Amorfrutin B is a selective modulator of the PPARγ receptor, which has recently been identified as an effective neuroprotective compound that protects brain neurons from hypoxic and ischemic damage. Our study demonstrated for the first time that a 6-h delayed post-treatment with amorfrutin B prevented hypoxia/ischemia-induced neuronal apoptosis in terms of the loss of mitochondrial membrane potential, heterochromatin foci formation, and expression of specific genes and proteins. The expression of all studied apoptosis-related factors was decreased in response to amorfrutin B, both during hypoxia and ischemia, except for the expression of anti-apoptotic BCL2, which was increased. After post-treatment with amorfrutin B, the methylation rate of the pro-apoptotic Bax gene was inversely correlated with the protein level, which explained the decrease in the BAX/BCL2 ratio as a result of Bax hypermethylation. The mechanisms of the protective action of amorfrutin B also involved the inhibition of autophagy, as evidenced by diminished autophagolysosome formation and the loss of neuroprotective properties of amorfrutin B after the silencing of Becn1 and/or Atg7. Although post-treatment with amorfrutin B reduced the expression levels of Becn1, Nup62, and Ambra1 during hypoxia, it stimulated Atg5 and the protein levels of MAP1LC3B and AMBRA1 during ischemia, supporting the ambiguous role of autophagy in the development of brain pathologies. Furthermore, amorfrutin B affected the expression levels of apoptosis-focused and autophagy-related miRNAs, and many of these miRNAs were oppositely regulated by amorfrutin B and hypoxia/ischemia. The results strongly support the position of amorfrutin B among the most promising anti-stroke and wide-window therapeutics.

Amorfrutin B 是 PPARγ 受体的选择性调节剂，最近被鉴定为一种有效的神经保护化合物，可保护大脑神经元免受缺氧和缺血性损伤。我们的研究首次表明，用 amorfrutin B 延迟 6 小时后处理可以防止缺氧/缺血诱导的神经元凋亡，包括线粒体膜电位丧失、异染色质灶形成以及特定基因和蛋白质的表达。在缺氧和缺血期间，除抗凋亡 BCL2 的表达增加外，所有研究的细胞凋亡相关因子的表达均响应 amorfrutin B 而降低。amorfrutin B 后处理后，促凋亡Bax的甲基化率基因与蛋白质水平呈负相关，这解释了Bax高甲基化导致 BAX/BCL2 比率降低。amorfrutin B 的保护作用机制还涉及自噬的抑制，表现为自噬溶酶体形成减少以及Becn1和/或Atg7沉默后 amorfrutin B 神经保护特性的丧失。尽管用 amorfrutin B 后处理降低了缺氧期间Becn1、Nup62和Ambra1的表达水平，但它刺激了Atg5以及缺血期间 MAP1LC3B 和 AMBRA1 的蛋白质水平，支持自噬在脑病变发展中的模糊作用。此外，amorfrutin B 影响细胞凋亡和自噬相关 miRNA 的表达水平，并且许多这些 miRNA 受到 amorfrutin B 和缺氧/缺血的相反调节。结果有力地支持了 amorfrutin B 在最有前途的抗中风和宽窗疗法中的地位。