Regulation of the Immune Microenvironment by an NLRP3 Inhibitor Contributes to Attenuation of Acute Right Ventricular Failure in Rats with Pulmonary Arterial Hypertension

Figures

• 

  Figure 1. Activation of the NLRP3 pathway was associated with RVF in PAH rats. (A) PET-CT image of PAH rat hearts showing a thickened wall and dilated chamber of the right ventricle in a rat with pulmonary hypertension. (B) The RVSP of the PAH rats before and after LPS injection is shown. The significance of differences was determined with unpaired Welch’s t-test (n = 6). (C) The FAC [FAC = (end-diastolic area - end-systolic area)/end-diastolic area×100%] in the PAH rats before and after LPS injection is shown. The significance of differences was analysed with unpaired Welch’s t-test (n=6). (D) The IL-1β mRNA level in normal rats (SD rats were fed for 28 days after intraperitoneal injection of saline, and the samples were collected 6 hours after another intraperitoneal injection of saline), normal+LPS rats (SD rats were fed for 28 days after intraperitoneal injection of saline, and the samples were collected 6 hours after intraperitoneal injection of LPS), PAH rats (SD rats were fed for 28 days after intraperitoneal injection of MCT, and the samples were collected 6 hours after intraperitoneal injection of saline), and PAH+LPS rats (SD rats were fed for 28 days after intraperitoneal…

• 

  Figure 2. NLRP3 inhibition effectively protected right ventricular function, relieved the asymmetrical IL-1β elevation and erased the change in inflammation in the right ventricle. (A) The FAC in PAH+LPS rats (SD rats were fed for 28 days after intraperitoneal injection of MCT; intravenous injection of saline was performed 1 hour before LPS injection, and the samples were collected 6 hours after intraperitoneal injection of LPS) and PAH+MCC950+LPS rats (SD rats were fed for 28 days after intraperitoneal injection of MCT; intravenous injection of MCC950 was performed 1 hour before LPS injection, and the samples were collected 6 hours after intraperitoneal injection of LPS) before and after LPS injection is shown. The significance of differences was determined with two-way ANOVA (n = 6). (B) IL-1β immunofluorescence in the right ventricle of PAH+LPS rats and PAH+MCC950+LPS rats 6 h after LPS injection. (C) Pretreatment with MCC950 significantly alleviated the asymmetrically increased IL-1β expression in myocardial tissue of the right ventricle. The ratio of the IL-1β area% in the right ventricle to that in the left ventricle is shown. The significance of differences was determined with…

• 

  Figure 3. Changes in the proportion of immune cells in myocardial tissue were analysed via flow cytometry. (A) The proportion of CD45+ cells in each group was detected by flow cytometry. (B) The proportion of CD45+CD11b+ cells in each group was detected by flow cytometry. (C) The proportion of CD68+CD86+ cells in each group was detected by flow cytometry. (D) Pulmonary hypertension and LPS caused a significant increase in the proportion of CD45-positive inflammatory cells in right ventricular tissue (normal vs PAH, normal vs normal + LPS, n = 3). (E) CD45-positive/CD11b-positive mononuclear macrophage infiltration was significantly elevated in right ventricular tissues from rats subjected to PAH or LPS (normal vs PAH, normal vs normal + LPS, normal + LPS vs PAH + LPS, n = 3). (F) The proportion of CD68-positive/CD86-positive M1 macrophages was decreased in the hearts of PAH rats and significantly increased in the right ventricular tissues of PAH rats stimulated with LPS, but MCC950 application significantly inhibited the change in the proportion of CD68-positive/CD86-positive M1 macrophages (n = 3). The significance of the difference was analysed by one-way ANOVA, **p < 0.01, ***p < 0.001,…

• 

  Figure 4. Through the NLRP3 pathway, cardiomyocytes participate in the macrophage polarization process through increased MCP-1 expression and cell pyroptosis under LPS stimulation. (A) We exposed H9C2 cells to AVP (1 µmol/mL) for 72 h to simulate hypertrophy of the right ventricular myocardium in rats with pulmonary hypertension, to MCC950 (100 mM) for 1 h to inhibit the NLRP3 pathway, and to LPS (100 µg/mL) for 6 h to mimic the in vivo inflammation state in the RVF model. The NLRP3 mRNA level in the H9C2, H9C2+LPS, H9C2+AVP, H9C2+AVP+LPS, and H9C2+AVP+MCC950+LPS cell groups is shown. The significance of the difference was analysed by one-way ANOVA (n = 3). (B) The MCP-1 mRNA level in the five abovementioned groups of cells is shown. The significance of the difference was analysed by one-way ANOVA (n = 3). (C) We analysed the correlation between NLRP3 and MCP-1 mRNA expression in H9C2 cell samples from different treatment groups. MCP-1 expression was highly correlated with NLRP3 expression (n = 14, r2 = 0.5840, p < 0.001). (D) The NLRP3 mRNA level in the H9C2+AVP+LPS and H9C2+AVP+MCC950+LPS cell groups is shown. The significance of differences was determined with an unpaired Welch’s t-test…