Autophagy inducing compound and use thereof
A compound and composition technology, applied in medical preparations containing active ingredients, organic chemistry, drug combination, etc.
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Embodiment
[0071] In the following examples, the following materials were used; commercial sources of the various materials are provided. Details of the various experimental protocols are also given below:
[0072] Reagents and antibodies. Isorhynchophylline (APC-164) was purchased from Aktin Chemicals. 3-MA (M9281) and chloroquine (C6628) were purchased from Sigma-Aldrich. Rapamycin (R5000) was purchased from LC Laboratories. Lysosomal red fluorescent probe DND-99 (L-7528), goat anti-mouse (626520) and goat anti-rabbit (G21234) secondary antibodies were purchased from Invitrogen. Anti-β-actin (sc-47778), anti-GFP (sc-8334) and anti-tyrosine hydroxylase (sc-14007) antibodies were purchased from Santa Cruz Biotechnology. Anti-LC3 (2775), anti-phospho-mTOR (5536), anti-phospho-p70S6K (9234), anti-(3738) antibodies were purchased from Cell Signaling Technology. Anti-α-syn antibody (610786) was purchased from BD Transduction Laboratories. Instant Drosophila food (173212) was purchased f...
Embodiment I
[0082] Example 1: IsoRhy induces autophagy in neuronal cell lines
[0083] It has been shown that the induction of autophagy in neuronal cells is much more difficult than in non-neuronal cells. To confirm IsoRhy (whose chemical structure is shown in Figure 1A Induction activity of neuronal autophagy in mouse neuroblastoma N2a, rat pheochromocytoma PC12 and human neuroblastoma SH-SY5Y were treated with different concentrations of IsoRhy for 24 hours, and the cell lysates were subjected to Western blot analysis of expression of LC3-II, which is an autophagy-specific marker. showed that IsoRhy increased LC3-II levels in N2a, PC12 and SH-SY5Y cells in a dose-dependent manner, while LC3-I levels were not affected ( Figure 1B -D).
[0084] In addition, a neuroblastoma cell line N2a that constantly expresses GFP-LC3 (a standard autophagy marker protein) was established. The formation of GFP-LC3 puncta after IsoRhy treatment was observed under a confocal microscope. Data are pre...
Embodiment II
[0088] Example II: IsoRhy Induces Autophagy in Primary Mouse Cortical Neurons
[0089] To further confirm the pro-autophagic effect of IsoRhy on primary neurons, mouse primary cortical neurons isolated from E17 embryonic ICR mice were used in this study. Primary neurons were treated with different concentrations of IsoRhy for 24 hours, and the expression of autophagy marker GFP-LC3 was examined by Western blot analysis. Neurons were fixed in 4% paraformaldehyde and analyzed under a confocal microscope. The number of GFP-LC3 puncta in each GFP positive neuron was counted and at least 20 neurons in each group were counted. Data are expressed as mean ± SEM from one representative experiment out of three independent experiments (***p<0.001, Student's t-test).
[0090] Similarly, a dose-dependent increase in LC3-II induced by IsoRhy was observed in primary mouse cortical neurons ( Figure 3A ). In neurons transfected with the GFP-LC3 construct, IsoRhy induced the massive formatio...
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