Fig. 5: Overexpressed KLF4 attenuated TiPs-induced inflammation by suppressing NF-κB pathway and macrophages M1/M2 polarization.

A IHC staining result of human synovial membranes from femoral head necrosis (FHN) patients and prosthetic aseptic loosening (AL) patients to detect the expression of KLF4. The black bar, 50 μm. B Supernatant TNFα and IL-6 from KLF4-knockdown (si-KLF4) or KLF4-overexpressed (KLF4⁺) macrophages were analysed by ELISA assay after 8 h TiPs stimulation. C mRNA expressions of TNFα and IL-6 in KLF4-knockdown macrophages or KLF4-overexpressed macrophages were examined after 8 h TiPs activation. D Total p65, total IκBα, phosphorylated p65 and phosphorylated IκBα were detected by WB assay to determine the activation of NF-κB signaling pathway upon 1 h TiPs stimulation in KLF4-knockdown macrophages or KLF4-overexpressed macrophages. E After 1 h TiPs stimulation, nuclear translocation of p65 was observed by confocal microscopy in Vector or KLF4-overexpressed macrophages. The white bar, 10 μm. F Macrophage polarization upon 24 h TiPs stimulation was estimated by FCM assay with iNOS symbolizing M1 polarization and CD206 symbolizing M2 polarization. G Quantitative analysis of FCM assay to measure macrophages M1/M2 ratio in Vector or KLF4-overexpressed macrophages upon 24 h TiPs stimulation. H The expressions of macrophage M1 polarization relative genes (IFN-γ, IL-23, IL-17a) and M2 polarization relative genes (IL-10, Arg-1, Ym-1) were detected by qPCR assay. All data were concluded from at least three independent assays. Statistic data were displayed as mean ± SEM and were conducted unpaired t test analysis or one-way ANOVA analysis to determine significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the negative group.