Fig. 5

Overexpression of PRDX3 reverses inflammation-compromised macrophage polarization and mitochondrial function of RAW 264.7 cells. The cells were transfected with Adv-NC or Adv-PRDX3 and were incubated in medium with both LPS and IFN-γ for 24 h. The cells were incubated in basal medium in control group. (A) Overexpression efficiency of PRDX3 in RAW 264.7 cells validated by western blotting. Meanwhile, relative macrophage polarization related protein expression of M1 markers (iNOS and TNF-α) and M2 markers (CD206 and Arg-1) were detected. (B) Relative macrophage polarization-related gene expression level of CD206, Arg-1, iNOS, and TNF-α determined by qRT-PCR. (C) The MMP was determined using a JC-1 probe (immunofluorescence straining; scale bar: 10 μm). (D) Relative mitochondrial complex-related protein expression of NDUFB8 (subunit of complex I), SDHA (subunit of complex II), UQCR1 (subunit of complex III), COXIV (subunit of complex IV) and ATP5A (subunit of complex V) determined by western blotting. (E) Quantification of mitochondrial ROS levels were determined by MitoSOX (red) in various groups. Scale bar: 10 μm. (F) The level of MDA and the level of SOD (G) were measured in RAW 264.7 cells transfected with Adv-NC or Adv-PRDX3 and then incubated in medium with both LPS and IFN-γ for 24 h. (H) The level of ATP was measured. (I) The level of inflammatory factors (IL-6/IL-1β/TNF-α) was measured. (J) ROS staining in macrophages after PRDX3 and MitoQ interference. Scale bar: 10 μm. (K) 8-OHG staining in macrophages after PRDX3 and MitoQ interference. Scale bar: 10 μm. ^*P < 0.05, vs the Control group, ^**P < 0.05, vs the Adv-NC group