Therapeutic effect of various ginsenosides on rheumatoid arthritis

Abstract

Background: Rheumatoid arthritis (RA) is an autoimmune disease which causes disability and threatens the health of humans. Therefore, it is of great significance to seek novel effective drugs for RA. It has been reported that various ginsenoside monomers are able to treat RA. However, it is still unclear which ginsenoside is the most effective and has the potential to be developed into an anti-RA drug.

Methods: The ginsenosides, including Rg1, Rg3, Rg5, Rb1, Rh2 and CK, were evaluated and compared for their therapeutic effect on RA. In in vitro cell studies, methotrexate (MTX) and 0.05% dimethyl sulfoxide (DMSO) was set as a positive control group and a negative control group, respectively. LPS-induced RAW264.7 cells and TNF-α-induced HUVEC cells were cultured with MTX, DMSO and six ginsenosides, respectively. Cell proliferation was analyzed by MTT assay and cell apoptosis was carried out by flow cytometry. CIA mice model was developed to evaluate the therapeutic efficacy of ginsenosides. The analysis of histology, immunohistochemistry, flow cytometry and cytokine detections of the joint tissues were performed to elucidate the action mechanisms of ginsenosides.

Results: All six ginsenosides showed good therapeutic effect on acute arthritis compared with the negative control group, Ginsenoside CK provided the most effective treatment ability. It could significantly inhibit the proliferation and promote the apoptosis of RAW 264.7 and HUVEC cells, and substantially reduce the swelling, redness, functional impairment of joints and the pathological changes of CIA mice. Meanwhile, CK could increase CD8 + T cell to down-regulate the immune response, decrease the number of activated CD4 + T cell and proinflammatory M1-macrophages, thus resulting in the inhibition of the secretion of proinflammatory cytokine such as TNF-α and IL-6.

Conclusion: Ginsenoside CK was proved to be a most potential candidate among the tested ginsenosides for the treatment of RA, with a strong anti-inflammation and immune modulating capabilities.

Keywords: CIA; Ginsenoside compound K; Panax ginseng; Rheumatoid arthritis; Therapeutic effect.

Fig. 1

The chemical structures of six ginsenoside monomers (a). The MTT assay results of six ginsenosides on the viability of RAW 264.7 cells (b) and HUVEC cells (c) as a function of concentrations for 24 h. DMSO and MTX was served as a negative group and a non-ginsenoside positive control group, respectively. Data were presented as the mean ± SD (n = 6). *p < 0.05, vs DMSO group

Fig. 2

MTT assay results of six ginsenosides on the proliferation of RAW264.7 cells induced by LPS (a) and HUVEC cells induced by TNF-α (b) as a function of concentrations for 24 h. DMSO and MTX was served as a negative group and a non-ginsenoside positive control group, respectively. Data are presented as the mean ± SD (n = 6). *p < 0.05, vs DMSO group; ^ap < 0.05, vs CK group

Fig. 3

Effects of six ginsenosides on the apoptosis of RAW264.7 cells and HUVEC cells. Representative scatter plots of Annexin V/PI analysis of RAW264.7 cells (a) and HUVEC cells (c). Percentage of early apoptotic cells, late apoptotic cells and total apoptotic cells of RAW264.7 cells (c) and HUVEC cells (d) in each group. Data were presented as the mean ± SD (n = 3). *p < 0.05, vs DMSO group; ^ap < 0.05, vs CK group

Fig. 4

Effects of six ginsenosides on arthritis development in CIA mice after treatment. a The joint images of representative mice in each group at the end of administration. b The arthritis index treated with different ginsenoside as a function of time. Data were expressed as mean ± SD, n = 6. p < 0.05 for each ginsenoside group vs the control group. c Pathological sections of hematoxylin and eosin stained ankle joints in each group, the arrows indicate pannus formation and bone destruction. (Scale bar = 100 μm). PBS and 0.5% Tween was set as normal group and control group, respectively

Fig. 5

a Effects of ginsenosides on TNF-a and IL-6 protein expression in joint tissues of mice (Scale bar = 100 μm). Immunohistochemical staining of osteogenic nuclei is shown to be yellow or brown, indicating positive TNF-α or IL-6. The mean density of TNF-α (b) and IL-6 (c) protein expression in mouse joints after different treatments. The level of TNF-α (d) and IL-6 (e) proinflammatory factor in the supernatant of mouse joint after different treatments, respectively. Values were expressed as means ± SD, n = 6; (^##p < 0.01 vs. the normal group; * p < 0.05 and ** p < 0.01 vs the control group; ^ap < 0.05, vs CK group)

Fig. 6

Effects of ginsenosides on M1-type macrophages (a, b) and T cells (c, d, e) in arthritis. a Representative flow scatter plots of CD45 + CD86 and CD45 + F4/80 expressions in the joints of mice in each group. b The content of M1-type macrophages (co-expressed CD45 + CD86 and CD45 + F4/80) in the joints of mice in each group. c Representative flow scatter plots of CD45 + CD8+ and CD45 + CD4+ expressions in the joints of mice in each group. d Expression of CD45 + CD4 + in all groups. e Expression of CD45 + CD8 + in all groups. (^##p < 0.01, vs normal group; *p < 0.05, vs control group; ^ap < 0.05, vs CK group)

Fig. 7

Hematoxylin and Eosin stained sections of the heart, liver, spleen, lung, and kidney of the mice treated with six ginsenosides, PBS and 0.5% Tween was set as normal group and control group, respectively