Effects Of Mg²⁺ On The Proliferation And Migration Of PASMCs In Pulmonary Hypertension Rats

Pulmonary Hypertension?PH?is a seriously fatal disease characterized by increased pulmonary vascular resistance and vascular remodeling,which can be said to be a cancer in cardiovascular disease.Pulmonary vascular remodeling is mainly caused by elevated intracellular free Ca²⁺concentrations in pulmonary arterial smooth muscle cells?PASMCs?,Which can affect the proliferation and migration of vascular smooth muscle cells?VSMC?through a variety of specific regulatory mechanisms.However,Mg²⁺is a physiological Ca²⁺antagonist,which is affected by the concentration of extracellular magnesium.Low magnesium can increase the reactivity of vascular smooth muscle,increasing the level of extracellular magnesium can reduce the tension and arterial pressure of cardiovascular smooth muscle.In the pathogenesis of PH,the function of Mg²⁺and its transporter with its effect on the proliferation of PASMCs has been less reported.In this study,pulmonary hypertension model was male rats induced by Monocrotaline and Chronic Hypoxemia respectively.The aim was to study the functional changes of solute carrier family 41?SLC41?and TRPM7,and the effect of Mg²⁺on the proliferation and migration of PASMCs in PH rats,to further investigate the role of SLC41 and TRPM7 in the proliferation of PASMCs in PH rats,so as to provide theoretical basis for the pathogenesis of PH.Objective:In vitro study of the effect of Mg²⁺on the proliferation and migration of PASMCs in PH rats induced by MCT and CH,and to explore the relationship between the magnesium ion channel/transporter and the proliferation of PASMCs,and provide a new theoretical basis for the pathogenesis and clinical treatment of PH.Methods:?1?Construction of PH model:150¹60g healthy male SD rats were randomly divided into control group?Control,CON?and model group?MCT,CH?,the group of MCT rats were injected by MCT?50mg/kg?at one time,the group of CH SD rats were placed in anoxic box?about 10%?,and the model group was identified.?2?PASMCs culture:isolated the identified pulmonary arteries of PH rats,cultured in vitro PASMCs,the group of CON and MCT PASMCs in constant temperature constant oxygen incubator?37°C,5%CO2?,and CH group cultured in low oxygen incubator?3%O2?.?3?Western Blot and RT-PCR were used to detect the protein and mRNA expression levels of the main magnesium channel/transporter in the three groups of PASMCs.?4?the experiment of proliferation and migration:the pre-experiment groups were given the same concentration gradient MgCl₂ solution respectively.1%FBS and10%FBS were pretested first,and the effect of Mg²⁺was added to the 5%FBS experimental group.MTS method was used to detect the proliferation of PASMCs in the above experimental groups,and the effect of Mg²⁺on the migration of PASMCs was detected by the method of scratching or Transwell chamber.?5?When Mg²⁺acted,the functional changes of solute carrier family 41?SLC41?and TRPM7 on the above three groups of PASMCs.Results:?1?compared with CON,?1?the RVSP,RVP and RVMI of rats in the group of MCT and CH were significantly increased.The smooth muscle layer of the pulmonary arteriole was thickened and the lumen was narrowed,suggesting that PH rats induced by MCT and CH have been successfully constructed.?2?mRNA and protein expression levels of SLC41A1,SLC41A2 and TRPM7 in the PASMCs of MCT and CH group were significantly increased,while SLC41A3 mRNA and protein expression levels were significantly down regulated in the group.?3?the proliferative activity of PASMCs in model group was significantly enhanced,suggesting that MCT and CH can promote the proliferation of PASMCs.?4?the migration ability of PASMCs in model group is higher than that in group CON,suggesting that PH can promote PASMCs migration.?2?under the preconditioning experiment,eliminating the effect of FBS in need:?1?the effect of low level of high[Mg²⁺]_o:3mM?mmol/L?Mg²⁺on PASMCs proliferation,but the difference is not significant;when 10mM Mg²⁺action,PASMCs proliferation is slow,but there are also individual differences,the proliferation of PASMCs in the model group can not be significantly inhibited;?2?ultra high level[Mg²⁺]_o:30mM Mg²⁺action,can partially inhibit the proliferation of PASMCs in PH group,the difference is statistically significant;60mM Mg²⁺action,each group of PASMCs on proliferation is significantly inhibited,with statistical significance,but not of clinical significance.Conclusion:MCT and CH can induce PH in rats and up-regulate the expression of SLC41A1/2,TRPM7 and MAGT1 on the magnesium transporter/ion channel on PASMCs,down the expression of SLC41A3,inducing pulmonary vascular remodeling and right ventricular hypertrophy,promoting the proliferation and migration of PASMCs.Low level of high[Mg²⁺]_o can not significantly inhibit the proliferation and migration of PASMCs in PH rats,theoretically,the intervention concentration of magnesium in reversing pulmonary vascular remodeling,which required a higher level of[Mg²⁺]_o.