Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: Chemical structures of ASD and Semaglutide and their effects on glucose metabolism and renal function in diabetic rats. Two-dimensional structures of (A) ASD and (B) Semaglutide were retrieved from PubChem. Enzyme-linked immunosorbent assay analysis of (C) FBG, (D) FSI and (E) HOMA-IR. (F-I) Biochemical analysis of BUN, Cr, 24-up and renal index. * P<0.05, ** P<0.01 vs. Control; ## P<0.01 vs. MOD; Δ P<0.05, ΔΔ P<0.01 vs. M + A; ☆ P<0.05, ☆☆ P<0.01 vs. M + S. ASD, akebia saponin D; FBG, fasting blood glucose; FSI, fasting serum insulin; HOMA-IR, homeostatic model assessment of insulin resistance; BUN, blood urea nitrogen; Cr, creatinine; 24-up, 24-h urinary protein.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: ASD and Semaglutide alleviate renal pathological damage in diabetic rats. (A-C) H&E staining was used to observe kidney structure (magnification, ×200 and ×400) and perform GSI and RTI scoring. (D) PAS staining and (E) quantification of PAS-positive area. (F) Masson staining and (G) quantification of collagen deposition. (H) Western blot analysis of TGF-β1, α-SMA, Collagen I and Collagen IV. (I) Quantification of protein expression normalized to GAPDH. *P<0.05, ** P<0.01 vs. Control; ## P<0.01 vs. MOD; Δ P<0.05, ΔΔ P<0.01 vs. M + A; ☆ P<0.05, ☆☆ P<0.01 vs. M + S. ASD, akebia saponin D; H&E, hematoxylin and eosin; GSI, glomerulosclerosis index; RTI, renal tubular interstitial injury index; PAS, periodic acid-Schiff; α-SMA, α-smooth muscle actin.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Staining, Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: Effects of ASD and Semaglutide on calcium and phosphate metabolism in diabetic rats. (A-F) Biochemical analyzer was used to measure the levels of Ca 2+ and PO 4 3− in (A and B) serum, (C and D) urine and (E and F) femur. * P<0.05, ** P<0.01 vs. Control; ## P<0.01 vs. MOD; ΔΔ P<0.01 vs. M + A; ☆☆ P<0.01 vs. M + S. ASD, akebia saponin D.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: ASD and Semaglutide alleviate femoral damage in diabetic rats. (A and B) H&E staining was employed to examine femoral structure and count osteocytes and osteoblasts. (C and D) Micro-CT was adopted to assess trabecular microstructure of the femur and to perform quantitative analysis of related parameters. * P<0.05, ** P<0.01 vs. Control; # P<0.05, ## P<0.01 vs. MOD; Δ P<0.05, ΔΔ P<0.01 vs. M + A; ☆ P<0.05, ☆☆ P<0.01 vs. M + S. ASD, akebia saponin D; H&E, hematoxylin and eosin; Micro-CT, micro-computed tomography.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Staining, Micro-CT, Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: Effects of ASD and Semaglutide on bone metabolism and remodeling in diabetic rats. (A-C) Three-point bending tests measuring ultimate load, bending strength and Young's modulus of the femur. The serum levels of (D) ALP, (E) PINP, (F) CTX-1 and (G) TRAP were measured using ELISA. (H) Western blot and (I) quantification of femoral protein expression (OPG, OCN, RANKL and RUNX2). * P<0.05, ** P<0.01 vs. Control; # P<0.05, ## P<0.01 vs. MOD; Δ P<0.05, ΔΔ P<0.01 vs. M + A; ☆ P<0.05, ☆☆ P<0.01 vs. M + S. ASD, akebia saponin D; ALP, alkaline phosphatase; PINP, procollagen type I N-terminal propeptide; CTX-1, C-terminal telopeptide of type I collagen; TRAP, tartrate-resistant acid phosphatase; OPG, osteoprotegerin; OCN, osteocalcin; RANKL, receptor activator of nuclear factor κ-b ligand; RUNX2, Runt-related transcription factor 2.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: Network pharmacology analysis of ASD and Semaglutide. Identification of differentially expressed genes in the (A) GSE30528 and (B) GSE35958 datasets. (C) Venn diagram depicting the intersection of DEGs from both diseases and drug-related genes. (D) PPI network of 26 related genes. GO enrichment analysis for (E) biological process, (F) cellular component and (G) molecular function. (H) KEGG pathway enrichment analysis. ASD, akebia saponin D; DEGs, differentially expressed genes; PPI, protein-protein interaction; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: ASD and Semaglutide inhibit the p53 pathway and DNA damage in the kidney and femur tissues of diabetic rats. Immunohistochemical staining of p53 and γ-H2AX in (A) kidney and (B) femur tissues, respectively. (C-F) Western blotting and quantification analysis of DNA damage-related proteins, including γ-H2AX, ATM, p-ATM, p53 and p21, in (C and D) kidney and (E and F) femur tissues. * P<0.05, ** P<0.01 vs. Control; # P<0.05, ## P<0.01 vs. MOD; ΔΔ P<0.01 vs. M + A; ☆ P<0.05, ☆☆ P<0.01 vs. M + S. ASD, akebia saponin D; p-, phosphorylated; ATM, ATM, ataxia-telangiectasia mutated protein.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Immunohistochemical staining, Staining, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: Klotho knockdown abrogates the renoprotective effects of ASD and Semaglutide. (A) Western blot analysis of Klotho protein levels in rat kidney tissues. (B-E) Serum levels of (B) BUN, (C) Cr, (D) 24-up and (E) kidney index. (F) H&E, (G) PAS and (H) Masson staining of renal tissue, with corresponding quantification of GSI, RTI, PAS-positive area and fibrosis index. (I) Western blotting and (J) quantification of renal TGF-β1, α-SMA, Collagen I and Collagen IV protein levels. * P<0.05, ** P<0.01 vs. Control; ## P<0.01 vs. MOD; ΔΔ P<0.01 vs. M + A + S; ☆☆ P<0.01 vs. M + A + S + shNC. ASD, akebia saponin D; BUN Cr, creatinine; 24-up, 24-h urinary protein; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; GSI, glomerulosclerosis index; RTI, renal tubular interstitial injury index; α-SMA, α-smooth muscle actin; sh, short hairpin.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Knockdown, Western Blot, Staining, Control

Journal: International Journal of Molecular Medicine

Article Title: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho-p53 signaling axis

doi: 10.3892/ijmm.2025.5696

Figure Lengend Snippet: Knockdown of Klotho reverses the therapeutic effects of ASD and Semaglutide in the treatment of diabetic osteoporosis. (A) Western blot analysis of Klotho protein levels in rat femur tissue. (B and C) H&E staining to assess histological changes in rat femur tissues and cell counting of osteocytes and osteoblasts. (D) Micro-CT measurement of BMD in rat femur. (E and F) Western blot and quantification analysis of bone metabolic markers (OPG, OCN, RANKL and RUNX2). * P<0.05, ** P<0.01 vs. Control; # P<0.05, ## P<0.01 vs. MOD; Δ P<0.05, ΔΔ P<0.01 vs. M + A + S; ☆ P<0.05, ☆☆ P<0.01 vs. M + A + S + shNC. ASD, akebia saponin D; H&E, hematoxylin and eosin; Micro-CT, micro-computed tomography; OPG, osteoprotegerin; OCN, osteocalcin; RANKL, receptor activator of nuclear factor κ-b ligand; RUNX2, Runt-related transcription factor 2.

Article Snippet: STZ, ASD and Semaglutide were obtained from MedChemExpress and the chemical structures of ASD and Semaglutide are shown in .

Techniques: Knockdown, Western Blot, Staining, Cell Counting, Micro-CT, Control

Journal: bioRxiv

Article Title: GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

doi: 10.64898/2025.12.26.696603

Figure Lengend Snippet: (A) Percent of Glp1r -expressing cells per cell identity; (B) Average gene expression of Glp1r in LECs from WT female and WT male mice respectively; (C) Volcano plot displaying the differentially expressed genes in LECs expressing Glp1r vs. those that did not express this gene; (D) Gene ontology analyses displaying the top molecular functions and biological processes associated with the differentially upregulated genes shown in panel C; (E) Representative image of an inguinal axillary lymphatic vessel exposed to FITC-labeled semaglutide to determine GLP-1R expression and cellular localization; (F) UMAP displaying the different LEC subtypes contained within the employed scRNAseq dataset; (G) DotPlot displaying the expression of Glp1r , as well as known markers of various LEC-subtypes (in this panel, the percent of cells expressing a given feature and the average gene expression are encoded in the size and color of dots respectively); and (H) Percent of LECs expressing Glp1r in clusters linked to collecting (collLECs), pre-collecting (precLECs), or capillary (capLECs) lymphatics (cluster numbers are indicated under the horizontal axis).

Article Snippet: The cellular localization of GLP-1Rs in collecting lymphatic vessels was assessed using semaglutide conjugated with FITC (Cat. No.: HY-114118F, MedChemExpress).

Techniques: Expressing, Gene Expression, Labeling

Journal: bioRxiv

Article Title: GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

doi: 10.64898/2025.12.26.696603

Figure Lengend Snippet: (A) Representative diameter trace of an inguinal axillary lymphatic vessel displaying the functional response to increasing concentrations of semaglutide in the range of 1nM to 1µM; the contractile activity was assessed for 5 minutes at each concentration; (B-I) Mean contractile parameters as a function of semaglutide concentration (expressed as mean±SEM), including (B) amplitude, (C) end diastolic diameter (EDD), (D) end systolic diameter (ESD), (E) ejection fraction, (F) contraction frequency, (G) width, (H) fractional pump flow (FPF), and (I) volume displaced. A total of n=17 vessels were included and a one-way ANOVA test that included a correction for multiple comparisons using Dunnett’s test and a Geisser-Greenhouse correction as no equal variances were assumed (i.e., sphericity was not assumed) were used to determine differences in contractile function parameters, with statistical significance set at p<0.05.

Article Snippet: The cellular localization of GLP-1Rs in collecting lymphatic vessels was assessed using semaglutide conjugated with FITC (Cat. No.: HY-114118F, MedChemExpress).

Techniques: Functional Assay, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

doi: 10.64898/2025.12.26.696603

Figure Lengend Snippet: (A) Representative diameter trace displaying the contractile activity of an inguinal axillary lymphatic vessel from a WT mouse under control conditions and following stimulation with a single 5 nM dose of semaglutide. (B-I) Summary data of different contractile parameters before (Ctrl) and after 5nM stimulus with semaglutide (Sema), including (B) amplitude, (C) end diastolic diameter (EDD), (D) end systolic diameter (ESD), (E) ejection fraction, (F) contraction frequency, (G) width, (H) fractional pump flow (FPF), (I) volume displaced, and (J) volume displaced for each individual contraction in panel A. A total of n=11 vessels were used for these experiments and paired parametric T test was used to determine significance, with statistical significance set at p<0.05.

Article Snippet: The cellular localization of GLP-1Rs in collecting lymphatic vessels was assessed using semaglutide conjugated with FITC (Cat. No.: HY-114118F, MedChemExpress).

Techniques: Activity Assay, Control

Journal: bioRxiv

Article Title: GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

doi: 10.64898/2025.12.26.696603

Figure Lengend Snippet: (A) Representative diameter trace displaying the contractile activity of an inguinal axillary lymphatic vessel from a WT mouse under control conditions, during a 20-minute pre-treatment with L-NAME (100µM) and indomethacin (10µM), and after stimulation with a single 5 nM dose of semaglutide. (B-I) Summary data of different contractile parameters under these 3 paired conditions, including (B) amplitude, (C) end diastolic diameter (EDD), (D) end systolic diameter (ESD), (E) ejection fraction, (F) contraction frequency, (G) width, (H) fractional pump flow (FPF), and (I) volume displaced. A total of n=11 vessels from WT mice were used for these experiments. A one-way ANOVA corrected with a Geisser-Greenhouse correction as no equal variances were assumed (i.e., sphericity was not assumed) was used to determine differences in contractile function parameters, with statistical significance set at p<0.05.

Article Snippet: The cellular localization of GLP-1Rs in collecting lymphatic vessels was assessed using semaglutide conjugated with FITC (Cat. No.: HY-114118F, MedChemExpress).

Techniques: Activity Assay, Control

Journal: bioRxiv

Article Title: GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

doi: 10.64898/2025.12.26.696603

Figure Lengend Snippet: (A) Representative diameter trace displaying the contractile activity of an inguinal axillary lymphatic vessel from a WT mouse under control conditions, during a 20-minute pre-treatment with L-NAME (100µM), indomethacin (10µM), and apocynin (100µM), and after stimulation with a single 5 nM dose of semaglutide. (B-I) Summary data of different contractile parameters under these 3 paired conditions, including (B) amplitude, (C) end diastolic diameter (EDD), (D) end systolic diameter (ESD), (E) ejection fraction, (F) contraction frequency, (G) width, (H) fractional pump flow (FPF), and (I) volume displaced. A total of n=11 vessels from WT mice were used for these experiments. A one-way ANOVA corrected with a Geisser-Greenhouse correction as no equal variances were assumed (i.e., sphericity was not assumed) was used to determine differences in contractile function parameters, with statistical significance set at p<0.05.

Article Snippet: The cellular localization of GLP-1Rs in collecting lymphatic vessels was assessed using semaglutide conjugated with FITC (Cat. No.: HY-114118F, MedChemExpress).

Techniques: Activity Assay, Control

Journal: bioRxiv

Article Title: GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

doi: 10.64898/2025.12.26.696603

Figure Lengend Snippet: Summary data of contractile parameters characterizing the contractile function of lymphatic vessels from WT, DIO, and ApoE KO mice under control conditions and following perfusion with a Krebs buffer containing 5 nM semaglutide. These contractile parameters are expressed as mean±SEM and include (A) contraction amplitude, (B) end diastolic diameter (EDD), (C) end systolic diameter (ESD), (D) ejection fraction, (E) contraction frequency, (F) contraction width (at half maximum), (G) fractional pump flow (FPF), and (H) volume displaced. A total of n=17 (control) and n=10 (sema) vessels from WT mice, n=13 (control) and n=12 (sema) vessels for DIO mice, and n=19 (control) and n=9 (sema) vessels from ApoE KO mice were used for these experiments. A two-way ANOVA was utilized to assess differences in contractile parameters with statistical significance set as p<0.05.

Article Snippet: The cellular localization of GLP-1Rs in collecting lymphatic vessels was assessed using semaglutide conjugated with FITC (Cat. No.: HY-114118F, MedChemExpress).

Techniques: Control