ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.