Sinomenine Inhibits the Progression of Rheumatoid Arthritis by Regulating the Secretion of Inflammatory Cytokines and Monocyte/Macrophage Subsets

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory arthropathy associated with articular damage and attendant comorbidities. Even although RA treatment has advanced remarkably over the last decade, a significant proportion of patients still do not achieve sustained remission. The cause of RA is not yet known despite the many potential mechanisms proposed. It has been confirmed that RA is associated with dysregulated immune system and persistent inflammation. Therefore, management of inflammation is always the target of therapy. Sinomenine (SIN) is the prescription drug approved by the Chinese government for RA treatment. A previous study found that SIN was a robust anti-inflammation drug. In this study, we screened the different secretory cytokines using inflammation antibody arrays and qRT-PCR in both LPS-induced and SIN-treated RAW264.7 cells followed by evaluation of the ability of SIN to modulate cytokine secretion in a cell model, collagen-induced arthritis (CIA) mouse model, and RA patients. Several clinical indexes affecting the 28-joint disease activity score (DAS28) were determined before and after SIN treatment. Clinical indexes, inflammatory cytokine secretion, and DAS28 were compared among RA patients treated with either SIN or methotrexate (MTX). To explore the mechanism of SIN anti-inflammatory function, RA-associated monocyte/macrophage subsets were determined using flow cytometry in CIA mouse model and RA patients, both treated with SIN. The results demonstrated that SIN regulated IL-6, GM-CSF, IL-12 p40, IL-1α, TNF-α, IL-1β, KC (CXCL1), Eotaxin-2, IL-10, M-CSF, RANTES, and MCP-1 secretion in vivo and in vitro and reduced RA activity and DAS28 in a clinical setting. Furthermore, SIN attenuated CD11b⁺F4/80⁺CD64⁺ resident macrophages in the synovial tissue, CD11b⁺Ly6C⁺CD43⁺ macrophages in the spleen and draining lymph nodes of CIA mice. The percentage of CD14⁺CD16⁺ peripheral blood mononuclear cells was reduced by SIN in RA patients. These data indicated that SIN regulates the secretion of multiple inflammatory cytokines and monocyte/macrophage subsets, thereby suppressing RA progression. Therefore, along with MTX, SIN could be an alternative cost-effective anti-inflammatory agent for treating RA.

Keywords: cytokine; inflammatory; macrophages; monocytes; rheumatoid arthritis; sinomenine.

Figure 1

SIN prevents LPS-induced cell injury. Cell viability was determined by the CCK-8 assay, and the viability of untreated RAW264.7 was assessed as 100%. (A) Chemical structure of Sinomenine (SIN), CAS number: 115-53-7. (B) RAW264.7 cells were incubated with SIN (0–1000 μg/mL) for 24 h. (C) RAW264.7 cells were treated with various concentrations of LPS (0–20 μg/mL) for 24 h (*P < 0.05 vs. control). (D) RAW264.7 cells were pre-treated with 50 μg/mL SIN for 0–4 h and then co-stimulated with LPS (1 μg/mL) for another 24 h (*P < 0.05 vs. 2 h pre-treated). (E) RAW264.7 cells were pre-incubated with 0, 1, 10, 50 μg/mL SIN for 2 h and co-stimulated with 1 μg/mL LPS for another 24 h. Data are presented as mean ± SD values of four independent experiments (*P < 0.05 vs. control, ^#P < 0.05 vs. LPS treated RAW264.7 cells).

Figure 2

Hierarchical cluster of cytokines in the inflammatory array and the 12 cytokines which were significantly regulated by SIN (at least one comparison P < 0.05, 10 μg/mL SIN + LPS vs. LPS or 50 μg/mL SIN + LPS vs. LPS). (A) Hierarchical cluster of the 40 cytokines. (B) Hierarchical cluster of the 12 selected secreted cytokines (IL-6, GM-CSF, IL-12 p40, IL-1α, TNF-α, IL-1β, KC, Eotaxin-2, IL-10, M-CSF, RANTES, and MCP-1). (C) Color bar of the cluster analysis. Red, upregulated; green, downregulated. All the signal density values of cytokines were transformed to Log₂ (signal density) and subtracted the density value-wise mean from the values of each cytokine, so that the mean value of each group was 0. Multiply all values in each group of data by scale factor S, so that the sum of the squares of the values in each row is 1.0. Cluster type, average linkage clustering. Figures were generated by Cluster 3.0 (Stanford University) and TreeView (Alok, version: 1.1.6r4). (D) Detailed relative signal density normalized by background of 12 selected cytokines. (*P < 0.05 vs. untreated; ^#P < 0.05 vs. LPS treated RAW264.7).

Figure 3

RAW 264.7 cells were incubated with serum-free medium for 12 h in 6-well plates, pre-treated with SIN (10 or 50 μg/mL) for 2 h, and finally co-stimulated with LPS (1 μg/mL) for another 24 h. (A) IL-6, GM-CSF, IL-12 p40, IL-1α, TNF-α, IL-1β, KC, Eotaxin-2, IL-10, M-CSF, RANTES, and MCP-1 release from conditioned medium was measured by ELISA, respectively. The values represent the means ± SD of triplicate experiments (*P < 0.05, **P < 0.01). (B) Relative mRNA levels of the 12 selected cytokines by arrays were determined using RT-PCR. The results of RT-PCR were normalized to β-actin and expressed as fold change to the control. The values represent the means ± SD of triplicate experiments. *P < 0.05 vs. LPS treated RAW264.7.

Figure 4

SIN rescues inflammation and cartilage damage in collagen-induced arthritis DBA/1 mice. (A) H&E staining and Safranin-O staining of the ankle joints of each group. (B) Clinical arthritis score was evaluated every 3 days in each group. From day 30 after immunization or the first time, SIN could ameliorate clinical arthritis scores of CIA. The values represent the means ± SD (n = 6). Significance was analyzed by Wilcoxon rank-sum test (*P < 0.05, **P < 0.01). (C) Body weight from each group had no significant difference. However, SIN slightly led to body weight gain in CIA mice. The values represent the means ± SD (n = 6). Significance was analyzed by unpaired 2-sided t-test. (D) Swelling of paws, (E) inflammatory cell infiltrations, (F) cartilage damage could also be attenuated by SIN. Data are presented as the mean ± SD (n = 6). Significance was analyzed by Wilcoxon rank-sum test (*P < 0.05, **P < 0.01).

Figure 5

Cytokines screened from LPS-induced RAW264.7 and SIN treatment in vitro were validated in vivo. Serum levels of (A) IL-6, (B) GM-CSF, (C) IL12-p40/p70, (D) IL-1α, (E) IL-1β, (F) TNF-α, (G) KC, (H) Eotaxin-2, (I) IL-10, (J) MCSF, (K) RANTES, (L) MCP-1 from mice in each group were measured by ELISA, respectively. Data are presented as the mean ± SD (n = 6). Significance was analyzed by unpaired two-sided t-test (*P < 0.05, **P < 0.01).

Figure 6

SIN reduces the percentage of CD11b⁺F4/80⁺CD64⁺ synovial macrophages and CD11b⁺Ly6C⁺CD43⁺ monocytes/macrophages in CIA mice. (A) A representative flow cytometry analysis of ankle macrophages in CIA mice under steady state conditions presenting the gating strategy. After exclusion of doublets and debris, and gating out granulocytes, dendritic cells, and B cells, synovial macrophages were identified as CD11b⁺F4/80⁺CD64⁺. After removing Ly6C⁺ cells, synovial tissue resident macrophages were identified as CD11b⁺F4/80⁺ CD64⁺ (Ly6C⁻). Arrow denotes a parent population being displayed in a subsequent plot. Cells positive for CD11b and F4/80 are gated and displayed in a plot of CD64 vs. Ly6C, in which the CD11b⁺F4/80⁺CD64⁺ cell population is present in left delineation gate. (B) Analysis of the total synovial macrophages (CD11b⁺F4/80⁺) and the percentage of synovial tissue resident macrophages from ankles of placebo-treated, CIA, CIA plus 50 or 100 mg/kg/d SIN treated mice. (C) Spleen cells were depleted of T, B, and red blood cells and stained with antibodies to CD11b, CD43, and Ly6C. Spleen monocytes/macrophages were identified as CD11b⁺Ly6C⁺CD43⁺. CD11b⁺ cells were initially gated on the basis of a monocyte/macrophage phenotype. Gating strategies were based on fluorescence minus one control, and numbers in gates represent % specific binding. A representative gating result about CIA mice is shown. Arrow denotes a parent population being displayed in a subsequent plot. Cells positive for CD11b is gated and displayed in a plot of CD43 vs. Ly6C, in which the CD11b⁺Ly6C⁺CD43⁺ cell population is present in right delineation gate. (D) Analysis of the percentage of CD11b⁺Ly6C⁺CD43⁺ monocytes/macrophages from spleens of placebo-treated, CIA, CIA plus 50 or 100 mg/kg/d SIN treated mice. (E) Draining lymph nodes (inguinal and popliteal) were isolated and prepared for staining CD11b, CD43, and Ly6C. CD11b⁺Ly6C⁺CD43⁺ monocytes/macrophages were identified in the same manner as those of the spleen. A representative gating result about CIA mice is shown. Arrow denotes a parent population being displayed in a subsequent plot. Cells positive for CD11b is gated and displayed in a plot of CD43 vs. Ly6C, in which the CD11b⁺Ly6C⁺CD43⁺ cell population is present in right delineation gate. (F) Analysis of the percentage of CD11b⁺Ly6C⁺CD43⁺ monocytes/macrophages from draining lymph nodes (inguinal and popliteal) of placebo-treated, CIA, CIA plus 50 or 100 mg/kg/d SIN treated mice. Data are presented as the mean ± SD (n = 6). Significance was analyzed by unpaired 2-sided t-test (**P < 0.01 vs. Placebo; ^#P < 0.05, ^##P < 0.01 vs. CIA.).

Figure 7

SIN modulates the secretory levels of inflammatory cytokines in the plasma of RA patients. (A) IL-6, (B) GM-CSF, (C) IL12-p40, (D) IL-1α, (E) IL-1β, (F) TNF-α, (G) GROα(CXCL1), (H)Eotaxin-2, (I) IL-10, (J) MCSF, (K) RANTES, and (L) MCP-1 secretion was detected by ELISA in heathy donors, before and after SIN and MTX treatments, respectively. Each dot represents one sample. Significance was analyzed by unpaired two-sided t-test (*P < 0.05, **P < 0.01).

Figure 8

Linear regression and coloration analysis between cytokine changes and ΔDAS28 in RA patients before and after treated with SIN or MTX. (A) IL-6, (B) GM-CSF, (C) IL12-p40, (D) IL-1α, (E) IL-1β, (F) TNF-α, (G) CXCL1, (H) Eotaxin-2, (I) IL-10, (J) MCSF, (K) RANTES, (L) MCP-1. Positively correlated with ΔDAS28: IL-6, GM-CSF, IL-12 p40 (SIN P = 0.216, MTX P = 0.0013), IL-1α, IL-1β, TNF-α, CXCL1 (GROα), Eotaxin-2 (SIN P = 0.0254, MTX P = 0.059); negatively correlated with ΔDAS28: IL-10; no correlation with ΔDAS28: M-CSF, RANTES, and MCP-1. r, Pearson correlation coefficient. Red, SIN therapy; blue, MTX therapy. Round dot, good responders; square with white cross, moderate responders; inverted triangle, non-responders. Red line, correlation of SIN; blue line, correlation of MTX.

Figure 9

SIN reduced the percentage of CD14⁺CD16⁺ monocytes in RA patient PBMCs. A representative flow cytometry analysis of CD14⁺CD16⁺ percentage in PBMCs of (A) heathy donors (B) patient with active RA (C) after SIN treatment RA patient (D) after MTX treatment RA patient. (E) Analysis of the percentage of CD14⁺CD16⁺ monocytes in PBMCs of each group. Heathy donors, n = 20, SIN before treatment, n = 25, SIN after treatment, n = 25, MTX before treatment, n = 24, MTX after treatment, n = 24. Each dot represents one sample. Significance was analyzed by unpaired two-sided t-test. **P < 0.01.