Docosahexaenoic Acid Inhibits Pulmonary Vascular Remodeling Via Attenuating Inflammation And Endoplasmic Reticulum Stress

Backgrounds and ObjectivesPulmonary hypertension (PH) is a progressive hemodynamic condition that ultimately leads to elevated pulmonary vascular resistance,right ventricular failure and death. Endoplasmic reticulum (ER) stress and inflammation contribute to PH pathogenesis. Previously, we confirmed docosahexaenoic acid (DHA) could improve hypoxic-induced PH.However, little is known about the link between DHA and monocrotaline (MCT)-induced PH. Our aims were, therefore, to evaluate the effects and molecular mechanisms of DHA on MCT-induced PH in rat.MethodsMale Sprague-Dawley (200-250g) rats were divided into four groups randomly: control group (n=10), MCT group (n=10),MCT+DHA-prevention group (n=10) and MCT+DHA-reversal group(n=10). Rat PH was induced by MCT. Rats were treated with DHA daily in prevention group (following MCT injection) and reversal group(after MCT injection for 2 weeks) by gavage.① After 4 weeks, mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index and morphologicaland immunohistochemical analysis were measured.②Real-time PCR was performed to quantify mRNA levels of the proinflammatory cytokines, IL1β, IL-6, MCP-1 and TNFCα.Immunohistochemistry was applied to detect the CD68 and CD3 positively stained cells in whole-lung tissue sections and peri-vessels.③ Western blot analysis and immunofluorescence staining were applied to detect the expression of ER stress markers in the lung tissue.Rat pulmonary artery smooth muscle cells (PASMCs) were used to investigate the effects of DHA on cell proliferation stimulated by PDGF-BB.① Western blot analysis and immunofluorescence staining were applied to detect the expression of ER stress markers in PASMC.② Real-time PCR was performed to quantify mRNA levels of the NFATc1, NFATc1, NFATc2, NFATc3 and NFATc4 in PASMC.③ Western blot analysis was applied to determine the expression and activation of NFATc1 in PASMC.④ Cell Counting Kit-8 test and BrdU incorporation were applied to dectect PASMC proliferation. Flow cytometry cell cycle analysis was determined by staining cells with propidium iodide (PI).Results① MCT treatment resulted in an increase in mean PAP (MCT: 48.2±3.1 mmHg vs control: 20.5± 1.3 mmHg;p<0.05),which was prevented and partially reversed by DHA (25.3±2.1 mmHg and 31.5±3.4 mmHg,respectively; p<0.05 vs MCT group). MCT-induced RV hypertrophy was increased compared with control group (0.55±0.01 vs 0.26±0.01;p<0.05). This increase was also prevented and partially reversed by DHA (0.37±0.02 and 0.42±0.01, respectively; p<0.05 vs MCT group).② The mRNA levels of IL-6, MCP-1, IL-1β and TNPα were significantly increased in the lungs of MCT group compared with those in control group, and MCT induced expression of these cytokines mRNA was attenuated by DHA. DHA also inhibited both lymphocytes and macrophages accumulation in the lung and pulmonary perivasculature of the MCT-injury rats.③ Compared with control group, protein expressions of ER stress markers GRP78, PDI and p-IRE1α were upregulated in the lung tissues from MCT group; by contrast, the expression of these ER stress markers was not increased in the DHA-treated group. Immunofluorescence staining revealed an increased expression of PDI in smooth muscle cells of the resistance PAs in MCT group compared with control group, which was partly suppressed in DHA reversal group and nearly absent in the prevention group.④ DHA reduced ER stress marker upregulation induced by chronic stimulation of PDGF-BB, which was consistent with the results of immunofluorescence.⑤Treatment of PASMC with PDGF-BB caused a significant increase in the mRNA levels of NFATc1, NFATc2 and NFATc3; preincubation with DHA inhibited the PDGF-BB-induced increase of NFATc1and NFATc2 and there was a lack of change in NFATc3.⑥ Treatment with PDGF-BB triggered an increase in total protein expression of NFATc1 and its nuclear translocation. Both DHA and 4-PBA inhibited NFATc1 expression and activation induced by PDGF-BB.⑦ PDGF-BB enhanced cell proliferation by 50% at 72 hour as compared to the control condition. However, cell proliferation was decreased by either DHA preincubation or NFATcl siRNA. Flow cytometry analysis showed that more PASMCs entered S phase from G0/G1 phase upon stimulation by PDGF-BB, compared to control cells. Consistent with cell cycle progression, protein expression level of cyclin D1, a key protein that promotes G1/S phase transition, was significantly increased in PDGF-BB-stimulated PASMCs, whereas the expression of p21 and p27, the cyclin-dependent kinase inhibitors, were significantly decreased in PDGF-BB treated cells. PDGF-BB-mediated p21 and p27 downregulation and cyclin D1 upregulation were reversed by DHA and NFTAc1 siRNA.Conclusions These findings suggest that DHA could protect against MCT-injured PH by reducing ER stress, suppressing cell proliferation and inflammation.