The etiological role of endoplasmic reticulum stress in acute lung injury-related right ventricular dysfunction in a rat model

Abstract

This study aimed to ascertain whether endoplasmic reticulum (ER) stress participates in acute lung injury (ALI) and related right ventricular dysfunction (RVD) as well as to explore the underlying mechanisms of these conditions. A single intratracheal instillation of lipopolysaccharide (LPS) (10 mg/kg) was used to establish the RVD model. The ER stress inhibitor, 4-PBA (500 mg/kg), was administered using a gavage 2 hours before and after the LPS treatment for prevention and treatment, respectively. At 12 hours post-LPS exposure, mRNA and protein expressions of ER stress-specific biomarkers, glucose regulating protein 78 (GRP78) and CCAAT/enhancer binding protein homology (CHOP), were significantly upregulated. This effect was inhibited by both 4-PBA prevention and treatment. In addition, echocardiography showed that 4-PBA improved the LPS-induced abnormality in the tricuspid annular plane systolic excursion (TAPSE) and the right ventricular end-diastolic diameter (RVEDD), however not in the pulmonary artery acceleration time (PAAT). Furthermore, hematoxylin and eosin staining (HE) and terminal transferase dUTP nick end labeling (TUNEL) assays revealed that the proportion of proapoptotic cells was higher in RVD rats. This was prominently ameliorated by 4-PBA treatment. Moreover, 4-PBA had a similar reverse effect on the LPS-induced increase in the Bax/Bcl-2 ratio, caspase-12, and caspase-3 expressions as revealed by western blotting. Furthermore, 4-PBA improved LPS-induced right ventricle (RV) myeloperoxidase (MPO)-positive neutrophil infiltration percentage, inhibited nuclear factor kappa B (NF-κB) activity, and reduced the expressions of inflammatory cytokines, TNF-α, IL-1β, and IL-6, in serum and RV. Taken together, our results indicated that ER stress-mediated apoptosis and inflammation might contribute to the development of ALI-related RVD induced by intratracheal LPS instillation. Gavage-administered 4-PBA could improve right ventricle (RV) systolic dysfunction and dilation, plausibly by blocking ER stress.

Keywords: 4-phenyl butyric acid; Endoplasmic reticulum stress; acute lung injury; apoptosis; inflammation; right ventricular dysfunction.

Figure 1

LPS intratracheal instillation induced obvious RV cardiomyocyte ER stress, which could be blocked by either 4-PBA prevention or treatment. An RVD rat model was established with intratracheal instillation of LPS 10 mg/kg. The ER stress inhibitor, 4-PBA (500 mg/kg), was administered using a gavage 2 hours before and after LPS in the prevention and treatment groups, respectively. (A, B) Relative expressions of GRP78 and CHOP mRNAs were detected using qRT-PCR. (C, D) Western blot bands of GRP78 and CHOP, with statistical results shown in (E and F), respectively. Data are presented as mean ± SEM and analyzed by ANOVA followed by Bonferroni post-hoc comparison. *P < 0.05 vs. con group; ^# P < 0.05 vs. RVD group, n = 3 in each group.

Figure 2

4-PBA improved LPS-induced RV systolic dysfunction and dilation. In RVD model rats, we observed a decrease in PAAT, which was not affected by 4-PBA. RV systolic dysfunction and dilation were observed as well, as reflected by a decrease in TAPSE and an increase in RVEDD, respectively. Both these effects could be improved by both 4-PBA prevention and treatment. A-D. Echocardiographic images in the parasternal view of mid-systolic pulmonary artery notching and pulse-wave Doppler. E-H. Representative M-mode measurement of TAPSE in the four groups. I-L. Apical 4-chamber views of rats in the four groups. M-P. Statistical analysis of four echocardiographic parameters, including PAAT, TAPSE, RVEDD, and RVFWT. Data are presented as mean ± SEM and analyzed by ANOVA followed by Bonferroni post-hoc comparison. *P < 0.05 vs. con group; ^# P < 0.05 vs. RVD group, n = 3 in each group.

Figure 3

Intratracheal instillation of LPS increased myocardial injury, which could be ameliorated by either 4-PBA prevention or treatment. Representative HE images of the four groups are shown. Scale bar = 50 μm.

Figure 4

Intratracheal instillation of LPS upregulated TUNEL-positive cardiomyocytes percentage, Bax/Bcl-2 ratio, and expressions of caspase-12 and caspase-3. The effect could be blocked by either 4-PBA prevention or treatment. (A) TUNEL staining of cardiomyocytes in the four groups. (B) Western blots of Bax, Bcl-2, caspase-12, and caspase-3 in the four groups. Statistical analysis of TUNEL-positive myocytes as well as expressions of Bax/Bcl-2, caspase-12, and caspase-3 are shown in (C-F) with data presented as mean ± SEM. *P < 0.05 vs. con group; ^# P < 0.05 vs. RVD group, n = 3 in each group. Scale bar = 50 μm.

Figure 5

4-PBA prevention or treatment inhibited LPS-induced MPO neutrophil infiltration, NF-κB signaling pathway activation, and inflammatory cytokine expression. LPS instillation significantly increased RV neutrophil infiltration and upregulated cytokine expression. (A) MPO staining in four groups with statistical analysis shown in (B). (C) RV NF-κB activity. RV and serum cytokine expression are shown in (D-F) and (G-I), respectively. Data in (B-I) are presented as mean ± SEM. *P < 0.05 vs. Con group; ^# P < 0.05 vs. RVD group, n = 3 in each group. Scale bar = 20 μm.