Yan Shu-Xun, Wang Ying
Department of Endocrinology and Metabolism, The First Affiliated Hospital, Henan College of Traditional Chinese Medicine, Zhengzhou City 450000, Henan Province, China.
Clin Exp Ophthalmol. 2006 Apr;34(3):265-71. doi: 10.1111/j.1442-9071.2006.01190.x.
To explore the effects of Triptolide, the principal active diterpenoid from the Chinese Medicinal Herb Tripterygium Wilfordii Hook F that has immunosuppressive and anti-inflammatory properties, on cell proliferation, hyaluronic acid (HA) synthesis, and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) and CD40 on cultured retro-ocular fibroblasts (RFs) from patients with Graves' ophthalmopathy.
After two to five passages, cultured RFs were incubated for 48 h within a medium alone or in the presence of recombinant human interferon-gamma (IFN-gamma) and various concentrations of Triptolide. Cell viability was assessed by MTT (3-[4.5-dimethylahiazol-2-yl]-2,5-diphenyltetrazolium Bromide). RFs proliferation was assessed by [(3)H]-thymidine incorporation assay. Flow cytometry was used to investigate the amount of HLA-DR, ICAM-1 and CD40. HA synthesis was measured by radioimmunoassay.
Cell viability was not detrimentally affected when incubated with Triptolide from 0.01 microg/L to 10 microg/L for 48 h, and decreased with 20 microg/L Triptolide. The incorporation of [(3)H]-thymidine of RFs was 55 476 +/- 15 842 cpm incubated with medium alone or 18 352 +/- 3568 cpm with 10 microg/L Triptolide (t = 5.600, P < 0.01). Initially, the percentage of positive cells of HLA-DR, ICAM-1 and CD40 on RFs were 4.75 +/- 2.13%, 17.53 +/- 10.12% and 6.38 +/- 2.23%, respectively, and the synthesis of HA was 100 +/- 12%. Compared with basal values, 48-h incubation with IFN-gamma (100 U/mL) significantly enhanced the amount of HLA-DR, ICAM-1 and CD40, and HA synthesis. The values were 60.58 +/- 10.12% (t = 13.224, P < 0.01), 62.66 +/- 18.17% (t = 5.315, P < 0.01), 57.67 +/- 13.61% (t = 9.110, P < 0.01) and 164 +/- 22% (t = 9.238, P < 0.01), respectively. Triptolide 0.01 microg/L had little effect on IFN-gamma-induced HLA-DR, ICAM-1 and CD40 amounts, as well as HA synthesis. When the concentration ranged from 0.1 microg/L to 10 microg/L, Triptolide inhibited IFN-gamma-induced RFs activation in a dose-dependent manner. It was also found that Triptolide had the same inhibiting effects on IFN-gamma-induced RFs and skin fibroblasts from patients with normal individual conditions.
Triptolide could inhibit IFN-gamma-induced activation of RFs derived from patients with Graves' ophthalmopathy.
雷公藤甲素是中草药雷公藤中主要的具有免疫抑制和抗炎特性的活性二萜类化合物，本研究旨在探讨其对Graves眼病患者培养的眼后成纤维细胞（RFs）的细胞增殖、透明质酸（HA）合成以及人类白细胞抗原-DR（HLA-DR）、细胞间黏附分子-1（ICAM-1）和CD40表达的影响。
培养的RFs传代2至5次后，在单独培养基中或在重组人干扰素-γ（IFN-γ）和不同浓度雷公藤甲素存在的情况下孵育48小时。通过MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四氮唑）评估细胞活力。通过[³H]-胸腺嘧啶核苷掺入试验评估RFs增殖。使用流式细胞术研究HLA-DR、ICAM-1和CD40的含量。通过放射免疫测定法测量HA合成。
当与0.01μg/L至10μg/L的雷公藤甲素孵育48小时时，细胞活力未受到不利影响，但在20μg/L雷公藤甲素作用下细胞活力降低。单独培养基孵育的RFs的[³H]-胸腺嘧啶核苷掺入量为55476±15842 cpm，而10μg/L雷公藤甲素作用下为18352±3568 cpm（t = 5.600，P < 0.01）。最初，RFs上HLA-DR、ICAM-1和CD40的阳性细胞百分比分别为4.75±2.13%、17.53±10.12%和6.38±2.23%，HA合成量为100±12%。与基础值相比，用IFN-γ（100 U/mL）孵育48小时显著增加了HLA-DR、ICAM-1和CD40的含量以及HA合成。其值分别为60.58±10.12%（t = 13.224，P < 0.01）、62.66±18.17%（t = 5.315，P < 0.01）、57.67±13.61%（t = 9.110，P < 0.01）和164±22%（t = 9.238，P < 0.01）。0.01μg/L的雷公藤甲素对IFN-γ诱导的HLA-DR、ICAM-1和CD40含量以及HA合成影响不大。当浓度范围为0.1μg/L至10μg/L时，雷公藤甲素以剂量依赖性方式抑制IFN-γ诱导的RFs活化。还发现雷公藤甲素对IFN-γ诱导的Graves眼病患者的RFs和正常个体的皮肤成纤维细胞具有相同的抑制作用。
雷公藤甲素可抑制IFN-γ诱导的Graves眼病患者来源的RFs活化。
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