Figures

Figure. 1. AEBS suppresses inflammatory arthritis in CIA mice. Both the arthritis score and the incidence of arthritis were reduced in CIA mice given AEBS. Mice were immunized with CII. Seven days later, mice were given oral AEBS (60 mg/kg) or vehicle, daily, for 8 weeks. (A) The mean arthritis scores ±± SDs (left panel) and arthritis incidence scores (right panel). (B) Histological data on joints from CIA mice given AEBS or vehicle. Mice were sacrificed on day 49 after immunization. Joint tissue sections were stained with H&E, toluidine blue, and safranin O. Joints from mice given AEBS exhibited mild erosive arthritis, whereas those from vehicle-treated mice exhibited markedly erosive destructive arthritis. Representative photographs are shown. Original magnifications ×40 or ×200, as indicated. The histological scores (measuring inflammation and the extent of cartilage damage) in mice given AEBS (n = 5) or vehicle (n = 5) are shown in the right graph. Data are expressed as means ± ± SDs. ***P < 0.01 compared to the vehicle-treated control group. (C) Tissue sections from joints of CIA mice given AEBS or vehicle were stained with anti-nitrotyrosine and anti-iNOS antibodies. Cells stained with either antibody are brown in color. Original magnification ×400. The cells showing positive nitrotyrosine, and iNOS were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (D) Levels of total circulating IgG, IgG1, and IgG2a in CIA mice given AEBS (n = 8) or vehicle (n = 8). Total IgG, IgG1, and IgG2a levels were determined in sera of individual mice via ELISA. Data are expressed as means ±± SDs. * P < 0.05, *** P < 0.001 compared to the vehicle-treated group. Each experiment was performed 3 times. Figure. 2. AEBS reduces Th17 cell numbers in CIA mice. Tissues were obtained and stained when the mice were sacrificed (8weeks from immunization). (A) AEBS reduced the expression levels of IL-17, IL-6, TNF-α, and IL-1β in synovial tissues. Tissue sections from joints of CIA mice given AEBS (n = 5) or vehicle (n = 5) were stained with anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-1β antibodies; or anti-isotype control antibodies. Stained cells are brown in color. Original magnification ×400. The cells showing positive IL-17, IL-6, TNF- α, and IL-1β were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (B) Pooled splenocytes from CIA mice given AEBS were cultured with PMA (25 ng/ml) and ionomycin (250 ng/ml) for 30 min and p-IκB levels determined via Western blotting. Data are expressed as means ±± SDs. *P < 0.05 compared to the vehicle-treated group. Each experiment was performed 3 times. Figure. 3. AEBS decreases Th17 cell numbers in CIA mice. (A) Spleens from each mouse were stained for CD4+p-STAT3(Y705)+, CD4+p-STAT3(S727)+, CD4+STAT3+, and CD4+IL-17+ T cells using antibodies specific for CD4, STAT3Y705, STAT3S727, and IL-17. The cell populations were analyzed via laser confocal microscopy (original magnification ×400). (B) The numbers of T cells positive for CD4+STAT3Y705+, CD4+STAT3S727+, CD4+STAT3+, and CD4+IL-17+ in each mouse were visually counted at a higher magnification (after projection of fields onto a screen) and mean values are shown. (C) Spleens from each mouse were stained for CD4+p-STAT5(Y694)+, CD4+STAT5+, and CD4+CD25+Foxp3+. (D) The numbers of T cells positive for CD4+p-STAT5(Y694)+, CD4+STAT5+, and CD4+CD25+Foxp3+ in each mouse were visually counted at a higher magnification (after projection of fields onto a screen) and mean values are shown. (Number of AEBS treated mice = 5, Number of vehicle treated mice = 5) Data are expressed as means ±± SDs. *P < 0.05, **P < 0.01. Each experiment was performed 3 times. Figure. 4. AEBS represses IL-17 synthesis by mouse CD4+ T cells. (A) CD4+ T cells isolated from the spleens of naïve DBA/1J mice were cultured under Th17-polarizing conditions in the presence or absence of AEBS (50–200μg/ml). Three days later, the cells were stained with antibodies against CD4 and IL-17, as described in Materials and Methods. (B) The IL-17 levels in the culture supernatants described in (A) above were measured via ELISA. (C) The levels of IL-17 and Ahr mRNAs were determined by real-time PCR. (Number of AEBS treated mice = 3, Number of vehicle treated mice = 3) Each experiment was performed 3 times. Figure. 5. AEBS represses IL-17 synthesis by human PBMCs. (A) CD4+ T cells isolated from PBMCs of normal healthy volunteers were cultured under Th17-polarizing conditions in the presence or absence of AEBS (50–200μg/ml). Three days later, the cells were stained with antibodies against CD4 and IL-17, as described in the Materials and Methods section. The proportion of Th17 cells were measured by Side-scattered light (SSC) method. (B) The levels of IL-17 in the culture supernatants described in (A) above were measured using sandwich ELISA (R&D Systems, Minneapolis, MN). (C) The levels of IL-17 and RORc mRNAs were determined using real-time PCR. (Number of AEBS group = 3, Number of vehicle group = 3) Each experiment was performed 3 times. Figure. 6. AEBS inhibits osteoclast formation in DBA/1J mice. (A) In vitro, AEBS inhibited osteoclast formation in a dose-dependent manner. BMM cells from naïve DBA/1J mice treated with vehicle were cultured in the presence of M-CSF (10 ng/ml), and/or RANKL (50 ng/ml), and/or AEBS (50–150 μg/ml). The medium was changed every 2 days. After 7 days, cells were stained to detect TRAP activity. Original magnification ×100. ***P < 0.001. (B) The levels of mRNAs encoding the osteoclastogenic markers MMP-9, the calcitonin receptor, TRAP, OSCAR, and NFATc1 were measured via real-time PCR under the experimental conditions described in (A) above. **P < 0.01, ***P < 0.001. (Number of AEBS treated mice = 3, Number of vehicle treated mice = 3) Each experiment was performed 3 times. Figure. 7. AEBS inhibits the differentiation of human monocytes into osteoclasts. (A) AEBS inhibited osteoclast formation, in a dose-dependent manner, in the presence of M-CSF and RANKL. Human monocytes from healthy volunteers (n = 3) were cultured in the presence of M-CSF (25 ng/ml), and/or RANKL (30 ng/ml), and/or AEBS (50–150 μg/ml). After 9 days, cells were stained to detect TRAP activity. Representative photographs from each group are shown in the left panels. The numbers of multinucleated TRAP+ cells are shown in the right panel. Original magnification ×100. ***P < 0.001. (B) The levels of mRNAs encoding TRAP, MMP-9, and cathepsin K were quantified via real-time PCR. **P < 0.01, ***P < 0.001, compared to treatment with M-CSF and RANKL. (Number of AEBS group = 3, Number of vehicle group = 3) Each experiment was performed 3 times.图1 AEBS可抑制胶原诱导性关节炎（Collagen-Induced Arthritis, CIA）小鼠的炎性关节炎。给予AEBS的CIA小鼠的关节炎评分与关节炎发病率均有所降低。实验中，小鼠先经II型胶原（CII）免疫，7天后每日经口给予AEBS（60 mg/kg）或溶剂对照，持续8周。(A) 平均关节炎评分±标准差（左图）与关节炎发病率评分（右图）。(B) 给予AEBS或溶剂对照的CIA小鼠的关节组织学结果。免疫后第49天处死小鼠，取关节组织切片，分别用苏木精-伊红（H&E）、甲苯胺蓝与番红O染色。给予AEBS的小鼠关节仅表现为轻度侵蚀性关节炎，而溶剂对照组小鼠则出现显著的侵蚀破坏性关节炎，图中展示为代表性图像。放大倍数为×40或×200，如图标注。右侧柱状图展示了AEBS处理组（n=5）与溶剂对照组（n=5）的组织学评分（评估炎症程度与软骨损伤范围），数据以平均值±标准差表示，与溶剂对照组相比，***P<0.01。(C) 给予AEBS或溶剂对照的CIA小鼠的关节组织切片，分别用抗硝基酪氨酸与抗诱导型一氧化氮合酶（iNOS）抗体染色，阳性染色细胞呈棕褐色，放大倍数为×400。由4名研究者在高倍镜下（投影至屏幕）对硝基酪氨酸与iNOS阳性细胞进行计数，结果以平均值表示（细胞数/视野）。(D) 给予AEBS（n=8）或溶剂对照（n=8）的CIA小鼠的循环总免疫球蛋白G（IgG）、IgG1与IgG2a水平。通过酶联免疫吸附试验（ELISA）检测个体小鼠血清中的总IgG、IgG1与IgG2a水平，数据以平均值±标准差表示，与溶剂对照组相比，*P<0.05，***P<0.001。所有实验均重复3次。 图2 AEBS可降低CIA小鼠体内Th17细胞数量。小鼠处死后（免疫后8周）获取组织并进行染色。(A) AEBS可降低滑膜组织中IL-17、IL-6、TNF-α与IL-1β的表达水平。取给予AEBS（n=5）或溶剂对照（n=5）的CIA小鼠的关节组织切片，分别用抗IL-17、抗IL-6、抗TNF-α、抗IL-1β抗体或同型对照抗体染色，阳性染色细胞呈棕褐色，放大倍数为×400。由4名研究者在高倍镜下（投影至屏幕）对IL-17、IL-6、TNF-α与IL-1β阳性细胞进行计数，结果以平均值表示（细胞数/视野）。(B) 从给予AEBS的CIA小鼠体内获取混合脾细胞，用佛波酯（PMA，25 ng/ml）与离子霉素（ionomycin，250 ng/ml）刺激30分钟，通过蛋白质印迹法（Western blotting）检测磷酸化IκB（p-IκB）水平。数据以平均值±标准差表示，与溶剂对照组相比，*P<0.05。所有实验均重复3次。 图3 AEBS可减少CIA小鼠体内Th17细胞数量。(A) 分别使用针对CD4、STAT3Y705、STAT3S727与IL-17的特异性抗体，对每只小鼠的脾脏进行CD4+p-STAT3(Y705)+、CD4+p-STAT3(S727)+、CD4+STAT3+与CD4+IL-17+ T细胞染色，通过激光共聚焦显微镜（laser confocal microscopy）分析细胞群（放大倍数×400）。(B) 在高倍镜下（视野投影至屏幕后）对每只小鼠体内CD4+STAT3Y705+、CD4+STAT3S727+、CD4+STAT3+与CD4+IL-17+阳性T细胞数量进行人工计数，结果以平均值表示。(C) 对每只小鼠的脾脏进行CD4+p-STAT5(Y694)+、CD4+STAT5+与CD4+CD25+Foxp3+染色。(D) 在高倍镜下（视野投影至屏幕后）对每只小鼠体内CD4+p-STAT5(Y694)+、CD4+STAT5+与CD4+CD25+Foxp3+阳性T细胞数量进行人工计数，结果以平均值表示。（AEBS处理组小鼠数=5，溶剂对照组小鼠数=5）数据以平均值±标准差表示，*P<0.05，**P<0.01。所有实验均重复3次。 图4 AEBS可抑制小鼠CD4+ T细胞合成IL-17。(A) 从未免疫的DBA/1J小鼠脾脏中分离CD4+ T细胞，在Th17细胞极化条件下，分别加入或不加入AEBS（50–200μg/ml）进行培养。3天后，按照材料与方法部分所述，使用抗CD4与抗IL-17抗体对细胞进行染色。(B) 通过ELISA检测上述(A)中培养上清液中的IL-17水平。(C) 通过实时荧光定量PCR（real-time PCR）检测IL-17与芳香烃受体（Ahr）的mRNA水平。（AEBS处理组小鼠数=3，溶剂对照组小鼠数=3）所有实验均重复3次。 图5 AEBS可抑制人外周血单个核细胞（PBMCs）合成IL-17。(A) 从健康志愿者的外周血单个核细胞中分离CD4+ T细胞，在Th17细胞极化条件下，分别加入或不加入AEBS（50–200μg/ml）进行培养。3天后，按照材料与方法部分所述，使用抗CD4与抗IL-17抗体对细胞进行染色，通过侧向散射光（SSC）法检测Th17细胞比例。(B) 通过夹心ELISA（sandwich ELISA，R&D Systems公司，明尼阿波利斯，明尼苏达州）检测上述(A)中培养上清液中的IL-17水平。(C) 通过实时荧光定量PCR（real-time PCR）检测IL-17与维甲酸相关孤儿受体γt（RORc）的mRNA水平。（AEBS组例数=3，溶剂对照组例数=3）所有实验均重复3次。 图6 AEBS可抑制DBA/1J小鼠体内破骨细胞形成。(A) 体外实验中，AEBS以剂量依赖性方式抑制破骨细胞形成。将未免疫的DBA/1J小鼠的骨髓巨噬细胞（BMM cells）在溶剂对照条件下，分别加入巨噬细胞集落刺激因子（M-CSF，10 ng/ml）、核因子κB受体活化因子配体（RANKL，50 ng/ml）与/或AEBS（50–150 μg/ml）进行培养，每2天更换一次培养基。7天后，对细胞进行抗酒石酸酸性磷酸酶（TRAP）染色以检测破骨细胞。放大倍数为×100，***P<0.001。(B) 在上述(A)的实验条件下，通过实时荧光定量PCR（real-time PCR）检测破骨细胞生成相关标记物MMP-9、降钙素受体、TRAP、破骨细胞相关受体（OSCAR）与活化T细胞核因子c1（NFATc1）的mRNA水平。**P<0.01，***P<0.001。（AEBS处理组小鼠数=3，溶剂对照组小鼠数=3）所有实验均重复3次。 图7 AEBS可抑制人单核细胞向破骨细胞分化。(A) 在M-CSF与RANKL存在的条件下，AEBS以剂量依赖性方式抑制破骨细胞形成。从健康志愿者（n=3）体内获取人单核细胞，分别加入M-CSF（25 ng/ml）、RANKL（30 ng/ml）与/或AEBS（50–150 μg/ml）进行培养。9天后，对细胞进行TRAP染色以检测破骨细胞，各组代表性图像展示于左侧面板，多核TRAP阳性细胞数量展示于右侧面板，放大倍数为×100，***P<0.001。(B) 通过实时荧光定量PCR（real-time PCR）定量检测TRAP、MMP-9与组织蛋白酶K的mRNA水平。与仅用M-CSF与RANKL处理的组相比，**P<0.01，***P<0.001。（AEBS组例数=3，溶剂对照组例数=3）所有实验均重复3次。