Effects And Mechanism Of Niclosamide On Proliferation And Migration Of Pulmonary Artery Smooth Muscle Cells

Background: Excessive proliferation and migration and apoptosis resistance of PASMCs are the hallmarks in the vascular remolding of PAH.Wnt/?-catenin signaling pathway plays a crucial role in the proliferation of PASMCs in PAH.Nicolsamide,a widely used antihelminthic medicine for over fifty years,which shows multiple biological functions in cancer cells,including inhibition of proliferation and migration,induction of apoptosis,and Nicolsamide influences various signaling pathways like Wnt/ ?-catenin,P53,ROS and so on.However,little is known to us that whether Nicolsamide could influence the proliferation,migration and apoptosis of PASMCs.In this study,we investigate the functions of Nicolsamide on proliferation and migration as well as apoptosis of PASMCs and determine the underlying mechanism.Methods: In this study,we treated pulmonary artery smooth muscle cells with different concentrations and different time of niclosamide.First,we carried out CCK-8proliferation experiments to see if niclosamide may affect the proliferation of pulmonary artery smooth muscle cells,and The EdU proliferation experiment was performed to understand whether niclosamide can affect DNA synthesis in pulmonary artery smooth muscle cells.Second,we performed the wound healing assay to see if niclosamide can affect the migration of pulmonary artery smooth muscle cells.We performed Hoechst apoptosis staining and Annexin V-FITC flow cytometry analysis to see if niclosamide can induce apoptosis of pulmonary artery smooth muscle cells.We performed cell cycle analysis by using flow cytometry to explore whether niclosamide interferes with cell proliferation by interfering with the cell cycle of pulmonary artery smooth muscle cells.Finally,To understand whether niclosamide affects apoptosis by targeting reactive oxygen species and mitochondria,we analyzed the effects of niclosamide on the production of reactive oxygen species in pulmonary artery smooth muscle by flow cytometry analysis,fluorescence microscopy and flow cytometry analysis were used to examine the influence of niclosamide on mitochondrial membrane potential of pulmonary artery smooth muscle cells;Western blot analysis of the effect of niclosamide on cycLin D1 protein expression in pulmonary artery smooth muscle cells to understand whether niclosamide affects cycLin D1 protein expression to intervene in the cell cycle.The effect of niclosamide on the expression of apoptosis-related proteins in pulmonaryartery smooth muscle cells was analyzed by Western blot to understand the mechanisms affecting apoptosis,including caspase-3,caspase-9,Bax,Cytochrome c,and p53.To explore the mechanism of niclosamide on anti-proliferation of pulmonary arteries smooth muscle cell,Western blot analysis was performed to examine the protein expression of Wnt / ?-catenin signaling pathway in pulmonary artery smooth muscle cells,including?-catenin,Wnt5 a,DVL2,DVL3,Axin1,P-LRP6.Results: It was found that niclosamide inhibited the proliferation of pulmonary artery smooth muscle cells by CCK-8.The EdU experiment showed that niclosamide treatment for 24 hours could reduce the DNA synthesis of pulmonary artery smooth muscle cells,the percentage of EdU positive cells in the control group was(9.8±0.7)%,the percentage of EdU positive cells in the 0.3 ?M niclosamide-treated group was(3.4±0.7)%,and the percentage of EdU positive cells in the treated group was decreased by 65%.niclosamide can inhibit the migration of pulmonary artery smooth muscle cells.Niclosamide can cause a disorder in the cell cycle of pulmonary artery smooth muscle,The percentage of cells in G0/G1 phase and S phase of the control group were(61.98±1.04)%,(27.08 ± 2.88)% respectively.The percentage of cells in G0/G1 phase and S phase of treatment group were(72.85 ± 0.52)%,(14.26 ± 1.23)%,respectively.Niclosamide induces apoptosis in pulmonary artery smooth muscle cells.Niclosamide causes an increase in the production of reactive oxygen species in pulmonary artery smooth muscle cells,whereas niclosamide has no effect on the mitochondrial membrane potential of pulmonary artery smooth muscle cells.Niclosamide causes a decrease in the expression level of cycLin D1 protein in pulmonary artery smooth muscle cells.Niclosamide increase the expression of caspase-3,caspase-9,Bax,Cytochrome c,and p53 proteins in pulmonary artery smooth muscle cells.Niclosamide inhibits the expression of ?-catenin,Wnt5 a,DVL2,DVL3,Axin1,and P-LRP6 proteins in pulmonary artery smooth muscle cells.Conclusion: our study demonstrates that Nicolsamide can inhibit the proliferation and migration of HPASMCs,Nicolsamide can induce the apoptosis of HPASMCs,which is mediated by ROS-mediated apoptosis and upregulation of P53,apoptosis of HPASMCs induced by Nicolsamide is not mediated via influencing the Mitochondrial membrane potential;The anti-proliferation effect of nicolsamide on HPASMCs is mediated by cell cycle arrest at G0/G1 phase and suppression of Wnt/?-catenin signaling pathway and protein expression of cycLin D1;.