Study On The Effect And Related Mechanisms Of Myricetin-induced Autophagy On HepG2Cells

ObjectiveThe present study aimed to examine the effects of myricetin on autophagy and related molecular mechanisms in HepG2cells.MethodsThe cytotoxic effect of Myricetin on HepG2cells was determined using both the MTT assay and observation of their morphological changes under microscopy after treated with varying concentrations of Myricetin (10,20,50,100μM) for12,24and36hours. For myricetin-induced autophagy, fluorescence microscope was used to observe the green puncta in GFP-LC3stable transfected MEF cell lines, and the changes of LC3-Ⅱ and p62protein level were measured using immunoblotting. Detecting cell apoptosis of HepG2treated with different concentrations of myricetin alone or in combination with chloroquine by flow cytometry.Results1. Myricetin at the concentration of50-200μM could inhibit cell proliferation and induce cell shrinkage. Low concentrations of myricetin (10or20μM) had no obvious influence on cell proliferation even after36hours; high concentration (>50μM) of myricetin inhibited cell proliferation in dose (10-200μM) dependent and time (12h, 24h,36h) dependent.2. As shown by immunoblotting analysis, after treatment with myriceitn of different concentrations (10-100μM) for24h, the protein level of LC3-Ⅱ increased, especially at the concentration of50and100μM. Myricetin at100μM could up-regulate the expression of LC3-Ⅱ and decrease p62level in a time dependent manner. In combination with chloroquine, myricetin further increased the LC3-Ⅱ level, which was higher than myricetin or chloroquine alone.3. The number of GFP-LC3puncta in GFP-LC3stable transfected MEF cells significantly rised after myricetin treatment4. Myricetin at concentration of10μM,20μM had no evident effects on cell apoptosis, while higher concentrations (50μM or higher) were able to induce apoptosis. Chloroqunie was found to enhance the myricetin-induced apoptosis and inhibition of proliferation.5. Compared to the control, myricetin was able to reduce p-mTOR, p-S6and Bcl-2, but increased the protein level of class Ⅲ PI3K.Conclusions1. Myricetin treatment results in a significant dose-and time-dependent inhibition in the growth of HepG2cells. Myricetin at higher concentrations induces cell apoptosis.2. Myricein stimulates autophagy in HepG2cells.3. Autophagy induced by myricetin plays pro-survival function against cell death.4. Myricetin induces autophagy by suppressing mTOR activity and up-regulating class Ⅲ PI3K signaling.