Research Paper Volume 15, Issue 10 pp 4429—4443

Figure 6. Metformin inhibits ROS production, oxidative stress, and KGN cell pyroptosis by increasing miR-670-3p. (A) Cells were pretreated with or without 10 μM LPS for 24 h and then incubated with 20 μM metformin for another 12 h. miR-670-3p levels were detected using RT-PCR; (B–L) Cells were pretreated with 10 μM LPS for 24 h or transfected with miR-670-3p inhibitor for 48 h and then incubated with 20 μM metformin for another 12 h; (B) Quantification of mRNA expression of NOX2 using RT-PCR; (C) Analysis of NOX2 protein level using Western blot; (D–H) Intracellular levels of ROS, MDA, SOD, GSH, and GSH-px were measured using commercial kits; (I) Levels of LDH in the cell culture medium were examined using an LDH cytotoxicity detection kit; (J) Caspase-1 activity was assessed using a colorimetric caspase-1 activity assay kit; (K) Representative micrographs of PI staining (Red) and Hoechst 33342 staining (Blue); (L) The levels of IL-1β, IL-18, IL-6, and TNF-α in the cell culture supernatant were determined by ELISA. Data are represented as the means ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar=20 μm.