Puerarin Influences Hypoxia-induced PASMCs Proliferation Through Energy Metabolism

Pulmonary arterial hypertension is a fatal disease that leads to right heart failure by pulmonary vascular resistance and elevated pulmonary arterial pressure.It is main caused by pulmonary vasoconstriction and pulmonary arterial lumen narrowing due to proliferation of pulmonary artery endothelial cells and smooth muscle cells.The development of drug therapy has improved the prognosis of pulmonary hypertension resently,although it remains poor.Puerarin is an isoflavone extracted from the Chinese medicine Pueraria,and exhibits the functions of anti-proliferation,the cardiovascular system,nervous system,osteoporosis,liver injury,and inflammation in vivo and in vitro.With the development of pulmonary hypertension,the transition of energy metabolism is also an important pathological process.Studies in recent years have also shown that puerarin has a certain therapeutic effect on pulmonary hypertension and anti-proliferation,but its specific anti-proliferative mechanism is still unknown.In this experiment,an animal model of pulmonary hypertension was first evaluated.The rats were diagnosed with hypoxia to induce pulmonary hypertension.The right heart catheterization method was used to evaluate the physiological parameters such as hemodynamics and Vessel diameter in the model rats.Histochemical method and Western Blot were used to detect the expressions of hexokinase II,pyruvate kinase 2 and pyruvate dehydrogenase in rat lung tissues,and to determine the expression of key enzymes for energy metabolism under hypoxic conditions.Pulmonary arterial smooth muscle cells were then cultured in vitro,and the effects of puerarin on cell proliferation under hypoxia were analyzed by CCK8 and KI-67 experiments.Western Blot was used to detect changes in the expression of key proteins for energy metabolism,and ATP production was measured using ATP detection kit To further determine the effect of puerarin on energy metabolism of pulmonary artery smooth muscle cells under hypoxia.At the same time,chemical inhibitors were used to inhibit the process of energy metabolism.The expression of PCNA in proliferating cells was measured by Western Blot.The change of cell proliferation was measured by CCK-8 method.It was further observed whether puerarin could exert anti-proliferative effects after inhibiting energy metabolism.The above experimental results show that puerarin may play a protective role in inhibiting the proliferation of pulmonary artery smooth muscle cells both in vivo and in vitro through the process of energy metabolism.In the hypoxic rat model,the pulmonary artery vessel wall was significantly thickened,and the pulmonary artery pressure was increased(P<0.01).The expression of key proteins hexokinase II and pyruvate kinase 2 was significantly down-regulated,and pyruvate dehydrogenase was significantly up-regulated(P<0.01).In the cell model,the detection of cell viability showed that puerarin can inhibit cell viability.The results on cellular ATP production also showed that puerarin has the effect of restoring cellular ATP production under hypoxic conditions(P<0.01).At the same time,the results of expression of key proteins of energy metabolism in cells are consistent with those in rat tissues.It indicates that puerarin has a significant effect on the energy metabolism of pulmonary artery smooth muscle in rats.The above results prove that puerarin has a certain therapeutic effect on pulmonary artery smooth muscle cells under hypoxic conditions.Finally,by inhibiting the cell's energy metabolism process,it was found that the expression of proliferating cell nuclear antigen PCNA did not change significantly in cells,and CCK8 results also showed that puerarin was difficult to exert anti-proliferative effects after adding inhibitors(P<0.01).This study proves that puerarin can alleviate pulmonary hypertension by affecting energy metabolism,and it is difficult for puerarin to exert its therapeutic effect when inhibiting the process of energy metabolism.Energy metabolism may be the site of action of puerarin,and it will provide some benefits for puerarin treatment of PAH Pharmacological basis.