| Pulmonary hypertension(PH) is characterized by increased resting intracellular free Ca2+ concentration([Ca2+]i) of pulmonary arterial smooth muscle cells(PASMCs), enhanced vascular reactivity and increased vascular remodeling. Recent studies have shown that canonical transient receptor potential(TRPC) proteins, stromal interaction molecule(STIM) and Orai are important molecules in activation of sotre-operated calcium entry(SOCE), nuclear factor of activated T cells(NAFTc) related to cell proliferation, and TRPC/SOCE is augmented in chronic hypoxia(CH)-induced PH(CHPH) and monocrotaline-induced PH rats. This study further on the basis of CHPH rat model, observed the effect of STIM-Orai/TRPC-NFATc signaling pathway in the pathogenesis of CHPH. And pretreatment with NFATc indirect inhibiter cyclosporine A(Cs A), observe its effect on CHPH rats to further clarify the role of STIM-Orai/TRPC-NFATc signaling pathway. We further examined the effect of ginsenoside Rb1(Rb1) on SOCE in pulmonary arteries(PAs) of CHPH rat, in order to provide a new targeted drug for curing PH.1. Alteration of STIM-Orai/TRPC-NFATc signaling pathway in PAs of CHPH ratsObjective: To observe the role and the pathophysiological significance of STIM-Orai/TRPC-NFATc signaling pathway in the pathogenic process of CHPH.Methods: On the basis of CHPH rat model induced by Sprague-Dawley rats exposed to normobaric hypoxia for 21 d, using: ①hemodynamics and PAs morphology detection methods, determination the changes of rat right ventricular systolic pressure( RVSP), right ventricular mass index(RVMI) and PAs morphology; ②quantitative real-time PCR and Western blot method, determination the expression levels of STIM-Orai/TRPC-NFATc m RNA in rat PAs, and the time course of upregulation of m RNA time curve; ③vascular ring tone detection methods, determination the effects of CPA-induced PAs contraction, and gadolinium(Gd3+)-induced vasorelaxation on PAs precontracted with endothelin-1(ET-1) in rats; ④Mn2+ quenching of Fura-2 and Fluo-3 fluorescence detection [Ca2+]imethods, determination the effects of CPA-induced Ca2+ transients and Ca2+ entry in PASMCs; ⑤MTT assay cell viability method, determination the proliferative activity of rat PASMCs; ⑥time course and linear correlation analysis method, the correlation was observed among RVSP, CPA-induced PAs contraction and upregulated m RNA.Results: In comparison with the control rats, ①CH-exposed rats’ RVSP and RVMI were markedly elevated and pulmonary arterial wall thickening and stenosis, indicated that CH-exposed rats exhibited profound PH; ②the expressions of STIM2-Orai2-TRPC1/6-NFATc2/3 m RNA and STIM2-Orai2-TRPC1/6 protein were significantly increased in PAs of CH-exposed rats; ③ET-1-induced PAs contraction was greater and Gd3+ significantly decreased ET-1-primed contraction PAs in CH-exposed rats, suggested that SOCE upregulation in PAs of CH-exposed rats; ④CPA-induced PAs contraction was increased in CH-exposed rats, also suggested that SOCE upregulation in PAs of CH-exposed rats; ⑤resting [Ca2+]i, CPA-induced Ca2+ transients and Ca2+ entry were increased in PASMCs of CH-exposed rats, suggested that SOCE upregulation in PASMCs of CH-exposed rats; ⑥PASMCs proliferative activity were enhanced of CH-exposed rats, suggested that NFATc upregulation in PASMCs of CH-exposed rats; ⑦upregulating Orai2-TRPC1-NFATc3 expression and CPA-induced PAs contraction were earlier than that of RVSP, and Orai2-TRPC1 and CPA-induced PAs contraction were strongly correlated with that of STIM2, TRPC6, NFATc2/3 and RVSP, indicating that Orai2/TRPC1 and enhancement of SOCE may be the core links and initiation factors of this signaling pathway.Conclusion: CH resulting in increased resting [Ca2+]i, enhanced vascular reactivity, increased vascular smooth muscle cell proliferation and remodeling, and PH by upregulating STIM2-Orai2-TRPC1-NFATc2/3 expression and by enhancing SOCE and PASMCs proliferation activity. Orai2/TRPC1 and enhancement of SOCE may be the core links and initiation factors in the pathogenic process of CHPH.2. Effect of Cs A-pretreatment on STIM-Orai/TRPC-NFATc signaling pathway in PAs of CHPH ratObjective: To further observed the contribution of STIM-Orai/TRPC-NFATc signaling pathway in the pathogenic process of CHPH, in the case of Cs A pretreatment.Methods: On the basis of pretreatment with 25 mg/kg Cs A intraperitoneal injection on alternate days in CHPH rat model, using: ①hemodynamics and PAs morphology detection methods, determination the changes of rat right ventricular systolic pressure( RVSP), right ventricular mass index(RVMI) and PAs morphology; ②quantitative real-time PCR method, determination the expression levels of STIM-Orai/TRPC-NFATc m RNA in rat PA; ③vascular ring tone detection methods, determination the changes of CPA-induced PAs contraction, and gadolinium(Gd3+)-induced vasorelaxation on PAs precontracted with endothelin-1(ET-1) in rats; ④Mn2+ quenching of Fura-2 and Fluo-3 fluorescence detection [Ca2+]imethod, determination the changes of CPA-induced Ca2+ transients and Ca2+ entry in PASMCs; ⑤MTT assay cell viability method, determination the proliferative activity of rat PASMCs; ⑥time course analysis method, determination the alteration of RVSP or RVMI.Results: In comparison with the CH-exposed rats, ①pretreatment with Cs A, RVSP and RVMI were markedly decreased and pulmonary arterial wall thinning and increased lumen in CH-exposed rats, indicated that Cs A inhibited CHPH; ②pretreatment with Cs A downregulated Orai2-TRPC1-NFATc2/3 m RNA expression levels but not STIM2 m RNA in PAs of CH-exposed rats; ③pretreatment with Cs A reduced ET-1-induced PAs contraction and Gd3+ decreased ET-1-primed contraction PAs in CH-exposed rats, suggested that Cs A downregulated SOCE in PAs of CH-exposed rats; ④CPA-induced PAs contraction was decreased in CH-exposed rats pretreatment with Cs A, further supporting the effect of Cs A inhibiting SOCE; ⑤pretreatment with Cs A decreased resting [Ca2+]i, CPA-induced Ca2+ transients and Ca2+ entry in PASMCs of CH-exposed rats, suggested that Cs A downregulated SOCE in PASMCs of CH-exposed rats; ⑥PASMCs proliferative activity were attenuated of CH-exposed rats pretreatment with Cs A, suggested that Cs A downregulated NFATc in PASMCs of CH-exposed rats; ⑦pretreatment with Cs A blunted RVSP increasing amplitude, advanced RVSP maxlmum values, inhibited RVMI augmentation of CH-exposed rats, suggested that enhancing vascular reactivity may be the initiation factor, and increasing vascular proliferation may be the strengthen and potentiation factor in the pathogenic process of CHPH.Conclusion: Pretreatment with Cs A decreased resting [Ca2+]i, attenuated vascular reactivity, lessened vascular smooth muscle cell proliferation and remodeling, and inhibited PH by downregulating Orai2-TRPC1-NFATc2/3 expression and by blunting SOCE and PASMCs proliferation activity. Enhancing vascular reactivity may be the initiation factor, and increasing vascular proliferation may be the strengthen and potentiation factor in the pathogenic process of CHPH.3. Effect of Rb1 on SOCE in PAs of CHPH ratObjective: To investigate the vascular effects and underlying mechanisms of Rb1 on PAs in normal and CHPH rats, and find a new targeted drug for curing PH.Methods: On the basis of CHPH rat model, using PAs vascular ring tone detection, Mn2+ quenching of Fura-2 and Fluo-3 fluorescence detection [Ca2+]i methods, observed: ①vascular effect of Rb1 on PAs in normal rats; ②effect of Rb1 on ET-1-induced PAs contraction in control and CH-exposed Rats; ③effect of Rb1 on CPA-induced PAs contraction in control and CH-exposed Rats; ④effect of Rb1 on CPA-induced Ca2+ entry in PASMCs of control and CH-exposed Rats; ⑤effect of Rb1 on CPA-induced Ca2+ transients in PASMCs of control and CH-exposed Rats.Results: ①Rb1-induced concentration-dependent vasodilation on PAs precontracted with KCl, ET-1 and ET-1 pretreated with Nif, and the vasodilation on ET-1-induced contraction was most significant, suggesting that Rb1 induced vasorelaxation via inhibiting voltage-independent Ca2+ influx; ②pretreatment with Rb1 inhibited the ET-1-induced contraction in control and CH-exposed Rats, further suggesting that Rb1 induced vasorelaxation via inhibiting voltage-independent Ca2+ influx; ③Rb1 decreased ET-1-primed PAs contraction in control and CH-exposed Rats. And in the presence of Gd3+ decreased ET-1-primed contraction, ginsenoside Rb1 did not significantly reduce the contraction, suggesting that Rb1 induced vasorelaxation via inhibiting SOCE in rats PAs; ④pretreatment with Rb1 inhibited the CPA-induced contraction in control and CH-exposed Rats, and the inhibition on PAs was greater in CH-exposed Rats, suggesting that Rb1 induced vasorelaxation via inhibiting SOCE, and blunted SOCE in CH-exposed Rats; ⑤ Rb1 decreased CPA-primed PAs contraction in control and CH-exposed Rats, further supporting the effect of Rb1 inhibiting SOCE; ⑥Rb1 decreased CPA-induced Ca2+ entry by Mn2+ quenching and Ca2+ transients in PASMCs of control and CH-exposed rats, suggesting that Rb1 inhibited SOCE in rat PASMCs.Conclusion: Rb1 attenuates PAs contraction response to agonist via inhibiting SOCE in normal and CHPH rats.In summary, STIM-Orai/TRPC-NFATc signaling pathway plays a key role in the pathogenic process of CHPH. CH resulting in increased resting [Ca2+]i, enhanced vascular reactivity, increased vascular smooth muscle cell proliferation and remodeling, and PH by upregulating STIM2-Orai2-TRPC1-NFATc2/3 expression and by enhancing SOCE and PASMCs proliferation activity. Pretreatment with Cs A decreased resting [Ca2+]i, attenuated vascular reactivity, lessened vascular smooth muscle cell proliferation and remodeling, and inhibited PH by downregulating Orai2-TRPC1-NFATc2/3 expression and by blunting SOCE and PASMCs proliferation activity. Ginsenoside Rb1 attenuates PAs contraction response to agonist via inhibiting SOCE in normal and CHPH rats. These findings may provide a new insight, target, and targeted drug for curing PH. |
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