The Senescence Of PASMCs Promotes The Proliferation Of Pulmonary Artery Smooth Muscle Cells In Pulmonary Hypertension By Paracrine IL-6

Background and purpose The main cause of pulmonary vascular remodeling of pulmonary hypertension is the media thickening of pulmonary vascular which can be caused by the proliferation and secretion of pulmonary artery smooth muscle cells?PASMCs?.Cell senescence is a process characterized by cell growth and irreversible loss of proliferative capacity.It is reported that the senescence of PASMCs significantly increased when pulmonary vascular remodeling in chronic obstructive pulmonary disease?COPD?,but the mechanism has not been clarified.We found that the positive cells stained by?-galactosidase and the expression of p21 significantly increased or up-regulated after hypoxia induction.It suggests that hypoxia can promote the senescence of PASMCs.However,it is not clear how the senescence of PASMCs promotes the proliferation of pulmonary artery smooth muscle cells.It has been proven that the senescence of aortic smooth muscle cells promote the occurrence and development of atherosclerosis by secreting SASP?IL-1??;In addition,And the secretion of IL-6 and the production of CCL2 in aortic smooth muscle cells will be up-regulated with the senescence,which will cause chronic inflammation promoting atherosclerosis.It is important that PASMCs senescence is associated with up-regulation of IL-6 expression in chronic obstructive pulmonary disease?COPD?,which regulates the proliferation of PASMCs.We found that the senescence of PASMCs under hypoxia-induced condition accompanied with a significant increase in the expression of IL-6.Therefore,we suggest that the senescence of PASMCs may promote the proliferation of PASMCs by paracrine IL-6 in hypoxia-induced PASMCs senescence.Thereby,it can promoting the pulmonary vascular remodeling of PH.we proved that PASMCs senescence plays an important role in pulmonary vascular remodeling of PH under hypoxia-induced by the cell culture experiment.It was further verified that senescence of PASMCs may promote the proliferation of PASMCs by paracrine IL-6 and lead to pulmonary vascular remodeling of PH.The PASMCs senescence may be a new pathophysiological mechanism of pulmonary vascular remodeling in pulmonary hypertension to reveal the effect of PASMCs senescence on hypoxia-induced pulmonary vascular remodeling in PH.It aims to provide direction for further study about how to reduce the senescence of pulmonary artery smooth muscle cells to inhibit the proliferation of pulmonary artery smooth muscle cells,and then to inhibit pulmonary hypertension pulmonary vascular remodeling.Methods The model of PASMCs hypoxia was established under normal oxygen?21%O₂?and hypoxia?3%O₂?taking the primary PASMCs of the third to eighth generation SD rats.?1?The alpha-smooth muscle actin??-SMA?was identified by immunehistochemical method.?2?The activity of?-galactosidase was detected by cell senescence?-galactosidase staining kit,and the positive rate was calculated.Western blot was used to detect the levels of cell cyclin protein kinase inhibitor p21,p16 and cell senescence.?3?The proliferation rate of PASMCs was etected by CCK8.Western blot detected the expression level of proliferating cell nuclear antigen?PCNA?protein.The expression level of proliferating cell nuclear antigen?PCNA?mRNA was detected by the method of qPCR.?4?The paracrine of IL-6 was detected by ELISA kit.Result:?1?PASMCs were cultured by isolated.After 3 days,PASMCs were adherent to the wall with the shape of long spindle shape.After 7 days,it presented typical“peak-valley”growth.About 10 days,the cells were more than 90%fused.Morphological analysis of cells under inverted microscope and positive results of immunohistochemistry were performed to determine that the cultured primary cells were PASMCs?P<0.05?.?2?The expression of?-galactosidase in hypoxia group?3%O₂?was significantly higher than that in normal oxygen group?P<0.05?.The expression of cyclin protein kinase inhibitor p21 and p16was significantly increased?P<0.05,P<0.01?.?3?In hypoxia group?3%O₂?,the proliferation rate of PASMCs and PCNA expression level of PASMCs increased significantly?P<0.05?.The expression of PCNA mRNA in hypoxia group?3%O₂?PASMCs increased significantly?P<0.05?.?4?The expression of IL-6 in the supernatant of hypoxia group?3%O₂?was significantly higher than that of normoxic group?P<0.01?.?5?After treatment with IL-6 inhibitor AX-024?P<0.001?,the expression level of IL-6 in supernatant of hypoxic group?3%O₂?was significantly decreased?P<0.01?,and it will had the best inhibitory effect when the concentration of 10 nmoL/mL?P<0.05?.Conclusions1.Hypoxia-induced senescence of PASMCs2.PASMCs senescence promotes the proliferation of PASMCs by secreting cytokine IL-6.