Role And Mechanism Of NAADP/TPCs/[Ca2+]i Signaling Pathway In Hypoxic Pulmonary Hypertension

Objective To study the changes of NAADP/TPCs/Ca2+ signaling pathway in pulmonary artery smooth muscle under chronic hypoxia and the relationship between this change and the proliferation of pulmonary artery smooth muscle,thus providing a new theoretical basis for the study of hypoxic pulmonary vascular remodeling(HPSR).Methods 1.A rat model of chronic hypoxic pulmonary hypertension was established.Mean pulmonary arterial pressure(m PAP)was measured by right heart catheterization.The Fulton index(RV/LV+S)was calculated by weighting method.HPSR markers were observed and Western-blot was used.real-time PCR,immunohistochemistry and ELISA were used to detect the m RNA and protein expression of NAADP and TPC1/2 in lung tissue,and their correlations were analyzed.2.The lentiviral vector containing the silenced TPC1/2 si RNA gene and the negative control lentivirus were injected into the normoxic and hypoxic groups,respectively,and the mean pulmonary artery pressure(m PAP)was measured by a right heart catheterization method.The Fulton index was calculated using a weighing method(RV/LV+S)and observation of HPSR indicators;Simultaneous detection of m RNA and protein expression of TPC1/2 in lung tissues by Western-blot,real-time PCR and immunohistochemistry.3.Real-time PCR and Western-blot were used to detect the Ca2+ concentration and Ca MKII,Calcineurin m RNA and protein expression in the silenced TPC1/2 group and negative control group.Results 1.The results of ELISA showed that NAADP concentration,m RNA and protein expression in TPC1 and TPC2 were significantly up-regulated in hypoxia group compared with normoxic group(P<0.05).2.Through Spearman rank correlation analysis,we observed that NAADP content was positively correlated with TPC1 and TPC2 expression(P<0.05);TPC1 expression was also positively correlated with TPC2 expression(P<0.05).3.Silencing TPC1/2 significantly down-regulated m RNA and protein expression in TPC1/2(P<0.05).In chronic hypoxic conditions,the silenced TPC1/2 group had better lung morphology than the negative control group(P<0.05).4.Pulmonary arterial pressure and Fulton index(RV/LV+S)were significantly increased in the hypoxic group compared with the normoxic group,while there was a statistically significant difference in the hypoxic+silencing TPC1/2 group between the low oxygen+negative control group(P <0.01).5.In the pulmonary arterial smooth muscle cells,silencing of TPC1/2 group compared with the negative control group significantly inhibited NAADP-AM-activated [Ca2+]i m RNA and protein expression of Ca MKII and Calcineurin,and the inhibition by TPC1 silencing was stronger(P<0.05).Conclusion:1.Hypoxia up-regulated the expression of NAADP and TPC1/2 in rat lung tissue,and NAADP content was positively correlated with the expression of TPC1 and TPC2.Both of them were involved in the pathogenesis of hypoxic pulmonary hypertension.2.Intervention of TPC1/2 could improve NAADP-induced [Ca2+]i release and proliferation in pulmonary artery smooth muscle cells,and TPC1 played a leading role in this process.