Effect Of Rutaecarpin On Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells And Possible Mechanism

BackgroundPulmonary hypertension(PAH)is a malignant and fatal disease.Pulmonary vascular remodeling,characterized by unbalances of proliferation and apoptosis in human pulmonary artery smooth muscle cells(HPASMCs),is considered a major cause for the development of HPH,and makes it difficult to be reversed.Therefore,to reverse pulmonary vascular remodeling by inhibiting the abnormal proliferation of HPASMCs is expected to be another breakthrough for the treatment of HPH.In addition,the damaged cells of patients with PAH have glycolysis effect which is similar to tumor cells,and glycolysis is closely related to the rapid proliferation of cells.Therefore,it would be a new therapy direction for the treatment of PAH from the perspective of energy metabolism to study the mechanism of pulmonary vascular remodeling.Rut carries out a wide range of biological and pharmacological activities,such as vasodilation,anti-angiogenesis,anti-oxidative stress,and regulating proliferation and apoptosis.Therefore,it has a wide range of applications in cardiovascular diseases.However,the role of Rut in HPH remains to be elucidated.In this study,we developed an in vitro model of HPH based on hypoxia-induced HPASMCs in order to investigate the effect of Rut on hypoxia-induced HPASMCs and possible mechanism.Methods1.Determination of IC50 of Rut in HPASMCsThe IC50 of Rut in HPASMCs was determined by CCK-8 method under normoxia and the optimal concentration of drug was explored.2.Established hypoxia model of HPH model in vitroHypoxia model of HPASMCs was established in vitro.Cell proliferation rate of hypoxia-induced HPASMCs was measured by CCK-8 and cell counting,and apoptosis was detected by flow cytometry.Lactate assay kit was used to determine the content of lactic acid in cell supernatant.3.The effect of Rut on hypoxia-induced HPASMCsHPASMCs were treated with indicated concentration(0,0.5,5,10,and 50μmol·L-1)of Rut in normoxia or hypoxia.Cell proliferation rate,apoptosis rate,and the content of lactic acid in cell supernatant were determined according to the above method.The mRNA expression of HIF-la was detected by qRT-PCR.The protein expressions of HIF-la and its target genes p53,p21,GLUT-1,PCNA,VEGF,and EPO were measured by Western-Blot.4.The mechanism of the effect of Rut in hypoxia-induced HPASMCsHPASMCs were treated with Rut(5μmol·L-1)in combination with siRNA against HIF-la or a control vector(scrambled siRNA)in hypoxia.Cell proliferation rate,apoptosis rate,and the content of lactic acid in cell supernatant,the protein expression of HIF-lα,p53,p21,GLUT-1 and PCNA of HPASMCs in hypoxia were measured according to the above method.5.The effect of GLUT-1 on hypoxia-induced HPASMCsAfter the intervention of GLUT-1 with siRNA,the cell proliferation rate of hypoxia-induced HPASMCs were measured by CCK-8 and cell counting method.The protein expression of PCNA was measured by Western-Blot.ResultsThe IC50 of Rut in HPASMCs under normoxia was 43.5 μmol-L-1.Hypoxia significantly increased proliferation and the content of lactic acid in cell supernatant and decreased apoptosis in HPASMCs,whereas Rut in the concentration above 5μmol·L-1 could rescue this phenomenon.Meanwhile,Rut effectively decreased the protein and mRNA expressions of HIF-la,and simultaneously reduced GLUT-1 and PCNA,whereas increased p53 and p21 protein levels.Knockdown of HIF-1a expression by small interfering RNA(siRNA)significantly enhanced the proapoptotic effect rather than antiproliferation effect of Rut in HPASMCs.No significant difference was observed in the content of lactic acid in cell supernatant and the protein levels of p53,p21,GLUT-1,and PCNA.After the intervention of GLUT-1 with siRNA,the proliferation of HPASMCs under hypoxia decreased significantly.Conclusion:In hypoxia-induced HPASMCs,Rut can decrease the expression of HIF-1α,thus,inhibiting proliferation by down-regulating the expression of downstream proliferation-related genes GLUT-1 and PCNA.And promoting apoptosis by up-regulating the expression of apoptosis-related genes p53 and p21.In addition,Rut also inhibits glucose transport and glycolysis by decreasing the expression of HIF-1α.