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目的探讨莱菔硫烷(sulforaphane,SFN)对脂多糖(LPS)诱导的星形胶质细胞(astrocyte,AST)增殖的影响及其机制。方法体外培养小鼠原代星形胶质细胞,给予0.1μg/m L LPS刺激星形胶质细胞活化增殖;同时分别给予不同浓度的莱菔硫烷(10、15、20μmol/L)处理星形胶质细胞24 h后,CCK8法检测细胞增殖情况;选取20μmol/L作为干预剂量行GFAP、Ki67双标免疫荧光染色及流式细胞仪细胞周期检测,进一步验证莱菔硫烷对LPS诱导星形胶质增殖的作用;Western blot检测细胞周期相关蛋白P27kip1、Cyclin D1、PCNA的表达情况。结果 CCK8法检测显示0.1μg/m L LPS能显著促进星形胶质细胞增殖(P<0.01),而加入20μmol/L莱菔硫烷能显著抑制LPS诱导的星形胶质细胞增殖(P<0.01),并能减少LPS刺激下的Ki67阳性细胞(P<0.05);细胞周期分析提示20μmol/L莱菔硫烷可减少LPS诱导下的星形胶质细胞S期比例,增加G1期细胞比例(P<0.05);Western blot提示20μmol/L莱菔硫烷能下调LPS刺激下PCNA、Cyclin D1的表达,上调P27kip1表达(P<0.05)。结论莱菔硫烷可以通过调控星形胶质细胞细胞周期,抑制体外LPS诱导的星形胶质细胞增殖。
Objective To investigate the effects of sulforaphane (SFN) on the proliferation of astrocyte (AST) induced by lipopolysaccharide (LPS) and its mechanism. Methods Primary mouse astrocytes were cultured in vitro and stimulated with 0.1 μg / mL LPS to stimulate astrocyte activation and proliferation. At the same time, different concentrations of Sulforaphane (10, 15 and 20 μmol / L) Glial cells 24 h, CCK8 assay cell proliferation; selected 20μmol / L as an intervention dose of GFAP, Ki67 double-labeled immunofluorescence staining and flow cytometry cell cycle test to further verify the effect of sulforaphane on LPS-induced starches The effects of P27kip1, Cyclin D1 and PCNA on cell cycle were analyzed by Western blot. Results CCK8 assay showed that 0.1μg / ml LPS could significantly promote astrocyte proliferation (P <0.01), while 20μmol / L sulfasalazine could significantly inhibit LPS-induced astrocyte proliferation (P <0.01) ) And Ki67 positive cells (P <0.05). The cell cycle analysis indicated that 20μmol / L sulforaphane could reduce the proportion of S phase of astrocytes induced by LPS and increase the proportion of cells in G1 phase <0.05). Western blot suggested that 20μmol / L sulfasalazine could down-regulate the expression of PCNA and Cyclin D1 and up-regulate the expression of P27kip1 (P <0.05). Conclusion Sulforaphane can inhibit the proliferation of astrocytes induced by LPS in vitro by regulating the cell cycle of astrocytes.