Total Alkaloids of Sophora alopecuroides Linn. Attenuates Rheumatoid Arthritis Through Regulating Follicular Helper T Cells

Abstract

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease with abnormal differentiation of follicular helper T (Tfh) cells, Total alkaloids of Sophora alopecuroides Linn. (Leguminosae) (TASA) have potential effects on collagen-induced arthritis (CIA) mice, while the mechanism needs further elucidation. The purpose of this study is to explore the regulation of TASA on rheumatoid arthritis and related mechanism.

Methods: The proportion of Tfh and B lymphocytes in peripheral blood lymphocytes of RA patients was examined by flow cytometry. We constructed the collagen induced arthritis DBA/1J mice model. Between days 15 and 45 following the first immunization, the mice were treated intraperitoneally with saline, TASA (100, 50, and 25 mg/kg), and dexamethasone (DXM) for 30 days. Molecular biological techniques such as FCM, PCR, ELISA, and Western-blotting were used to examine Tfh cells and associated signal pathways.

Results: Our results indicated that the follicular helper T cells and B lymphocytes in rheumatoid arthritis patients were significantly increased compared with the healthy control. The percentage of Tfh cells are correlated with RA related inflammatory factors. Total alkaloids of Sophora alopecuroides Linn. could significantly attenuate joint swelling. Meanwhile, it reduced the frequencies of spleen Tfh, B lymphocytes and the expression of TLR2, TLR9, p-NF-κBp65, CXCR5, Bcl-6, ICOS of ankle joints in CIA mice.

Conclusion: Total alkaloids of Sophora alopecuroides Linn. may down-regulate the frequency and function of Tfh cells and inhibit GCB cells via TLRs/NF-κB signal pathway to relieve the immune-pathological progression of CIA mice.

Keywords: C-X-C chemokine receptor type 5; collagen-induced arthritis; toll-like receptors; total alkaloids of Sophora alopecuroides Linn..

Figure 1

The number of B lymphocytes and Tfh cells in RA patients was detected by flow cytometry. a. Classification strategy and flow chart of Tfh cell and B lymphocytes phenotypes by flow cytometry. Tfh cells (CD4⁺CD3⁺ICOS⁺CXCR5⁺), B lymphocytes (CD19⁺). b. The frequency of Tfh cells (*P<0.05) and B lymphocytes (*P<0.05) in RA patients and healthy controls.

Figure 2

Correlation analysis of inflammatory factors and the percentage of Tfh cells to B lymphocytes in RA patients. (a) B lymphocytes (r = 0.4238, P = 0.0196). (b) RF (r = 0.4468, P = 0.0133).(c) IL-21 (r = 0.4518, P = 0.0122). (d) IL-10 (r = −0.4704, P = 0.0087). Serum levels of Tfh cell related cytokines IL-21 and IL-10 in healthy controls and RA patients were detected by ELISA. (e) Serum IL-21 level. (f) Serum IL-10 level. **P < 0.01 compared with healthy controls.

Figure 3

Determination of aloperine, sophoridine, oxymatrine, oxysophoridine, matrine, sophocarpine in total alkaloids of Sophora alopecuroides L. by HPLC. (From left to right: 1. Matrine 2. Oxysophoridine 3. Sophoridine 4. Oxymatrine 5. Sophocarpine).

Figure 4

TASA therapy relieve the development of arthritis in CIA mice. (a)Representative picture of hind paw (A. Model. B. TASA-L. C. TASA-M. D. TASA-H. E. DXM. F. Normal). (b)Hind joint swelling is measured every three days. (c)Arthritis score is measured every three days. (d) H & E stained pictures of joints (A. Model. B. TASA-L. C. TASA-M. D. TASA-H. E. DXM. F. Normal) (n=6). TASA, Total alkaloids of Sophora alopecuroides L. CIA, collagen-induced arthritis. DXM, dexamethasone. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-M, Medium dose Total alkaloids of Sophora alopecuroides. TASA-H, High dose Total alkaloids of Sophora alopecuroides.

Figure 5

Effects of TASA treatment on Tfh and B lymphocytes in CIA mice. (a)Typical FACS plots from the FCM analysis of the B220⁺Gl-7⁺B lymphocytes obtained from the spleen of CIA mice. (b)Typical FACS plots of CD4⁺ICOS⁺CXCR5⁺ T cells from the spleen lymphocytes. (c)The percentage of Tfh cells (CD4⁺ICOS⁺CXCR5⁺). (d)The percentage of B lymphocytes (B220⁺Gl-7⁺) (n=6). Data are expressed use mean ± SD. ^▲P<0.05 vs the TASA-L group.*P<0.05 vs the Model group. TASA, Total alkaloids of Sophora alopecuroides L. CIA, collagen-induced arthritis. DXM, dexamethasone. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-M, Medium dose Total alkaloids of Sophora alopecuroides. TASA-H, High dose Total alkaloids of Sophora alopecuroides.

Figure 6

The effects of TASA on serum concentrations of IgG1, IL-21, IgG2a, and CII-Ab. (a)IL-21. (b) IgG1. (c)IgG2a. (d)CII-Ab. Levels of IgG1, IL-21, IgG2a were decreased by TASA in a dose-dependent manner (n=6). Data are expressed use mean ± SD. ^▲P<0.05 vs the TASA-L group. *P<0.05 vs the Model group.TASA, Total alkaloids of Sophora alopecuroides L. CIA, collagen-induced arthritis. DXM, dexamethasone. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-M, Medium dose Total alkaloids of Sophora alopecuroides. TASA-H, High dose Total alkaloids of Sophora alopecuroides.

Figure 7

TASA inhibited the protein expression of TLR9, TLR4, TLR2, p-NFκBp65, NF-κBp65, Bcl-6, CXCR5, and ICOS in spleen Tfh cells and ankle joints. (a)Examination of TLR2, TLR4, TLR9, NF-κBp65, p-NFκBp65, CXCR5, ICOS, and Bcl-6 in spleen Tfh using Western blotting and quantification by optical density analysis. (b)Examination of TLR2, TLR4, TLR9, NF-κBp65, p-NFκBp65, CXCR5, ICOS, and Bcl-6 in ankle joints using Western blotting and quantification by optical density analysis (n=6). Data are expressed use mean ± SD. ^▲P<0.05, ^▲▲P<0.01 vs the TASA-L group.*P<0.05, **P<0.01 vs the Model group.TASA, Total alkaloids of Sophora alopecuroides L. CIA, collagen-induced arthritis. DXM, dexamethasone. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-M, Medium dose Total alkaloids of Sophora alopecuroides. TASA-H, High dose Total alkaloids of Sophora alopecuroides.

Figure 8

Effects of TASA on the activation of NF-κB-signaling pathways and Tfh cell-associated factor in ankle joints. (a-g) Levels of NF-κB-signaling pathways and Tfh cell-associated factor were tested by Immunohistochemistry. (a) TLR2. (b) TLR9. (c)NF-κBp65. (d)p-NF-κBp65. (e) CXCR5. (f)ICOS. (g)Bcl-6. (h)Quantitation result of immunohistochemistry (n=6). Data are expressed as mean ± SD, ^▲P<0.05, ^▲▲P<0.01 vs the TASA-L group. *P<0.05, **P<0.01 vs the Model group. TASA, Total alkaloids of Sophora alopecuroides L. CIA, collagen-induced arthritis. DXM, dexamethasone. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-M, Medium dose Total alkaloids of Sophora alopecuroides. TASA-H, High dose Total alkaloids of Sophora alopecuroides. ((a-g), (A) Model. B. TASA-L. C TASA-M. D. TASA-H. E. DXM. F. Normal).

Figure 9

TASA repressed mRNA levels of TLR2, TLR4, TLR9, CXCR5, ICOS, Bcl-6 in spleen Tfh cells. (a) TLR2. (b) TLR4. (c) TLR9. (d) CXCR5. (e) ICOS. (f) Bcl-6. (n=6). Data are expressed as mean ± SD, ^▲P<0.05, ^▲▲P<0.01 vs the TASA-L group. *P<0.05, **P<0.01 vs the Model group.TASA, Total alkaloids of Sophora alopecuroides L. CIA, collagen-induced arthritis. DXM, dexamethasone. TASA-L, Low dose Total alkaloids of Sophora alopecuroides. TASA-M, Medium dose Total alkaloids of Sophora alopecuroides. TASA-H, High dose Total alkaloids of Sophora alopecuroides.

Figure 10

The molecular mechanism of TASA against RA. TASA may regulate the Tfh cells of CIA mice through the TLRs/NF-κB signaling pathway, thereby inhibiting the proliferation and differentiation of germinal center B lymphocytes and reducing the production of autoantibodies to alleviate rheumatoid arthritis.