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: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

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: PI3K/AKT1 mediates H 2 O 2 -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 H 2 O 2 . (D) Molecular docking between H 2 O 2 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 H 2 O 2 -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 PI3K/AKT1 signaling, RRM2 levels, and oxeiptosis regulation under GLU stimulation. (M) Quantitative analysis of protein levels shown in (L). (N) AND-dependent attenuation of GLU-induced apoptosis detected by Annexin V-mCherry/SYTOX Green. (O) Quantification of Annexin V-mCherry-positive expression from (N). (P) AND-mediated modulation of AIFM1 (pS116) expression and its co-localization with mitochondrial under GLU stress. (Q) Quantitative analysis of AIFM1 (pS116) expression from (P). (R) AND suppresses H 2 O 2 upregulation and modulates the high GLU-promoted PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis. Scale bar: 10 µM. 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: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Expressing, Phospho-proteomics, Staining

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: 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 pathway activity, RRM2 expression, and oxeiptosis-associated proteins. (N) Quantitative analysis of protein levels in (M). (O) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining under RRM2 overexpression and GLU treatment. (P) Quantification of Annexin V-mCherry-positive expression from (O). (Q) RRM2 overexpression modulates AIFM1 (pS116) phosphorylation and mitochondrial co-localization during GLU exposure. (R) Quantitative analysis of AIFM1 (pS116) levels in (Q). (S) Impact of RRM2 inhibition on GLU-driven PI3K/AKT1 signaling, RRM2 expression, and oxeiptosis markers. (T) Quantitative analysis of protein levels in (S). (U) RRM2 knockdown attenuates GLU-induced apoptosis detected by Annexin V-mCherry/SYTOX Green. (V) Quantification of Annexin V-mCherry-positive expression from (U). (W) RRM2 inhibition-mediated modulation of AIFM1 (pS116) expression and its co-localization with mitochondrial under GLU stress. (X) Quantitative analysis of AIFM1 (pS116) levels in (W). Scale bar: 10 µM. 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: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Over Expression, Activity Assay, Expressing, Staining, Phospho-proteomics, Knockdown, Inhibition

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: 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 staining in RRM2-overexpressing cells treated with Miltefosine and 50 mM GLU. (L) Quantitative analysis of Annexin V-mCherry-positive expression from (K). (M) RRM2 overexpression combined with Miltefosine regulates AIFM1 (pS116) expression and mitochondrial co-localization during 50 mM GLU treatment. (N) Quantitative analysis of AIFM1 (pS116) levels in (M). (O) Analysis of the interaction between RRM2 and KEAP1 in HEK293T cells. (P) Protein-protein docking of RRM2 and KEAP1. (Q) Effect of Hydroxyurea (HU) on the interaction between RRM2 and KEAP1. (R) Effect of HU on the expression of oxeiptosis-related proteins promoted by RRM2 overexpression and 50 mM GLU. (S) Quantitative analysis of the related protein levels from (R). (T) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in RRM2-overexpressing cells treated with HU and 50 mM GLU. (U) Quantitative analysis of Annexin V-mCherry-positive cells from (T). (V) High GLU induced oxeiptosis via the PI3K/AKT1 signaling pathway in an RRM2-dependent manner. Scale bar: 10 µM. 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: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Over Expression, Expressing, Activity Assay, Staining, Phospho-proteomics

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: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

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

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. The expression of RRM2 was detected via western blotting in synovial specimens from patients with RA and normal controls. The relative expression of RRM2 was shown in histogram. **p<0.01, compared to normal controls. B. MH7A cells were treated with TNF-α (10 ng/mL) and IL-1β (10 mg/L) for 24 h, and the expression of RRM2 was determined via western blotting. The relative expression of RRM2 was shown in histogram. **p<0.01, compared with untreated group. ##p<0.01, compared with untreated group. C. MH7A cells were treated with RRM2 shRNA lentivirus and lentiviral controls for 24 h. The relative expression of RRM2 was determined via western blotting and shown in histogram. **p<0.01, compared with control lentivirus group. D. MTT assay. MH7A cells were infected with RRM2 shRNA lentivirus and lentiviral controls for 24, 48, and 72 h, and cell viability was determined via MTT assay. **p<0.01. E. Clone formation assay. MH7A cells were infected with RRM2 shRNA and control shRNA lentivirus and cultured for 2 weeks. Colony formation assay was performed as described in Materials and Methods. The colony number was shown in histogram. **p<0.01, compared with control shRNA group. F. Migration and invasion assays. MH7A cells were infected with RRM2 shRNA and control shRNA lentiviruses and treated with 10 ng/mL of TNF-α. The migratory and invasive abilities were detected using transwell Boyden chamber coated without or with a Matrigel basement membrane matrix after 48 h. Original magnification ×100.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Expressing, Western Blot, shRNA, MTT Assay, Infection, Tube Formation Assay, Cell Culture, Colony Assay, Migration, Membrane

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: IGF2BP3-RRM2 association.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques:

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. IGF2BP3 RIP assay. IGF2BP3 immunoprecipitation was performed and detected by Western blotting in TNF-α or IL-1β-treated MH7A cells. GAPDH and IgG was used as the negative control. B. RIP-qPCR assay demonstrated the enrichment of RRM2 mRNA in in anti-IGF2BP3 precipitates of TNF-α or IL-1β-treated MH7A cells(**p<0.01). C. MeRIP-qPCR assay. The m6A enrichment of RRM2 mRNA was shown by using anti-IgG and anti-m6A antibodies in TNF-α or IL-1β-treated MH7A cells after knocking down IGF2BP3. **P < 0.01.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Immunoprecipitation, Western Blot, Negative Control

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. MH7A cells were treated with TNF-α (10 ng/mL) and IL-1β (10 mg/L) for 24 h, and the expression of RRM2 was determined via western blotting. B. The relative expression of IGF2BP3, RRM2, MMP-9 and MMP-1 was shown in histogram. **p<0.01, compared with control group. C. MH7A cells were infected with IGF2BP3 shRNA and control shRNA lentiviruses and treated with 10 ng/mL of TNF-α for 24 h. The expression of IGF2BP3, RRM2, MMP-9, and MMP-1 was detected via western blotting. D. The relative expression of IGF2BP3, RRM2, MMP-9 and MMP-1 was shown in histogram. **p<0.01, compared with control shRNA group.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Expressing, Western Blot, Infection, shRNA

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. MTT assay. MH7A cells were infected with IGF2BP3 shRNA lentivirus and control shRNA lentivirus was treated with TNF-α (10 ng/mL) for 24, 48, and 72 h. The cell viability was determined using MTT assay. **p<0.01, compared with control shRNA group. B. Clone formation assay. MH7A cells were infected with IGF2BP3 shRNA and control shRNA lentivirus and cultured for 2 weeks. The colony number was shown in histogram. **p<0.01, compared with control shRNA group. C. Migration and invasion assays. The effects of IGF2BP3 knockdown on invasion and migration were detected using transwell Boyden chamber coated with or without a Matrigel basement membrane matrix after 48 h. Original magnification ×100. D. MTT assay. MH7A cells were treated with IGF2BP3 shRNA lentivirus and overexpressed control lentivirus, control shRNA lentivirus and overexpressed RRM2 lentivirus, IGF2BP3 shRNA lentivirus and overexpressed control lentivirus, and IGF2BP3 shRNA lentivirus and overexpressed RRM2 lentivirus for 24, 48, and 72 h. Cell viability was determined using MTT assay. **p<0.01, compared with control group.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: MTT Assay, Infection, shRNA, Tube Formation Assay, Cell Culture, Migration, Membrane

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. MH7A cells were infected with RRM2 shRNA or control shRNA lentiviruses for 48 h. The expression of RRM2, phosphorylated Akt, total Akt, and MMP-9 was detected via western blotting. The relative expression of RRM2, p-Akt, Akt and MMP-9 was shown in histogram. **p<0.01, compared with control shRNA group. B. The MH7A cells infected with RRM2 shRNA or control shRNA lentiviruses were treated with Akt inhibitor for 24 h. The expression of pAkt-S473, total Akt, and MMP-9 was detected via western blotting. The relative expression of RRM2, p-Akt, Akt, MMP-1 and MMP-9 was shown in histogram. **p<0.01, ##p<0.01, compared with control group.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Infection, shRNA, Expressing, Western Blot