金银花抗氧化作用的分子学机理研究

The molecular mechanism of anti-oxidative effects of honeysukle: An experimental study

目的探讨金银花抗氧化作用分子学机理。方法采用过氧化氢(H2O2)作用于人正常RBL肝细胞,制作氧化应激损伤的细胞模型。将培养的细胞分为四个实验组:正常对照组(C),H2O2损伤组(H2),金银花前保护组(Jb),金银花后保护组(Ja)。采用TUNEL和DNALadder法,检测各组细胞凋亡情况。应用免疫组化和Westernblot观察各组细胞的HSP-70、NF-kB、Bcl-2、Bax及Caspase-3的表达及免疫反应活性。应用MTT实验检测RBL细胞的生存率。结果Jb组的细胞凋亡率明显低于H2和Ja组,Bcl-2表达增高,HSP-70、NF-kB、Bax和Caspase-3的表达降低。结论金银花对RBL细胞氧化应激损伤具有一定保护作用,且前保护作用效果好于后保护作用,其保护作用机理可能是通过抑制HSP-70和NF-kB的表达,阻抑NF-kB信号传导途径并提高细胞内抗氧化防御酶系水平。

Objective To explore the underlying mechanism of anti-oxidative effects of the honeysukle. Methods The human RBL cells in logarithmic growth phase incubated in DMEM were divided into 4 groups： 1） J pre-protection group （Jb）; prior to incubation with 0.5mmol/L H2O2 for 30min, the RBL cells were incubated with 0.25mg/ml honeysukle for 24h. 2） J post-protection group （Ja）： after incubation with 0.5mmol/L H2O2 for 30 min, the RBL cells were incubated with 0.25mg/ml honeysukle for 24h. 3） H2O2 damage group （H2）： the RBL cells were incubated with 0.5mmol/L H2O2 for 30 min followed by subsequent incubation with 10% FBS for 24h. 4） The control group （C）： the apoptosis was detected by DNA ladder and TNNEL. The expressions of HSP-70, NF-kB,Bcl-2, Bax, and Caspase-3 were demonstrated with immunocytochemistry and Western blotting. The RBL cell survival rate was detected by MTT assay. Results The apoptosis in the Jb group was decreased than that in the J, or H2 group. When the Jb in comparison with Ja and H2 cells, the expressions of Bcl-2 was increased; HSP-70,NF- kB, Bax and Caspase-3 were attenuated. Conclusion The anti-oxidative effect of honeysukle against the H2O2 damage product of ROS on the RBL cells may act through inhibiting expressions of HSP-70 and NF-kB.