知母须根总皂苷提取工艺的优化及其对糖氧剥夺损伤的PC12细胞保护作用研究

目的优化知母须根总皂苷提取工艺,探讨其对糖氧剥夺(oxygen-glucose deprivation,OGD)损伤的PC12细胞的保护作用。方法在单因素实验基础上,利用响应面法优化知母须根总皂苷提取工艺;建立OGD损伤的PC12细胞模型,采用终质量浓度为20、40、80 mg/L的知母须根总皂苷进行干预。分别用倒置显微镜观察细胞形态,MTT法测定细胞存活率,荧光探针二氯荧光素-双乙酸盐(DCF-DA)法测定细胞内活性氧(ROS)含量,Annexin V/PI双染法测定细胞凋亡率,蛋白免疫印记法检测凋亡信号蛋白Bcl-2、Bax表达情况。结果知母须根总皂苷提取的最优条件为乙醇体积分数72.22%,料液比1∶11,提取时间73.33 min,在此条件下,理论提取率为8.38%,实测提取率为8.33%。PC12细胞经OGD损伤4 h后细胞活力显著降低,知母须根总皂苷对其呈现保护作用,并随着知母须根总皂苷质量浓度的升高而增强;流式细胞术分析结果表明知母须根总皂苷能明显减少OGD损伤PC12细胞中ROS含量及凋亡率,免疫印迹实验结果表明知母须根总皂苷可上调PC12细胞Bcl-2表达并下调Bax表达。结论通过响应面法优化的工艺得率高、提取效果好。知母须根总皂苷对OGD损伤的PC12细胞具有一定的保护作用,其机制可能与减少OGD诱导PC12细胞ROS含量、抑制线粒体凋亡通路有关。

Optimization of extraction process for total saponins from fibrous root of Anemarrhena asphodeloides and its protective effect on PC12 cells induced by oxygen-glucose deprivation

Objective To optimize the extraction of total saponins from fibrous root of Anemarrhena asphodeloides (TSFAA) and explore its protective effect on PC12 cells induced by oxygen-glucose deprivation (OGD). Methods Single factor experiment and Box-Benhken response surface method were used to select the best extraction technology. The model of PC12 cells induced by OGD was established and treated with the total concentration of 20, 40, and 80 mg/L TSFAA. Inverted microscope was used to observe the morphology of PC12 cells, and cell viability was measured by MTT assay. Fluorescence probe was used to detect the intracellular oxygen free radical (ROS), and Annexin V/PI double staining method was performed to measure the apoptotic rate. The apoptotic protein Bcl-2 and Bax expression were detected by Western blotting. Results The optimum conditions were as follows:The concentration of solvent was 72.22%; The ratio of material to liquid was 1:11; And the extraction time was 73.33 min. Under this condition, the theoretical calculated extraction rate was 8.38% and the measured value was 8.33%. PC12 cells viability was significantly decreased after OGD injury for 4 h. However, TGA showed a dose-dependent protective effect on OGD-induced cell damage; Flow cytometry analysis showed that TSFAA significantly reduced the content of ROS and apoptotic rate of PC12 cells. Also, Western blotting showed that TSFAA up-regulated the expression of Bcl-2 and down-regulated Bax expression. Conclusion The extraction process for TSFAA optimized by response surface method has high yield and good extraction effect. TSFAA has protective effect on PC12 cells injured by OGD. The mechanism may be related to the decrease in the content of ROS in PC12 cells induced by OGD and the inhibition of mitochondrial apoptosis pathway.