Dan-Shen-Yin Granules Prevent Hypoxia-Induced Pulmonary Hypertension via STAT3/HIF-1α/VEGF and FAK/AKT Signaling Pathways.

Traditional Chinese medicine (TCM) plays an important role in the treatment of complex diseases, especially cardiovascular diseases. However, it is hard to identify their modes of action on account of their multiple components. The present study aims to evaluate the effects of Dan-Shen-Yin (DSY) granules on hypoxia-induced pulmonary hypertension (HPH), and then to decipher the molecular mechanisms of DSY. Systematic pharmacology was employed to identify the targets of DSY on HPH. Furthermore, core genes were identified by constructing a protein-protein interaction (PPI) network and analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analysis. Related genes and pathways were verified using a hypoxia-induced mouse model and hypoxia-treated pulmonary artery cells. Based on network pharmacology, 147 potential targets of DSY on HPH were found, constructing a PPI network, and 13 hub genes were predicted. The results showed that the effect of DSY may be closely associated with AKT serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription 3 (STAT3), and HIF-1 signaling pathways, as well as biological processes such as cell proliferation. Consistent with network pharmacology analysis, experiments in vivo demonstrated that DSY could prevent the development of HPH in a hypoxia-induced mouse model and alleviate pulmonary vascular remodeling. In addition, inhibition of STAT3/HIF-1α/VEGF and FAK/AKT signaling pathways might serve as mechanisms. Taken together, the network pharmacology analysis suggested that DSY exhibited therapeutic effects through multiple targets in the treatment of HPH. The inferences were initially confirmed by subsequent in vivo and in vitro studies. This study provides a novel perspective for studying the relevance of TCM and disease processes and illustrates the advantage of this approach and the multitargeted anti-HPH effect of DSY.

Long-term administration of salvianolic acid A promotes endogenous neurogenesis in ischemic stroke rats through activating Wnt3a/GSK3ß/ß-catenin signaling pathway.

Stroke is the major cause of death and disability worldwide. Most stroke patients who survive in the acute phase of ischemia display various extents of neurological deficits. In order to improve the prognosis of ischemic stroke, promoting endogenous neurogenesis has attracted great attention. Salvianolic acid A (SAA) has shown neuroprotective effects against ischemic diseases. In the present study, we investigated the neurogenesis effects of SAA in ischemic stroke rats, and explored the underlying mechanisms. An autologous thrombus stroke model was established by electrocoagulation. The rats were administered SAA (10 mg/kg, ig) or a positive drug edaravone (5 mg/kg, iv) once a day for 14 days. We showed that SAA administration significantly decreased infarction volume and vascular embolism, and ameliorated pathological injury in the hippocampus and striatum as well as the neurological deficits as compared with the model rats. Furthermore, we found that SAA administration significantly promoted neural stem/progenitor cells (NSPCs) proliferation, migration and differentiation into neurons, enhanced axonal regeneration and diminished neuronal apoptosis around the ipsilateral subventricular zone (SVZ), resulting in restored neural density and reconstructed neural circuits in the ischemic striatum. Moreover, we revealed that SAA-induced neurogenesis was associated to activating Wnt3a/GSK3ß/ß-catenin signaling pathway and downstream target genes in the hippocampus and striatum. Edaravone exerted equivalent inhibition on neuronal apoptosis in the SVZ, as SAA, but edaravone-induced neurogenesis was weaker than that of SAA. Taken together, our results demonstrate that long-term administration of SAA improves neurological function through enhancing endogenous neurogenesis and inhibiting neuronal apoptosis in ischemic stroke rats via activating Wnt3a/GSK3ß/ß-catenin signaling pathway. SAA may be a potential therapeutic drug to promote neurogenesis after stroke.

Pinocembrin attenuates hemorrhagic transformation after delayed t-PA treatment in thromboembolic stroke rats by regulating endogenous metabolites.

Hemorrhagic transformation (HT) is a common serious complication of stroke after thrombolysis treatment, which limits the clinical use of tissue plasminogen activator (t-PA). Since early diagnosis and treatment for HT is important to improve the prognosis of stroke patients, it is urgent to discover the potential biomarkers and therapeutic drugs. Recent evidence shows that pinocembrin, a natural flavonoid compound, exerts anti-cerebral ischemia effect and expands the time window of t-PA. In this study, we investigated the effect of pinocembrin on t-PA-induced HT and the potential biomarkers for HT after t-PA thrombolysis, thereby improving the prognosis of stroke. Electrocoagulation-induced thrombotic focal ischemic rats received intravenous infusion of t-PA (10 mg/kg) 6 h after ischemia. Administration of pinocembrin (10 mg/kg, iv) prior t-PA infusion significantly decreased the infarct volume, ameliorated t-PA-induced HT, and protected blood-brain barrier. Metabolomics analysis revealed that 5 differential metabolites in the cerebral cortex and 16 differential metabolites in serum involved in amino acid metabolism and energy metabolism were significantly changed after t-PA thrombolysis, whereas pinocembrin administration exerted significant intervention effects on these metabolites. Linear regression analysis showed that lactic acid was highly correlated to the occurrence of HT. Further experiments confirmed that t-PA treatment significantly increased the content of lactic acid and the activity of lactate dehydrogenase in the cerebral cortex and serum, and the expression of monocarboxylate transporter 1 (MCT 1) in the cerebral cortex; pinocembrin reversed these changes, which was consistent with the result of metabolomics. These results demonstrate that pinocembrin attenuates HT after t-PA thrombolysis, which may be associated with the regulation of endogenous metabolites. Lactic acid may be a potential biomarker for HT prediction and treatment.

Salvianolic acid A prevented cerebrovascular endothelial injury caused by acute ischemic stroke through inhibiting the Src signaling pathway.

Stroke is an acute cerebrovascular disease caused by ruptured or blocked blood vessels. For the prevention of ischemic stroke, the coagulation state of blood and cerebrovascular protection should be considered. Our previous study has shown that salvianolic acid A (SAA), which is a water-soluble component from the root of Salvia Miltiorrhiza Bge, prevents thrombosis with a mild inhibitory effect on platelet aggregation. In this study we investigated the preventive effects of SAA on cerebrovascular endothelial injury caused by ischemia in vivo and oxygen-glucose deprivation (OGD) in vitro, and explored the underlying mechanisms. An autologous thrombus stroke model was established in SD rats by electrocoagulation. SAA (10 mg/kg) was orally administered twice a day for 5 days before the operation. The rats were sacrificed at 24 h after the operation. We showed that pretreatment with SAA significantly improved the neurological deficits, intracerebral hemorrhage, BBB disruption, and vascular endothelial dysfunction as compared with model group. In human brain microvascular endothelial cells (HBMECs), pretreatment with SAA (10 µM) significantly inhibited OGD-induced cell viability reduction and degradation of tight junction proteins (ZO-1, occludin, claudin-5). Furthermore, we found that SAA inhibited the upregulation of Src signaling pathway in vivo and vitro and reversed the increased expression of matrix metalloproteinases (MMPs) after ischemic stroke. In conclusion, our results suggest that SAA protects cerebrovascular endothelial cells against ischemia and OGD injury via suppressing Src signaling pathway. These findings show that pretreatment with SAA is a potential therapeutic strategy for the prevention of ischemic stroke.

QTL mapping and genetic effect of chromosome segment substitution lines with excellent fiber quality from Gossypium hirsutum × Gossypium barbadense.

Chromosome segment substitution lines (CSSLs) are ideal materials for identifying genetic effects. In this study, CSSL MBI7561 with excellent fiber quality that was selected from BC4F3:5 of CCRI45 (Gossypium hirsutum) × Hai1 (Gossypium barbadense) was used to construct 3 secondary segregating populations with 2 generations (BC5F2 and BC5F2:3). Eighty-one polymorphic markers related to 33 chromosome introgressive segments on 18 chromosomes were finally screened using 2292 SSR markers which covered the whole tetraploid cotton genome. A total of 129 quantitative trait loci (QTL) associated with fiber quality (103) and yield-related traits (26) were detected on 17 chromosomes, explaining 0.85-30.35% of the phenotypic variation; 39 were stable (30.2%), 53 were common (41.1%), 76 were new (58.9%), and 86 had favorable effects on the related traits. More QTL were distributed in the Dt subgenome than in the At subgenome. Twenty-five stable QTL clusters (with stable or common QTL) were detected on 22 chromosome introgressed segments. Finally, the 6 important chromosome introgressed segments (Seg-A02-1, Seg-A06-1, Seg-A07-2, Seg-A07-3, Seg-D07-3, and Seg-D06-2) were identified as candidate chromosome regions for fiber quality, which should be given more attention in future QTL fine mapping, gene cloning, and marker-assisted selection (MAS) breeding.

Chemokines play complex roles in cerebral ischemia.

Ischemic stroke (IS) is a disease caused by deficiency of blood and oxygen in focal or complete brain, followed by inflammation cascade and other pathological reactions, which finally lead to irreversible damage to the cerebrum. For the inflammation is a key progress at the initiation of ischemia and poststroke, and chemokines work as vital cytokines in inflammation, we focus the roles of chemokines in IS. Studies have shown cerebral ischemia is associated with marked induction of both CXC and CC chemokines which resulting in extensive leukocyte infiltration in the ischemic brain, and neutrophil infiltration may increase cerebral edema inducing injury in the ischemic area. In addition, chemokines also shows other functions such as promote neuroblast migration, hematogenous cell recruitment and functional brain repair. Thus, a similar chemokine ligand/chemokine receptor pair can mediate both beneficial and detrimental effects depending on the window of observation and pathophysiological conditions. This manuscript reviews the studies about chemokine-mediated effects in cerebral ischemia/reperfusion and discusses the potential significance of these interactions in injury and repair of ischemic tissues. We also refer drug development based on the chemokines and clinical applications using chemokines as diagnostic or prognostic biomarkers in ischemic stroke.

IMM-H004, A New Coumarin Derivative, Improved Focal Cerebral Ischemia via Blood-Brain Barrier Protection in Rats.

OBJECTIVE: IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a novel coumarin derivative that showed better effect in improving global cerebral ischemia in rats. However, the effects and mechanisms in focal cerebral ischemia were not clear. Blood-brain barrier (BBB) protection is a vital strategy for the treatment of cerebral ischemia. This study is to investigate whether IMM-H004 improves brain ischemia injury via BBB protection. METHODS: Focal brain ischemia model was induced by middle cerebral artery occlusion for 1 hour and reperfusion (MCAO/R) for 24 hours in rats. IMM-H004 (1.5, 3, 6 mg/kg) and edaravone (positive drug, 6 mg/kg) were administered after 5 minutes of occlusion. Neurological score and TTC staining were used to evaluate the effect of IMM-H004. Evans Blue (EB) staining and electron microscopy were used to assess BBB permeability. Western blot, reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect the expression of BBB structure-related proteins. RESULTS: Compared with the model group, IMM-H004 in the focal brain ischemia model improved neurological function and reduced cerebral infarction size and edema content. IMM-H004 sharply reduced the EB content and alleviated BBB structure. In addition, IMM-H004 increased the level of zonula occludens (ZO-1) and occluding, decreased the level of aquaporin 4 and matrix metalloproteinase 9, either in cortex or in hippocampus. And all of these changed were related to BBB protection. CONCLUSION: IMM-H004 improved cerebral ischemia injury via BBB protection. For a potential therapy drug of cerebral ischemia, IMM-H004 merits further study.

Inhibition of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 Regulates the Hepatobiliary Excretion and Plasma Exposure of Thienorphine and Its Glucuronide Conjugate.

Thienorphine (TNP) is a novel partial opioid agonist that has completed phase II clinical evaluation as a promising drug candidate for the treatment of opioid dependence. Previous studies have shown that TNP and its glucuronide conjugate (TNP-G) undergo significant bile excretion. The purpose of this study was to investigate the roles of efflux transporters in regulating biliary excretion and plasma exposure of TNP and TNP-G. An ATPase assay suggested that TNP and TNP-G were substrates of P-gp and MRP2, respectively. The in vitro data from rat hepatocytes showed that bile excretion of TNP and TNP-G was regulated by the P-gp and MRP2 modulators. The accumulation of TNP and TNP-G in HepG2 cells significantly increased by the treatment of mdr1a or MRP2 siRNA for P-gp or MRP2 modulation. In intact rats, the bile excretion, and pharmacokinetic profiles of TNP and TNP-G were remarkably changed with tariquidar and probenecid pretreatment, respectively. Tariquidar increased the Cmax and AUC0-t and decreased MRT and T1/2 of TNP, whereas probenecid decreased the plasma exposure of TNP-G and increased its T1/2. Knockdown P-gp and MRP2 function using siRNA significantly increased the plasma exposure of TNP and TNP-G and reduced their mean retention time in mice. These results indicated the important roles of P-gp and MRP2 in hepatobiliary excretion and plasma exposure of TNP and TNP-G. Inhibition of the efflux transporters may affect the pharmacokinetics of TNP and result in a drug-drug interaction between TNP and the concomitant transporter inhibitor or inducer in clinic.

Inhibition of chemokine-like factor 1 improves blood-brain barrier dysfunction in rats following focal cerebral ischemia.

Disruption of blood-brain barrier (BBB) and subsequent edema are major contributors to the pathogenesis of ischemic stroke, and the current clinical therapy remains unsatisfied. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, plays important roles in immune response. The expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 activity showed neuroprotective effect by alleviating infiltration of neutrophil and neuron apoptosis in cerebral ischemia. However, few studies have focused on the role of CKLF1 on BBB integrity. The objective of present study was to investigate the role of CKLF1 on BBB integrity by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Brain water content, Evans blue leakage and the expression of aquaporin-4 (AQP-4), matrix metalloproteinase-9 (MMP-9), Zonula Occludens-1 (ZO-1) and Occludin were measured. After treatment with anti-CKLF1 antibody, brain water content and Evans blue leakage in ipsilateral hemisphere were decreased in a dose-dependent manner at 24h after reperfusion, but not changed in contralateral hemisphere. Anti-CKLF1 antibody reduced the expression of AQP-4 and MMP-9, and upregulated the expression of ZO-1 and Occludin. These results suggest that CKLF1 is involved in BBB disruption after reperfusion. Inhibition of CKLF1 protects against cerebral ischemia by maintaining BBB integrity, possibly via inhibiting the expression of AQP-4 and MMP-9, and increasing the expression of tight junction protein.

Inhibition of P-glycoprotein Gene Expression and Function Enhances Triptolide-induced Hepatotoxicity in Mice.

Triptolide (TP) is the major active principle of Tripterygium wilfordii Hook f. and very effective in treatment of autoimmune diseases. However, TP induced hepatotoxicity limited its clinical applications. Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes. In this study, small interfering RNA (siRNA) and specific inhibitor tariquidar were used to investigate the impact of P-gp down regulation on TP-induced hepatotoxicity. The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers (ALT and AST) and pathological changes in liver. The other toxicological indicators (MDA, SOD and Bcl-2/Bax) were also significantly changed by P-gp inhibition. The data analysis showed that the increase of TP exposure in mice was quantitatively correlated to the enhanced hepatotoxicity, and the hepatic exposure was more relevant to the toxicity. P-gp mediated clearance played a significant role in TP detoxification. The risk of herb-drug interaction likely occurs when TP is concomitant with P-gp inhibitors or substrates in clinic.

[The evaluation of efflux transporter model based on RNA interference technology in vitro].

In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.

Inhibition of chemokine-like factor 1 protects against focal cerebral ischemia through the promotion of energy metabolism and anti-apoptotic effect.

Chemokine-like factor 1 (CKLF1) is a novel C-C chemokine, and plays important roles in immune response and brain development. In previous study, we have found that the expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 using antagonist C19 peptide protected against cerebral ischemia. However, few studies have focused on the role of CKLF1 on neuronal apoptosis. The objective of present study is to investigate the role of CKLF1 on neuronal apoptosis by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Antibodies against CKLF1 was applied to the right cerebral ventricle immediately after transient middle cerebral artery occlusion (MCAO), and infarct volume, neurological score, glucose metabolism and apoptosis-related protein were measured. Treatment with anti-CKLF1 antibody decreased infarct volume and neurological score, and inhibited neuronal apoptosis in a dose-dependent manner at 24h after reperfusion. Anti-CKLF1 antibody also reduced the level of phosphorylation of Akt (P-Akt), and led to decrease of pro-apoptotic protein Bcl-2 associated X protein (Bax) and increase of anti-apoptotic protein B cell lymphoma-2 protein (Bcl-2) and the ratio of Bcl-2/Bax, and inhibited caspase-3 at last. In addition, positron emission tomography (PET) indicated that anti-CKLF1 antibody increased glucose metabolism in ischemic hemisphere. These results suggest that CKLF1 is associated with neuronal apoptosis after cerebral ischemia and reperfusion. Neutralization of CKLF1 with antibodies shows neuroprotective effects against cerebral ischemia, which may be involved in inhibition of Akt pathway, regulation of apoptosis-related protein expression, and improvement glucose metabolism in ischemic hemisphere. Therefore, CKLF1 may be a novel target for the treatment of stroke.

Neutralization of chemokine-like factor 1, a novel C-C chemokine, protects against focal cerebral ischemia by inhibiting neutrophil infiltration via MAPK pathways in rats.

BACKGROUND: Inflammation plays a key role in the pathophysiology of ischemic stroke. Some proinflammatory mediators, such as cytokines and chemokines, are produced in stroke. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, displays chemotactic activities in a wide spectrum of leukocytes and plays an important role in brain development. In previous studies, we have found that the expression of CKLF1 increased in rats after focal cerebral ischemia and treatment with the CKLF1 antagonist C19 peptide decreased the infarct size and water content. However, the role of CKLF1 in stroke is still unclear. The objective of the present study was to ascertain the possible roles and mechanism of CKLF1 in ischemic brain injury by applying anti-CKLF1 antibody. METHODS: Male Sprague-Dawley rats were subjected to one-hour middle cerebral artery occlusion. Antibody to CKLF1 was applied to the right cerebral ventricle immediately after reperfusion; infarct volume and neurological score were measured at 24 and 72 hours after cerebral ischemia. RT-PCR, Western blotting and ELISA were utilized to characterize the expression of adhesion molecules, inflammatory factors and MAPK signal pathways. Immunohistochemical staining and myeloperoxidase activity was used to determine the extent of neutrophil infiltration. RESULTS: Treatment with anti-CKLF1 antibody significantly decreased neurological score and infarct volume in a dose-dependent manner at 24 and 72 hours after cerebral ischemia. Administration with anti-CKLF1 antibody lowered the level of inflammatory factors TNF-α, IL-1ß, MIP-2 and IL-8, the expression of adhesion molecules ICAM-1 and VCAM-1 in a dose-dependent manner. The results of immunohistochemical staining and detection of MPO activity indicated that anti-CKLF1 antibody inhibited neutrophil infiltration. Further studies suggested MAPK pathways associated with neutrophil infiltration in cerebral ischemia. CONCLUSIONS: Selective inhibition of CKLF1 activity significantly protects against ischemia/reperfusion injury by decreasing production of inflammatory mediators and expression of adhesion molecules, thereby reducing neutrophils recruitment to the ischemic area, possibly via inhibiting MAPK pathways. Therefore, CKLF1 may be a novel target for the treatment of stroke.

C19, a C-terminal peptide of chemokine-like factor 1, protects the brain against focal brain ischemia in rats.

There has been accumulating evidence that chemokines play an important role in cerebral ischemia. Chemokines-like factor 1 (CKLF1), as a novel C-C chemokine, is upregulated in cerebral ischemia. In this study we examined the protective effect of C19, a C-terminal peptide of chemokine-like factor 1, in an animal model of cerebral ischemia. Adult rats were anesthetized with chloral hydrate. C19 was given intracerebroventricularly immediately after 60 min middle cerebral artery occlusion. Animals were examined for their neurological function, infarct size and water content at 24h after reperfusion. The result showed that C19 (0.1, 1, 10 or 50 µg) significantly improved neurological function, decreased infarct size and water content in a dose-dependent manner. In contrast, application of C19 (50 µg) showed no effect in normal rats. Taken together, our data suggest that C19 is protective against ischemic brain injury, and may be an ideal peptide drug for the treatment of cerebral ischemia.

Expression of chemokine-like factor 1 after focal cerebral ischemia in the rat.

Cerebral ischemic injury is associated with the induction of a series of pro-inflammatory mediators such as cytokines and chemokines. The chemokine-like factor 1(CKLF1), as a novel human cytokine, displays chemotactic activities in a wide spectrum of leukocytes. The present study was conducted to determine if CKLF1 was produced in the brain of rats subjected to transient middle cerebral artery occlusion (TMCAO). Therefore, RT-PCR, Western blot and immunohistochemistry were utilized to characterize the expression of CKLF1 at different times after TMCAO. The result showed that almost no expression of CKLF1 was found in the sham-operated or contralateral cerebral cortex and hippocampus. CKLF1 expression significantly increased in the ischemic cerebral cortex and hippocampus, elevating at 12 h and peaking at 2 days after reperfusion. CKLF1 positive staining was mainly present in the cerebral cortex, hippocampus, thalamus, and hypothalamus. These results demonstrate that the expression of CKLF1 increases after focal cerebral ischemia in rat brain. Thus, CKLF1 may be a potential therapeutic target for cerebral ischemia.

Inhibition of endothelin-1 and hypoxia-induced pulmonary pressor responses in the rat by a novel selective endothelin-A receptor antagonist, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe.

Pulmonary hypertension is a kind of disease associated with a very high rate of mortality, and there are not many effective drugs for the treatment. Today, endothelin (ET)-1 receptor antagonists were proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin-A receptor (ETA) antagonist for treatment of pulmonary hypertension, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe, named GF063, was synthesized at base of selective ETA receptor antagonist BQ485 and selected for the further pharmacological characterization. The preliminary pharmacodynamics of GF063 was evaluated by radioligand receptor binding assay and test of antivasoconstriction effects in vitro and in vivo. The integrative pharmacodynamics was evaluated in hypoxia-induced rat pulmonary hypertension. In vitro, GF063 bound to ETA receptor with 100,000-fold higher affinity than to ETB receptor. GF063 concentration dependently inhibited contraction of isolated rat aortic ring induced by ET-1 and shifted the cumulative concentration-contraction response curve to right with no change in the maximal response. In vivo, GF063 inhibited the increase of mean systemic arterial pressure induced by ET-1 in anesthetized rat. In hypoxia-induced rat pulmonary hypertension model, pretreatment with GF063 (40 mg/kg, s.c.) significantly decreased pulmonary artery pressure and right ventricular hypertrophy, also significantly inhibited the increase of ET-1 level in lung, improved hemodynamics, and alleviated the wall thickness of pulmonary vessels. This study indicated that GF063, as a selective ETA receptor antagonist, could inhibit vasoconstriction effects in vivo and in vitro, could prevent pulmonary hypertension induced by hypoxia, and may have great potential to be developed as a new drug of antipulmonary hypertension.