Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: Upregulation of PI3K/AKT1 Pathway, RRM2, and oxeiptosis in type 2 diabetic nephropathy (T2DN). (A) Renal transmission electron micrographs of each groups in animal studies. Scale bar: 2 μm and zoomed scale bar: 500 nm. (B) Schematic illustration of the core scientific questions in this study. (C) Expression levels of CAT and GPX1 in animal experimental groups. (D) Quantitative analysis of CAT and GPX1 protein levels based on (C). (E) Hydrogen peroxide levels in renal tissues. (F) Reactive oxygen species (ROS) levels in renal tissues. (G) Hydrogen peroxide levels in MDCK cells treated with 50 mM glucose (GLU) and the regulatory effects of andrographolide (AND). (H) ROS levels in MDCK cells treated with 50 mM GLU and the regulatory effects of AND. Scale bar: 100 μm. (I) Quantitative analysis of ROS levels based on (H). (J) Protein expression levels of PI3K/AKT1 pathway, RRM2, and oxeiptosis-related proteins in renal tissues. (K) Quantitative analysis of target protein expression levels based on (J). (L) Immunohistochemical (IHC) staining of p-AKT1 (PI3K/AKT1 pathway), RRM2, and oxeiptosis-related proteins. Scale bar: 200 μm. (M) Quantitative analysis of protein expression levels based on (L). (N) Expression profiles of oxeiptosis-related proteins in MDCK cells exposed to 50 mM GLU. (O) Quantitative analysis of oxeiptosis-related protein levels based on (N). (P) Effects of EUK-134 on oxeiptosis in MDCK cells treated with 50 mM GLU. (Q) Quantitative analysis of oxeiptosis-related protein levels based on (P). All data are presented as mean ± SD, n = 3.

Article Snippet: To activate or inhibit the PI3K/AKT1 signaling pathway, MDCK cells were treated with Recilisib (HY-101625, MCE, China) or Miltefosine (HY-13685, MCE, China), respectively.

Techniques: Transmission Assay, Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: Andrographolide (AND) inhibits hyperglycemia-induced oxeiptosis through dual suppression of PI3K/AKT1 and RRM2 and demonstrates enhanced potential for ameliorating type 2 diabetic nephropathy (T2DN) in combination with metformin (MET). (A) Effects of AND combined with AKT1 overexpression on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis-associated proteins under 50 mM glucose (GLU) treatment. (B) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels from (A). (C) Effects of AND combined with si-AKT1 on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis markers under 50 mM GLU. (D) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels from (C). (E) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in cells treated with AND combined with AKT1 overexpression or si-AKT1 under 50 mM GLU. Scale bar: 10 µM. (F) Quantitative analysis of Annexin V-mCherry-positive expression from (E). (G) Effects of AND combined with AKT1 overexpression or si-AKT1 on AIFM1 (pS116) expression and mitochondrial co-localization under 50 mM GLU. Scale bar: 10 µM. (H) Quantitative analysis of AIFM1 (pS116) levels from (G). (I) Effects of AND combined with RRM2 overexpression or si-RRM2 on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis markers under 50 mM GLU. (J) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels from (I). (K) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in cells treated with AND combined with RRM2 overexpression or si-RRM2 under 50 mM GLU. Scale bar: 10 µM. (L) Quantitative analysis of Annexin V-mCherry-positive expression from (K). (M) Effects of AND combined with RRM2 overexpression or si-RRM2 on AIFM1 (pS116) expression and mitochondrial co-localization under 50 mM GLU. Scale bar: 10 µM. (N) Quantitative analysis of AIFM1 (pS116) levels from (M). (O) Schematic diagram illustrating the mechanism by which AND suppresses high glucose-induced oxeiptosis through dual inhibition of PI3K/AKT1 and RRM2. The AND + MET combination provided superior improvement in HOMA-IR (P) and fasting blood glucose levels (Q) compared to MET alone. (R) Representative images of kidney sections subjected to H&E staining (Scale bar: 50 µm; blue arrows indicate cellular vacuolization, green arrows indicate proteinaceous mucus, purple arrows indicate mesangial expansion), MASSON staining (Scale bar: 100 µm; black arrows indicate collagen fiber deposition), PAS staining (Scale bar: 50 µm; blue arrows indicate glycogen deposition), and PASM staining (Scale bar: 50 µm; blue arrows indicate glomerular basement membrane thickening). (S) Effect of the AND + MET combination on the oxeiptosis-related protein expression in renal tissues. (T) Relative quantification of oxeiptosis-related protein levels from (S). (U) Effect of the AND + MET combination on the oxeiptosis-relate protein expression in MDCK cells treated with 50 mM GLU. (V) Relative quantification of oxeiptosis-related protein levels from (U). (W) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in cells treated with AND combined with MET under 50 mM GLU. Scale bar: 10 µM. (X) Quantitative analysis of Annexin V-mCherry-positive expression from (W). (Y) Effects of AND combined with MET on AIFM1 (pS116) expression and mitochondrial co-localization under 50 mM GLU. Scale bar: 10 µM. (Z) Quantitative analysis of AIFM1 (pS116) levels from (Y). All data are presented as mean ± SD, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To activate or inhibit the PI3K/AKT1 signaling pathway, MDCK cells were treated with Recilisib (HY-101625, MCE, China) or Miltefosine (HY-13685, MCE, China), respectively.

Techniques: Over Expression, Expressing, Activity Assay, Staining, Inhibition, Membrane, Quantitative Proteomics