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

Figures

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  Fig. 1. AKT1 serves as a potential target for AND in the treatment of type 2 diabetic nephropathy (T2DN) and is capable of modulating the clinical characteristics of T2DN. (A) AKT1 as the hub target for AP (Andrographis paniculata) in T2DN treatment. AKT1 was identified as the hub gene among the intersection targets of AP and T2DN using Centiscape, CytoHubba, and MCODE. (B) Molecular docking between AND and AKT1. (C) Molecular dynamics of AND and AKT1. The root mean square fluctuation (RMSF) of residues reflects the stability of the binding between AND and AKT1. (D) Kinetic fitting of the Surface Plasmon Resonance (SPR) assay indicated a strong binding affinity between AND and AKT1. (E) Experimental timeline schematic. (F) Dynamic HOMA-IR profiling. (G) Fasting glycemia surveillance post-streptozotocin (STZ) intravenous challenge. (H) Fructosamine (FUN) quantification for intermediate glycemic control assessment. (I) HbA1C longitudinal metabolic memory evaluation. (J) Oral glucose tolerance test (OGTT) kinetic patterns. (K) Insulin secretory dynamics across intervention phases. All data are presented as mean ± SD, n = 3.

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  Fig. 2. Renal biochemical profiles and pathological features across experimental groups: Control (CON), type 2 diabetic nephropathy (T2DN), 18 mg/kg andrographolide (AND) + T2DN, 12 mg/kg AND + T2DN, 6 mg/kg AND + T2DN, Metformin (MET), and 18 mg/kg AND monotherapy control groups. (A) Phosphorylation status of IRS-1 at Tyr612 and Ser307 across groups. (B) Quantification of IRS-1 phosphorylation levels corresponding to (A). (C) Longitudinal serum creatinine (CREA) monitoring post-streptozotocin (STZ) intravenous challenge. (D) Dynamic cystatin C (Cys-C) profiling as a glomerular filtration rate biomarker. (E) Blood urea nitrogen (BUN) metabolic surveillance following STZ induction. (F) Ketone body (KET) metabolic surveillance. (G) Uric acid (UA) metabolic surveillance. (H) Urinary glucose excretion dynamics. (I) Quantitative urinary protein (UPRO) tracking. (J) Urinary creatinine (UCRE) tracking. (K) Urine protein-to-creatinine ratio (UPC) trajectory analysis. (L) Renal ultrasonography and digital radiography at baseline (0d) and endpoint (120d) post-STZ administration. (M) Histopathological features were characterized by H&E staining (purple arrows indicating mesangial expansion; blue…

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  Fig. 3. Identification of type 2 diabetic nephropathy (T2DN)-related pathways and hub genes through transcriptomics for andrographolide (AND) therapy. (A) Volcano plot displaying differentially expressed genes (DEGs) between control (CON) and T2DN groups. (B) Heatmap illustrating the distribution of DEGs across CON and T2DN groups. (C) KEGG pathway enrichment analysis of DEGs in CON vs. T2DN comparison. (D) GO functional annotation of DEGs in CON vs. T2DN comparison. (E) Volcano plot showing DEGs between T2DN + AND and T2DN groups. (F) Heatmap demonstrating the distribution of DEGs in T2DN + AND vs. T2DN groups. (G) KEGG pathway analysis of DEGs from T2DN + AND vs. T2DN comparison. (H) GO term enrichment of DEGs from T2DN + AND vs. T2DN comparison. (I) Venn diagram identifying overlapping genes between T2DN targets, CON-T2DN DEGs, and T2DN + AND-T2DN DEGs. (J) Heatmap displaying expression patterns of overlapping genes. (K) KEGG pathway analysis of overlapping genes. (L) GO functional classification of overlapping genes. (M-N) Hub gene selection using Least Absolute Shrinkage and Selection Operator (LASSO) regression algorithm. (O) ROC curve validation of LASSO algorithm reliability. (P)…

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  Fig. 4. 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…

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  Fig. 5. PI3K/AKT1 mediates H2O2-induced oxeiptosis, while andrographolide (AND) reverses hyperglycemia-triggered upregulation of PI3K/AKT1 signaling, RRM2, and oxeiptosis. (A) Effect of miltefosine on hydrogen peroxide-induced alterations in the PI3K/AKT1 signaling pathway, RRM2 expression, and oxeiptosis-associated proteins. (B) Quantitative analysis of protein levels shown in (A). (C) Phosphorylation kinetics of the PI3K/AKT pathway in response to H2O2. (D) Molecular docking between H2O2 and PI3K. (E) Expression profiles of PI3K/AKT1 pathway components, RRM2, and oxeiptosis markers under 50 mM glucose (GLU) stimulation. (F) Quantitative analysis of protein levels shown in (E). (G) Pan-caspase inhibitor Z-VAD-FMK (Z-VAD) and necroptosis inhibitor Necrostatin-1 (Nec-1) fail to completely reverse apoptosis induced by H2O2-triggered oxeiptosis. (H) Apoptosis detection using Annexin V-mCherry/SYTOX Green staining in cells exposed to 50 mM GLU. (I) Quantification of Annexin V-mCherry-positive expression from (H). (J) GLU-induced mitochondrial co-localization and expression of AIFM1 (pS116). (K) Quantitative analysis of AIFM1 (pS116) expression from (J). (L) AND-mediated restoration of…

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  Fig. 6. Regulation of oxeiptosis by the PI3K/AKT1 pathway and RRM2 (A) Effect of AKT1 overexpression on 50 mM glucose (GLU)-modulated PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-associated proteins. (B) Quantitative analysis of protein levels in (A). (C) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining under AKT1 overexpression and GLU treatment. (D) Quantification of Annexin V-mCherry-positive expression from (C). (E) AKT1 overexpression modulates AIFM1 (pS116) phosphorylation and mitochondrial co-localization during GLU exposure. (F) Quantitative analysis of AIFM1 (pS116) levels in (E). (G) Impact of AKT1 knockdown on GLU-driven PI3K/AKT1 signaling, RRM2 expression, and oxeiptosis markers. (H) Quantitative analysis of protein levels in (G). (I) AKT1 knockdown attenuates GLU-induced apoptosis detected by Annexin V-mCherry/SYTOX Green. (J) Quantification of Annexin V-mCherry-positive expression from (I). (K) AKT1 inhibition-mediated modulation of AIFM1 (pS116) expression and its co-localization with mitochondrial under GLU stress. (L) Quantitative analysis of AIFM1 (pS116) levels in (K). (M) Effect of RRM2 overexpression on 50 mM GLU-modulated PI3K/AKT1…

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  Fig. 7. High glucose (GLU) activates oxeiptosis through the PI3K/AKT1/RRM2 axis. (A) Interaction between RRM2 and AKT1 in HEK293T cells. (B) Protein-protein docking of RRM2 and AKT1. (C) Effects of RRM2 overexpression combined with Recilisib on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis-associated proteins under 50 mM GLU treatment. (D) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels in (C). (E) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in RRM2-overexpressing cells treated with Recilisib and 50 mM GLU. (F) Quantitative analysis of Annexin V-mCherry-positive expression from (E). (G) RRM2 overexpression combined with Recilisib modulates AIFM1 (pS116) phosphorylation and mitochondrial co-localization during GLU exposure. (H) Quantitative analysis of AIFM1 (pS116) levels in (G). (I) Effects of RRM2 overexpression combined with Miltefosine 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 in (I). (K) Apoptosis detection via Annexin V-mCherry/SYTOX Green…

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  Fig. 8. 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)…

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