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

Abstract

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.

Keywords: Radix Angelicae biseratae; anti- inflammation; molecular mechanism; osteoarthritis; systematic pharmacology; traditional Chinese medicine.

Figure 1

Flowchart of the experimental procedures. OA, osteoarthritis; TCMSP, Traditional Chinese Medicine Systems Pharmacology.

Figure 2

Venn analysis and network analysis of active components and potential targets of Radix Angelicae biseratae in the treatment of OA. (A) Venn diagram of target proteins of Radix Angelicae biseratae and OA-related proteins based on the Open Targets Platform and Drugbank database. (B) The compound-target network of Radix Angelicae biseratae to treat OA, the square nodes represent the compounds, the circular nodes represent the targets, and the node size is proportional to the degree. OA, osteoarthritis; PTGS2, prostaglandin-endoperoxide synthase 2; NOS3, nitric oxide synthase 3; CYP2B6, cytochrome P450 2B6; ACHE, acetylcholinesterase; ESR1, estrogen receptor 1; SLC6A4, solute carrier family 6 member 4; CYP2D6, cytochrome P450 2D6; NOS2, nitric oxide synthase 2; OPRM1, opioid receptor µ1; CASP1, caspase 1; AR, androgen receptor; PTGS1, prostaglandin-endoperoxide synthase 1; PON1, paraoxonase 1; ADRA1A, adrenoceptor α1A; POR, cytochrome P450 oxidoreductase; ADRB2, adrenoceptor β2; KCNH2, potassium voltage-gated channel subfamily H member 2; BCL2, B-cell lymphoma 2; SCN5A, sodium voltage-gated channel α subunit 5.

Figure 3

‘Target-target’ interaction network analysis. (A) Target-target interaction network constructed using Cytoscape. The sizes of the nodes are proportional to their degrees. (B) Barplot of the degrees of connection between the targets. PTGS2, prostaglandin-endoperoxide synthase 2; NOS3, nitric oxide synthase 3; CYP2B6, cytochrome P450 2B6; JUN, jun proto-oncogene; ACHE, acetylcholinesterase; ESR1, estrogen receptor 1; SLC6A4, solute carrier family 6 member 4; CYP2D6, cytochrome P450 2D6; NOS2, nitric oxide synthase 2; OPRM1, opioid receptor µ1; CASP1, caspase 1; AR, androgen receptor; PTGS1, prostaglandin-endoperoxide synthase 1; PON1, paraoxonase 1; ADRA1A, adrenoceptor α1A; POR, cytochrome P450 oxidoreductase; ADRB2, adrenoceptor β2; KCNH2, potassium voltage-gated channel subfamily H member 2; BCL2, B-cell lymphoma 2; SCN5A, sodium voltage-gated channel α subunit 5.

Figure 4

Top 20 biological process terms from the Gene Ontology analysis. The names of the path are specified on the vertical axis, while the horizontal axis represents the ratio of the number of enriched genes to the total number of uploaded genes. The color of the bars indicates the significance of the P-values.

Figure 5

Volcano plot of all genes. The red dots indicate the upregulated genes (adj.P.Val <0.05 and logFC >1), the green dots indicate the significantly downregulated genes (adj.P.Val <0.05 and logFC <-1) and the gray dots indicate the genes with no obvious change. PTGS2, prostaglandin-endoperoxide synthase 2; NOS3, nitric oxide synthase 3; CYP2B6, cytochrome P450 2B6; FC, fold change; adj.P.Val, adjusted P-value.

Figure 6

Molecular docking between active components and potential targets. (A) A three-dimensional model of the binding pattern of sitosterol at the active site of the protein PTGS2. Active-site amino acid residues are represented as tubes, while the compound is presented using a stick-ball model. (B) A two-dimensional diagram of Sitosterol at the active site of the protein PTGS2. (C) A three-dimensional model of the binding pattern of Sitosterol at the active site of protein NOS3. (D) A two-dimensional diagram of sitosterol at the active site of the protein NOS3. PTGS2, prostaglandin-endoperoxide synthase 2; NOS3, nitric oxide synthase 3.

Figure 7

Schematic diagram of HPLC analysis. (A) Chromatogram of sitosterol. (B) Chromatogram of 80% ethanol extract of Radix Angelicae biseratae. 1, sitosterol; mAU, milli-absorbance units.

Figure 8

Radix Angelicae biseratae extract inhibits the expression of inflammatory factors in rat chondrocytes induced by IL-1β. Chondrocytes were treated with 10 ng/ml IL-1β for 24 h and then treated with Radix Angelicae biseratae extract (1, 5 and 25 µM) for 24 h. The mRNA levels of PTGS2 in chondrocytes were detected by reverse transcription-quantitative PCR. ^#P<0.05; ^*P<0.05. PTGS2, prostaglandin-endoperoxide synthase 2.

Figure 9

Protein expression of PTGS2 in chondrocytes treated with IL-1β and/or Radix Angelicae biseratae extract. (A) Representative western blot image and (B) quantified expression levels. ^##P<0.01; ^*P<0.05. PTGS2, prostaglandin-endoperoxide synthase 2.