盐酸小檗碱对肝星状细胞自噬和凋亡的影响*

Effect of berberine hydrochloride on autophagy and apoptosis in hepatic stellate cells*

摘要:

目的 研究盐酸小檗碱对肝星状细胞（HSCs）自噬和凋亡的影响。方法 取指数生长期HSC-LX2细胞，实验分组：溶剂对照组（1‰ DMSO）、盐酸小檗碱5?μg/ml组、盐酸小檗碱10?μg/ml组和盐酸小檗碱20?μg/ml组。盐酸小檗碱干预24?h。流式细胞术检测细胞凋亡、半胱天冬氨酸蛋白酶-3（Caspase-3） 阳性细胞荧光强度。通过实时荧光定量聚合酶链反应（qRT-PCR）检测Caspase-3、与Bcl-2相互作用的肌球蛋白样卷曲螺旋蛋白1（Beclin1）、自噬相关基因5（Atg5）、丝氨酸/苏氨酸蛋白激酶（Akt）和c-Jun氨基末端激酶（JNK）mRNA的表达，Western blotting检测Beclin1、Atg5、Akt、p-Akt、JNK及p-JNK蛋白的表达。结果 与溶剂对照组比较，不同盐酸小檗碱浓度干预组HSC-LX2凋亡增加（P?<0.05），Caspase-3 mRNA表达增加（P?<0.05），Caspase-3阳性细胞荧光强度及蛋白活性升高（P?<0.05）；Beclin1、Atg5 mRNA及蛋白水平下降（P?<0.05）；Akt、JNK mRNA水平下降（P?<0.05），Akt、p-Akt、JNK、p-JNK蛋白水平下降（P?<0.05）。结论 盐酸小檗碱可能通过抑制Akt信号通路促进凋亡，通过抑制JNK信号传导途径抑制自噬。

Abstract:

Objective To investigate the influence of berberine hydrochloride on autophagy and apoptosis in hepatic stellate cells- LX2 (HSC-LX2). Methods HSC-LX2 at logarithmic growth stage were used in the study. The cells were divided into blank control group (1‰ DMSO) and different concentrations of berberine hydrochloride groups (5, 10, 20?mg/ml). The cells were intervened with berberine hydrochloride for 24?h. Cell apoptosis and the fluorescence intensity in Caspase-3 positive cells were accessed by flow cytometry. Caspase-3, Coiled-coil myosin-like Bcl-2 interaetig protein (Beclin1), autophagy-related gene 5 (Atg5), Akt and c-Jun N-terminal kinase (JNK) transcriptions were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western-blot analysis was also used to check the protein expression of Beclin1, Atg5, Akt, p-Akt, JNK and p-JNK. Results Compared with the control group, berberine hydrochloride could increase cell apoptosis, Caspase-3 transcription, the fluorescence intensity in Caspase-3 positive cells and Caspase-3 protein activity (P?