Research Paper Volume 15, Issue 22 pp 13384—13410

Figure 3. BDH1-mediated βOHB metabolism reverses HG or PA-induced ROS overproduction and inflammation. (A) Representative WB image showing the protein level of BDH1 in HK-2 cells transfected with NC siRNA or Bdh1 siRNA. (B) DCFH-DA probe was used to identify the level of ROS in HK-2 cells transfected with NC siRNA or Bdh1 siRNA. (C) Representative WB image showing the protein level of IL-1β in HK-2 cells transfected with NC siRNA or Bdh1 siRNA. (D, E) ELISA showing the levels of IL-1β (D) and IL-18 (E) in the cell culture supernatant of HK-2 cells transfected with NC siRNA or Bdh1 siRNA. (F) Representative WB image showing the protein level of BDH1 in HK-2 cells transfected with vector or Flag-BDH1. (G) DCFH-DA probe was used to identify the level of ROS in HK-2 cells based on the indicated treatment. (H) Representative WB image showing the protein level of IL-1β in HK-2 cells based on the indicated treatment. (I, J) ELISA showing the levels of IL-1β and IL-18 in the cell culture supernatant of BDH1-overexpressed HK-2 cells treated with HG (I) or PA (J). For samples used in G-J, HK-2 cells were transfected with the plasmids overexpressing Flag-BDH1 or vector for 6 h and then treated with HG or PA for 48 h. (K) DCFH-DA probe was used to identify ROS levels in HK-2 cells based on the indicated treatment. (L) Representative WB image showing the protein level of IL-1β in HK-2 cells based on the indicated treatment. (M, N) ELISA showing the levels of IL-1β and IL-18 in the cell culture supernatant of βOHB-supplemented HK-2 cells treated with HG (M) or PA (N). For samples used in K-N, HK-2 cells were treated with vehicle or βOHB and HG or PA for 48 h. All results are representative of three independent experiments. Values are presented as mean ± standard deviation. Bar: 50 μm in B, 50 μm in top panels and 100 μm in bottom panels in G, 50 μm in K. Abbreviations: BDH1: β-hydroxybutyrate dehydrogenase 1; βOHB: β-hydroxybutyrate; HG: high glucose; PA: palmitic acid; ROS: reactive oxygen species; WB: western blot. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.