Inhibitory Effect Of Sodium Tanshinone ?A Sulfonate On Hypoxia-induced Proliferation Of Rat Pulmonary Arterial Smooth Muscle Cells In Vitro

ObjectivePulmonary arterial hypertension is a common complication of chronic lung disease.The pulmonary vascular remodeling caused by abnormal proliferation of pulmonary artery smooth muscle cells plays a key role in the pathophysiological process of pulmonary arterial hypertension.Although the treatment of underlying lung disease can reduce mild pulmonary arterial hypertension,pulmonary hypertension in the end-stage lung disease is irreversible.Currently,the drugs for idiopathic pulmonary arterial hypertension and pulmonary hypertension associated with chronic lung disease.do not have satisfactory effect in clinical trials.Studies have shown that the effective ingredient of Salvia miltiorrhiza,tanshinone II A,for the treatment of pulmonary arterial hypertension can reduce mean pulmonary artery pressure,relieve clinical symptoms and increase exercise tolerance,but the specific mechanism is unknown.This study was to observe the effect of sodium tanshinone ?A sulfonate(STS)on the proliferation of rat pulmonary arterial smooth muscle cells(PASMCs)induced by hypoxia and further to explore its molecular mechanism.Methods(1)Rat pulmonary artery smooth muscle cell lines after passage were randomly divided into normoxic group,normoxic + STS group(5 ng/ml,10 ng/ml,20 ng/ml),hypoxic group,hypoxic + STS group(5 ng/ml,10 ng/ml,20 ng/ml).The absorbance values of each group measured by CCK-8 method represent the cell variability of pulmonary artery smooth muscle cells.(2)Similarly,Pulmonary arterial smooth muscle cells were randomly divided into normoxic group,hypoxic group,and hypoxic + STS group(10 ng/ml).Relative mRNAs expression level of mTOR,eIF2?,and c-myc were determined by fluorescent quantitative PCR.(3)Consistent with the culture method above,pulmonary arterial smooth muscle cells were randomly divided into normoxic group,hypoxic group,hypoxic + STS group(10 ng/ml),hypoxia + rapamycin group(20 nmol/L).The protein expression of mTOR,p-mTOR,e IF2?,p-eIF2? and c-myc in each group was detected by Western blotting.Results(1)CCK-8 results showed that compared with the normoxic group,the cell variablity in the hypoxic group was significantly higher,and the difference was statistically significant(P<0.05);compared with the hypoxic group,the cell variablity in the hypoxic group treated with STS was significantly reduced(P<0.05).However,compared with the normoxic group,the cell variablity in the normoxic group treated with STS was not significantly different,and the difference was not statistically significant(P>0.05).(2)Analysis by PCR showed that the relative mRNA expressions of mTOR,eIF2?,and c-myc in the hypoxic group were significantly higher than those in the normoxic control group(all P<0.05);compared with the hypoxic group,The expressions of all three mRNAs in the hypoxic group treated with STS decreased,and the difference was statistically significant(all P<0.05).(3)The protein expression of each group was determined by Western blotting: Compared with the normoxic group,the expression of mTOR,p-mTOR,eIF2?,p-eIF2? and c-myc in the hypoxic group was significantly increased,the difference was statistically significant(all P <0.05).Compared with the hypoxic group,The protein levels of mTOR,p-mTOR,eIF2?,p-eIF2? and c-myc in hypoxic+STS group and hypoxia+rapamycin group were significantly increased and the difference was statistically significant(P<0.05).However,There was no significant difference in protein expression between rapamycin group and STS group(P>0.05).Conclusion(1)Hypoxia can upregulate mTOR,eIF2 a and c-myc proteins and promote the proliferation of rat pulmonary arterial smooth muscle cells.(2)Tanshinone ?A sodium sulfonate may reduce the expression of eIF2 a and c-myc protein through the mTOR signaling pathway and inhibit hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells.Consequently,STS has potential therapeutic effects on pulmonary hypertension.