目的:淫羊藿苷(ICA)具有良好的神经保护作用,可以上调自然衰老大鼠的神经元基底自噬。线粒体功能障碍与脑衰老相关的神经退行性疾病有关。线粒体通透性转换孔(mPTP)的异常开放是线粒体功能障碍的关键因素,并与过度自噬有关。本研究旨在探讨ICA在D-半乳糖(D-gal)诱导的细胞损伤模型中通过阻断mPTP开放和下调自噬水平对神经元损伤的保护作用。
方法:用200mmol/LD-gal处理大鼠嗜铬细胞瘤细胞(PC12细胞)48h,建立神经元损伤的细胞模型。PC12细胞用不同浓度的ICA预处理24h。MTT用于检测细胞活力。采用衰老相关β-半乳糖苷酶(SA-β-Gal)染色观察细胞衰老。进行蛋白质印迹分析以检测衰老相关蛋白(p21)的表达水平,自噬标记(LC3B,P62,Atg7,Atg5和Beclin1),线粒体裂变和融合相关蛋白(Drp1,Mfn2和Opa1),和线粒体自噬标记(Pink1和Parkin)。用mRFP-GFP-LC3腺病毒检测自噬血流的变化。透射电镜观察细胞内超微结构。免疫荧光法检测mPTP,线粒体膜电位(MMP),线粒体活性氧(mtROS)和ROS水平。流式细胞术检测ROS和细胞凋亡水平。
结果:D-gal处理显著降低了PC12细胞的活力,与对照组相比,SA-β-Gal阳性细胞明显增加。有了D-gal刺激,p21的表达明显上调。此外,D-gal刺激导致LC3BII/I比率升高和p62表达降低。同时,自噬体和自溶酶体显著增加,表明自噬水平异常激活。此外,在这种D-gal诱导的细胞损伤模型中,mPTP异常开放,ROS的产生不断增加,MMP逐渐降低,细胞凋亡增加。ICA可有效改善线粒体功能障碍,以防止D-gal诱导的细胞损伤和凋亡。它通过阻断mPTP的开放强烈抑制过度的自噬。ICA和mPTP抑制剂(环孢素A)共同治疗不能改善线粒体功能障碍。然而,通过与ICA和mPTP激活剂(lonidamine)共同处理,保护作用减弱。
结论:ICA抑制过度自噬的激活,从而通过阻断mPTP开放改善线粒体功能障碍。
OBJECTIVE: Icariin (ICA) has a good neuroprotective effect and can upregulate neuronal basal autophagy in naturally aging rats. Mitochondrial dysfunction is associated with brain aging-related neurodegenerative diseases. Abnormal opening of the mitochondrial permeability transition pore (mPTP) is a crucial factor in mitochondrial dysfunction and is associated with excessive autophagy. This study aimed to explore that ICA protects against neuronal injury by blocking the mPTP opening and down-regulating autophagy levels in a D-galactose (D-gal)-induced cell injury model.
METHODS: A cell model of neuronal injury was established in rat pheochromocytoma cells (PC12 cells) treated with 200 mmol/L D-gal for 48 h. In this cell model, PC12 cells were pre-treated with different concentrations of ICA for 24 h. MTT was used to detect cell viability. Senescence associated β-galactosidase (SA-β-Gal) staining was used to observe cell senescence. Western blot analysis was performed to detect the expression levels of a senescence-related protein (p21), autophagy markers (LC3B, p62, Atg7, Atg5 and Beclin 1), mitochondrial fission and fusion-related proteins (Drp1, Mfn2 and Opa1), and mitophagy markers (Pink1 and Parkin). The changes of autophagic flow were detected by using mRFP-GFP-LC3 adenovirus. The intracellular ultrastructure was observed by transmission electron microscopy. Immunofluorescence was used to detect mPTP, mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (mtROS) and ROS levels. ROS and apoptosis levels were detected by flow cytometry.
RESULTS: D-gal treatment significantly decreased the viability of PC12 cells, and markedly increased the SA-β-Gal positive cells as compared to the control group. With the D-gal stimulation, the expression of p21 was significantly up-regulated. Furthermore, D-gal stimulation resulted in an elevated LC3B II/I ratio and decreased p62 expression. Meanwhile, autophagosomes and autolysosomes were significantly increased, indicating abnormal activation of autophagy levels. In addition, in this D-gal-induced model of cell injury, the mPTP was abnormally open, the ROS generation was continuously increased, the MMP was gradually decreased, and the apoptosis was increased. ICA effectively improved mitochondrial dysfunction to protect against D-gal-induced cell injury and apoptosis. It strongly inhibited excessive autophagy by blocking the opening of the mPTP. Cotreatment with ICA and an mPTP inhibitor (cyclosporin A) did not ameliorate mitochondrial dysfunction. However, the protective effects were attenuated by cotreatment with ICA and an mPTP activator (lonidamine).
CONCLUSIONS: ICA inhibits the activation of excessive autophagy and thus improves mitochondrial dysfunction by blocking the mPTP opening.