The mechanism of the anti-inflammatory effect of Oldenlandia diffusa on arthritis model rats: a quantitative proteomic and network pharmacologic study

Abstract

Background: In China, Oldenlandia diffusa (OD) has been prescribed as a therapeutic herb for rheumatoid arthritis (RA). We previously conducted a preliminary study of the anti-inflammatory effect of OD, and the purpose of this study is to further investigate its mechanism.

Methods: We performed a quantitative proteomic analysis of synovium, identified the differentially expressed proteins, and performed bioinformatics analyses. With the help of network pharmacology, we aimed to find the key synovial proteins which OD or its key compound might influence. To verify the result, liquid chromatography-mass spectrometry (LC-MS) was applied to quantify and qualify the absorbable potential compounds of OD. The anti-inflammatory effect was evaluated by morphological, histopathological, and cytokine analyses. Target proteins were observed by immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA).

Results: MMP3 and CAV1 were identified as 2 of the differentially expressed proteins in RA synovium, and might be influenced by quercetin, the active compound of OD. MMP3 might be altered through atherosclerosis signaling, while CAV1 might be altered through caveolar-mediated endocytosis signaling. According to our verification, quercetin was identified as the absorbed and effective compound of OD, and it could exert an anti-inflammatory effect on the collagen-induced arthritis (CIA) model, including serum cytokine expression, synovial hyperplasia and lymphocyte infiltration, articular cartilage lesion. Quercetin could also down-regulate the synovial expression of MMP3 and CAV1, and could exert better effects at a high dose.

Conclusions: Quercetin was the main active compound of OD in the treatment of RA. OD might alleviate inflammatory responses in CIA rats by suppressing the expression of MMP3 and CAV1 through quercetin, and at a high dose, quercetin could exert a better anti-inflammatory effect.

Keywords: Isobaric tags for relative and absolute quantification (iTRAQ); Oldenlandia diffusa (OD); network pharmacology; rheumatoid arthritis.

Figure 1

Herb-compound-target network. Green: OD; Blue: active compounds of OD; Pink: target proteins which were relevant to the active compounds. OD, Oldenlandia diffusa; FER, ferulic acid; p-MCA, 4-methoxycinnamic acid.

Figure 2

Intersected targets among the targets of DEPs, OD, RA, and S&V. Two were found to be related, namely MMP3 and CAV1. DEPs, differentially expressed proteins; OD, Oldenlandia diffusa; RA, rheumatoid arthritis; S&V, synovitis & vasculitis.

Figure 3

Canonical pathways associated with the 67 DEPs. Their expression was significant (threshold of 1.3 corresponds to P value of 0.05) as identified by IPA. Among them, 2 significant pathways were identified (black columns), namely atherosclerosis signaling for MMP3 and caveolar-mediated endocytosis signaling for CAV1. DEPs, differentially expressed proteins; IPA, ingenuity pathway analysis.

Figure 4

Ion chromatograms of the mixed standard substance by MRM in negative mode. (A) XIC of the mixed standard substance; (B) XIC of quercetin; (C) XIC of beta-sitosterol; (D) TIC of the mixed standard substance. MRM, multiple reaction monitoring; XIC, extracted ion chromatogram; TIC, total ion chromatogram.

Figure 5

Ion chromatograms of the OD decoction by MRM in negative mode. (A) XIC of the OD decoction; (B) XIC of quercetin; (C) XIC of beta-sitosterol; (D) TIC of the OD decoction. OD, Oldenlandia diffusa; MRM, multiple reaction monitoring; XIC, extracted ion chromatogram; TIC, total ion chromatogram.

Figure 6

Ion chromatograms of drug serum by MRM in negative mode. (A) XIC of the drug serum; (B) XIC of quercetin; (C) XIC of beta-sitosterol; (D) TIC of drug serum. MRM, multiple reaction monitoring; XIC, extracted ion chromatogram; TIC, total ion chromatogram.

Figure 7

Standard curve of the ion pair from the quercetin standard in MRM−. The x-axis refers to the concentration of quercetin (µg/mL) and the y-axis refers to peak area (cps). MRM, multiple reaction monitoring.

Figure 8

Morphological changes of the posterior limb in each group (A-L) from day 1 to 28. Control group (A). Model group on day 14 (B), 21 (C), and 28 (D). CIA rats treated with OD decoction (OD group) on day 21 (E) and 28 (F). CIA rats treated with Que-LD (Que-LD group) on day 21 (G) and 28 (H). CIA rats treated with Que-MD (Que-MD group) on day 21 (I) and 28 (J). CIA rats treated with Que-HD (Que-HD group) on day 21 on day 21 (K) and 28 (L). CIA, collagen induced arthritis; OD, Oldenlandia diffusa; Que-LD, quercetin in low dose; Que-MD, quercetin in medium dose; Que-HD, quercetin in high dose.

Figure 9

Means of arthritis index (AI) in each group, from day 1 to 28. In comparison with the model group (*, P<0.05); in comparison with the OD group (^#, P<0.05). CIA rats treated with OD decoction (OD group). CIA rats treated with Que-LD (Que-LD group). CIA rats treated with Que-MD (Que-MD group). CIA rats treated with Que-HD (Que-HD group). CIA, collagen induced arthritis; OD, Oldenlandia diffusa; Que-LD, quercetin in low dose; Que-MD, quercetin in medium dose; Que-HD, quercetin in high dose.

Figure 10

Histopathological changes (hematoxylin-eosin staining) of knee joint in each group (A-F) on day 28. In each image, left: regular scope (×40); right: magnified scope (×200). Control group (A). Model group (B). CIA rats treated with OD decoction (OD group) (C). CIA rats treated with Que-LD (Que-LD group) (D). CIA rats treated with Que-MD (Que-MD group) (E). CIA rats treated with Que-HD (Que-HD group) (F). CIA, collagen induced arthritis; OD, Oldenlandia diffusa; Que-LD, quercetin in low dose; Que-MD, quercetin in medium dose; Que-HD, quercetin in high dose.

Figure 11

Serum cytokines (TNF-α, IL-1β, IL-6) in each group on day 28. In comparison with the model group (*, P<0.05); in comparison with the OD group (^#, P<0.05). CIA rats treated with OD decoction (OD group). CIA rats treated with Que-LD (Que-LD group). CIA rats treated with Que-MD (Que-MD group). CIA rats treated with Que-HD (Que-HD group). CIA, collagen induced arthritis; OD, Oldenlandia diffusa; Que-LD, quercetin in low dose; Que-MD, quercetin in medium dose; Que-HD, quercetin in high dose.

Figure 12

The expression of MMP3 (A,C,E,G) and CAV1 (B,D,F,H) in each group (immunohistochemistry staining) on day 28. In each image, left: regular scope (×100); right: magnified scope (×400). Control group (A,B). Model group (C,D). CIA rats treated with OD decoction (OD group) (E,F). CIA rats treated with Que-HD (Que-HD group) (G,H). CIA, collagen induced arthritis; OD, Oldenlandia diffusa; Que-HD, quercetin in high dose.

Figure 13

Synovial proteins MMP3 (A) and CAV1 (B) in each group on day 28. In comparison with the model group (*, P<0.05); in comparison with the OD group (^#, P<0.05). CIA rats treated with OD decoction (OD group). CIA rats treated with Que-HD (Que-HD group). CIA, collagen induced arthritis; OD, Oldenlandia diffusa; Que-HD, quercetin in high dose.