In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Essential Oil from Alpiniae Zerumbet Fructus Alleviates High Glucose-induced HUVEC Injury via TXNIP Signaling Pathway / 中国实验方剂学杂志

Objective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus （EOAZF） against high glucose （HG）-induced injury of human umbilical vein endothelial cells （HUVECs） <italic>in vitro</italic>， so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF. Method:The cells were divided into the normal group， model group （25 mmol·L^-1 glucose）， positive control group （100 mg·L^-1 vitamin C）， and the low- （0.25 μg·L^-1）， medium- （1 μg·L^-1）， and high-dose （4 μg·L^-1） EOAZF groups. The HUVECs were damaged by HG. The secretion amounts of malondialdehyde （MDA）， nitric oxide （NO）， and endothelin-1 （ET-1） in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury. The effects of EOAZF on the apoptosis and reactive oxygen species （ROS） generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） staining and dichloro-dihydro-fluorescein diacetate （DCFH-DA） assay. The protein and mRNA expression levels of thioredoxin interacting protein （TXNIP） and thioredoxin 1 （Trx-1） were determined by Western blot and Real-time polymerase chain reaction （Real-time PCR）， followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method. Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged. Compared with the model group， EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner. The secretion amounts of MDA and ET-1 （<italic>P</italic><0.05） in the model group were increased in contrast to those in the control group， while the NO level was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at all the three concentrations， especially at 4 μg·L^-1， obviously reduced the secretion of MDA and ET-1 （<italic>P</italic><0.05）， but elevated NO after HG induction （<italic>P</italic><0.05）. The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in the model group were higher than those in the control group （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly lowered the ROS level and apoptosis （<italic>P</italic><0.05） of HUVECs damaged by HG. The Western blot assay and Trx-1 activity detection uncovered that the protein and mRNA expression levels of TXNIP in the model group were significantly up-regulated as compared with those in the control group （<italic>P</italic><0.05）， whereas the Trx-1 activity was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly down-regulated the mRNA and protein （<italic>P</italic><0.05） expression levels of TXNIP and enhanced the total Trx-1 activity （<italic>P</italic><0.05） in HUVECs， thus suppressing the oxidative stress. Conclusion:EOAZF exerts the protective effects against HG-induced injury in HUVECs by improving the endothelial function and reducing intracellular ROS and apoptosis. Its efficacy in anti-oxidative stress may be related to the down-regulation of mRNA and protein expression levels of TXNIP and the enhancement of Trx-1 activity.

Effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma Extract on HUVECs Senescence Mediated by Endothelial Microparticles / 中国实验方剂学杂志

Objective:To investigate the effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract on endothelial microparticles （EMPs）-induced vascular endothelial cell senescence， and explore the possible mechanism. Method:Human umbilical vein endothelial cells （HUVECs） were used as the research objects， and the aged model was established with 10-12 passages of replicative senescence cells. The experimental cells were divided into young group （2-4 passage cells）， aged group （10-12 passage cells）， only EMPs intervention group （extract EMPs produced by aged cells to intervene young cells） and low dose， middle dose and high dose drug intervention groups （200， 300， 400 mg·L^-1）. Senescence related <italic>β</italic>-galactosidase （SA-<italic>β</italic>-gal） staining and cell cycle propidium iodide （PI） staining were used to determine cell senescence. Cell counting kit-8 （CCK-8） assay was used to screen the drug concentration. EMPs were extracted by two-step centrifugation， EMPs labeled with phycoerythrin （PE） anti-human CD31 antibody or fluorescein isothiocyanate （FITC） annexin V were detected by flow cytometry， intracellular reactive oxygen species （ROS） were detected by 2'，7'- dichlorofluorescein diacetate （DCFDA） staining. Result:After treatment with the drug， SA-<italic>β</italic>-gal activity of the aged cells significantly decreased （<italic>P</italic><0.01）， the S phase arrest was restored （<italic>P</italic><0.01）， and the number of CD31⁺ EMPs and annexin V⁺ EMPs secreted by aged cells decreased （<italic>P</italic><0.05）. Compared with the young group， only EMPs intervention group could induce increased SA-<italic>β</italic>-gal activity and S phase arrest in young cells （<italic>P</italic><0.05，<italic>P</italic><0.01）. However， after intervention of EMPs and the drug， EMPs-mediated increase of SA-<italic>β</italic>-gal activity was significantly inhibited and S phase arrest was restored （<italic>P</italic><0.05）. The increase of intracellular ROS induced by EMPs was also significantly inhibited by the drug （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract can delay the senescence of vascular endothelial cells by influencing EMPs， and the mechanism may be related to the inhibition of increased intracellular ROS induced by EMPs.

Rhein inhibits hydrogen peroxide-induced apoptosis of human umbilical vein endothelial cells and its mechanism / 中国中药杂志

This study investigated the effect of rhein(RH) on the apoptosis and autophagy of human umbilical vein endothelial cells(HUVECs) induced by hydrogen peroxide(H_2O_2) and its underlying mechanism. The oxidative damage model in HUVECs was established and the cells were divided into different treatment groups. Cell survival rate was detected by MTT assay, apoptosis by Annexin V-FITC/PI double staining and Hoechst 33258 fluorescence staining, autophagy by Ad-mCherry-GFP-LC3 B adenovirus transfection, and protein expression by Western blot. The results showed that RH could protect cells by increasing the cell survival rate in a dose-dependent manner, decreasing the expression of apoptosis-related proteins(Bax and cleaved caspase-3) and the ratio of Bax/Bcl-2, elevating the expression of Bcl-2, up-regulating the expression of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ, and down-regulating the expression of p62. Adenovirus transfection results showed that RH could increase the green and red spots, as well as the yellow spots. However, after the addition of autophagy inhibitor 3-MA, autophagy was reduced and apoptosis was increased. RH could enhance the expression of silent information regulator 2 related enzyme 1(SIRT1). The addition of SIRT1 inhibitor EX-527 reduced the protective effect of RH and cell viability. The addition of 3-MA had no effect on the expression of SIRT1 protein, but the expression of SIRT1 and LC3-Ⅱ proteins decreased and the expression of p62 increased after the addition of EX-527. After RH treatment, the phosphorylation of adenosine monophosphate-activated protein kinase(AMPK) increased, while that of the mechanistic target of rapamycin(mTOR) decreased in a dose-dependent manner. Moreover, this effect could be weakened by the AMPK inhibitor compound C. RH may enhance autophagy through SIRT1/AMPK/mTOR pathway to reduce H_2O_2-induced apoptosis of HUVECs.

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

LncRNA XIST knockdown ameliorates oxidative low-density lipoprotein-induced endothelial cells injury by targeting miR-204-5p/TLR4

Oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury is a key contributor toatherosclerosis development. However, the role and mechanism of long noncoding RNA X-inactive specifictranscript (XIST) in atherosclerosis remain largely unknown. The ox-LDL-induced human umbilical veinendothelial cells (HUVECs) injury was analyzed by cell viability, apoptosis, inflammatory cytokines secretionand oxidative stress. The expression levels of XIST, microRNA-204-5p (miR-204-5p) and toll-like receptor 4(TLR4) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. Thetarget interaction between miR-204-5p and XIST or TLR4 was explored by bioinformatics analysis, luciferaseassay and RNA immunoprecipitation. The expression of XIST was enhanced in ox-LDL-treatedHUVECs. Knockdown of XIST attenuated ox-LDL-induced viability inhibition, apoptosis production,inflammatory response and oxidative stress in HUVECs. XIST was validated as a sponge of miR-204-5pand TLR4 acted as a target of miR-204-5p. Knockdown of miR-204-5p reversed silence of XISTmediated suppressive role in ox-LDL-induced injury. TLR4 alleviated miR-204-5p-mediated inhibitiveeffect on ox-LDL-induced injury. Moreover, XIST could regulate TLR4 expression by spongingmiR-204-5p. In conclusion, silence of XIST displayed a protective role in ox-LDL-induced injury inHUVECs by regulating miR-204-5p/TLR4 axis, providing a novel mechanism for understanding thepathogenesis of atherosclerosis.

Linc-OIP5 in the breast cancer cells regulates angiogenesis of human umbilical vein endothelial cells through YAP1/Notch/NRP1 signaling circuit at a tumor microenvironment

BACKGROUND: LincRNAs have been revealed to be tightly associated with various tumorigeneses and cancer development, but the roles of specific lincRNA on tumor-related angiogenesis was hardly studied. Here, we aimed to investigate whether linc-OIP5 in breast cancer cells affects the angiogenesis of HUVECs and whether the linc-OIP5 regulations are involved in angiogenesis-related Notch and Hippo signaling pathways. METHODS: A trans-well system co-cultured HUVECs with linc-OIP5 knockdown breast cancer cell MDA-MB-231 was utilized to study the proliferation, migration and tube formation abilities of HUVECs and alterations of related signaling indicators in breast cancer cells and their conditioned medium through a series of cell and molecular experiments. RESULTS: Overexpressed linc-OIP5, YAP1, and JAG1 were found in breast cancer cell lines MCF7 and MDA-MB-231 and the expression levels of YAP1 and JAG1 were proportional to the breast cancer tissue grades. MDA-MB-231 cells with linc-OIP5 knockdown led to weakened proliferation, migration, and tube formation capacity of co-cultured HUVECs. Besides, linc-OIP5 knockdown in co-cultured MDA-MB-231 cells showed downregulated YAP1 and JAG1 expression, combined with a reduced JAG1 level in conditioned medium. Furthermore, a disrupted DLL4/Notch/NRP1 signaling in co-cultured HUVECs were also discovered under this condition. CONCLUSION: Hence, linc-OIP5 in MDA-MB-231 breast cancer cells may act on the upstream of the YAP1/Notch/NRP1 signaling circuit to affect proliferation, migration, and tube formation of co-cultured HUVECs in a non-cellular direct contact way through JAG1 in conditioned medium. These findings at least partially provide a new angiogenic signaling circuit in breast cancers and suggest linc-OIP5 could be considered as a therapeutic target in angiogenesis of breast cancers.

Effects of leptin-modified human placenta-derived mesenchymal stem cells on angiogenic potential and peripheral inflammation of human umbilical vein endothelial cells (HUVECs) after X-ray radiation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-KB (NF-kB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-KB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.

Comparative transcriptomic analysis of vascular endothelial cells after hypoxia/re-oxygenation induction based on microarray technology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Objective: To provide comprehensive data to understand mechanisms of vascular endothelial cell (VEC) response to hypoxia/re-oxygenation. Methods: Human umbilical vein endothelial cells (HUVECs) were employed to construct hypoxia/re-oxygenation-induced VEC transcriptome profiling. Cells incubated under 5% O2, 5% CO2, and 90% N2 for 3 h followed by 95% air and 5% CO2 for 1 h were used in the hypoxia/re-oxygenation group. Those incubated only under 95% air and 5% CO2 were used in the normoxia control group. Results: By using a well-established microarray chip consisting of 58 339 probes, the study identified 372 differentially expressed genes. While part of the genes are known to be VEC hypoxia/re-oxygenation-related, serving as a good control, a large number of genes related to VEC hypoxia/re-oxygenation were identified for the first time. Through bioinformatic analysis of these genes, we identified that multiple pathways were involved in the reaction. Subsequently, we applied real-time polymerase chain reaction (PCR) and western blot techniques to validate the microarray data. It was found that the expression of apoptosis-related proteins, like pleckstrin homology-like domain family A member 1 (PHLDA1), was also consistently up-regulated in the hypoxia/re-oxygenation group. STRING analysis found that significantly differentially expressed genes SLC38A3, SLC5A5, Lnc-SLC36A4-1, and Lnc-PLEKHJ1-1 may have physical or/and functional protein-protein interactions with PHLDA1. Conclusions: The data from this study have built a foundation to develop many hypotheses to further explore the hypoxia/re-oxygenation mechanisms, an area with great clinical significance for multiple diseases.

Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells / 生物医学与环境科学（英文）

OBJECTIVE@#Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs).@*METHODS@#HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively.@*RESULTS@#Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown.@*CONCLUSION@#Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.

The Effect of Abnormal Blood Flow on Human Umbilical Vein Endothelial Cells / 医用生物力学

Objective To study the effects of abnormal blood flow on the secretion of ET-1/NO and the expression of the mRNA and the protein of ET-1, eNOS, VCAM-1, ICAM-1 and MCP-1 in human umbilical vein endothelial cells (HUVECs), so as to explore the mechanism of atherosclerosis （AS） caused by abnormal hemodynamics. MethodsThe HUVECs were divided into stress group, wall pressure group and normal group according to the different stress. The HUVECs were cultured under the corresponding stress for 24 hours and then collected. The secretion levels of NO and ET-1 were detected by enzyme method and radioimmunoassay method. The mRNA expression levels of eNOS and ET-1 were detected by qPCR. The expression levels of the mRNA and the protein of VCAM-1, ICAM-1, MCP-1 were detected by qPCR and Western blot. Results Compared with normal group, the secretion level and the mRNA expression level of ET-1 in wall pressure group increased significantly (P＜0.01), and the secretion level of NO and the mRNA expression level of eNOS in stress group also increased significantly (P＜0.01), The expressions level of the mRNA and the protein of VCAM-1, ICAM-1 and MCP-1 obviously increased in stress group and wall pressure group (P＜0.01). Conclusions Stress or wall pressure acting on HUVECs alone could lead to its dysfunction of the secretion and the expression of gene and protein. The mechanism of AS caused by abnormal blood flow was related to these dysfunction of HUVEC.

The Effect of Abnormal Blood Flow on Human Umbilical Vein Endothelial Cells / 医用生物力学

Objective To study the effects of abnormal blood flow on the secretion of ET-1/NO and the expression of the mRNA and the protein of ET-1, eNOS, VCAM-1, ICAM-1 and MCP-1 in human umbilical vein endothelial cells (HUVECs), so as to explore the mechanism of atherosclerosis （AS） caused by abnormal hemodynamics. MethodsThe HUVECs were divided into stress group, wall pressure group and normal group according to the different stress. The HUVECs were cultured under the corresponding stress for 24 hours and then collected. The secretion levels of NO and ET-1 were detected by enzyme method and radioimmunoassay method. The mRNA expression levels of eNOS and ET-1 were detected by qPCR. The expression levels of the mRNA and the protein of VCAM-1, ICAM-1, MCP-1 were detected by qPCR and Western blot. Results Compared with normal group, the secretion level and the mRNA expression level of ET-1 in wall pressure group increased significantly (P＜0.01), and the secretion level of NO and the mRNA expression level of eNOS in stress group also increased significantly (P＜0.01), The expressions level of the mRNA and the protein of VCAM-1, ICAM-1 and MCP-1 obviously increased in stress group and wall pressure group (P＜0.01). Conclusions Stress or wall pressure acting on HUVECs alone could lead to its dysfunction of the secretion and the expression of gene and protein. The mechanism of AS caused by abnormal blood flow was related to these dysfunction of HUVEC.

Effect of Periplaneta Aamericana Polypeptide on Angiogenesis / 中国实验方剂学杂志

Objective: To investigate the effect of the Periplaneta Americana polypeptide on the angiogenesis. Method:Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cell scratch assay were used to observe effect of different concentration (6.25,12.5,25,50,100 mg·L-1) of the Periplaneta Americana polypeptide, CⅡ-3 and skimmed cream on the proliferation and migration of human umbilical vein endothelial cells (HUVECs), and a normal group and a thalidomide group were also established in this study. The tubule formation assay was used to detect the effect of different concentration (25, 50, 100 mg·L-1) of the Periplaneta Americana extracts on the formation of tubules in HUVECs cells. The adhesion between HepG2 cells and HUVECs cells was observed by cell adhesion assay. The expression of vascular endothelial growth factor (VEGF) proteins in HUVECs was detected by immunocytochemical staining and enzyme linked immunosorbent assay (ELISA). Result:MTT results showed that the Periplaneta Americana polypeptide could inhibit the proliferation of HUVECs in a dose-dependent manner (PPPPPPPPPConclusion:The Periplaneta Americana polypeptide can inhibit the invasion, metastasis and tube formation of HUVECs, and down-regulate the expression of VEGF in HUVECs. The effect of Periplaneta Americana polypeptide is better than CⅡ-3 and skimmed cream, and the among the polypeptide, the effect of PAP-2 is superior to the other two.

Protective effect and mechanism of extracts and main components from stem and leaves of Salvia miltiorrhiza on HUVECS / 中草药

Objective: To investigate the protective effects of the extracts and active components from stems and leaves of Salvia miltiorrhiza on oxidative stress and high glucose-injured human umbilical endothelial cells (HUVECs). Methods: The models of oxidative stress and high glucose injury in HUVECs were established. The ethanol extract of S. miltiorrhiza stems and leaves (CJ), ethanol extract of S. miltiorrhiza roots (CG), water extract of S. miltiorrhiza stems and leaves (SJ), water extract of S. miltiorrhiza roots (SG), rosmarinic acid, salvianolic acid B, rutin, isoquercitrin, cryptotanshinone, aminoguanidine and VC were administrated to cells. MTT were used to observe the cell viability. The levels of glutathione peroxidase (GSH-Px), catalase (CAT), nitric oxide (NO), endothelin (ET-1), ICAM-1 and TNF-α were detected. Result:s Compared with the control group, H2O2 decreased the levels of GSH-Px, CAT and NO (P < 0.01) and increased the level of ET-1 (P < 0.01), glucose increased the levels of ICAM-1 and TNF-α (P < 0.01) and decreased the level of NO (P < 0.01). Compared with the model group, the levels of GSH-Px, CAT and NO in CJ, CG, SJ and SG groups were increased, and the levels of ET-1, ICAM-1 and TNF-α were decreased (P < 0.05, P < 0.01). VC, rosmarinic acid, salvianolic acid B and rutin high and medium dose groups, and isoquercitrin, cryptotanshinone high dose group significantly increased the levels of GSH-Px, CAT and NO (P < 0.05, P < 0.01) and decreased the level of ET-1 (P < 0.05, P < 0.01). ICAM-1 levels were significantly decreased in the high dose groups of salvianolic acid B, isoquercitrin and rutin as well as in the high and medium dose groups of rosmarinic acid (P < 0.01). In addition to the aminoguanidine group, the levels of TNF-α of other groups were significantly lower than the model group (P < 0.05, P < 0.01). Except the aminoguanidine group and isoquercetin low dose group, the levels of NO of other groups were significantly higher than the model group (P < 0.05, P < 0.01). Conclusion: In certain concentration range of alcohol extract and water extract of stems, leaves and roots of S. miltiorrhiza, rosmarinic acid, salvianolic acid B, rutin and isoquercitrin have protective effects on HUVECs injured by H2O2 and glucose. And the mechanisms are related to inhibition of intercellular adhesion molecule expression and regulation of NO and TNF-α production. This study will provide reference for the discovery and transformation of the resource value of non-medicinal stems and leaves produced during the production of S. miltiorrhiza.

Effects of rice peptide YGEGSSEEG on improving endothelial cell injury induced by tumor necrosis factor / 中国药理学通报

Aim To investigate the peptides and its protection for vascular endothelial cells, derived from the absorbed components of rice α-globulin,which was shown to be effective in anti-atherosclerosis. Methods The amino acid sequence was purified by gel chro-matography and RP-HPLC, and determined by ESI/MS. Then the peptide was chemically synthesized. Hu-man umbilical vein endothelial cell injury model was induced by tumor necrosis factor-α. The cell viability was measured by cell counting kit to screen the appro-priate peptide intervention concentration. The apoptotic rate was detected by flow cytometry. Bcl-2, Bax, p-p38, vascular cell adhesion molecule and the protein expression level of NF-κB signaling pathway were de-tected by Western blot and immunofluorescent stai-ning. Results Apoptosis of HUVECs induced by TNF-α was significantly increased by YGEGSSEEG, which also regulated expression of Bcl-2/Bax proteins and inhibited phosphorylation of p38 protein. Besides, the peptide suppressed the production of VCAM-1, ICAM-1 and activation of NF-κB pathway. While it did not significantly improve the oxidative stress response in HUVECs. Conclusion Peptide YGEGSSEEG pro-tects vascular endothelial cells through suppressing ap-optosis and expression of adhesion molecules.

The protective effects of Astragaloside Ⅳ on diastolic function of rat thoracic aortic rings impaired by microvesicles / 中国应用生理学杂志

OBJECTIVES@#To investigate the effects of Astragaloside IV (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs), and the mechanism of AST.@*METHODS@#H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs under the condition of hypoxia for 12 hour/Reoxygenation for 4 hour, H/R-EMVs were stored in D-Hank's solution. Male Wistar rats were underwent thoracotomy, the thoracic aorta with intact endothelium were carefully removed and cut into 3~4 mm rings. The experiment was divided into six groups. H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10g/ml; different doses of AST groups:thoracic aortic rings of rats were treated with 10, 20, 40, 60 mg/L AST co-incubated with 10g/ml H/R-EMVs respectively; control group were treated with the same volume of D-Hank's solution. Duration of incubation was 4 h, each group was tested in five replicate aortic rings. Effects of AST on endothelium-dependent relaxation were detected. The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinase (Akt), phosphorylated Akt (p-Akt, Ser-473), extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2, Thr202/Tyr204) of rat thoracic aortic rings were detected.@*RESULTS@#Teng/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (<0.01). Compared with H/R-EMVs group, relaxation of rat thoracic aortic rings was increased by 20, 40 and 60 mg/L AST in a concentration-dependent manner (<0.01), the level of NO production was also enhanced (<0.05, <0.01). The level of t-eNOS, t-Akt and ERK1/2 was not changed, but the level of p-eNOS, p-Akt and p-ERK1/2 increased by the treatment with AST (<0.01).@*CONCLUSIONS@#AST could effectively ameliorate endotheliumdependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner, the mechanism might involve the increase in production of NO, and the protein level of p-eNOS, p-Akt and p-ERK1/2.

Effect of procyanidine on the function and expression of miR-221 in human umbilical vein endothelial cells / 解放军医学杂志

Objective To study the effect ofprocyanidine (PC) on the proliferation and migration of human umbilical vein endothelial cells (HUVECs),determine the expression changes of miR-221,and to investigate the mechanism involved.Methods HUVECs were cultured in vitro and treated with PC (5,25,50,75,100μg/ml) for 24 hours,and a PC concentration of 50μg/ml was screened by CCK-8 assay for the follow up experiment,then the cell proliferation activity was detected by the wound healing assay.The expression of miR-221 in HUVECs was detected by real-time quantitative PCR;MiR-221 target genes were predicted in miRWalk database,and the target genes were analyzed by GO and KEGG pathway.Results Compared with the control group,the HUVECs treated with PC for 24h,their proliferation activity in PC 5μg/ml group did not change obviously (P＞0.05),and in PC 25,50,75 and 100μg/ml groups decreased in a concentration dependent manner (P＜0.01).Compared with the control group,the migration ability of PC 50μg/ml group decreased markedly (P＜0.01).Compared with the control group,the expression of miR-221 increased after treatment with PC 50μg/ml (P＜0.01).Go analysis indicated that the target genes of miR-221 were mainly related to cell proliferation,migration,gene translation and so on.The target genes related to cell proliferation and migration were ADAM17,KIT,PDGFD and so on.KEGG pathway analysis showed that the target genes of miR-221 enriched 5 signal pathways,such as FoxO,PI3K-Akt and so on.Conclusions Low concentration of PC has no effect on the proliferation activity of HUVECs.A certain concentration of PC can inhibit the proliferation and migration of HUVECs,of which the mechanism may be involved with the up-regulation of miR-221 and FoxO,PI3K-Akt and other signaling pathways.

Observation of Proliferation of Human Umbilical Vein Endothelial Cells Treated with Serum Containing Yang-warming, Blood-activating, and Toxin-removing Recipe Sampled Under Different Conditions / 广州中医药大学学报

Objective To study the influences of Yang-warming, Blood-activating, and Toxin-removing Recipe(YBTR)-containing serum sampled under different conditions on the proliferation of human umbilical vein endothelial cells(HUVECs). Methods HUVECs were cultured in vitro for the experiment. Pharmacological serum for the experiment was prepared as follows: Thirty-two SD rats (male in half) were randomly divided into 4 groups, namely normal saline group and low-, middle-, and high-dose YBTR groups (at the intragastric dosage of 10.35, 31.05, 93.15 g·kg-1 respectively, twice a day). The pharmacological serum was taken from one female rat and one male rat in various groups at 4 time points, i.e. at hour 1, 2 after first intragastric administration on the fourth feeding day, and at hour 1, 2 after first intragastric administration on the sixth feeding day(abbreviated as D3H1, D3H2, D5H1, D5H2 respectively). The effects of YBTR-containing serum on the proliferation of HUVECs were observed by CCK-8 assay method. Results The difference of proliferation-inhibition rate of HUVECs was statistically significant after treated with YBTR-containing serum prepared from rats of different genders at different time(Pgender=0.000<0.01, Ptime=0.000<0.01). The difference of interaction of time and gender was also significant (Ptime × gender=0.001<0.01), and the effect at D3H1 and D5H1 varied with the gender (PD3H2×gender = 0.000 < 0.01, PD5H1×gender = 0.002 < 0.01). The inhibitory action of YBTR-containing serum became stronger with the increase of the dosage of serum of female rat at D3H2, and the inter-group difference was statistically significant (P < 0.05), the effect showing concentration-dependent tendency. The inhibition of HUVECs proliferation reached the peak after treated by various doses of YBTR-containing serum from the female rat at D3H2, while the inhibition arrived to the peak after treated by low- and middle-dose YBTR-containing serum from the male rat at D5H1, and the inhibition arrived to the peak in the group of high-dose YBTR-containing serum from the male rat at D3H1. Conclusion The inhibitory action of YBTR-containing serum on the proliferation of HUVECs was stronger when the serum was taken from the female rat at D3H2.

HUVECs extraction and UHPLC LTQ Orbitrap analysis for screening vascular endothelium protective components in Kudiezi injection / 中国中药杂志

An effective method has been employed as a tool for screening active components in Kudiezi injection by using cell chromatography and sensitive UHPLC-HR-MSn method. The potential bioactive components in Kudiezi injection could be selectively bound to the HUVECs target cells first. After cell target desensitization and inactivation, the chemical constituents with cell target affinity were identified by LC-MS, so as to screen the possible active components in Kudiezi injection. Based on the accurate mass measurements and the retention time, in total, 9 compounds were tentatively identified and characterized, including 4 sesquiterpene lactones, 3 phenolic acids and 2 flavonoids. HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis could provide a rapid and efficient method for the identification of potential bioactive components in Kudiezi injection, and provide the reference for further research on its effective materials basis.

Role of HCMV infection in the activation and oxidative stress of cultured human umbilical vein endothelial cells / 中华实验和临床病毒学杂志

Objective@#To investigate the role of the activation and oxidative stress of cultured human umbilical vein endothelial cells (HUVEC) after HCMV infection.@*Methods@#HUVECs were divided into four groups: control, HCMV(+ ), after HCMV AD169 infection, and the supernatant of the culture was extracted, and the cells were lysed. The levels of vascular cellular adhesion molecule-1 (VCAM-1) in HUVEC were measured by real-time PCR. And the content of nitrogen monoxide (NO) of the supernatant was detected by nitrate reductasemethod accordingly.@*Results@#Twenty-four hours after infection, the mRNA expression of VCAM-1 in HUVECs of HCMV infected group increased obviously compared to control, and NO quantity increased accordingly and time-dependently. There was significant difference between groups(P<0.01).@*Conclusions@#HCMV increases the mRNA expression of VCAM-1 and quantity of NO, which may contribute to the formation of AS.