Journal of Traditional Chinese Medicine ›› 2022, Vol. 42 ›› Issue (6): 932-939.DOI: 10.19852/j.cnki.jtcm.20220815.004
• Research Articles • Previous Articles Next Articles
HE Lianhua1, LUAN Huijie2, QIN Qingxia2, HE Juan2, CHEN Jian2, HU Yiping2, CAI Yueming2, SUN Desheng3, SHI Yu3(), WANG Qingwen2()
Received:
2021-11-13
Accepted:
2022-01-21
Online:
2022-12-15
Published:
2022-08-15
Contact:
SHI Yu,WANG Qingwen
About author:
Prof. WANG Qingwen, Department of Rheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen 518036, China. wqw_sw@163.com,Telephone: +86-13688802429; +86-13823578405Supported by:
HE Lianhua, LUAN Huijie, QIN Qingxia, HE Juan, CHEN Jian, HU Yiping, CAI Yueming, SUN Desheng, SHI Yu, WANG Qingwen. Shikonin alleviates collagen-induced arthritis mice by inhibiting M1 macrophage polarization[J]. Journal of Traditional Chinese Medicine, 2022, 42(6): 932-939.
Figure 1 SKN inhibits M1 macrophage polarization in the joints of CIA mice (via immunofluorescence, ×40) A, C: photomicrographs of iNOS+ M1 macrophage immunofluorescence stained synovial membrane tissues in knee joints of control, CIA, 4 mg/kg SKN-treated, and 0.5 mg/kg MTX-treated CIA mice are shown, respectively;the timing of the dosage for 23 d from day 21 after primary immunization. A1-A3: the F4/80+ cells, iNOS+ cells, iNOS+ F4/80+ cells in control group; A4-A5: the F4/80+ cells, iNOS+ cells, iNOS+ F4/80+ cells in CIA group; A6-A9: the F4/80+ cells, iNOS+ cells, iNOS+ F4/80+ cells in SKN group; A10-A12: the the F4/80+ cells, iNOS+ cells, iNOS+ F4/80+ cells in MTX group; B, C: photomicrographs of CD68+ M1 macrophage immunofluorescence-stained ankle joints of control, CIA, SKN-treated, and MTX-treated CIA mice are shown, respectively. B1-B3: the F4/80+ cells, CD68+ cells, CD68+ F4/80+ cells in control group; B4-B5: the F4/80+ cells, CD68+ cells, CD68+ F4/80+ cells in CIA group; B6-B9: the F4/80+ cells, CD68+ cells, CD68+ F4/80+ cells in SKN group; B10-B12: the the F4/80+ cells, CD68+ cells, CD68+ F4/80+ cells in MTX group. All data are present as mean ± standard error of mean (n = 6). aP < 0.001, compared to the control group; bP < 0.01, and cP < 0.05, compared to the CIA group. SKN: shikonin; CIA: collagen-induced arthritis; iNOS: nitric oxide synthase; MTX: methotrexate.
Figure 2 Cell morphology of BMDM A: the morphology of BMDMs at 10 × magnification; B: the morphology of BMDMs at 20× magnification; C: the morphology of BMDMs at 40× magnification; D: DAPI in BMDMs via immunofluorescence; E: F4/80 in BMDMs via immunofluorescence; F: the positive rate of F4/80 in BMDMs via immunofluorescence. BMDM: bone marrow-derived-macrophages; DAPI: diphenylindole.
Figure 3 SKN inhibits M1 polarization induced by LPS and IFN-γ in bone marrow-derived macrophages (via immunofluorescence, ×20) BMDMs were pre-treated with LPS 10 μg/L + IFN-γ 20 μg/L or IL-4 20 μg/L + IL-13 20 μg/L for 1 h, and incubated with or without different concentrations of SKN (0.05, 0.1, and 0.2 μM) for 24 h. A, C: effect of SKN on polarization of iNOS+M1 induced by LPS and IFN-γ.A1-A3: the F4/80+ cells, iNOS+ cells, the iNOS+F4/80+ cells in BMDMs; A4-A6: the F4/80+ cells, iNOS+ cells, the iNOS+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs; A7-A9: the F4/80+ cells, iNOS+ cells, the iNOS+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs with SKN 0.05 μM; A10-A12: the F4/80+ cells, iNOS+ cells, the iNOS+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs with SKN 0.1 μM; A13-A15: the F4/80+ cells, iNOS+ cells, the iNOS+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs with SKN 0.05 μm. ; B, C: effect of SKN on polarization of CD68+M1 induced by LPS and IFN-γ. B1-B3: the F4/80+ cells, CD68+ cells, CD68+F4/80+ cells in BMDMs; B4-B6: the F4/80+ cells, CD68+ cells, CD68+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs; B6-B9: the F4/80+ cells, CD68+ cells, CD68+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs with SKN 0.05 μM; B10-B12: the F4/80+ cells, CD68+ cells, CD68+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs with SKN 0.1 μM; B13-B15: the F4/80+ cells, CD68+ cells, CD68+F4/80+ cells in LPS 10 μg/L + IFN-γ 20 μg/L induced BMDMs with SKN 0.2 μm. All data are expressed as mean ± standard error of mean (n = 12). aP < 0.001, compared with the control group. bP < 0.05, cP < 0.01, and dP < 0.001, compared to the vehicle group. SKN: shikonin; iNOS: nitric oxide synthase; LPS: lipopolysaccharide; IFN: interferon; BMDMs: bone marrow-derived-macrophages.
Figure 4 SKN inhibits M1 polarization in CIA mice and LPS + IFN-γ- or IL4 + IL13-induced BMDMs The mice were divided into four groups: the control group (Control), CIA model group (CIA), CIA mice treated with 4 mg/kg SKN group, and CIA mice treated with 0.5 mg/kg MTX group. The timing of the dosage for 23 d from day 21 after primary immunization. BMDMs were pre-treated with LPS 10 μg/L + IFN-γ 20 μg/L or IL-4 20 μg/L + IL-13 20 μg/L for 1 h, and incubated with or without different concentrations of SKN (0.05, 0.1, and 0.2 μM) for 24 h. SKN significantly inhibited the expression of iNOS and CD68, but not arginase and CD206 in LPS + IFN-γ- or IL4 + IL13-induced BMDMs (A and B) (aP < 0.001, compared with the control group. bP < 0.05, cP < 0.01, and dP < 0.001, compared to the vehicle group). Additionally, after SKN and MTX treatment, the expression levels of iNOS and CD68, rather than arginase and CD206, were significantly decreased (C and D). All of the experiments were performed in triplicate. Mean ± standard error of mean was calculated for independent experiments (aP < 0.001, compared to the control group; bP < 0.01, and cP < 0.05, compared to the CIA group.). SKN: shikonin; iNOS: nitric oxide synthase; LPS: lipopolysaccharide; IFN: interferon; IL: interleukin; BMDMs: bone marrow-derived-macrophages; MTX: methotrexate.
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