Figure 1.
IL-1/TLR-induced NFκB signaling pathway.
After the stimulation, IL-1R/TLR recruits adaptor molecule myeloid differentiation factor 88(MyD88) to their TIR domain, which further recruits and activates IRAK4. Then IRAK4, TRAF6 and IRAKs combine into a complex. After the coalition of Pellino2 and TAK1, the new complex is divided into at least two parts: complex including TAK1, activating NFκB through IκBα phosphorylation and degradation, and complex including IRAK4 which phosphorylates p38 and binds to the ARE-binding proteins like HuR and c-Myc. Two complexes both promote the release of cytokines and chemokines like IL-6 to further promote inflammation response. During the process EMR1 keeps growing.
Table 1.
Levels of serum GLU, CHOL and TG in GK rats and Wistar rats.
Figure 2.
EVG staining of common carotid arteries in Wistar and GK rats (100×).
EVG staining of uninjured (A-a, A-b), Day 3 post-injury (A-c, A-d) and Day 7 post-injury (A–e, A–f) in Wistar and GK rats. Neointimal emerged at Day 7 post-injury (B), the media peaked at Day 3 post-injury (C) and N/M ratio in GK rats was higher than that in Wistar rats (D) at Day 7 post-injury. White arrows pointed to the media. Data are represented as mean ± SEM. ΔΔΔindicates versus uninjured, p<0.001; *indicates versus Day 3 post-injury, p<0.05, **p<0.01; #indicates versus Day 7 post-injury, p<0.05, ###p<0.001.
Figure 3.
Representative images of EdU staining of common carotid arteries in Wistar and GK rats (400×).
EdU staining (A–a, b, c, d), Hoechst33342 staining (A–e, f, g, h), images of merging EdU staining and Hoechst33342 staining (A–j, k, m, n). Percentage of EdU-positive cells of Day 3 post-injury were more than those of Day 7 post-injury in both rats and percentage of EdU-positive cells in GK rats were more than those in Wistar rats (B). Data are represented as mean ± SEM. **indicates versus Day 3 post-injury, p<0.01, ***p<0.001; #indicates versus Day 7 post-injury, p<0.05.
Figure 4.
Immunohistochemistry for the expression of PCNA and NFκBp65 in common carotid arteries.
PCNA-positive cells of uninjured in both rats (A–a, A–b). PCNA-positive cells of Day 3 post-injury (A–c, A–d) were more than those of Day 7 post-injury (A–e, A–f) in both rats and PCNA-positive cells in GK rats were more than those in Wistar rats (B). NFκBp65-positive cells (C) in GK rats were more than those in Wistar rats at Day 7 post-injury. Data are represented as mean ± SEM. ΔΔΔindicates versus uninjured, p<0.001; ***indicates versus Day 3 post-injury, p<0.001; ##indicates versus Day 7 post-injury, p<0.01.
Figure 5.
Agilent Whole Genome Oligo Microarray of common carotid arteries in Wistar and GK rats.
Through three pairs of comparison (Wistar uninjured versus Wistar Day 7 post-injury (A), GK uninjured versus Day 7 post-injury (B), Wistar Day 7 post-injury versus GK Day 7 post-injury(C)), IL-1/TLR-induced NFκB activation signaling pathway was filtered out.
Figure 6.
qRT-PCR of factors from common carotid arteries in Wistar and GK rats.
mRNA levels of TLR4, IRAK4, HuR, IκBα, EMR1 and c-Myc in Wistar rats and GK rats(A, B, C, D, E, F). Data are represented as mean ± SEM. Δindicates versus uninjured p<0.05, ΔΔp<0.01, ΔΔΔp<0.001; *indicates versus Day 3 post-injury, p<0.05, **p<0.01, ***p<0.001; #indicates versus Day 7 post-injury, p<0.05, ###p<0.001.
Figure 7.
Changes of proteins included in IL-1/TLR-induced NFκB signaling pathway by western blotting analysis invivo.
Representative western blotting of TLR4 and IRAK4 (A), EMR1 and IκBα (B), p-p38 and HuR(C). Bar diagrams depicting the relative protein level of TLR4, IRAK4, EMR1, IκBα, p-p38 and HuR (D, E, F, G, H, I). Data are represented as mean ± SEM. ΔΔΔindicates versus uninjured, p<0.001; *indicates versus Day 3 post-injury, p<0.05, **p<0.01, ***p<0.001; #indicates versus Day 7 post-injury, p<0.05, ##p<0.01, ###p<0.001.
Figure 8.
LPS-induced activation of factors in BMM.
Representative western blotting bands of TLR4, IRAK4, IκBα, p-p38, HuR in BMM from Wistar rats and GK rats (A). Bar diagrams depicting the relative protein levels after normalization to β-actin (B, C, D, E, F). Changes of IL-6 with the LPS stimulation by ELISA in both rats (G). Data are represented as mean ± SEM. Δindicates versus 0 h, p<0.05, ΔΔΔp<0.001; *indicates versus 2 h, p<0.05, **p<0.01, ***p<0.001; # indicates versus 4 h, p<0.05, ##p<0.01, ###p<0.001; $indicates 12 h, p<0.05, $$p<0.01, $$$ p<0.001; ☆indicates 24 h, p<0.05, ☆☆☆p<0.001.