Nephroprotective mechanism of Kunkui Baoshen decoction in diabetic kidney disease: Targeting the HERC2/NCOA4-mediated autophagy-dependent ferroptosis pathway

Figure 10  Kunkui Baoshen decoction modulates ferroptosis by regulating nuclear receptor coactivator 4-mediated ferritinophagy. A-G: Western blot and quantitative reverse transcription-polymerase chain reaction analyses of autophagy related 7 (ATG7), nuclear receptor coactivator 4 (NCOA4), microtubule-associated protein light chain 3 (LC3), sequestosome 1 (SQSTM1/p62), and ferritin heavy chain 1 (FTH1) in normal and high glucose groups; H: Co-localization of ferritinophagy-related proteins observed by immunofluorescence double staining in human kidney-2 cells (scale bars = 50 μm); I-O: Western blot and quantitative reverse transcription-polymerase chain reaction analyses of ATG7, NCOA4, LC3, SQSTM1/p62, and FTH1 in the four groups; P-V: Western blot analysis of ATG7, glutathione peroxidase 4, solute carrier family 7 member 11, LC3, SQSTM1/p62, and FTH1 after NCOA4 knockdown. NG: Normal group; HG: High glucose; ATG7: Autophagy related 7; NCOA4: Nuclear receptor coactivator 4; LC3: Microtubule-associated protein light chain 3; SQSTM1/p62: Sequestosome 1; FTH1: Ferritin heavy chain 1; Fer-1: Ferrostatin-1; KKBS:: Kunkui Baoshen decoction; GPX4: Glutathione peroxidase 4; SLC7A11: Solute carrier family 7 member 11; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; si-NC: Negative control small interfering RNA; si-NCOA4: Nuclear receptor coactivator 4-targeting small interfering RNA. ^aP < 0.05 vs normal group, ^bP < 0.01 vs normal group, ^dP < 0.0001 vs normal group; ^eP < 0.05 vs high glucose group, ^fP < 0.01 vs high glucose group, ^gP < 0.001 vs high glucose group, ^hP < 0.0001 vs high glucose group; ⁱP < 0.05 vs high glucose + ferrostatin-1 group, ^jP < 0.01 vs high glucose + ferrostatin-1 group, ^kP < 0.001 vs high glucose + ferrostatin-1 group, ^lP < 0.0001 vs high glucose + ferrostatin-1 group; ^mP < 0.05 vs negative control small interfering RNA group; ⁿP < 0.05 vs nuclear receptor coactivator 4-targeting small interfering RNA group, and ^oP < 0.01 vs nuclear receptor coactivator 4-targeting small interfering RNA group.