紫铆花素抑制脂多糖诱导的骨髓源性巨噬细胞炎症反应的研究

目的：研究紫铆花素（butein）对脂多糖（LPS）诱导小鼠骨髓源性巨噬细胞炎症反应的抑制效应，并探讨JAK2-STAT3通路在其中的作用。方法：分离C57BL/6小鼠骨髓，用40 ng/mL的巨噬细胞集落刺激因子刺激7 d，诱导为骨髓源性巨噬细胞（BMDM）。采用500 ng/mL的LPS刺激BMDM 12 h建立炎症模型，butein干预组采用5、10、20 μmol/L butein与LPS共处理，butein单独处理组为20 μmol/L的butein处理12 h，并设立空白对照组。ELISA法检测BMDM培养液中TNF-α、IL-6和NO的水平；流式细胞术检测细胞内ROS水平；Western blot法检测细胞内iNOS、p-JAK2、JAK2、p-STAT3和STAT3蛋白表达水平，并分析P-JAK2/JAK2和P-STAT3/STAT3蛋白表达水平的比值变化。结果：ELISA实验结果显示，LPS刺激BMDM后，培养液中的TNF-α、IL-6和NO含量显著升高，而5、10、20 μmol/L的butein干预可抑制上述促炎因子的分泌，且呈现剂量-效应关系。流式细胞术检测结果显示，butein抑制了LPS活化BMDM的ROS水平升高。Western blot检测结果表明，LPS刺激后BMDM内iNOS蛋白表达水平升高，JAK2和STAT3蛋白表达水平虽未明显变化，但磷酸化水平显著增加。而butein干预可有效抑制JAK2和STAT3蛋白磷酸化。结论：Butein可抑制LPS诱导的巨噬细胞炎症反应，JAK2-STAT3信号通路可能参与调控这一效应，提示butein是一种炎症相关疾病的候选药物。

Inhibition of LPS-induced inflammatory response by butein in bone marrow primary macrophages

OBJECTIVE: To investigate whether butein protects against LPS-induced inflammation in primary bone marrow macrophages,and to analyze the role of JAK2-STAT3 in this process. METHODS: In order to obtain bone marrow-derived macrophages (BMDM),bone marrows samples from C57BL/6 mice were treated with 40 ng/mL GM-CSF for 7 d. BMDM cells were stimulated with 500 ng/mL LPS for 12 h in the presence or absence of 5,10,20 μmol/L butein. Levels of TNF-α,IL-6 and NO in the culture medium were assessed by ELISA specific kits. To detect intracellular levels of reactive oxygen species (ROS),BMDM were stained with DCFH-DA and analyzed by flow-cytometry. Moreover,Western blot assay was used to detect protein expression of iNOS,p-JAK2,JAK2,p-STAT3 and STAT3. Ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were determined. RESULTS: Exposure to LPS significantly increased the levels of TNF-α,IL-6 and NO in the culture medium of BMDM cells. However,administration of 5,10 and 20 μmol/L butein inhibited the production of these proinflammatory factors in a dose-dependent manner. Flow cytometry assays indicate that butein attenuated ROS generation in LPS-activated BMDM macrophages. In addition,Western blot results suggest that LPS promoted iNOS,p-JAK2 and p-STAT3 protein expression,and butein blunted this process. CONCLUSION: Butein inhibited LPS-induced inflammation in BMDM cells by regulating expressions in the JAK2-STAT3 pathway. Therefore,butein may be useful as a drug for control of inflammatory diseases.