Bone marrow mesenchymal stem cells promote uterine healing by activating the PI3K/AKT pathway and modulating inflammation in rat models

Figure 4 Influence of the microenvironment on uterine smooth muscle cells and bone marrow mesenchymal stem cells. A: Representative optical micrographs of P1 uterine smooth muscle cells (USMCs) cultured for 7 d (scale bar = 100 μm); B: Immunofluorescence staining for USMC markers, with green fluorescence indicating positivity (scale bar = 200 μm); C: Bright-field image showing USMC migration at 2 h, 24 h, and 48 h, with red dashed lines delineating migration contours (scale bar = 1000 μm); D: Quantification of the residual USMC area. ^bP < 0.01 vs the USMCs, ^cP < 0.001 vs the USMCs; E: Fluorescence images depicting the 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation assay used to detect USMC proliferation, where red fluorescence indicates EdU-positive proliferating cells and blue fluorescence indicates Hoechst 33342-positive nuclei (scale bar = 400 μm); F: Percentage of EdU-positive USMCs. ^cP < 0.001 vs USMCs; G: Immunofluorescence staining of alpha-smooth muscle actin (α-SMA) and calponin in bone marrow mesenchymal stem cells (BMSCs). Green indicates positivity, and the fluorescence intensity reflects the expression level (scale bar = 200 μm); H: Western blot analysis of α-SMA, calponin, and the Kiel-67 marker of proliferation (Ki67) expression in BMSCs cocultured with USMCs for 1, 3, or 5 d; I: Statistical analysis of relative protein levels at different coculture times. ^aP < 0.05 vs the 3-d group, ^bP < 0.01 vs the 1-d group, ^cP < 0.001 vs the 1-d and 3-d groups, ^dP < 0.001 vs the 1-d group. ImageJ software was used to process the images. The data are presented as the mean ± SD, n = 3 per group. P values for D were determined via two-way ANOVA, and those for I were determined via one-way ANOVA. Comparisons between two groups in F were conducted via a t test. ER: Estrogen receptor.