ABSTRACT
OBJECTIVE@#To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22).@*METHODS@#Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe2+ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells.@*RESULTS@#Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05).@*CONCLUSION@#Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.
Subject(s)
Animals , Mice , Berberine/pharmacology , Ferroptosis , Fluorescent Dyes , Hippocampus/metabolism , Iron/metabolism , NF-E2-Related Factor 2/metabolism , Piperazines , Reactive Oxygen Species/metabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of chloroquine in inducing apoptosis of human hepatocellular carcinoma cells and explore the possible mechanism.</p><p><b>METHODS</b>MTT assay and flow cytometry were used to evaluate chloroquine-induced growth inhibition and apoptosis in human hepatocellular carcinoma HepG2 cells, respectively. The ATP levels in chloroquine-treated cells were detected using an ATP assay kit. PCR and Western blotting were used to detect the expression levels of miR-26b and Mcl-1 in the cells, respectively.</p><p><b>RESULTS</b>Chloroquine inhibited the proliferation of HepG2 cells in a time- and concentration-dependent manner. Treatments with 80 µmol/L chloroquine for 24, 48, and 72 h induced survival rates of (71.59∓0.2)%, (45.40∓0.5)%, and (26.34∓1.4)% in the cells. Treatments with chloroquine at 40, 80, and 160 µmol/L for 5 h resulted in obviously lowered intracellular ATP levels in the cells to 87.80%, 71.29%, and 38.02% of the control level, respectively. At 80 µmol/L, chloroquine significantly increased the expression of miR-26b and down-regulated the expression of Mcl-1 in HepG2 cells, and the application of the miR-26b inhibitor increased the cellular expression of Mcl-1.</p><p><b>CONCLUSION</b>s Chloroquine can inhibit the cell proliferation, reduce ATP level and induce apoptosis in HepG2 cells possibly through miR-26b-mediated regulation of Mcl-1.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of small interfering RNA (siRNA)-mediated silencing of monocarboxylate transporter 1 (MCT1) on the sensitivity of drug-resistant nasopharyngeal carcinoma HNE1/DDP cells to cisplatin (DDP)-induced apoptosis and explore the possible mechanism.</p><p><b>METHODS</b>The expression of MCT1 was analyzed in HNE1 and HNE1/DDP cells and in HNE1/DDP cells transfected with siRNA using Western blot. MTT assay was used to assess the inhibitory effect of different concentrations of DDP alone or in combination with MCT1 siRNA on the proliferation of HNE1/DDP cells. The apoptosis of cells treated with MCT1 siRNA or/and DDP (8 µmol/L) was assessed using flow cytometry with PI staining, and the mitochondrial membrane potential was detected using JC-1 staining assay; the expressions of Mcl-1, Bak, Bcl-2, and Bax were analyzed using Western blotting.</p><p><b>RESULTS</b>HNE1/DDP cells showed a high expression of MCT1, and MCT1 silencing using siRNA significantly increased the sensitivity of HNE1/DDP cells to DDP (P<0.05) and partly reversed DDP resistance of the cells. MCT1 silencing enhanced the sensitivity of HNE1/DDP cells to DDP-induced apoptosis. Treatment of HNE1/DDP cells with MCT1 siRNA combined with 8 µmol/L DDP for 24 h resulted in an apoptotic rate of (51.23∓2.86)%, significantly higher than that in cells treated with MCT1 siRNA or DDP alone (P<0.05). The combined treatment also reduced the mitochondrial membrane potential, down-regulated the expression of Mcl-1 and Bcl-2, and up-regulated the expression of Bax in the DDP-resistant cells.</p><p><b>CONCLUSION</b>MCT1 siRNA can enhance the sensitivity of HNE1/DDP cells to DDP-induced apoptosis, the mechanism of which may involve the down-regulation of Mcl-1 and Bcl-2 and up-regulation of Bax expression.</p>