龟板提取物靶向BMP4通路抑制PC12细胞凋亡

Inhibition of Testudinis Carapax et Plastrum extracts targeting BMP4 pathway on PC12 cell apoptosis

目的探讨龟板提取物(Testudinis Carapax et Plastrum extracts,TCPE)对血清饥饿诱导的PC12细胞凋亡的保护作用及其机制。方法采用血清饥饿3 d的方法建立PC12细胞凋亡模型,并将细胞分为对照组,模型组,TCPE低、高剂量(3、30μg/mL)组。在施加处理因素3 d后,用MTT比色分析测定细胞吸光度值,Annexin V/PI双染流式细胞术测定细胞凋亡率,Western blotting检测Caspase-3、BMPs信号通路的表达水平,并检测BMPs信号通路阻断后TCPE的抗凋亡作用。用Bio-Rad Quantity One凝胶分析系统对条带进行半定量分析。结果 MTT与流式细胞术结果显示,TCPE能提高PC12细胞活性,降低PC12细胞凋亡率,并呈剂量依赖性,TCPE组与模型组相比差异具有统计学意义(P<0.05、0.01)。Western blotting结果显示,TCPE能降低Caspase-3表达,增加BMP4、BMPR-IA、p-Smad1/5/8的表达,TCPE组与模型组相比,差异具有统计学意义(P<0.05、0.01)。TCPE对BMP2、BMP7、BMPR-II的表达没有影响,BMPR-IB没有被检测出。BMP4中和抗体减弱了TCPE的抗凋亡活性。结论 TCPE具有抑制血清饥饿诱导PC12细胞凋亡的作用,且这种作用呈剂量依赖性,其作用机制可能与激活BMP4信号通路表达有关。

Objective To observe the protection of Testudinis Carapax et Plastrum extracts（TCPE） on serum starvation-induced PC12 cell apoptosis and explore its mechanism.Methods The PC12 apoptosis model was established by serum starvation for 3 d.The cells were randomly divided into four groups： control group,model group,low-dose and high-dose（3 and 30 μg/mL） TCPE groups.In the three days of the treatment,cell absorbance was determined by MTT,ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry（FCM）,Caspase-3,BMP4,BMPR-IA,and p-Smad1/5/8 signaling molecular expression were detected by Western blotting,and the anti-apoptotic effect of TCPE was observed after blocking BMPs signal pathway.Semi-quantitative analysis of bands was carried out by Bio-Rad Quantity One gel analysis system.Results MTT and FCM analyses demonstrated that TCPE could increase PC12 cell viability and decrease their apoptotic ratios in a dose dependent manner.Western blotting results showed that TCPE could decrease Caspase-3 expression,promote the expression of BMP4,BMPR-IA,and p-Smad1/5/8.There was statistically significant difference between TCPE（3 and 30 μg/mL） groups and model group（P0.05,P0.01） in all above results.While TCPE had no effect on the expression of BMP2,BMP7,and BMPR-II.BMPR-IB hadn’t been detected.The anti-apoptotic activity was partially mitigated by neutralizing BMP4 antibody.Conclusion TCPE has the capacity to inhibit the apoptosis of PC12 induced by serum starvation in a dose dependent manner and its mechanism may be associated with partially activating and up-regulating the expression of BMP4 signaling pathway.