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

Figure 11  Kunkui Baoshen decoction inhibits autophagy-dependent ferroptosis by enhancing the ubiquitination of nuclear receptor coactivator 4 via homologous to E6-AP C-terminus and RCC1-like domain-containing E3 ubiquitin protein ligase. A-D: Western blot analysis of nuclear receptor coactivator 4 (NCOA4) protein levels in human kidney-2 (HK-2) cells under high glucose conditions following treatment with Kunkui Baoshen decoction (KKBS), MG132 (10 μM), and 3-MA (5 mmol/L); E: Co-immunoprecipitation was performed using NCOA4 or immunoglobulin G antibodies to evaluate NCOA4 ubiquitination in high glucose-treated HK-2 cells with or without KKBS and MG132 pretreatment. Ubiquitination levels were assessed by Western blot; F-H: KKBS significantly upregulates homologous to E6-AP C-terminus and RCC1-like domain-containing E3 ubiquitin protein ligase (HERC2) expression at both the protein and mRNA levels; I: Co-localization of NCOA4 (red) and HERC2 (green) was observed via immunofluorescence double staining. DAPI (blue) was used for nuclear staining (scale bars = 50 μm); J: Co-immunoprecipitation analysis was conducted using NCOA4 and HERC2 antibodies to confirm protein-protein interactions; K and L: Protein and mRNA expression levels of interleukin-6, tumor necrosis factor-α, epithelial-cadherin, α-smooth muscle actin, and transforming growth factor-β1 in HK-2 cells after HERC2 overexpression; M-O: Expression of NCOA4 following HERC2 overexpression; P: After KKBS intervention, immunoblotting of HK-2 cells was performed using His- and Myc-tag antibodies to evaluate tagged protein expression; Q: Ubiquitination assay evaluating changes in NCOA4 ubiquitination levels following ectopic transfection with different ubiquitin lysine-mutant constructs; R: Molecular docking analysis validating the interaction between HERC2 (PDB ID: 7Q40) and NCOA4 (PDB ID: 1T5Z). NCOA4: Nuclear receptor coactivator 4; HG: High glucose; KKBS: Kunkui Baoshen decoction; UB: Ubiquitin; NG: Normal group; HERC2: Homologous to E6-AP C-terminus and RCC1-like domain-containing E3 ubiquitin protein ligase; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; E-cadherin: Epithelial-cadherin; TGF-β1: Transforming growth factor-β1; α-SMA: α-smooth muscle actin; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; His-UB: Histidine-ubiquitin; OE-HERC2: Overexpression of homologous to E6-AP C-terminus and RCC1-like domain-containing E3 ubiquitin protein ligase. ^aP < 0.05 vs normal group, ^bP < 0.01 vs normal group; ^fP < 0.01 vs high glucose group, ^gP < 0.001 vs high glucose group; ^pP < 0.01 vs high glucose + Kunkui Baoshen decoction group; ^qP < 0.05 vs vector group, and ^rP < 0.01 vs vector group.