Zuo Gui pill alleviates M1 macrophage polarization in type 2 diabetic osteoporosis through the Nrf2/HO-1/XCT/GPX4 pathway

Figure 1 Network pharmacological analysis. A: Venn diagram depicting the common target of Zuo Gui pill and type 2 diabetic osteoporosis; B: Protein-protein interaction network of intersecting targets; C: Kyoto Encyclopedia of Genes and Genomes enrichment analysis and WikiPathways analysis; D: Gene Ontology enrichment analysis; E: Binding energy of the active ingredient to the target protein. The darker blue indicates higher binding energies (unit: Kcal/mol). T2DOP: Type 2 diabetic osteoporosis; ZGP: Zuo Gui pill.

Figure 2 Results of molecular docking analysis. HO-1: Hemoxygenase-1; IL-6: Interleukin-6; Nrf2: Nuclear factor E2-related factor 2; TNF-α: Tumor necrosis factor-alpha.

Figure 3 Zuo Gui pill cooperates with Fer-1 to reduce symptoms and oxidative stress in type 2 diabetic osteoporosis. A: Weekly changes in rat blood glucose after type 2 diabetic osteoporosis model construction; B: Weekly changes in rat body weight after type 2 diabetic osteoporosis model construction; C: Serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol; D: Serum International Federation of Clinical Chemistry and Laboratory Medicine-glycated hemoglobin, insulin levels; E: Levels of malondialdehyde, superoxide dismutase, and glutathione/glutathione disulfide in serum. Data were expressed as mean ± SD (n = 3). ^aP < 0.05,^bP < 0.01,^cP < 0.001. GSH/GSSH: Glutathione/glutathione disulfide; IFCC-HbA1c: International Federation of Clinical Chemistry and Laboratory Medicine-glycated hemoglobin; LDL-C: Low-density lipoprotein cholesterol; MDA: Malondialdehyde; MET: Metformin; SOD: Superoxide dismutase; TC: Total cholesterol; TG: Triglycerides; ZGP: Zuo Gui pill.

Figure 4 Effect of Zuo Gui pill and Fer-1 on histopathology in type 2 diabetic osteoporosis rats. A: Distal femur hematoxylin and eosin staining (scale bar: 200 µm); B: Micro-CT scan of distal femur; C: Micro-CT showed distal femur bone mineral density, bone surface/total volume, bone surface/bone volume, bone volume/total volume, trabecular number, trabecular thickness, and trabecular spacing; D: Bone metabolism propeptide of type I procollagen and Tracp5b expression in serum; E: Expression of inflammatory factors interleukin-6 and tumor necrosis factor-alpha in serum. Data were expressed as mean ± SD (n = 3). ^aP < 0.05,^bP < 0.01,^cP < 0.001. BMD: Bone mineral density; BS: Bone surface; BV: Bone volume; IL-6: Interleukin-6; MET: Metformin; PINP: Propeptide of type I procollagen; Tb.N: Trabecular number; Tb.Th: Trabecular thickness; Tb.Sp: Trabecular spacing; TNF-α: Tumor necrosis factor-alpha; TV: Total volume; ZGP: Zuo Gui Pill.

Figure 5 Zuo Gui pill and Fer-1 regulate M1 macrophage polarization and nuclear factor E2-related factor 2 signaling pathway in type 2 diabetic osteoporosis bone marrow. A: M1 macrophage polarization level in bone marrow; B: Nuclear factor E2-related factor 2 (Nrf2), hemoxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), XCT, FTH1, and related mRNA expression of bone metabolism proteins receptor activator of nuclear factor-kappa B and RUNX2 in bone tissue; C: Protein associated with Nrf2, HO-1, GPX4, and XCT in bone tissue; D: Immunohistochemical staining of Nrf2, HO-1, and GPX4 in bone tissue visualized under a microscope (scale bar: 200 µm). Data were expressed as mean ± SD (n = 3). ^aP < 0.05,^bP < 0.01,^cP < 0.001. GPX4: Glutathione peroxidase 4; HO-1: Hemoxygenase-1; MET: Metformin; Nrf2: Nuclear factor E2-related factor 2; ZGP: Zuo Gui pill; Rankl: Receptor Activator of Nuclear Factor κB Ligand; Runx2: Runt-related Transcription Factor 2.