Ming-Qing Dong

Beta-estradiol attenuates hypoxic pulmonary hypertension by stabilizing the expression of p27kip1 in rats

BackgroundPulmonary vascular structure remodeling (PVSR) is a hallmark of pulmonary hypertension. P27kip1, one of critical cyclin-dependent kinase inhibitors, has been shown to mediate anti-proliferation effects on various vascular cells. Beta-estradiol (β-E2) has numerous biological protective effects including attenuation of hypoxic pulmonary hypertension (HPH). In the present study, we employed β-E2 to investigate the roles of p27kip1 and its closely-related kinase (Skp-2) in the progression of PVSR and HPH.MethodsSprague-Dawley rats treated with or without β-E2 were challenged by intermittent chronic hypoxia exposure for 4 weeks to establish hypoxic pulmonary hypertension models, which resemble moderate severity of hypoxia-induced PH in humans. Subsequently, hemodynamic and pulmonary pathomorphology data were gathered. Additionally, pulmonary artery smooth muscle cells (PASMCs) were cultured to determine the anti-proliferation effect of β-E2 under hypoxia exposure. Western blotting or reverse transcriptional polymerase chain reaction (RT-PCR) were adopted to test p27kip1, Skp-2 and Akt-P changes in rat lung tissue and cultured PASMCs.ResultsChronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of right ventricle/left ventricle plus septum (RV/LV+S) ratio, medial width of pulmonary arterioles, accompanied with decreased expression of p27kip1 in rats. Whereas, β-E2 treatment repressed the elevation of RVSP, RV/LV+S, attenuated the PVSR of pulmonary arterioles induced by chronic hypoxia, and stabilized the expression of p27kip1. Study also showed that β-E2 application suppressed the proliferation of PASMCs and elevated the expression of p27kip1 under hypoxia exposure. In addition, experiments both in vivo and in vitro consistently indicated an escalation of Skp-2 and phosphorylated Akt under hypoxia condition. Besides, all these changes were alleviated in the presence of β-E2.ConclusionsOur results suggest that β-E2 can effectively attenuate PVSR and HPH. The underlying mechanism may partially be through the increased p27kip1 by inhibiting Skp-2 through Akt signal pathway. Therefore, targeting up-regulation of p27kip1 or down-regulation of Skp-2 might provide new strategies for treatment of HPH.

ERK/MAPK activation involves hypoxia‐induced MGr1‐Ag/37LRP expression and contributes to apoptosis resistance in gastric cancer

We previously demonstrated that hypoxia increased the hypoxia‐inducible factor (HIF‐1)–dependent MGr1‐Ag/37LRP expression, which enhanced adhesion of gastric cancer cells to laminin, inhibited drug‐induced apoptosis and caused cell adhesion–mediated drug resistance (CAM‐DR). Here, we investigated the role of extracellular‐regulated kinase (ERK) 1/2 in the signaling mechanisms underlying these events. We found that hypoxia activated ERK activity in vitro and in vivo. Overexpression of mitogen‐activated protein kinase (MAPK) kinase (MEK), which preferentially activated ERK, mimics, in a nonadditive way, hypoxia‐induced activity of MGr1‐Ag/37LRP promoter and expression of MGr1‐Ag/37LRP. Furthermore, U0126, the MEK inhibitor, inhibited hypoxia‐ and MEK‐induced MGr1‐Ag/37LRP promoter activity in a dose‐dependent manner. MEK inhibition also reversed hypoxia‐ and MEK‐induced HIF‐1 protein and its activity in a dose‐dependent manner. We also investigated reactive oxygen species signaling this response. Exogenous addition of H2O2 was sufficient to activate ERK in a dose‐dependent profile. Reactive oxygen species scavengers of H2O2 significantly inhibited hypoxia‐induced ERK or HIF‐1 activation and sequential expression of MGr1‐Ag/37LRP. We also investigated the signaling in hypoxia‐induced cell adhesion and apoptosis induced by vincristine. Hypoxia significantly enhanced adhesion of SGC7901 cells to laminin in a time‐dependent manner, which might be inhibited by the MEK inhibitor U0126 and MGr1‐Ag/37LRP siRNA. Consistent with results of adhesion assay, hypoxia‐resistant apoptosis might be reversed by U0126 in a dose‐dependent manner. Our results suggest that hypoxia‐elicited MGr1‐Ag/37LRP expression activated by HIF‐1 depends on ERK activation. These events are dependent of reactive oxygen intermediates.

Tanshinone IIA Inhibits Hypoxia-Induced Pulmonary Artery Smooth Muscle Cell Proliferation via Akt/Skp2/p27-Associated Pathway

We previously showed that tanshinone IIA ameliorated the hypoxia-induced pulmonary hypertension (HPH) partially by attenuating pulmonary artery remodeling. The hypoxia-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the major causes for pulmonary arterial remodeling, therefore the present study was performed to explore the effects and underlying mechanism of tanshinone IIA on the hypoxia-induced PASMCs proliferation. PASMCs were isolated from male Sprague-Dawley rats and cultured in normoxic (21%) or hypoxic (3%) condition. Cell proliferation was measured with 3 - (4, 5 - dimethylthiazal - 2 - yl) - 2, 5 - diphenyltetrazoliumbromide assay and cell counting. Cell cycle was measured with flow cytometry. The expression of of p27, Skp-2 and the phosphorylation of Akt were measured using western blot and/or RT-PCR respectively. The results showed that tanshinone IIA significantly inhibited the hypoxia-induced PASMCs proliferation in a concentration-dependent manner and arrested the cells in G1/G0-phase. Tanshinone IIA reversed the hypoxia-induced reduction of p27 protein, a cyclin-dependent kinase inhibitor, in PASMCs by slowing down its degradation. Knockdown of p27 with specific siRNA abolished the anti-proliferation of tanshinone IIA. Moreover, tanshinone IIA inhibited the hypoxia-induced increase of S-phase kinase-associated protein 2 (Skp2) and the phosphorylation of Akt, both of which are involved in the degradation of p27 protein. In vivo tanshinone IIA significantly upregulated the hypoxia-induced p27 protein reduction and downregulated the hypoxia-induced Skp2 increase in pulmonary arteries in HPH rats. Therefore, we propose that the inhibition of tanshinone IIA on hypoxia-induce PASMCs proliferation may be due to arresting the cells in G1/G0-phase by slowing down the hypoxia-induced degradation of p27 via Akt/Skp2-associated pathway. The novel information partially explained the anti-remodeling property of tanshinone IIA on pulmonary artery in HPH.

Tanshinone IIA modulates pulmonary vascular response to agonist and hypoxia primarily via inhibiting Ca2+ influx and release in normal and hypoxic pulmonary hypertension rats

The present study was designed to investigate the vascular effects and underlying mechanisms of tanshinone IIA on isolated rat pulmonary artery. Isometric tension was recorded in the arteries from normal and hypoxic pulmonary hypertension rats under normoxia or hypoxia condition. The results showed that tanshinone IIA exerted a biphasic effect on rat pulmonary artery. The constriction was attenuated by endothelium-denudation but was enhanced by inhibition of nitric oxide synthase. Pretreatment with tetraethylammonium (Ca2+-activated K+ channel inhibitor) upward shifted the concentration-response curve without affecting the maximum dilatation. Pretreatment with zinc protoporphyrin IX (heme oxygenase-1 inhibitor), 4-aminopyridine (KV channel inhibitor), glibenclamide (KATP channel inhibitor) or BaCl2 (inwardly rectifying K+ channel inhibitor) did not affect the vasoreactivity. Meanwhile, tanshinone IIA almost abolished vasoconstriction induced by extracellular Ca2+. Under hypoxia condition, tanshinone IIA eliminated acute hypoxia-induced initial contraction, potentiated following vasorelaxation, attenuated and reversed sustained contraction to relaxation in pulmonary artery from normal rats, and reversed phenylephrine-induced sustained constriction to sustained relaxation in remodeled pulmonary artery from hypoxic pulmonary hypertension rats. We concluded that the mild constrictive effect induced by tanshinone IIA was affected by integrity of endothelium and production of nitric oxide, while the potent dilative effect was endothelium-independent and produced primarily by inhibiting extracellular Ca2+ influx and partially by inhibiting intracellular Ca2+ release, as well as activating Ca2+-activated K+ channels. The modulation of tanshinone IIA on pulmonary vasoreactivity under both acute and chronic hypoxia condition may provide a new insight for curing hypoxic pulmonary hypertension.

Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells

Daidzein, a phytoestrogen, has been reported to produce vasodilation via inhibition of Ca(2+) inflow. However, the involvement of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the effect of daidzein is debated. Therefore, the present study was designed to investigate the effect of daidzein on the rat cerebral basilar artery and the underlying molecular mechanisms. Isolated cerebral basilar artery rings and single vascular smooth muscle cells (VSMCs) were used for vascular reactivity and electrophysiology measurements, to investigate the effect of daidzein on BK(Ca) channels in cerebral basilar artery smooth muscle. In addition, the human BK(Ca) channel alpha-subunit gene (hslo) was transfected into HEK293 cells, to directly assess whether daidzein activates BK(Ca) channels. The results showed that daidzein produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Paxilline, a selective BK(Ca) channel blocker, significantly inhibited the daidzein-induced vasodilation, whereas NS1619, a selective BK(Ca) channel opener, enhanced the vasodilation. In the whole-cell configuration, daidzein increased noisy oscillation currents in cerebral basilar artery VSMCs in a concentration-dependent manner, and washout of daidzein or blockade of BK(Ca) channels with paxilline fully reversed the increase. However, daidzein did not substantially affect hSlo currents in HEK293 cells when applied to the outside of the cell membrane. In conclusion, these results indicate that the activation of BK(Ca) channels in VSMCs at least partly contributes to the daidzein-induced vasodilation of the rat cerebral basilar artery. The beta1-subunit of BK(Ca) channels plays a critical role in the activation of BK(Ca) currents by daidzein.

Tanshinone IIA–Induced Attenuation of Lung Injury in Endotoxemic Mice Is Associated with Reduction of Hypoxia-Inducible Factor 1α Expression

Inhibiting hypoxia-inducible factor (HIF)-1α activity has been proposed as a novel therapeutic target in LPS-induced sepsis syndrome. We have reported that tanshinone IIA (TIIA) can reduce LPS-induced lethality and lung injury in mice, but the precise mechanisms have not been fully described. Therefore, the present study investigated whether the protective effect of TIIA was related to the inhibition of LPS-induced HIF-1α expression and what mechanisms accounted for it. This study showed that TIIA pretreatment improved LPS-induced biochemical and cellular changes and reduced the production of inflammatory cytokines. Pretreatment with TIIA decreased LPS-induced HIF-1α expression in vivo and in vitro. TIIA did not affect the LPS-induced HIF-1α mRNA level but inhibited HIF-1α protein translation by the inhibition of the PI3K/AKT and MAPK pathways and related protein translational regulators, such as p70S6K1, S6 ribosomal protein, 4E-BP1, and eIF4E, and promoted HIF-1α protein degradation via the proteasomal pathway in LPS-stimulated macrophages. These observations partially explain the antiinflammatory effects of TIIA, which provides scientific basis for its application for the treatment of acute lung injury/acute respiratory distress syndrome or sepsis.

1α,25-dihydroxyvitamin D3 ameliorates seawater aspiration-induced acute lung injury via NF-κB and RhoA/Rho kinase pathways.

Introduction Inflammation and pulmonary edema are involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have reported that 1α,25-Dihydroxyvitamin D3 (calcitriol) suppresses inflammation, it has not been confirmed to be effective in seawater aspiration-induced ALI. Thus, we investigated the effect of calcitriol on seawater aspiration-induced ALI and explored the probable mechanism. Methods Male SD rats receiving different doses of calcitriol or not, underwent seawater instillation. Then lung samples were collected at 4 h for analysis. In addition, A549 cells and rat pulmonary microvascular endothelial cells (RPMVECs) were cultured with calcitriol or not and then stimulated with 25% seawater for 40 min. After these treatments, cells samples were collected for analysis. Results Results from real-time PCR showed that seawater stimulation up-regulated the expression of vitamin D receptor in lung tissues, A549 cells and RPMVECs. Seawater stimulation also activates NF-κB and RhoA/Rho kinase pathways. However, we found that pretreatment with calcitriol significantly inhibited the activation of NF-κB and RhoA/Rho kinase pathways. Meanwhile, treatment of calcitriol also improved lung histopathologic changes, reduced inflammation, lung edema and vascular leakage. Conclusions These results demonstrated that NF-κB and RhoA/Rho kinase pathways are critical in the development of lung inflammation and pulmonary edema and that treatment with calcitriol could ameliorate seawater aspiration-induced ALI, which was probably through the inhibition of NF-κB and RhoA/Rho kinase pathways.

Epigallocatechin-3-gallate ameliorates seawater aspiration-induced acute lung injury via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats.

Signal transducers and activators of transcriptions 1 (STAT1) play an important role in the inflammation process of acute lung injury (ALI). Epigallocatechin-3-gallate (EGCG) exhibits a specific and strong anti-STAT1 activity. Therefore, our study is to explore whether EGCG pretreatment can ameliorate seawater aspiration-induced ALI and its possible mechanisms. We detected the arterial partial pressure of oxygen, lung wet/dry weight ratios, protein content in bronchoalveolar lavage fluid, and the histopathologic and ultrastructure staining of the lung. The levels of IL-1, TNF-α, and IL-10 and the total and the phosphorylated protein level of STAT1, JAK1, and JAK2 were assessed in vitro and in vivo. The results showed that EGCG pretreatment significantly improved hypoxemia and histopathologic changes, alleviated pulmonary edema and lung vascular leak, reduced the production of TNF-α and IL-1, and increased the production of IL-10 in seawater aspiration-induced ALI rats. EGCG also prevented the seawater aspiration-induced increase of TNF-α and IL-1 and decrease of IL-10 in NR8383 cell line. Moreover, EGCG pretreatment reduced the total and the phosphorylated protein level of STAT1 in vivo and in vitro and reduced the phosphorylated protein level of JAK1 and JAK2. The present study demonstrates that EGCG ameliorates seawater aspiration-induced ALI via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats.

Oxymatrine prevents hypoxia- and monocrotaline-induced pulmonary hypertension in rats.

Pulmonary hypertension is a progressive disease characterized by marked pulmonary arterial remodeling and increased vascular resistance. Inflammation and oxidative stress promote the development of pulmonary hypertension. Oxymatrine, one of the main active components of the Chinese herb Sophora flavescens Ait. (Kushen), plays anti-inflammatory and antioxidant protective roles, which effects on pulmonary arteries remain unclear. This study aimed to investigate the effects of oxymatrine on pulmonary hypertension development. Sprague-Dawley rats were exposed to hypoxia for 28 days or injected with monocrotaline, to develop pulmonary hypertension, along with administration of oxymatrine (50mg/kg/day). Hemodynamics and pulmonary arterial remodeling data from the rats were then obtained. The antiproliferative effect of oxymatrine was verified by in vitro assays. The inflammatory cytokine mRNA levels and leukocyte and T cell accumulation in lung tissue were detected. The antioxidative effects of oxymatrine were explored in vitro. Our study shows that oxymatrine treatment attenuated right-ventricular systolic pressure and pulmonary arterial remodeling induced by hypoxia or monocrotaline and inhibited proliferation of pulmonary arterial smooth muscle cells (PASMCs). Increased expression of inflammatory cytokine mRNA and accumulation of leukocytes and T cells around the pulmonary arteries were suppressed with oxymatrine administration. Under hypoxic conditions, oxymatrine significantly upregulated Nrf2 and antioxidant protein SOD1 and HO-1 expression, but downregulated hydroperoxide levels in PASMCs. In summary, this study indicates that oxymatrine may prevent pulmonary hypertension through its antiproliferative, anti-inflammatory, and antioxidant effects, thus providing a promising pharmacological avenue for treating pulmonary hypertension.

Protective effect of bicyclol on lipopolysaccharide-induced acute lung injury in mice.

Bicyclol is synthesized based on schisandrin, which is one of the main active components of Chinese herb Fructus Schisandrae. The purpose of this study is to investigate whether bicyclol has a beneficial effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Bicyclol was given to mice by gavage for three times. ALI was induced by vena caudalis injection of LPS. The last dose of bicyclol was administrated 1 h before LPS given. Mice in each group were sacrificed at different time point after LPS administration. As revealed by survival study, pretreatment with high doses of bicyclol reduced the mortality of mice from ALI. Bicyclol pretreatment significantly improved LPS-induced lung pathological changes, inhibited myeloperoxidase (MPO) activity, and reduced lung/body and lung wet/dry weight ratios. Bicyclol also inhibited the release of TNF-α, IL-1β and HMGB1, whereas simultaneously increased the expression of IL-10. Furthermore, the phosphorylation level of NF-κB p65 was markedly decreased by bicyclol. Taken together, our study showed that bicyclol improves survival rate and attenuates LPS-induced ALI. The protective mechanism may be due to the inhibition of NF-κB activation and regulation of cytokine secretion.