The Role And Mechanism Of Reactive Oxygen Species In Alcoholic Pulmonary Hypertension

OBJECTIVE:To study the mechanism and effect of reactive oxygen species（ROS）on the increase of calcium concentration[Ca²⁺]_i in pulmonary artery smooth muscle cells induced by alcohol stimulation and the mechanism of pulmonary hypertension induced by chronic alcohol stimulation.METHODS:Primary cultured pulmonary artery smooth muscle cells（PASMC）from SD rats;cultured pulmonary artery smooth muscle cells with mitochondrial DNA depleted;specific plasmids were transfected into pulmonary artery smooth muscle cells;H₂-DCFDA fluorescent probe and confocal microscopy were used to detect reactive oxygen species in PASMC;The formation of the calcium concentration in PASMC was measured by Fura-2/AM fluorescent probe and confocal microscopy;the SD rat alcohol model was fed with Lieber-DeCarli fodder;and the hemodynamics of the model animals were measured.Results:（1）The increase of[Ca²⁺]_i caused by alcohol stimulation can be inhibited by mitochondrial DNA depleted;（2）Alcohol stimulation can increase the production of reactive oxygen species（ROS）in pulmonary artery smooth muscle cells,and with alcohol concentration increased intracellular ROS production increased;（3）,simulated by exogenous H₂O₂,alcohol-induced ROS produced by PASMCs is equivalent to 23.46μM H₂O₂ stimulated reactive oxygen species;（4）alcohol-induced induction of reactive oxygen species is mitochondrial origin;（5）,23.46μM H₂O₂ stimulates pulmonary artery smooth muscle cells to cause[Ca²⁺]_i elevation,and can be inhibited by calcium-sensitive receptor（CaSR）specific inhibitors Calhex231 and CaSR shRNA;（6）Pretreatment with PEG-catalase can inhibit the increase of[Ca²⁺]_i stimulated by alcohol,indicating that the main component of reactive oxygen species in alcohol-stimulated cells is hydrogen peroxide;（7）using CaSR knockout SD rats to construct chronic alcohol poisoning model,CaSR gene knockout can inhibit alcohol-induced pulmonary hypertension.Conclusion:Alcohol stimulation can increase the production of mitochondria-derived reactive oxygen species in pulmonary artery smooth muscle cells,and the main component of reactive oxygen species is hydrogen peroxide.The increased hydrogen peroxide increases the intracellular calcium concentration through calcium-sensitive receptors.Calcium-sensitive receptors can inhibit pulmonary hypertension caused by alcohol stimulation.