天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (11): 1258-1262.doi: 10.11958/j.issn.0253-9896.2015.11.010

• 细胞与分子生物学 • 上一篇    下一篇

荔枝核总黄酮对 TGF-β1 诱导的大鼠肝星状细胞内NF-κB、 α-SMA 表达的影响#br#

刘燕秀， 赵永忠△， 李彩， 董勇， 覃桂金， 成秋宸， 郑清华#br#   

  1. 桂林医学院附属医院消化内科 （邮编541001）
  • 收稿日期:2015-05-06 修回日期:2015-06-13 出版日期:2015-11-15 发布日期:2015-11-15
  • 通讯作者: 赵永忠 E-mail： 13607736670@163.com E-mail:lyanx1989@163.com
  • 作者简介:刘燕秀 （1989）， 女， 硕士在读， 主要从事慢性肝病的基础与临床研究
  • 基金资助:
    国家自然科学基金资助项目 （81360659）； 广西自然科学基金项目 （2012GXNSFAA053105）

Effects of the total flavone of litchi chinensis sonn on expressions of NF-κB and α-SMA in TGF-β1 activated rat hepatic stellate cells#br#

LIU Yanxiu, ZHAO Yongzhong△, LI Cai, DONG Yong, QIN Guijin, CHENG Qiuchen, ZHENG Qinghua#br#   

  1. Department of Digestive Medicine, The Affiliated Hospital of Guilin Medical University, Guangxi Guilin 541001, China
  • Received:2015-05-06 Revised:2015-06-13 Published:2015-11-15 Online:2015-11-15
  • Contact: ZHAO Yongzhong E-mail： 13607736670@163.com E-mail:lyanx1989@163.com

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摘要： 目的 研究荔枝核总黄酮(TFL)对转化生长因子-β1(TGF-β1)诱导的大鼠肝星状细胞 T6(HSC-T6)细胞增殖的影响及其相关分子机制。方法 0.25%胰蛋白酶消化收集细胞， 用含 10%FBS 的 DMEM+5 μg/L TGF-β1 培养液配成单细胞悬液。（1） MTT 法检测细胞增殖活力。将细胞接种于 96 孔培养板中， 设 TGF-β1 组， 对照组 （含 5‰DMSO）， TFL80、160、 320、 640、 800 组 （80、 160、 320、 640、 800 mg/L TFL），每组设 3 个复孔。加药 24、 48、 72 h 后， 采用酶标仪测定 490 nm处各孔吸光度 （A） 值并计算细胞抑制率； 根据半数抑制质量浓度 （IC50） 确定后续实验的药物浓度组及药物作用时间。（2） 采用 PCR 和 Western blot 分别检测 HSC-T6 细胞核转录因子 （NF） -κB、 α-平滑肌肌动蛋白 （α-SMA） mRNA 和蛋白的表达。将细胞接种于 10 cm 培养皿中， 设 TGF-β1 组，对照组 （含 5‰DMSO）， TFL125、 250、 500 组 （125、 250、 500 mg/LTFL），分组培养 48 h 后测定。结果 同一作用时间点， 随着 TFL 浓度的增高， HSC-T6 细胞的 A 值基本逐渐降低，细胞抑制率逐渐上升。TGF-β1 组 NF-κB、 α-SMA mRNA 和蛋白表达量与对照组差异均无统计学意义， TFL125 组与 TGF-β1 组、对照组差异均无统计学意义。随着 TFL 浓度的增高， HSC-T6 细胞 NF-κB、 α-SMA mRNA 和蛋白表达量基本逐渐降低。结论 TFL 可抑制活化的 HSC-T6 细胞增殖， 其可能通过抑制NF-κB、 α-SMA 的表达来发挥抗肝纤维化作用。
 

关键词: 黄酮类, 肝硬化, 大鼠, NF-κB, 荔枝核总黄酮, 肝星状细胞, α-平滑肌肌动蛋白

Abstract: Objective To investigate the effects of total flavonoids of litchi chinensis sonn (TFL) on cell proliferation and the molecular mechanism in rat hepatic stellate cells (HSC-T6) activated by growth factor-β1 (TGF-β1). Methods HSC-T6 cells were treated by 0.25% Trypsin-EDTA and then were digested into single cell suspension by DMEM (10% FBS included), which were mixed with TGF-β1 (5 μg/L). (1) MTT method was used to detect the proliferation of HSC-T6 cells. Cells were cultured in 96-well plate and were treated by different concentrations of TFL including TGF-β1 group, the control group (5‰ DMSO included), and different concentrations of TFL groups (80, 160, 320, 640 and 800 mg/L TFL). Each group has three wells. The absorbance (A) value was measured by enzyme standard meter at the 490 nm wavelength after 24 h, 48 h and 72 h treatment. The cell inhibitory rate was calculated. The subsequent experimental drug concentration and drug treatment time were determined according to half inhibitory concentration (IC50). (2) The expression levels of NF-κB and α-SMA mRNA were detected by PCR (for mRNA) and Western blot assay (for protein). Cells were cultured in the 10 cm culture dish and were divided into different TGF-β1 groups, including TGF-β1 group, the control group (5‰ DMSO included), and different concentrations of TFL groups (125, 250 and 500 mg/L TFL). After 48 h, related indicators were measured. Results At the same treatment time point, with the increased concentrations of TFL, A values were gradually decreased, and the cell inhibitory rates were gradually increased. There were no significant differences in the expressions of NF-κB and α-SMA mRNA between TGF-β1 group and control group. And there were no significant differences in the expressions of NF-κB and α-SMA mRNA between TFL125 group, TGF-β1 group and control group. There was a gradually decrease in the expressions of NF-κB and α-SMA mRNA and protein with the increased concentrations of TFL. Conclusion TFL can inhibit TGF-β1-induced HSC-T6 cell proliferation, which is involved in the inhibited expressions of NF-κB and α-SMA to anti-fibrotic effects in liver fibrosis.

Key words: flavones, liver cirrhosis, rats, NF-kappa B, total flavone of litchi chinensis sonn, hepatic stellate cell, α-smooth muscle actin