Yu Mengyao, Xu Xiaoyan, Jiang Nan, Wei Wei, Li Fang, He Liming, Luo Xia
Sichuan Academy of Chinese Medical Sciences, No. 51, 4th section of South Renmin Road, Chengdu 610041, PR China.
Sichuan Academy of Chinese Medical Sciences, No. 51, 4th section of South Renmin Road, Chengdu 610041, PR China.
J Ethnopharmacol. 2017 Feb 23;198:167-173. doi: 10.1016/j.jep.2017.01.007. Epub 2017 Jan 7.
Fuling, the sclerotium of Poria cocos, was frequently used in traditional Chinese medicine (TCM) formulae for Alzheimer's disease (AD) intervention over the past 10 centuries. And its extracts exhibited significant effects in both cellular and animal models of AD in previous studies. However, its mechanisms on prevention and treatment of AD have not been well elucidated yet.
To investigate the effect and corresponding mechanisms of dehydropachymic acid, which is one of the major triterpenes in P. cocos, on the clearance of β-amyloid accumulation in bafilomycin A1 induced PC12 cells.
MTT assay was used to examine the DPA effect on the viability of PC12 cells stable transfected with pCB6-APP (PC12-APP). PC12-APP cells were treated with DPA at the concentration of 6.25, 12.5, 25μg/mL for 4h, and then co-treated with 50nmol/L bafilomycin A1 for 48h except the controls. The Aβ content in culture medium was determined by ELISA. The intracellular amount of APP, Aβ, LC3, cathepsin D was measured by Western blotting and normalized to GAPDH loading control. The PC12 cells stable transfected with pSelect-LC3-GFP (PC12-LC3-GFP) was used in the fluorescence microscopy estimation of autophagosomes accumulation. The internal pH in lysosome was detected by LysoTracker Red staining.
DPA had no significant effect on the cell viability but could significantly decrease Aβ content in culture medium and eliminate the intracellular accumulation of APP and Aβ in bafilomycin A1 induced PC12-APP cells. Furthermore, DPA lowered the LC3-II/LC3-I ratio and reduced the GFP-labeled LC3 puncta which were elevated by bafilomycin A1. And the increase in internal pH of lysosome and decrease in mCatD amount in Bafilomycin A1 induced PC12-APP cells were restored by DPA treatment. These results indicated that DPA could restore the lysosomal acidification and recover the autophgic flux which is impaired by bafilomycin A1.
DPA could effectively clear the accumulation of Aβ in bafilomycin A1 impaired PC12 cells through restoring the lysosomal acidification and recovering the autophgic flux. And these results highlight its therapeutic potential for AD treatment.
茯苓，即多孔菌的菌核，在过去10个世纪中常用于中药配方中以干预阿尔茨海默病（AD）。并且其提取物在先前的研究中在AD的细胞和动物模型中均表现出显著效果。然而，其预防和治疗AD的机制尚未得到充分阐明。
研究茯苓中的主要三萜之一去氢茯苓酸对巴弗洛霉素A1诱导的PC12细胞中β-淀粉样蛋白积累清除的作用及相应机制。
采用MTT法检测去氢茯苓酸（DPA）对稳定转染pCB6-APP（PC12-APP）的PC12细胞活力的影响。PC12-APP细胞分别用浓度为6.25、12.5、25μg/mL的DPA处理4小时，然后除对照组外，与50nmol/L巴弗洛霉素A1共同处理48小时。采用ELISA法测定培养基中Aβ含量。通过蛋白质印迹法测定细胞内APP、Aβ、LC3、组织蛋白酶D的含量，并以GAPDH作为内参进行标准化。使用稳定转染pSelect-LC3-GFP（PC12-LC3-GFP）的PC12细胞通过荧光显微镜评估自噬体的积累。通过溶酶体红色荧光探针染色检测溶酶体内的pH值。
DPA对细胞活力无显著影响，但能显著降低培养基中Aβ含量，并消除巴弗洛霉素A1诱导的PC12-APP细胞中APP和Aβ的细胞内积累。此外，DPA降低了LC3-II/LC3-I比值，并减少了巴弗洛霉素A1升高的GFP标记的LC3斑点数量。DPA处理可恢复巴弗洛霉素A1诱导的PC12-APP细胞中溶酶体内pH值的升高和mCatD含量的降低。这些结果表明，DPA可以恢复溶酶体酸化并恢复被巴弗洛霉素 A1 损害的自噬通量。
DPA可通过恢复溶酶体酸化和自噬通量有效清除巴弗洛霉素A1损伤的PC12细胞中Aβ的积累。这些结果突出了其在AD治疗中的潜在治疗价值。
