Exploring the Mechanism of Topical Application of Clematis Florida in the Treatment of Rheumatoid Arthritis through Network Pharmacology and Experimental Validation

Abstract

Clematis Florida (CF) is a folk medicinal herb in the southeast of China, which is traditionally used for treating osteoarticular diseases. However, the mechanism of its action remains unclear. The present study used network pharmacology and experimental validation to explore the mechanism of CF in the treatment of rheumatoid arthritis (RA). Liquid chromatography-mass spectrometry (LC-MS/MS) identified 50 main compounds of CF; then, their targets were obtained from TCMSP, ETCM, ITCM, and SwissTargetPrediction databases. RA disease-related targets were obtained from DisGeNET, OMIM, and GeneCards databases, and 99 overlapped targets were obtained using a Venn diagram. The protein-protein interaction network (PPI), the compound-target network (CT), and the compound-potential target genes-signaling pathways network (CPS) were constructed and analyzed. The results showed that the core compounds were screened as oleanolic acid, oleic acid, ferulic acid, caffeic acid, and syringic acid. The core therapeutic targets were predicted via network pharmacology analysis as PTGS2 (COX-2), MAPK1, NF-κB1, TNF, and RELA, which belong to the MAPK signaling pathway and NF-κB signaling pathway. The animal experiments indicated that topical application of CF showed significant anti-inflammatory activity in a mouse model of xylene-induced ear edema and had strong analgesic effect on acetic acid-induced writhing. Furthermore, in the rat model of adjuvant arthritis (AA), topical administration of CF was able to alleviate toe swelling and ameliorate joint damage. The elevated serum content levels of IL-6, COX-2, TNF-α, IL-1β, and RF caused by adjuvant arthritis were reduced by CF treatment. Western blotting tests showed that CF may regulate the ERK and NF-κB pathway. The results provide a new perspective for the topical application of CF for treatment of RA.

Keywords: Clematis Florida; MAPK; NF-κB; network pharmacology; rheumatoid arthritis.

Figure 1

Flowchart of the methodology for studying the mechanism of action of topical application of CF in the treatment of RA.

Figure 2

Total ion current diagram of CF extraction solution: (a) positive ion detection mode; (b) negative ion detection mode.

Figure 3

Network pharmacological analysis of CF for treating RAL: (a) Venn diagram showing intersection of CF-related targets and RA-related predicted targets; (b) plot of association between CF compounds and RA. The 16 yellow nodes on the left represent the main compounds of CF, and the 99 CF targets for the treatment of RA are labeled in blue on the right-hand side; (c) CTS network of CF in treatment of RA. Yellow nodes indicate the main compounds of CF, red nodes denote the 9 signaling pathways screened from the KEGG analysis of the PPI network, and blue nodes symbolize the overlapped target genes linked to both CF and RA.

Figure 4

GO and KEGG functional analysis: (a) GO enrichment analysis. BP: GO with reference to biological processes; CC: gene ontology with reference to cellular components; MF: gene ontology with reference to molecular function; (b) KEGG enrichment analysis.

Figure 5

Pattern diagram of molecular docking: (a–c) molecular docking of dexamethasone, oleanolic acid, and syringic acid to NFκB1; (d) molecular docking of caffeic acid to TNF-α; (e–h) molecular docking of ulixertinib, oleanolic acid, ferulic acid and oleic acid to MAPK1; (i–n) molecular docking of rofecoxib, oleanolic acid, caffeic acid, ferulic acid, syringic acid, and oleic acid to PTGTS2. The yellow dashed line represents hydrogen bonds and the number next to the yellow dashed line represents the length of the hydrogen bond. The green part represents the amino acid residue. The yellow compounds were the core compounds screened from network pharmacology analysis. The red compound was inhibitor.

Figure 6

Results of HPLC: (a) HPLC chromatogram of the reference standard; (b) HPLC chromatograms of the CF sample.

Figure 7

Effects of CF on inflammation and pain in mice (mean ± SEM, n = 8): (a) anti-inflammatory effect of CF on xylene-induced ear edema in mice; (b) analgesic effect of CF in the acetic acid-induced writhing model. Abbreviations: DD, diethylamine diclofenate; CF-H, CF—high dose; CF-L, CF—low dose. ** p < 0.01, *** p < 0.001 vs. model group.

Figure 8

Amelioration effects of CF on arthritis in AA rats (mean ± SEM, n = 8): (a) macroscopic changes in arthritis of the hind limbs in rats were observed; (b) comparison of toe swelling in rats in each group at different times after dosage; (c) weight changes in rats from the induction of RA to the end of treatment. *** p < 0.001 vs. control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. model group.

Figure 9

Effects of CF extracts on serum inflammation factors and protein expression in RA rats: (a) serum IL-6 level; (b) serum TNF-α level; (c) serum COX-2 level; (d) serum IL-1β level; (e) serum RF level (mean ± SEM, n = 8); (f) Western blot analysis; (g) effect of CF on p-ERK, ERK proteins in rats; (h) effect of CF on NF-κB, p-NF-κB proteins in rats (mean ± SEM, n = 5). ** p < 0.01, *** p < 0.001 vs. control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. model group; ^&&& p < 0.001 vs. DD group.

Figure 10

Morphology of synovial tissue in ankle joints of rats in various groups. All figures were magnified by 100×. Scare Bar: 100 μm.