荔枝核总黄酮干预大鼠肝星状细胞核因子κB核转位及相关蛋白表达的研究

Study on the effects of total flavonoids from litchi nucleus on nuclear translocation of nuclear factor-kappa B and related protein expression in rat hepatic stellate cell

目的研究荔枝核总黄酮（TFL）对体外由转化生长因子p1（TGFD1）诱导活化的大鼠肝星状细胞T6细胞株（HSC-T6）中核因子KB（NF-κB）核转位及相关蛋白表达的影响，探究该药抗肝纤维化的作用机制。方法体外培养HSC-T6，通过TGFD1诱导24h后以125、250、500μg／ml的TFL干预48h，采用激光共聚焦显微镜观察TFL对HSC-T6内NF-κB核转位的影响，并以Westernblot法检测TFL对细胞中TLR4、P-IκBCt、P-NF-κBp65、NF-κB及CollagenI蛋白表达水平的影响，免疫荧光法检测TLR4、P-NF-κBp65的表达。数据采用均数±标准差表示，先进行方差齐性检验，再行单因素方差分析（ANOVA），组间多重比较采用LSD检验，P〈0．05为差异具有统计学意义。结果激光共聚焦显微镜可见TFL抑制活化HSC-T6中NF-KB的核转位，并具有浓度依赖性。TFL可下调HSC-T6中TLR4、P-IKBQ、p-NF-κBp65、NF-κB及CollagenI的蛋白表达水平，同时具有浓度依赖性。结论TFL的抗肝纤维化的机制可能与其抑制活化HSC-T6内NF-κB的核转位，下调HSC-T6中TLR4、P-IKBQ、D-NF-κBp65、NF-κB及CollagenI的表达相关。

Objective The effect of total flavonoids of litchi （TFL） on nuclear translocation of nuclear factor-kappa B （NF- kappa B） in rat hepatic stellate cell line （HSC-T6） induced by transforming growth factor - beta 1 （TGF- beta 1） in vitro was studied to explore the mechanism of action of anti-hepatic fibrosis drugs. Methods HSC-T6 was cultured in vitro, induced by TGFI31 for 24 h, and then treated with TFL at 125, 250 and 500 μg/ml for 48 h. The effect of TFL on NF-r,B nuclear translocation in HSC-T6 was observed by confocal laser microscopy. The effects of TFL on the expression of TLR4, p-1κB, Bct, p-NF-κB p65, NF-κB and Collagen I protein were detected by western blot. The expressions of TLR4 and p-NF-κB p65 were detected by immunofluorescence. Data were presented as mean＋SEM. Homogeneity test of variance was performed and then followed by one-way analysis of variance （ANOVA）. The multiple comparisons between groups were performed by LSD test. P 〈 0.05 was considered statistically significant. Results Confocal laser scanning microscopy showed TFL inhibited the nuclear translocation of NF-κB in activated HSC-T6 in a concentration-dependent manner and TFL down regulated the protein expression levels of TLR4, p-IκB α, p-NF-nB p65, NF-κB and collagen I protein in HSC-T6 in a concentration-dependent manner. Conclusion The mechanism of TFL against hepatic fibrosis may be related to the inhibition of nuclear translocation of NF- Kb in the activated HSC-T6 and the expression of TLR4, P-iKba, P-nf-κb p65, NF-κb and collagen I protein in HSC-T6.