confirmed that Radix Angelicae biseratae extract inhibited the expression of PTGS2 in the cartilage degeneration model in vitro, which indicates that certain active components of Radix Angelicae biseratae may have a role in the treatment of OA via anti-inflammatory mechanisms.
Combined with the results of molecular docking, the present results indicated that sitosterol may be a potential inhibitor of NOS3 and PTGS2 and may have an anti-inflammatory effect on OA. The levels of PTGS2 and NOS3 and the effect of sitosterol on their expression in animal models will be investigated in future experiments.
The results of the present pathway analysis indicated that Radix Angelicae biseratae exerted its pharmacological effects in OA by modulating multiple pathways, including drug metabolism, inflammation and immune modulation.
In conclusion, sitosterol was demonstrated to be a critical active component of Radix Angelicae biseratae in the treatment of OA. Radix Angelicae biseratae extract reduced the expression of PTGS2 in degenerative chondrocytes, which may be the underlying mechanism of action of Radix Angelicae biseratae in reducing inflammation in the treatment of OA. However, the present study is based on data mining analysis and in vitro experiments and the results require to be further verified by in vivo experiments.