Monomeric CRP Aggravates Myocardial Injury After Myocardial Infarction by Polarizing the Macrophage to Pro-Inflammatory Phenotype Through JNK Signaling Pathway

Figure 1. Effects of mCRP and the inhibitors in the expression of (A) IL-1β and (B) TNF-α in cultured macrophages. The mRNA expression of IL-1β and TNF-α in the THP-1 cells incubated with mCRP (50 μg/mL) for 24 h was blocked by the JNK inhibitor (SP600125,10 μM) instead of the NF-κB inhibitor (CAPE,10 μM) and lipid rafts disrupter (M-β-CD, 5 mM). Data are represented as mean ± SD, *p<0.05.

Figure 2. Effects of mCRP and the inhibitors on the protein levels of (A) IL-1β and (B) TNF-α in cultured macrophages measured by ELISA. The protein levels of IL-1β and TNF-α in the supernatant of cultured THP-1 cells incubated with mCRP (50 μg/mL) for 24 h was blocked by the JNK inhibitor (SP600125, 10 μM) instead of the NF-κB inhibitor (CAPE, 10 μM) and lipid rafts disrupter (M-β-CD, 5 mM). Data are represented as mean ± SD, *p<0.05.

Figure 3. Monomeric CRP polarized the mouse peritoneal macrophages to pro-inflammatory phenotypes in C57BL/6 mice in vivo. Peritoneal macrophages were collected and F4/80, CD11c, and CD206 expression were analyzed by flow cytometry at 24 h after intraperitoneal injection. (A) All cells are gated by F4/80 staining, and (B) then the subtypes were differentiated by CD11c and CD206 staining in each group as indicated respectively. (C) Data represented the mean ± SD of five groups. n = 3 per group. *p<0.05.

Figure 4. Effects of mCRP and the JNK inhibitor, SP600125 on the protein levels of (A) IL-1β and (B) TNF-α in mice peritoneal fluids measured by ELISA. The protein levels of IL-1β and TNF-α in the peritoneal fluid stimulated by mCRP (2.5 mg/kg) for 24h was blocked by the JNK inhibitor (SP600125, 10 μM). n = 3 per group. Data are represented as mean ± SD, *p<0.05.

Figure 5. Effects of mCRP and the JNK inhibitor, SP600125 on the protein levels of JNK signaling pathway. (A) Representative images of p-JNK, JNK, and ASK1 in THP-1 cells incubated with mCRP (50 μg/mL) and/or JNK inhibitor (SP600125, 10 μM) for 8 h. (B and C) Quantitative analysis of p-JNK, and ASK1 in cultured THP-1 cells. (D) Representative images of p-JNK, JNK, and Smad2/3 in infarcted myocardium 7 days after myocardial infarction. (E and F) Quantitative analysis of p-JNK, and Smad2/3 in the infarcted area in mice. n = 3 per group. Data are represented as mean ± SD, *p<0.05.

Figure 6. Effects of mCRP and the JNK inhibitor (SP600125) on the cardiac function and infarct size in MI mice. (A) Representative long-axis or short-axis echocardiographic images of mice at 7-days after myocardial infarction in each group. (B and C) Quantitative analysis of cardiac function of EF (%) and FS (%) in mice in each group. (D) Representative TTC stained image of each mice group. The area at risk is stained as red and the infarcted myocardium is stained as white. (E) Quantitative analysis of ratio of infarct size. n = 5–7 per group. Data are presented as mean ± SD. *p<0.05 vs sham group; #p<0.05.

Figure 7. Effects of mCRP and the JNK inhibitor (SP600125) on histopathological changes in mice. (A) Representative images of HE staining of infarction area (200× magnification) at 7d and 21d post-MI in mice in each group. Scale bars 100 μm. (B) Representative images of Masson trichrome staining of infarction tissue in each group (200× magnification) at 21 d post-MI in mice. Scale bars 100 μm.