Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 reduces Sema4D levels to elevate cortical strength in mice with type 2 diabetes mellitus (T2DM). (A) Schedule of enzyme-linked immunosorbent assay (ELISA) and the corresponding groups. (B) ELISA to determine the levels of ALP, Runx-2, OCN, OPN, and Sema4D in control mice, mice with T2DM, mice with T2DM treated with metformin (Met)/exendin-4 (Ex-4), and GLP-1R knockdown T2DM mice treated with Ex-4. n=6 mice per group. ALP: Alkaline Phosphatase; Runx-2: Runt-related transcription factor 2; OCN: Osteocalcin; OPN: Osteopontin; Sema4D: Semaphorin 4D. (C) Schedule of micro-CT to determine changes in trabecular bone. (D) Micro-CT images representing trabecular formation in control mice, mice with T2DM, mice with T2DM treated with Ex-4, mice with T2DM treated with anti-Sema4D, and mice with T2DM treated with a combination of Ex-4 and anti-Sema4D within a region of interest (ROI). n=6 mice per group. (E) Quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 mice per group. (F, G) Micro-CT images representing cortical bone formation in control mice, mice with T2DM, mice with T2DM treated with Ex-4, mice with T2DM treated with anti-Sema4D, and mice with T2DM treated with a combination of Ex-4 and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 mice per group. (H) Effects of exendin-4 or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To investigate the regulatory role of Sema4D in cancellous bone loss and impaired bone defect regeneration in mice, mice were randomly divided into 5 groups (n = 6/group): control group, Sema4D group (10 ng/mL), Sema4D group (15 ng/mL), Sema4D (10 ng/mL) + metformin (300 mg/kg/day, MCE) group and Sema4D (15 ng/mL) + metformin (300 mg/kg/day, MCE) group.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Knockdown, Micro-CT, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: The combination of metformin and exendin-4 can neutralize the effect of Sema4D. (A) Schematic chart of the sample preparation of micro-CT and 3-point bending experiments to determine the effect of the combination of metformin and exendin-4. (B) Micro-CT images representing trabecular formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within a region of interest (ROI), and quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 per group. (C) Micro-CT images representing cortical formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 per group. (D) Effects of metformin, exendin-4, the combination of metformin and exendin-4, or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To investigate the regulatory role of Sema4D in cancellous bone loss and impaired bone defect regeneration in mice, mice were randomly divided into 5 groups (n = 6/group): control group, Sema4D group (10 ng/mL), Sema4D group (15 ng/mL), Sema4D (10 ng/mL) + metformin (300 mg/kg/day, MCE) group and Sema4D (15 ng/mL) + metformin (300 mg/kg/day, MCE) group.

Techniques: Sample Prep, Micro-CT, Control, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 promotes pseudopodia numbers, leading to the stretching and wider spread of BMSCs via CRMP2. (A) Cell morphology observed using fluorescence microscopy. ImageJ was used to split color channels to observe the pseudopodium in human bone mesenchymal stem cells (hBMSCs). n=5 per group. White arrows indicate the pseudopodium in BMSCs. (B) Immunofluorescence staining to determine F-actin expression in BMSCs. n=5 per group. (C) Western blotting to determine F-actin expression in different groups. n=3 per group. (D) Proteomics analysis to determine the expression of different phosphorylated proteins. (E) Cell morphology observed using fluorescence microscopy. ImageJ was used to split color channels to observe the pseudopodium in BMSCs. n=5 per group. White arrows indicate the pseudopodium in BMSCs. (F) Immunofluorescence staining showing F-actin in different groups. n=5 per group. (G) Immunofluorescence staining indicating CRMP2 expression in BMSCs. n=5 per group. CRMP2: Collapsin Response Mediator Protein 2. (H) Western blotting to determine ALP, Runx-2, and Osterix expression in BMSCs. n=3 per group. (I) Alizarin red staining to analyze mineral deposits in BMSCs with DPYSL2 KD/OE. n=3 per group. DPYSL2 KD/OE: Dihydropyrimidinase like protein 2 knockdown/overexpression. (J) Western blotting to determine pCRMP2 expression in BMSCs with Sema4D or exendin-4. n=3 per group. (K) Immunofluorescence staining to determine pCRMP2 expression in BMSCs. n=5 per group. **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To investigate the regulatory role of Sema4D in cancellous bone loss and impaired bone defect regeneration in mice, mice were randomly divided into 5 groups (n = 6/group): control group, Sema4D group (10 ng/mL), Sema4D group (15 ng/mL), Sema4D (10 ng/mL) + metformin (300 mg/kg/day, MCE) group and Sema4D (15 ng/mL) + metformin (300 mg/kg/day, MCE) group.

Techniques: Fluorescence, Microscopy, Immunofluorescence, Staining, Expressing, Western Blot, Knockdown, Over Expression, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 activates bone formation via the GLP-1R/PI3K/GSK-3β/CRMP2 signaling pathway. (A) Western blotting results of pCRMP2 and F-actin expression in BMSCs with GSK-3β knockdown or overexpression. n=3 per group. GSK-3β: Glycogen Synthase Kinase 3 beta. (B) Immunofluorescence staining of F-actin and pCRMP2 expression in BMSCs. n=5 per group. (C) Western blotting to determine pCRMP2 expression after Sema4D treatment with or without GSK-3β knockdown. n=3 per group. (D) Western blotting to determine pCRMP2 expression after exendin-4 treatment with or without GSK-3β knockdown. n=3 per group. (E) Western blotting to determine the expression of pGSK-3β, pCRMP2, and F-actin after Akt knockdown or overexpression. n=3 per group. Akt: Protein Kinase B/ Ak strain transforming. (F) Western blotting to determine the expression of pGSK-3β, pCRMP2, and F-actin after PI3K knockdown or overexpression. n=3 per group. PI3K: Phosphoinositide 3-Kinase. (G) Western blotting to determine the expression of pGSK-3β after exendin-4 treatment with or without PI3K knockdown and overexpression. n=3 per group. (H) Western blotting to determine P-PI3K expression after exendin-4 treatment with or without GSK-3β knockdown and overexpression. n=3 per group. (I) Western blotting to determine pGSK-3β expression after Sema4D treatment with or without PI3K knockdown and overexpression. n=3 per group. (J) Western blotting to determine pPI3K expression after Sema4D treatment with or without GSK–3β knockdown or overexpression. n=3 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To investigate the regulatory role of Sema4D in cancellous bone loss and impaired bone defect regeneration in mice, mice were randomly divided into 5 groups (n = 6/group): control group, Sema4D group (10 ng/mL), Sema4D group (15 ng/mL), Sema4D (10 ng/mL) + metformin (300 mg/kg/day, MCE) group and Sema4D (15 ng/mL) + metformin (300 mg/kg/day, MCE) group.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Immunofluorescence, Staining, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Metformin modulates the expression of plexin B1 and GLP-1R in hBMSCs. (A) Differential expression of miRNAs among hBMSCs, hBMSCs+Sema4D, and hBMSCs+Sema4D+Met. MiR-140-3p and miR-3657 showing a significant increase and reduced expression in hBMSCs treated with Met. (B) Differential expression of miR-140-3p, plxnb1, and GLP-1R in different groups. n=3 per group. (C, D) Western blotting for Plexin B1 and GLP-1R expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (E) MiR-3657 expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (F, G) GLP-1R and Plexin B1 levels in BMSCs (con, miR-140-mimics/inhibitor, miR-3657-mimics/inhibitor). n=3 per group. (H) GLP-1R levels in BMSCs (con, miR-140-mimics, miR-140-mimics + miR-3657 inhibitor, miR-140-3p+miR-3657-mimics). n=3 per group.

Article Snippet: To investigate the regulatory role of Sema4D in cancellous bone loss and impaired bone defect regeneration in mice, mice were randomly divided into 5 groups (n = 6/group): control group, Sema4D group (10 ng/mL), Sema4D group (15 ng/mL), Sema4D (10 ng/mL) + metformin (300 mg/kg/day, MCE) group and Sema4D (15 ng/mL) + metformin (300 mg/kg/day, MCE) group.

Techniques: Expressing, Quantitative Proteomics, Western Blot