Combination Therapy of PEG-HM-3 and Methotrexate Retards Adjuvant-Induced Arthritis

Abstract

At present, the early phenomenon of inflammatory angiogenesis is rarely studied in Rheumatoid arthritis (RA). Previous research found that PEG-HM-3, an integrin inhibitor, possessed anti-angiogenesis and anti-rheumatic activity. In this study, the advantages of inhibiting angiogenesis and immune cell adhesion and migration, as well as the benefits of anti-arthritis effects, were evaluated using a combination of PEG-HM-3 and methotrexate (MTX). In vitro, spleen cell proliferation and the levels of tumor necrosis factor α (TNF-α) in macrophage supernatant were assessed. Hind paw edema, arthritis index, clinical score, body weight and immunohistochemistry (IHC) of the spleen, thymus, and joint cavity were evaluated in vivo in adjuvant-induced arthritis rats. Joints of the left hind paws were imaged by X-ray. The expression of the toll-like receptor 4 (TLR-4) protein was assessed in lipopolysaccharide (LPS)-induced synoviocytes. PEG-HM-3 combined with MTX significantly reduced primary and secondary swelling of the hind paws, the arthritis index, the clinical score and bone erosion. The results of IHC showed that the levels of interleukin-6 (IL-6) in spleens and the levels of TNF-α, CD31 (cluster of differentiation 31), and CD105 in the joint cavity were decreased. The body weight of rats was maintained during combination therapy. Ankle cavity integrity, and bone erosion and deformity were improved in combination treatment. The expression of TLR-4 was significantly reduced with combination treatment in rat synoviocytes. Co-suppression of both inflammation and angiogenesis in arthritis was achieved in this design with combination therapy. The activity of nuclear transcription factor (NF-κB) and the expression of inflammatory factors were down regulated via integrin α_vβ₃ and TLR-4 signaling pathways. In the future, the application of this combination can be a candidate in early and mid-term RA therapy.

Keywords: PEG-HM-3; angiogenesis; combination therapy; inflammation; methotrexate; peptide; rheumatoid arthritis.

Figure 1

Effect of PEG-HM-3 alone or in combination with Methotrexate (MTX) on lymphoproliferative responses to mitogen ConA and anti-inflammation activity. (A) Inhibited proliferation with PEG-HM-3 (1.13–7.2 μM) in ConA (5 μg·mL⁻¹)-induced splenocytes. (B) Inhibited proliferation with MTX (0.5–8 μM) in ConA (5 μg·mL⁻¹)-induced splenocytes. (C) Dose-dependent inhibited proliferation with MTX in combination with fixed PEG-HM-3 (18 μM) in ConA (5 μg·mL⁻¹)-induced splenocytes. (D) TNF-α levels in LPS (1 μg·mL⁻¹)-induced RAW264.7 macrophage supernatants treated by MTX (1 μM), PEG-HM-3 (18 μM) or their combination. Values are means and standard error of the mean (SD) (n = 3 in (A,B); n = 4 in (C); n = 3 in (D)). The one-way ANOVA was used for group comparison. Versus ConA group or LPS group, * p < 0.05, ** p < 0.01 or *** p < 0.001.

Figure 2

Curative effect of PEG-HM-3 alone or in combination with Methotrexate (MTX) on adjuvant-induced arthritis rats. All parameters were evaluated once every three days from the 13th day to the 28th day after disease onset (day 13, 16, 19, 22, 25, 28). (A) Swelling of the left-hind paws (mL); (B) Swelling of the right-hind paws (mL); (C) Arthritis index; (D) Clinical score; (E) Weight added (g) at the 28th day. MTX (1 mg·kg⁻¹), PEG-HM-3 (10 mg·kg⁻¹) and combination of MTX (1 mg·kg⁻¹) and PEG-HM-3 (10 mg·kg⁻¹) were used. Values are means and standard error of the mean (SD) ((A–E), n = 9 in each group); (F) Morphology of the left- and right-hinds paws in each group; (G) X-ray exhibition of the left-hind paws of rats at the end of the experiment; (H) The radiographic analysis of the left-hind paws (n = 8 in each group). Versus AIA model group. ** p < 0.01; *** p < 0.001.

Figure 3

Histological staining in arthritic rats. (A) Histological staining of spleens; (B) Histological staining of thymus. Pathology areas were indicated with black arrows (n = 5–8 in each group, ×200 magnification); (C) Histological staining of the left-hind ankles; (D) Histological staining of the right-hind ankles. Images were observed by hematoxylin-eosin (HE) staining under inverted microscope. Pathological regions of synovial hyperplasia, pannus, inflammation and bone erosion were indicated by red arrows (n = 5–8 in each group, ×100 magnification).

Figure 4

Immunohistochemical and western blot analysis of the levels of cytokines and proteins. (A) IL-6 expressions in spleens; (B) TNF-α expressions in hind ankles; (C) Levels of CD31 in joint cavity; (D) Levels of CD105 in joint cavity; (C,D) n = 5–8 in each group, ×200 magnification); (E) Western blot analysis of expressions of TLR-4 in synovial of rat (n = 3). The one-way ANOVA was used for group comparison. Versus LPS group, ** p < 0.01 or *** p < 0.001.

Figure 5

Immunohistochemical analysis in arthritic rats. (A) Expressions of IL-6 in spleens; (B) Expressions of TNF-α in hind ankles; (C) Expressions of CD31 in joint cavity; (D) Expressions of CD105 in joint cavity. The positive cells were stained brown and yellow and were indicated with black arrows. Values are means and standard error of the mean (SD) (A–D), n = 5–8 in each group, ×200 magnification).

Figure 6

Schematic diagram of the combination therapy for early stage of RA. Black arrow represents a down regulation in the phenomena of splenocyte proliferation,bone erosion and pannus, expression of the proteins and contents of the cytokines.