Studies On Cell Growth Inhibition And Apoptosis Inducement Of Celecoxib On Vascular Smooth Muscle Cells And Its Molecular Mechanism

Studies on cell growth inhibition and apoptosis inducement of celecoxib on vascular smooth muscle cells and its molecular mechanismBackground:Percutaneous transluminal coronary angioplasty (PTCA) has been widely accepted for the treatment of coronary artery disease, but the high rate of angiographic restenosis and recurrent symptoms at 6 months are up to 33%. It is demonstrated that the main mechanism involved in this process is vascular smooth muscle cell (VSMC) accumulation to form neointimal lesions. Cyclooxygenase-2 (COX-2) is the inducible isoform of cyclooxygenase which catalyzes the production of prostaglandin E₂ (PGE₂). COX-2 and PGE₂ are both related to inflammation and tumor's development. Recent reports show that the hyperplasia of VSMCs is inflammation events. Celecoxib, a selective COX-2 inhibitor approved by U.S Food and Drug Administration, has been shown preclinically and clinically to has efficacy comparable to that of NSAIDs for relief of pain and inflammation in osteoarthritis. However, the researches about the effects of celecoxib on VSMC_S are not well known. Therefore, in this article, we studied the biological effects of celecoxib on VSMC_S in vitro. We try to understand whether its selective inhibition might be an attractive therapeutic target in restenosis.Objectives: 1. To detect the expression of COX-2 protein in VSMC_S.2. To study the biological effects of celecoxib on cell growth inhibition and apoptosis induction.3. To investigate the functional mechanisms of celecoxib on the VSMC_S.4. Fabrication of celecoxib-PLGA microparticles.Methods:1. Cell culture: The primary and transfer culture of the rat VSMC.2. Western blot analysis was used to detect the expression of COX-2 protein.3. Morphological changes were observed under the inverted microscope.4. The effects of celecoxib on the proliferation of VSMCs were measured by cell viability and growth rate (WST-1) assay.5. Apoptotic index (AI) was detected by Flow Cytometry (FCM).6. Western blot analysis was used to detect the phosphorylation level of Akt (Thr³⁰⁸) and activation of caspase-3.7. The celecoxib-PLGA microparticles were prepared by emulsion solvent evaporation technique.Results:1. Western blot showed that COX-2 protein were detectable in VSMC_S.2. The morphological changes of the vascular smooth muscle cells became shrunken, round, and detached from the dish which treated with celecoxib.3. WST-1 assay showed that treated with 0,6.25,12.5,25,50μM/L celecoxib for 24 hours, the cell growth rate was 100%, 81.15%, 66.72%, 54.93% and 11.41%, respectively.4. Flow Cytometry analysis showed a result of 30.72% cell apoptosis ratio after 50μM/L celecoxib treated for 24 hours.5. Western blot analysis revealed celecoxib induced activation of caspase-3 after treatment with celecoxib for 24 hours in VSMCs. And western blot showed remarkable decreasing phosphorylation of Akt(Thr³⁰⁸) after treatment with celecoxib for 2 hours in VSMCs.6. The average size of the celecoxib-PLGA microparticles were 1～5μm under inverted microscope and the microparticle morphology kept stable for at least 14 days.Conclusion:1. COX-2 protein is detectable in vascular smooth muscle cells.2. Celecoxib inhibits VSMC_S proliferation and induces apoptosis in a dose dependent manner in vitro.3. The apoptosis induction by celecoxib was caused by the activation of caspase-3 through Akt signaling pathway.4. The celecoxib-PLGA microparticle morphology kept stable for at least 14 days..