Effects Of Hesperetin On Pulmonary Artery Smooth Muscle Cells Proliferation Induced By Platelet-derived Growth Factor-BB

Background:Pulmonary arterial hypertension (PAH) is recognized as a mean pulmonary artery pressure (mPAP) of 25 mm Hg or greater at rest. With complex etiology and pathogenesis, it is characterized by increased pulmonary vascular resistance that can be found in multiple clinical conditions. The histological findings in PAH include intimal hyperplasia, medial hypertrophy, adventitial proliferation/ fibrosis, occlusion of small arteries, thrombosis formed, and inflammatory cells infiltrated. The excessive of pulmonary aterial smooth muscle cells plays an important role in pulmonary vascular remodeling. PAH is thought to have a persistent elevation of pulmonary artery pressure at last leads to right ventricular failure and death, but there is no effective treatment until now, drugs frequently-used can only improve the patient's symptoms but can't make the process of PAH reversed. Therefore, it is important to find an effective treatment that can inhibit the excessive proliferation of PASMCs and improve the survival rate of PAH.Objective:to establish platelet-derived growth factor-BB stimulated pulmonary aterial smooth muscle cell proliferation in a rat model, and then investigate the effect and the possible mechanisms of hesperetin in different concentrations on PASMCs, so as to find a new treatment to prevent or reverse pulmonary vascular remodeling.Methods:Select Sprague Dawley (SD) rats weighed between 150 to 200 g, separate pulmonary artery and then use 0.2% Collagenase I digest the PASMCs. Use 20 ng/ml PDGF-BB to induce PASMCs as to establish models of the cell proliferation. Preincubation with diverse concentrations of hesperetin (12.5,25,50,100?mol/L), then treat PASMCs with 20 ng/ml PDGF-BB for 12,24 and 48 hours in the presence of hesperetin and detect the proliferation and DNA synthesis by CCK-8 and BRDU; Determine the toxicity of hesperetin on PASMCs by the trypan blue exclusion test. After 12,24 and 48 h reaction with hesperetin in various concentrations(12.5,25,50, 100?mol/L), the PASMCs were removed and excluded 0.4% trypan blue to evaluate cell viability. PASMCs were pretreated by hesperetin in 100? mol/L, then stimulated by 20 ng/mL PDGF-BB for 24 h, use flow cytometry to analyze the cell cycle progression and determine the mRNA expression of cyclin D1, cyclin E, CDK4, CDK2 and cyclin kinase inhibitor protein p27 by real-time reverse transcription PCR, The total and phosphorylated forms of ERK1/2, p38, JNK and AKT, proto-synthase kinase 3? (GSK3?) were tested in 100umol/L hesperetin pretreated PASMCs in 5,10 and 15 min after PDGF-BB induction by western blotting analysis.Results:CCK-8 and BRDU assays showed that hesperetin(12.5,25,50,100?mol/L) inhibited PDGF-BB-induced pulmonary artery smooth muscle cell proliferation and DNA synthesis in a dose- and time-dependent manner. Trypan blue staining result indicated that hesperetin did not induce cytoclasis of PASMCs with the concentration from 12.5 to 100?mol/L compared with untreated cells; Flow cytometry analysis showed that hesperetin at a concentration of 100?mol/L increased the percentages of cells in G0/G1 phase(62.0±0.6%?69.7±0.3%) and reduced the S phase(25.7± 0.9%?17.7±1.2%) populations in PDGF-BB-induced PASMCs; Real Time PCR results demonstrated that hesperetin inhibit the cell cycle progress was associated with reducing the mRNA expression of cyclin D1, cyclin E, CDK4, CDK2, as well as an increase of the mRNA expression of cyclin kinase inhibitor protein p27 in PDGF-BB-stimulated PASMCs. Further study showed that the beneficial effect of hesperetin on blocking the proliferation of PASMCs is related to suppress AKT/GSK3? and p38 signaling pathway, but little affect to extracellular signal-regulated kinase(ERK)1/2 and JNK signaling pathway.Conclusion:These results demonstrate that hesperetin can reduce the expression of cyclin D1, cyclin E, CDK4, CDK2, and increase the expression of p27 to inhibit the proliferation of PASMCs through G0/G1 to S phase of the cell cycle. Further study indicates that the anti-proliferation effect of hesperetin on PDGF-BB-induced PASMCs is associated with an inhibition of AKT/GSK3(3 and p38 signal pathway and hesperetin has therapeutic potential for pulmonary aterial hypertension.