Microvesicles derived from hypoxia/reoxYgenation-treated human umbilical vein endothellal cells impair relaxation of rat thoracic aortic rings

Objective To investigate the effects of microvesicles(MVs) derived from hypoxia/reoxygenation(H/R)-treated human umbilical vein endothelial cells(HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.Methods H/R injury model was established to induce HUVECs to release H/R-EMVs.H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium.H/R-EMVs were characterized using 1 urn latex beads and anti-PE-CD144 by flow cytometry.Thoracic aortic rings of rats were incubated with 2.5,5,10,20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours,and their endothelium-dependent relaxation in response to acetylcholine(ACh) or endothelium-independent relaxation in response to sodium nitroprusside(SNP) was recorded in vitro.The nitric oxide(NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent.The expression of endothelial NO synthase(eNOS) and phosphorylated eNOS(p-eNOS,Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting.Furthermore,the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.Results H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation.The membrane vesicles(< 1 urn) induced by H/R were CD144 positive.ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner(P<0.05,P<0.01).The expression of total eNOS(t-eNOS)was not affected by H/R-EMVs.However,the expression of p-eNOS decreased after treated with H/R-EMVs.The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings(P<0.01).Conclusion ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner.The mechanisms included a decrease in NO production,p-eNOS expression and an increase in oxidative stress.

Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs

Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10^(-7)-10^(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P<0.05, P<0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P<0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.

Flow cytometric analysis of circulating microvesicles derived from myocardial ischemic preconditioning and cardioprotection of ischemia/reperfusion injury in rats

Objective: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion(I/R) injury in rats. Methods: Myocardial IPC was elicited by three cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending(LAD) coronary artery. Platelet-free plasma(PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFP. PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 μm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure(MAP), heart rate(HR) and ST-segment of electrocardiogram(ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin(HE) staining. The activity of plasma lactate dehydrogenase(LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. Results: Total IPC-MVs and different phenotypes, including platelet-derived MVs(PMVs), endothelial cell-derived MVs(EMVs), leucocyte-derived MVs(LMVs) and erythrocyte-derived MVs(RMVs) were all isolated which were identified membrane vesicles(<1 μm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats(P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR(P<0.01), decreased ST-segment and LDH activity(P<0.05, P<0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment(P<0.01). Conclusion: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.

Effects of endothelial microvesicles induced by A23187 on H9c2 cardiomyocytes

Objective To investigate the effects of endothelial microvesicles(EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells(HUVECs) were treated with 10 μmol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 μm latex beads and antiPE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles(< 1 μm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner(P<0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.

Dynamic recombination of triplet excitons in polymer heterojunctions

The dynamic recombination of two triplet excitons with opposite spins in the heterojunction structure has been investigated using a nonadiabatic evolution method.We demonstrate that luminous composite states including the excited polaron and the biexciton can be formed efficiently via the triplet exciton-triplet exciton reaction in the heterojunction and therefore this reaction can enhance the electroluminescence efficiency considerably,which is consistent qualitatively with experimental observations.Meanwhile,we find that,although the heterojunctions are beneficial to the generation of luminescent particles,large band offset caused by the heterojunction structure is not helpful to improve the electroluminescence efficiency.In addition,the mechanism of the triplet exciton-triplet exciton reaction in heterojunction is different from that of two similar coupling chains.Our results may deepen the understanding of the electroluminescence mechanism in polymer light-emitting devices.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection

Mosquito microRNAs （miRNAs） are involved in host-virus interaction, and have been reported to be altered by dengue virus （DENV） infection in Aedes albopictus （Diptera： Culicidae）. However, little is known about the molecular mechanisms of Aedes albopictus midgut--the first organ to interact with DENV--involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA （mRNA） expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.

Effects of coke charging pattern on burden movement in COREX melter gasifier based on physical simulation

A three-dimensional semicircle cold model of COREX melter gasifier(MG)was established,and the effects of coke charging pattern on burden movement were investigated by adopting this model.The burden flow pattern of packed bed was measured with different coke charging amounts,coke charging positions,and coke sizes.The results show that the solid flow pattern presents a change from straight line type to reverse U type and finally to W type in MG with traditional coke charging,while the burden flow pattern experiences the change from straight line type to W type with platform and finally W type under center coke charging,and the solid flow pattern presents a change from straight line type to W type with platform and finally W type under intermediate coke charging.The difference demonstrates that coke charging pattern affects the uniform descending in MG to some degree.The burden in the coke charging zone keeps a straight line descending.No matter what kind of coke charging pattern is adopted,the burden residence time is shortened with the increase in coke charging amount,and the vertex of deadman decreases obviously,which is conducive to the renewal of the deadman.The appropriate coke charging amount and coke size should be well controlled at 8.7%and 5 mm,respectively.The results are useful not only in developing further understanding of solid flow in MG but also in verifying different mathematical models,particularly the coupled discrete element method with computational fluid dynamics simulation which has been increasingly used in the literature.

Bioactive xanthones from whole plants of Gentianella acuta

Objective： To study the constituents from the whole plants of Gentianella acuta and their biological activities.Methods： The compounds were isolated by multiple chromatographic methods and the structures of mentioned isolates were determined by routine NMR experiments and chemical methods.Results： A phytochemical investigation to obtain intestine motility inhibitor resulted in the isolation of one new xanthone glycoside,gentixanthonoside A（1）,along with nine tetrahydroxanthones,1,3,5R,8S-tetrahydroxy-5,6,7,8-tetrahydroxanthone（2）,1,3,5S,8S-tetrahydroxy-5,6,7,8-tetrahydroxanthone（3）,amarellin E（4）,amarellin F（5）,swertiachoside B（6）,amarellin D（7）,amarellin C（8）,amarellin A（9）,and amarellin B（10）from the whole plants of G.acuta.Conclusion： Compounds 2 –10 showed significant reduce effects on contraction tension at 40 μM.