Journal: Chinese Medicine

Article Title: Berberine attenuates diabetic kidney injury via FOXO1/SP1-associated regulation of renal SGLT-2 expression

doi: 10.1186/s13020-026-01533-7

Figure Lengend Snippet: Immunohistochemical and PCR analysis of key target gene expression in renal tissue. From 8 to 24 weeks of age, db/db mice received vehicle, EM (1 mg/kg/day), BBR (50, 100, or 200 mg/kg/day), or BBR (100 mg/kg/day) plus EM (1 mg/kg/day) by once-daily oral gavage for 16 weeks. A Representative immunohistochemical staining images of p-FOXO1, FOXO1, SP1, and SGLT-2 in renal tissues from different groups. B Quantitative analysis of immunohistochemical staining was performed using kidney sections from n = 5 mice per group. Three nonoverlapping fields were analyzed for each mouse, and the field-level measurements were averaged to obtain one value per mouse. C Renal tissue qPCR analysis was performed using n = 3 mice per group. Each biological sample was analyzed in technical triplicate, and the technical replicate values were averaged to obtain one value per mouse. Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 vs db/db; # P <0.05, ## P <0.01, ### P <0.001, #### P <0.0001 vs db/m

Article Snippet: Primary antibodies included phosphorylated Forkhead box O1 (p-FOXO1; Ser256; Cell Signaling Technology, 9461), FOXO1 (HUABIO, ET1608), SP1 (HUABIO, ET1702), SP1 (Proteintech, 66508-1-Ig), SGLT-2 (Immunoway, YT4274), and β-actin (Proteintech, 20536-1-AP).

Techniques: Immunohistochemical staining, Analysis, Targeted Gene Expression, Tissue, Staining

Journal: Chinese Medicine

Article Title: Berberine attenuates diabetic kidney injury via FOXO1/SP1-associated regulation of renal SGLT-2 expression

doi: 10.1186/s13020-026-01533-7

Figure Lengend Snippet: BBR regulates glucose uptake and the FOXO1/SP1/SGLT-2 axis in model cells. HK-2 cells were cultured in medium containing 50 mM D-glucose and treated with BBR (10 or 20 μM) for 24 h. The control group was maintained under an osmotically matched condition (5.5 mM D-glucose plus 44.5 mM mannitol), whereas the high-glucose group was exposed to 50 mM D-glucose. A Representative 2-NBDG fluorescence images showing glucose uptake in control, model, and BBR-treated cells. B Western blot analysis of FOXO1, p-FOXO1, SP1, and SGLT-2 expression in total cellular protein extracts. C Western blot analysis of FOXO1, p-FOXO1, and SP1 expression in nuclear and cytoplasmic fractions. D Quantitative analysis of FOXO1, p-FOXO1/FOXO1, and SP1 expression in total, nuclear and cytoplasmic fractions. E Representative immunofluorescence staining images of p-FOXO1, FOXO1, SP1, and SGLT-2 in different groups. F Relative mRNA expression levels of FOXO1, SP1 , and SLC5A2 determined by qPCR. Western blot and densitometric analyses were performed with n = 4 independent biological replicates; the 2-NBDG uptake, immunofluorescence, and qPCR experiments were performed with n = 3 independent biological replicates. Data are presented as the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001 vs model; # P <0.05, ## P <0.01, ### P <0.001 vs control

Article Snippet: Primary antibodies included phosphorylated Forkhead box O1 (p-FOXO1; Ser256; Cell Signaling Technology, 9461), FOXO1 (HUABIO, ET1608), SP1 (HUABIO, ET1702), SP1 (Proteintech, 66508-1-Ig), SGLT-2 (Immunoway, YT4274), and β-actin (Proteintech, 20536-1-AP).

Techniques: Cell Culture, Control, Fluorescence, Western Blot, Analysis, Expressing, Immunofluorescence, Staining

Journal: Chinese Medicine

Article Title: Berberine attenuates diabetic kidney injury via FOXO1/SP1-associated regulation of renal SGLT-2 expression

doi: 10.1186/s13020-026-01533-7

Figure Lengend Snippet: Functional involvement of FOXO1 and SP1 in BBR-mediated regulation of SGLT-2 expression. HK-2 cells were cultured under high-glucose conditions (50 mM D-glucose) and treated with BBR (20 μM) for 24 h. For the indicated experiments, cells were transduced with FOXO1-overexpressing lentivirus at an MOI of 50 or treated with MMA (50 nM) for 24 h. Dual-luciferase reporter activity was measured in HEK-293 T cells 48 h after transfection. A Western blot analysis of Flag, SP1, and SGLT-2 expression in HK-2 cells under high-glucose (HG) conditions with FOXO1 overexpression and BBR treatment. B Relative mRNA expression levels of FOXO1 , SP1 , and SLC5A2 in the indicated groups, determined by qPCR. C Representative reciprocal endogenous Co-IP assays supporting the association between FOXO1 and SP1 in HK-2 cells. Normal IgG served as a negative control. D Dual-luciferase promoter-reporter assays showing that FOXO1 overexpression reduced SP1 promoter-reporter activity, whereas SP1 overexpression reduced wild-type (WT) SLC5A2 promoter-reporter activity; this effect was no longer observed with the mutant (MUT) reporter. E Western blot analysis of SP1 and SGLT-2 expression in HK-2 cells treated with HG, BBR, and mithramycin A (MMA), a pharmacological inhibitor of SP1-dependent transcription. F Representative immunofluorescence staining images showing the expression of FOXO1/SP1 and SGLT-2/SP1 in HK-2 cells under the indicated treatment conditions. Nuclei were counterstained with DAPI. qPCR, dual-luciferase reporter, and immunofluorescence experiments were performed with n = 3 independent biological replicates. Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001

Article Snippet: Primary antibodies included phosphorylated Forkhead box O1 (p-FOXO1; Ser256; Cell Signaling Technology, 9461), FOXO1 (HUABIO, ET1608), SP1 (HUABIO, ET1702), SP1 (Proteintech, 66508-1-Ig), SGLT-2 (Immunoway, YT4274), and β-actin (Proteintech, 20536-1-AP).

Techniques: Functional Assay, Expressing, Cell Culture, Transduction, Luciferase, Activity Assay, Transfection, Western Blot, Analysis, Over Expression, Co-Immunoprecipitation Assay, Negative Control, Mutagenesis, Immunofluorescence, Staining

Journal: Chinese Medicine

Article Title: Berberine attenuates diabetic kidney injury via FOXO1/SP1-associated regulation of renal SGLT-2 expression

doi: 10.1186/s13020-026-01533-7

Figure Lengend Snippet: Proposed model of BBR-mediated regulation of the FOXO1/SP1/SGLT-2 axis in diabetic kidney injury. BBR reduced blood glucose levels and increased urinary glucose excretion in db/db mice, accompanied by reduced renal SGLT-2 expression and attenuation of diabetic kidney injury. In high-glucose-exposed HK-2 cells, BBR increased FOXO1 Ser256 phosphorylation, reduced nuclear FOXO1 accumulation, restored SP1 protein expression, decreased SGLT-2 expression, and reduced cellular glucose uptake. Collectively, these findings support a proposed FOXO1/SP1-associated regulatory mechanism linking BBR treatment to altered SGLT-2 expression and renal tubular glucose handling

Article Snippet: Primary antibodies included phosphorylated Forkhead box O1 (p-FOXO1; Ser256; Cell Signaling Technology, 9461), FOXO1 (HUABIO, ET1608), SP1 (HUABIO, ET1702), SP1 (Proteintech, 66508-1-Ig), SGLT-2 (Immunoway, YT4274), and β-actin (Proteintech, 20536-1-AP).

Techniques: Expressing, Phospho-proteomics