The Effect And Mechanism Of Prostacyclin Analogue Beraprost On Cardiac Fibroblasts Proliferation

Objective To investigate the effect and mechanism of prostacyclin analogue beraprost on cardiac fibroblasts proliferation. We also explore the effect of TGF-β-Smad signal on myocardial fibrosis.Methods CFs were isolated from the neonatal Sprague-Dawley(SD) rats. CFs were pre-treated with prostacyclin analogue beraprost followed by Ang II stimulation(100 nM, 4 h). Numbers of cardiac fibroblasts was determined. Content of hydroxyproline in culture mediums was measured. Real time PCR analysis was used to measure collagen I and collagen III mRNA expression. Western blot analysis and immunofluorescence staining were used to measure collagen protein expression. After prostacyclin receptor(IP) was knocked down with siRNA technology, numbers of CFs and content of hydroxyproline in culture mediums, collagen expression were measured to investigate the effect of beraprost again. Further more, pre-treatment of PPAR inhibitor was performed to observe which receptor was responsible for the anti-fibrosis effect of prostacyclin. Protein levels of TGF β,Smad2 and CREB were analyzed by western blot. Electrophoretic mobility shift assays(EMSA) was used to detected the combination between Smad and DNA. The interaction of CBP and Smad2 and p-CREB was investigated by co-immunoprecipitaion assays.Results 1. Beraprost can inhibit CFs proliferation induced by Ang II in a concentration and time depend manner, while 10 μM, 4 h has the most signification to control(P<0.01). 2. Ang II triggered significant enhancement expression of collagen I and III at both mRNA and protein level(P<0.01). Pre-treatment with beraprost(10 μM, 4 h) down-regulated collagen I expression(P<0.01), while level of collagen III showed no obvious alteration. 3. Immunofluorescence staining was carried out to further validate that beraprost inhibited collagen I expression in CFs. 4. SiRNA sequences specific for IP were transfected into CFs respectively. In those cells, Ang II-mediated proliferation and hydroxyproline content, and collagen I synthesis could no longer be ‘rescued’ by beraprost after IP was knocked down. 5. A potent PPAR inhibitor especially specific to PPARγ GW9662(10 μM), anatagonist specific to PPARβ/δ GSK0660(1 μM) or antagonist specific to PPARα GW6471(20 μM) were respectively pre-incubation for 4 h following with beraprost treatment and Ang II stimulation. Similar suppressive ability of beraprost on cell numbers and hydroxyproline secretion was still available after GW9662 pre-treated(P<0.01). 6. Exposure of CFs to Ang II(100 nM, 24 h) enhanced expression of TGF β and phosphorylation of Smad2. Moreover, treatment with bereprost significantly decreased both TGF β mRNA and protein expression(P<0.01). Beraprost also reduced Smad2 phosphorylation(P<0.01). 7. EMSA demonstrated that a stronger binding activity to Smad/DNA-binding sites has been detected in nuclear proteins from Ang II-stimulated cells, whereas beraprost weakened this binding activity. 8. Ang II significantly increased phosphorylation of CREB at ser133 but not ser142. There was an additional enhancement of phosphorylation of CREB at ser133 after beraprost pre-treatment. 9. The protein extracted from cytoplasm and nucleus was quantified with western blot respectively in CFs. It was found that phosphorylation of CREB at ser133 in cytoplasm enhanced significantly after Ang II stimulation, which translocated into nucleus in response to beraprost pre-treatment. 10. Immunofluorescence staining was performed to validate that beraprost enhanced the nuclear translocation of phosphorylation of CREB at ser133. 11. Co-immunoprecipitation analyses of nuclear protein indicated that more CREB but less Smad2 bound with CBP after beraprost pre-treatment.Conclusion Prostacyclin analogue beraprost inhibited cardiac fibroblast proliferation via activation of prostacyclin receptor but not PPAR, which might increase phosphorylation and accumulation of CREB in nucleus to bind with CBP, and suppress TGF β-Smad signal pathway, so that beraprost inhibits cardiac fibroblast proliferation.