Effects of GW501516 on the injury of pulmonary artery endothelial cells induced by hypoxia and its mechanism / 中国药房

OBJECTIVE To investigate the effects of the peroxisome proliferator-activated receptors δ （PPARδ） agonist GW501516 on the injury of pulmonary artery endothelial cells （PAECs） induced by hypoxia and its mechanism. METHODS The cytotoxic effects of GW501516 were observed by detecting the relative survival rate of PAECs； the protein expression of PPARδ was determined by Western blot assay. The cellular model of PAECs injury was established under hypoxic conditions； using antioxidant N-acetylcysteine （NAC） as positive control， the effects of GW501516 on cell injury and reactive oxygen species （ROS） production were investigated by detecting cell apoptotic rate， cell viability， lactate dehydrogenase （LDH） activity and ROS levels. Using nuclear factor erythroid 2-related factor 2（Nrf2） activator dimethyl fumarate （DMF） as positive control， PAECs were incubated with GW501516 and/or Nrf2 inhibitor ML385 under hypoxic conditions； the mechanism of GW501516 on PAECs injury induced by hypoxia was investigated by detecting cell injury （cell apoptosis， cell viability， LDH activity）， the levels of superoxide dismutase （SOD）， glutathione peroxidase （GPx）， catalase （CAT）， malondialdehyde （MDA） and ROS， the expressions of Nrf2， heme oxygenase-1 （HO-1） and cleaved-caspase-3 （C-caspase-3） protein. RESULTS The results demonstrated that hypoxia inhibited the protein expression of PPARδ （P＜0.05）， while GW501516 promoted the protein expression of PPARδ in hypoxia- exposed PAECs without obvious cytotoxic effects. GW501516 inhibited the apoptosis of PAECs， improved cell viability， and reduced LDH activity and ROS levels. GW501516 could up-regulate the protein expression of HO-1 in PAECs and the levels of SOD， GPx and CAT， while down-regulated the levels of MDA and ROS by activating the Nrf2 pathway （P＜0.05）； but Nrf2 inhibitor ML385 could reverse the above effects of GW501516 （P＜0.05）. GW501516 exerted similar effects to Nrf2 activator DMF in down-regulating the expression of C-caspase-3 and inhibiting the injury of PAECs under conditions of hypoxia （P＜0.05）. Moreover， Nrf2 inhibitor ML385 reversed the 163.com inhibition effects of GW501516 on PAECs injury （P＜0.05）. CONCLUSIONS GW501516 can relieve the hypoxia-induced injury of PAECs via the inhibition of oxidative stress， the mechanism of which may be associated with activating Nrf2.

Research progress in the regulation of hypoxic pulmonary hypertension by hypoxia-inducible factor-1 signaling pathway / 中国临床药理学与治疗学

Pulmonary hypertension (PH) is a rare and severe progressive disease. It results from hypertrophic remodeling of distal pulmonary arterioles that increases pulmonary arterial pressure and pulmonary vascular resistance in the absence of left heart, pulmonary parenchymal, or thromboembolic disease. Hypoxia-inducible factor-1 (HIF-1) regulates a large number of genes related to the occurrence and development of PH, and induces pulmonary angiogenesis, cell proliferation and migration, cellular energy metabolism and utilization. HIF-1 is an important component of the pathogenesis of hypoxic PH and plays an important role in driving the pathological process of pulmonary vascular and right ventricular remodeling. This article systematically elucidated the role and regulation of HIF-1 in hypoxic PH and its potential in targeted therapy of PH.

Calpain-1 accelerates endothelial cell apoptosis in pulmonary hypertension by inducing endoplasmic reticulum stress / 中国药理学通报

Aim To explore the mechanism by which calpain-1 promotes hypoxia-induced pulmonary hypertension pulmonary artery endothelial cell apoptosis through endoplasmic reticulum stress. Methods C57BL/6 wild-type (WT) and calpain-1 gene knockout mice (KO) were reared in a hypoxic chamber (10% O

Effect of Serum Containing Buyang Huanwu Tang on Endothelial-to- mesenchymal Transition of Human Pulmonary Artery Endothelial Cells / 中国实验方剂学杂志

Objective:To investigate the regulatory effect of serum containing Buyang Huanwu Tang on endothelial-to-mesenchymal transition(EndMT) in human pulmonary artery endothelial cells (HPAEC), and make further analysis on its mechanism from the perspective of the signal transduction of Jagged1/Notch1. Method:Rabbit serum containing Buyang Huanwu Tang was prepared by gavage with dosage of 53.36 g·kg-1·d-1, and blank serum was prepared by gavage with same volume of normal saline. The HPAECs cultured in vitro, EndMT model was established by the transforming growth factor-β1(TGF-β1) induced, which were divided into five groups:the control group (10%blank serum), the model group (10%blank serum+TGF-β1), the serum containing high-dose Buyang Huanwu Tang group (10%medicated serum + TGF-β1), the medium-dose group (5%medicated serum + 5%blank serum medicated + TGF-β1) and the low-dose group(2.5%medicated serum+7.5%blank serum+ TGF-β1). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method. Cell migration was detected by transwell and scratch assay. The endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial cadherin (VE-cadherin) and the mesenchymal markers fibroblast-specific protein 1 (FSP1), α-smooth muscle actin (α-SMA) were observed by immunofluorescence assay. The expression levels of Notch1, Jagged1 and CBF1 were detected by Western blot assay. Result:Compared with the control group, the proliferation and migration abilities of the HPAEC cells in model group were enhanced (Pα-SMA were increased. Further study found that the expressions of Notch1, Jagged1 and CBF1 were up-regulated (PPPα-SMA were on the decline. The expressions of Notch1, Jagged1 and CBF1 were also significantly lower than those in model group (PPConclusion:The serum containing Buyang Huanwu Tang can partly inhibit the EndMT in human pulmonary artery endothelial cells, which may be related to the regulation effect of Jagged1/Notch1 signaling.

Autophagy is involved in pulmonary artery endothelial cell apoptosis induced by cigarette smoke extract / 中国病理生理杂志

AIM: To investigate the role of autophagy in the apoptosis of human pulmonary artery endothelial cells (HPAECs) induced by cigarette smoke extract (CSE).METHODS: HPAECs were cultured routinely.HPAECs were treated with CSE at different concentrations, and the cell viability was detected by MTT assay.HPAECs were divided into control group, CSE group, 3-methyladenine (3-MA) group and 3-MA+CSE group.The autophagy was observed under fluorescence microscope with monodansylcadaverine (MDC) staining.Annexin V/propidium iodide staining and Hoechst 33342 staining were employed to detect apoptosis.In addition, the protein levels of LC3, beclin-1 and cleaved caspase-3 were determined by Western blot.RESULTS: MDC staining showed the increased production of autophagic vacuoles was observed in CSE group.The results of Western blot showed that the expression levels of autophagy-related proteins LC3 and beclin-1 were increased, while 3-MA pretreatment inhibited the expression of these proteins and the production of autophagic vacuoles.Observation with Annexin V/propidium iodide staining and Hoechst 33342 staining showed that the apoptotic rate in CSE group was significantly higher than that in control group, and pretreatment with 3-MA induced further increase in the cell apoptosis.The protein level of cleaved caspase-3 in CSE group was significantly higher than that in control group (P<0.05), and 3-MA+CSE treatment induced the further increase in the protein level of cleaved caspase-3.CONCLUSION: CSE induces autophagy and apoptosis in the HPAECs.Inhibition of autophagy promotes the apoptosis induced by CSE in HPAECs, which can be achieved through activation of caspase-3.

Effects of iptakalim on the functions of endothelin system in human pulmonary artery endothelial cells / 中国药理学通报

Aim To investigate the effects of iptakalim(IPT),a novel K_(ATP) opener,on the functions of endothelin system in human pulmonary artery endothelial cells.Methods Primary cultured human pulmonary artery endothelial cells were incubated with different concentrations iptakalim for 24 h.The levels of ET-1 in medium were observed by radioimmunoassay.Reverse transcription polymerase chain reaction(RT-PCR)was performed to analyze the expression of ET-1 and ECE.Results When endothelial cells were incubated with IPT at concentrations above 10 μmol·L~(-1),the levels of ET-1 release in medium and the levels of ET-1 mRNA were significantly inhibited.When endothelial cells were incubated with IPT at concentrations above 1 μmol·L~(-1),the levels of ECE mRNA were significantly inhibited.Conclusions IPT can inhibit the expression of ET-1 and ECE mRNA from human pulmonary artery endothelial cells, thus it inhibits the secretion of ET-1 from endothelial cells. Iptakalim may serve as a promising candidate drug to treat pulmonary hypertension.

YS 49, a Synthetic Isoquinoline Alkaloid, Protects Sheep Pulmonary Artery Endothelial Cells from tert-butylhydroperoxide-mediated Cytotoxicity

Endothelium, particularly pulmonary endothelium, is predisposed to injury by reactive oxygen species (ROS) and their derivatives. Heme oxygenase (HO) has been demonstrated to provide cytoprotective effects in models of oxidant-induced cellular and tissue injuries. In the present study, we investigated the effects of YS 49 against oxidant [tert-butylhydroperoxide (TBH) ]-induced injury using cultured sheep pulmonary artery endothelial cells (SPAECs). The viability of SPAECs was determined by quantifying reduction of a fluorogenic indicator Alamar blue. We found that TBH decreased cell viability in a time- and concentration-dependent manner. YS 49 concentration- and time-dependently increased HO-1 induction on SPAECs. As expected, YS 49 significantly decreased the TBH-induced cellular injury. In the presence of zinc protophorphyrin, HO-1 inhibitor, effect of YS 49 was significantly inhibited, indicating that HO-1 plays a protective role for YS 49. Furthermore, YS 49 showed free radical scavenging activity as evidenced by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and inhibition of lipid peroxidation. However, YS 49 did not inhibit apoptosis induced by lipopolysaccharide (LPS) in SPAECs. Taken together, HO-1 induction along with strong antioxidant action of YS 49 may be responsible for inhibition of TBH-induced injury in SPAECs.

Effect of taurine on proliferation of cultured pulmonary artery endothelial cells and pulmonary artery smooth muscle cells under hypoxia / 第三军医大学学报

Objective To investigate the effect of taurine and hypoxia(2.5% O_(2) or less than 1% O_(2)) on proliferation of bovine pulmonary artery endothelial cells(PAECs) and pulmonary artery smooth muscle cells(PASMCs).Methods ~(3)H-TdR incorporation method was used to detect the proliferation of PAECs and PASMCs and the proliferation changes by adding taurine.The PAECs and PASMCs were respectively cultured under normoxia(21% O_(2)),hypoxia Ⅰ(2.5% O_(2)),hypoxia Ⅱ(less than 1% O_(2)) and the effect of hypoxia on the cell proliferation was observed in 6,12,24 h.Meanwhile,the two kinds of cells were cultured under normoxia+taurine(0,2.5,5,10,20 mmol/L),hypoxia Ⅰ+taurine,hypoxia Ⅱ+taurine.Results Taurine inhibited the proliferation of PAECs under normoxia in a dose-dependent manner(P20 mmol/L) could inhibit the proliferation of both the cells that grow fast or slow,indicating that taurine may benefit in the prevention and treatment of hypoxic pulmonary hypertension.