School of Pharmacy, Guangxi University of Chinese Medicine, Nanning 530200, PR China.
School of Basic Medical Sciences, Guangxi University of Chinese Medicine, Nanning 530200, PR China.
J Ethnopharmacol. 2022 Mar 1;285:114859. doi: 10.1016/j.jep.2021.114859. Epub 2021 Nov 21.
Panax notoginseng (Burk) F. H. Chen is a well-known traditional Chinese medicine with a long history and is widely used in the treatment of cerebrovascular disease. Panax notoginseng saponins (PNS) are the main active ingredients in Panax notoginseng (Burk) F. H. Chen, and its injection is used to treat nerve damage caused by cerebral ischemia and other conditions. PNS is thought to alleviate cognitive impairment in patients with Alzheimer's disease; however, its mechanism of action is unclear.
We elucidated the role of PNS in attenuating cellular mitochondrial damage caused by amyloid β (Aβ) protein and in protecting cell viability from the perspective of regulating autophagy. By investigating the effects of PNS on the targets regulating mitophagy, we wanted to reveal the autophagy related mechanism by which PNS attenuated Aβ damage in neuronal cells.
The effect of PNS on the mitochondrial membrane potential of Aβ-injured PC12 cells was detected using flow cytometry, which reflected the alleviating effect of PNS on mitochondrial damage. Using mRFP-GFP-LC3-transfected PC12 cells, the effect of PNS on cellular autophagy flux was observed using laser confocal microscopy. Formation of the intracellular autophagosome was observed using transmission electron microscopy, which reflected the activation of autophagy by PNS. The siPINK1 lentivirus was used to silence the PINK1 gene in PC12 cells to obtain siPINK1-PC12 cells. The effects of PNS on the expression of the PINK1 gene and on the autophagy-related proteins LC3II/Ⅰ, p62, PINK1, parkin, NDP52, and OPTN were observed to reveal the possible targets of PNS in regulating autophagy.
After PNS treatment, the viability of Aβ-injured PC12 cells improved and the mitochondrial membrane potential was restored. PNS treatment significantly enhanced the autophagy flux of damaged cells and increased the levels of LC3II/Ⅰ protein and decreased p62 protein, while significantly improving the structure and mitochondrial morphology of PC12 cells injured by Aβ. These changes led to more autophagosomes wrapping around the damaged mitochondria and promoting the depletion of OPTN, a mitophagy receptor. After silencing the PINK1 gene, PNS could not alter the PINK1 gene and protein levels, but could still increase LC3II/Ⅰ, decrease p62 and OPTN, and significantly increase the amount of parkin.
PNS could enhance the autophagic activity of cells, alleviate mitochondrial damage caused by Aβ injury, and protect the activity of PC12 cells. It is possible that enhanced autophagy was achieved by promoting the recruitment of parkin protein to the mitochondrial receptors in a non-PINK1-dependent manner.
三七（Burk）F. H. Chen 是一种历史悠久的著名中药，广泛用于治疗脑血管疾病。三七总皂苷（PNS）是三七（Burk）F. H. Chen 的主要活性成分，其注射液用于治疗脑缺血等引起的神经损伤。PNS 被认为可以减轻阿尔茨海默病患者的认知障碍；然而，其作用机制尚不清楚。
本研究从调控自噬的角度，阐明 PNS 减轻淀粉样β（Aβ）蛋白引起的细胞线粒体损伤和保护细胞活力的作用。通过研究 PNS 对调节自噬的靶点的影响，我们希望揭示 PNS 通过减轻神经元细胞中 Aβ 损伤的自噬相关机制。
采用流式细胞术检测 PNS 对 Aβ 损伤 PC12 细胞线粒体膜电位的影响，反映 PNS 对线粒体损伤的缓解作用。利用 mRFP-GFP-LC3 转染的 PC12 细胞，通过激光共聚焦显微镜观察 PNS 对细胞自噬流的影响。透射电镜观察细胞内自噬体的形成，反映 PNS 对自噬的激活作用。用 siPINK1 慢病毒沉默 PC12 细胞中的 PINK1 基因，获得 siPINK1-PC12 细胞。观察 PNS 对 PINK1 基因表达和自噬相关蛋白 LC3II/Ⅰ、p62、PINK1、parkin、NDP52、OPTN 的影响，揭示 PNS 调节自噬的可能靶点。
PNS 处理后，Aβ 损伤 PC12 细胞活力提高，线粒体膜电位恢复。PNS 处理显著增强了受损细胞的自噬流，增加了 LC3II/Ⅰ 蛋白水平，降低了 p62 蛋白水平，同时显著改善了 Aβ 损伤的 PC12 细胞的结构和线粒体形态。这些变化导致更多的自噬体包裹在受损的线粒体周围，并促进了 OPTN（一种自噬受体）的耗竭。沉默 PINK1 基因后，PNS 不能改变 PINK1 基因和蛋白水平，但仍能增加 LC3II/Ⅰ，降低 p62 和 OPTN，并显著增加 parkin 的量。
PNS 能增强细胞自噬活性，减轻 Aβ 损伤引起的线粒体损伤，保护 PC12 细胞活性。这可能是通过促进 parkin 蛋白在非 PINK1 依赖的方式募集到线粒体受体上来实现的。
