Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

Figure 5 Effects of the supernatant of Ad-Null and Ad-hepatocyte growth factor modified dental pulp stem cells on the polarization and inflammatory cytokine expression of RAW264. 7 mouse macrophages under inflammatory stimulation. RAW264.7 cells were indirectly co-cultured with the supernatant of Ad-Null modified dental pulp stem cells (DPSCs) and Ad-hepatocyte growth factor modified DPSCs for 24 h under lipopolysaccharide (150 ng/mL) stimulation. The mean fluorescence intensity of the anti-CD86 antibody was detected by flow cytometry. A: Representative image; B: A statistical plot of the mean fluorescence intensity (MFI) of CD86 was generated (n = 3); C: The mRNA expression levels of the M1 macrophage markers tumor necrosis factor-α (TNF-α) and arginase 2 (Arg2) and the M2 macrophage marker Arg1 were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) under inflammatory stimulation (n = 3); D: The mRNA expression levels of the inflammatory factors interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 were detected by RT-PCR under inflammatory stimulation (n = 3); E: The expression of phosphorylated p65 and p65 in RAW264.7 cells under inflammatory stimulation conditions was investigated using western blotting; F: The gray values were analyzed; G: The expression levels of phosphorylated p65 and p65 in RAW264.7 cells under nuclear factor-κB (NF-κB) activator treatment were detected by western blotting; H: Analyzed for gray values; I: After NF-κB activator treatment, the CD86 MFI was detected by flow cytometry; J: A statistical plot of the MFI of CD86 was generated (n = 3). All results are representative of the mean ± SEM; ^bP < 0.01, ^cP < 0.001 vs. control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs. lipopolysaccharide group; ^gP < 0.05, ⁱP < 0.001 vs. supernatant of Ad-Null modified dental pulp stem cells group. LPS: Lipopolysaccharide; DPSC-Null-CM: Supernatant of Ad-Null modified dental pulp stem cells; DPSC-HGF-CM: Supernatant of Ad-hepatocyte growth factor modified dental pulp stem cells; IL: Interleukin; TNF: Tumor necrosis factor; Arg2: Arginase 2; COX-2: Cyclooxygenase-2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.