EGCG和PC-B2 联合用药对AFB1 致肝细胞损伤的保护作用及其机制

目的:探讨多酚类植物化学物表没食子儿茶素没食子酸酯(EGCG)与原花青素B2(PC-B2),对黄曲霉素B1(AFB1)起的肝细胞损伤保护作用及机制。方法:选用人胚肝细胞(L-02细胞)进行体外实验研究,实验分为6组,分别为空白组,溶剂对照组,AFB_1染毒组,AFB_1染毒+EGCG组,AFB_1染毒+PC-B_2组和AFB_1染毒+EGCG+PC-B_2组。采用MTT检测细胞存活度,通过单细胞凝胶电泳试验(SCGE)检测细胞DNA损伤,通过流式细胞法检测细胞凋亡,采用Western blot法检测凋亡信号通路相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Caspase-9,p53蛋白表达的水平。结果:EGCG与PC-B_2均能降低AFB_1所致L-02细胞的生长抑制,使AFB_1对肝细胞的生长抑制率从61.12%降至42.18%和46.72%;EGCG与PC-B_2联用则效果更佳。SCGE实验结果表明EGCG与PC-B_2单用与联用均能减小AFB_1引起的L-02细胞DNA损伤(P0.05)。EGCG与PC-B_2单用与联用均能显著降低AFB_1所致L-02细胞的早期凋亡率(P0.05)。Western blot实验结果表明,AFB_1染毒后,EGCG和/或PC-B_2处理均能显著降低Caspase-3,Caspase-9的表达(P0.01),提高Bcl-2/Bax比值,而对p53表达影响不明显。结论:EGCG与PC-B_2通过降低L-02细胞DNA损伤与细胞凋亡率,从而降低AFB_1对人肝细胞的损伤作用,其作用机制可能与下调Caspase-3,Caspase-9的表达,升高Bcl-2/Bax有关。

Protective Mechanism of Epigallocatechin Gallate (EGCG) and Procyanidins-B2 (PC-B2 ) on Liver Cell from AFB1 -induced DNA Damage

Objective:To evaluate the protective effects of (-)-epigallocatechin gallate (EGCG) combined with procyanidins B₂ (PC-B₂ ) on liver cell damage caused by aflatoxin B₁ (AFB₁ ). Method:L-02 cells were selected in an experimental study in vitro and divided into 6 groups:control group; solvent control group; AFB₁ group; EGCG+AFB₁ group; PC-B₂ +AFB₁ group; EGCG+PC-B₂ +AFB₁ group. The cells survival was determined by MTT; DNA damage was detected by single cell gel electrophoresis (SCGE); the flow cytometry method was used to detect cell apoptosis; and the expressions of protein about apoptosis signaling pathways (Bcl-2, Bax, Caspase-3, Caspase-9, P53) were detected with Western blot method. Result:EGCG and PC-B₂ can reduce liver cell growth inhibition caused by AFB₁ from 61.12% to 42.18% and 46.72%; EGCG combined with PC-B₂ made a better effect. SCGE results showed that either EGCG or PC-B₂ can reduce liver cell DNA damage caused by AFB₁ . Simple or combined administration of EGCG and PC-B₂ can decrease the early apoptosis rate of L-02 cells induced by AFB₁ (P<0.05). Western blot experiments showed that after treatment with AFB₁ , EGCG or (and) PC-B₂ treatment can reduce the expression of Caspase-3 and Caspase-9 significantly (P<0.01), and improve the ratio of Bcl-2 to Bax, but with no obvious expression of P53. Conclusion:EGCG and PC-B₂ can protect the human normal liver cells from the damage induced by AFB₁ reducing L-02 cell DNA damage and inhibiting the apoptosis, and the mechanism may be correlated with the down-regulation of Caspase-3 and Caspase-9 expressions and the increase in Bcl-2/Bax.