Amorfrutin B Protects Mouse Brain Neurons from Hypoxia/Ischemia by Inhibiting Apoptosis and Autophagy Processes Through Gene Methylation- and miRNA-Dependent Regulation

Abstract

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.

Posttreatment with Ospemifene Attenuates Hypoxia- and Ischemia-Induced Apoptosis in Primary Neuronal Cells via Selective Modulation of Estrogen Receptors

“…The molecular mechanism of interaction between BCL2 and BAX relies on BCL2-induced conformational changes of BAX that prevent its oligomerization, which is necessary to form pores in the mitochondrial membrane (Dlugosz et al 2006 ); in this way, the decrease in the BCL2/BAX ratio induces apoptosis. Upregulation of BCL2 in the ischemic model may seem surprising, but similar effects have already been observed in other cellular models of brain hypoxia and ischemia as well as Alzheimer’s and Huntington’s diseases (Satou et al 1995 ; O’Barr et al 1996 ; Kitamura et al 1998 ; Sulejczak et al 2004 ; Sassone et al 2013 ; Wnuk et al 2021a , 2021b ; Przepiórska et al 2023 ). The observed phenomenon is likely a survival mechanism.…”

“…Measurement of mitochondrial membrane potential was performed using a JC-10 Assay Kit (Abcam, Cambridge, UK) as previously described (Przepiórska et al 2023 ). JC-10 transpires into mitochondria and polymerizes there in cells with proper mitochondrial membrane potential, forming aggregates with characteristic red fluorescence.…”

Ursolic acid inhibits autophagy-dependent neuronal cell death by reducing oxidative stress levels in an in vitro model of Parkinson's disease

“…Overwhelming evidence has demonstrated that autophagy is required for the development, differentiation, tissue remodeling, and immune regulation of various organisms [10,11]. Therefore, autophagic dysfunction or impaired autophagic degradation is associated with the pathogenesis of many human diseases, including infections, cancer [12], neurodegeneration [13], aging [14], heart disease [15], and muscle disease [16].…”