| Background and objective The dominant hypotheses,response-to-injury and inflammatory hypothesis, indicate that the injury of endothelial cells, the migration of smooth muscle cells (SMC) from media and proliferation in intima are key steps in atherosclerosis (AS). Endothelin (ET), a vasoconstrictive peptide released from endothelial cells and smooth muscle cells (SMC), plays an important role in the occurrence and development of AS. Many evidences support that ET-1 induces the smooth muscle cells (SMC) proliferation. However, it is still unclear that what the effect of elevated ET on the endothelial cells is. We therefore used ECV304 cells treated with ET-1 in vitro to explore the influence of ET-1 on human vascular endothelial cells (HVECs).Sarafotoxin gene family diverges from the same ancestral gene as ET gene family. Sarafotoxin has a close similarity in molecular structure and biological actions to ET. Lobelia Chinensis Lour (Campanulaceae) has been used as fever reliever, antidote, diuretic, venom antagonist and maybe an ET antagonist. In 1993, JF Zhang found that Lobelia Chinensis Lour relieved the contraction of vascular ring both with and without endothelium induced by ET-1 and noradrenalin. Studies in hyperlipidemic rats have been carried in our laboratory for the protection effect of Lobelia Chinensis Lour on the injured endothelium. The conclusion was that Lobelia Chinensis Lour could not decrease the concentration of serum lipid but antagonizeET secreted and increase endothelial nitric oxide synthase synthesized by endothelium of hyperlipidic rats. The vascular endothelial injury induced by hyperlipidemia was relieved. In order to further explore the protective mechanism of Lobelia Chinensis Lour on endothelial cells and found its effective components, Lobelia Chinensis Lour alkaloids (LCLAs) were extracted, together with its main component, lobeline, administrated to vascular endothelial cells treated with ET and observed their effects on fibrinolytic system, NO system, apoptosis and adhesion molecules. It would provide experimental evidences for using LCLAs and lobeline in clinics to treat the patients with cardiovascular and cerebrovascular diseases.Part One: Gene expression profile of human vascular endothelial cells treated with endothelinMethodsCells count kit used to identify the proper concentration of ET-1 dose for injuring the cultured human endothelial cells. ECV304 cells were cultured in DMEM medium containing 10% fetal bull serum (FBS). Cells were trypsinized by 0.25% trypsin and resuspended by DMEM medium. Solution with single cells in suspension were plated in 96-well plates at a density of 4 X 104 cells/ml. Cells were treated with ET-1 (0.02, 0.1, 0.5 n mol/L) for 6 and 24 hours in experimental group, without ET-1 in Control group. CCK solution was administered to each well 2 hours before terminating medicine effect. Cells continued to be in culture for 2 hours and then their optical density (OD) was recorded using Enzyme Linked Immunosorbent Assay Plate Reader. Data were analyzed using ANOVA and q test. The significance was set at 0.05.GeneChip Microarray Analysis of gene expression profile in HVECs. The gene expression profile of ECV304 cells treated with ET-1 for 6 and 12 hours was assessed using microarray technology with the GEArry Q Series Human Endothelial Cell Biology Gene Array, which contain 96 genes. ECV304 cells were plated in 3 flasks of 25ml volume at the density of 4X 104 cells /ml, 4ml per flask. When cells attached fully in flasks, substituted medium without FBS for 6 hours to stop cells at Gl stageand then substituted medium containing 10% FBS. Cells were divided into 3 groups, including control group, ET-1 6-h group and ET-1 12-h group.Total RNA was extracted from ECV304 cells using TRIzol reagent and 5 u g of them as template to generate Biotin- 16-dUTP-labeled cDNA probes. The cDNA probes were denatured and hybridized with the SuperArray membrane, which was washed and exposed with a chemiluminescent substrate. To analyze the SuperArray membrane, we scanned the X-ray film and imported it into Adobe Photoshop as TIEF file. The image file was inverted, and the spots were digitized with ScanAlyze software, and normalized by subtraction of the background as the average intensity value of 3 spots containing plasmid DNA. The averages of 2 GAPDH or 4 cyclophilin A spots were used as positive controls and set as baseline values with which the signal intensity of other spots was compared. Ratio of two groups more than 2 was set as up-regulation standard, while more than zero and less than 0.5 was set as down-regulation standard.ResultsThe proper ET-1 dose to establish cells model. There was no significance between control group and all groups treated with ET-1 for 6 hours and groups treated with 0.02 U mol/L, 0.1 U mol/L ET-1 for 24 hours in optical density(.P>0.05). However, there was different significant between control group and group treated with 0.5 u mol/L ET-1 for 24 hours in optical density (PO.01). Therefore, 0.5 u mol/L ET-1 was chosen to establish the model of injured cells in all experiments.Gene expression profile of HVECs. Compared with control group, the expression of 67 genes was changed in ECV304 cells after treated with ET-1 for 6 hours. Among them, 61 genes including antithrombotic and antioxidant factors genes were downregulated and 6 genes including prothrombotic genes were upregulated. The expression of 53 genes was changed in ECV304 cells after exposed to ET-1 for 12 hours. 22 genes downregulated included antithrombotic and antioxidant genes and 31 genes upregulated included prothrombotic genes and inflammatory factor genes.ConclusionsET-1 could injure human vascular endothelial cells by downregulating antithrombotic and antioxidant genes and upregulating prothrombotic and inflammatory factors genes.Part Two: Influence of Lobelia Chinensis Lour alkaloids and Lobeline on fibrinolytic system of human vascular endothelial cells treated with endothelinMethodsAlkaloids extraction from Lobelia Chinensis Lour. 3kg Lobelia Chinensis Lour in a tissue bag was put into extraction utensil and ethanol was poured to immerge 3cm above the medium. Extraction utensil was closed to heat up for 2 hours. After medine components entered into ethanol, the extracted solution was condensed to ointment while ethanol retrieve. Repeated the procedure 3 times. The ointment was extracted with ether to remove organic acid after dissolved in 3% hydrochloric acid. Lobelia Chinensis Lour alkaloids were extracted with chloroform after basified using sodium carbonate. Column chromatography was carried out to purify Lobelia Chinensis Lour alkaloids and thin layer chromatography to identify them.MTT assay to screen doses of Lobelia Chinensis Lour alkaloids and Lobeline. After a 24-h incubation, attached ECV304 cells were collected by trypsinization, resuspended them in medium. Cells were plated in 96-well plates at density of 4X 104 cells/ml and treated with 6 doses (lOOmg/L, 50mg/L, 25mg/L,12.5mg/L, 6.25mg/L, 3.125mg/L) of Lobelia Chinensis Lour alkaloids or Lobeline respectively. MTT was administered at 4 hours before terminating the effect of medicine to cells, which continued to be in culture for 4 hours. After crystal was dissolved in dimethylsulphoxide, optical density was recorded using Enzyme Linked Immunosorbent Assay Plate Reader. Data were analyzed using ANOVA and q test. The significance was set at 0.05.ELISA technique to determine concentrations oft-PA and PAI-1. ECV304 cells were cultured in 24-well plates. There were 10 groups, including control group, ET-1group, BQ788 group, BQ123 group, 50mg/L LCLAs group, 25mg/L LCLAs group, 12.5 mg/L LCLAs group, 50mg/L Iobeline group, 25mg/L Iobeline group and 12.5mg/L Iobeline group. After a 24-h incubation, ECV304 culture supernatants were collected. The concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in supernatants were assayed using the enzyme linked immunosorbent assay technique. Data analysis was performed as mentioned above.RT-PCR analysis of the PAI-1 mRNA expression. The expression of PAI-1 mRNA in ECV304 cells was assayed using Reverse Transcription - Polymerase Chain Reaction (RT-PCR) technique. After removed the culture medium, ECV304 cells grown in 24-well plates were lysed with TRIzol reagent, and total RNA was isolated from them. The mRNA was reversibly transcribed into cDNA and then PCR was performed. After themocycling and electrophoresis of the PCR products on 1% agarose gel containing ethidium bromide, the bands were visualized using UV transilluminator and gel photographs were obtained. Ratio between mRNA of PAI-1 and beta-actin was calculated.ResultsIdentification of Lobelia Chinensis Lour alkaloids. Lobelia Chinensis Louralkaloids were identified using bismuth potassium iodide. Salmon pink spots presented as a proof of Lobelia Chinensis Lour alkaloids.Doses of Lobelia Chinensis Lour alkaloids and Iobeline for exepriments. Equal and less than the dose of 50mg/L LCLAs, and equal and less than the dose of lOOmg/L Iobeline could be used in experiments without cytotoxic effect on the cells.The concentrations of PAI-1 and t-PA. The concentration of PAI-1 were higher in endothelin group than in control group (P<0.0l). LCLAs and Iobeline lessened PAI-1 release significantly from ECV304 cells injured by ET-1 (P<0.01). Their effects were similar to that of ETb receptor antagonist, BQ788. But there was no significantly difference in the concentrations of t-PA among all groups.The expression of PA I mRNA. The expression of PAI mRNA was higher in ET-1 groups than in control group. LCLAs and Iobeline lessened PAI-1 expression inECV304 cells injured by ET-1. Their effects were similar to that of ETB receptor antagonist, BQ788.ConclusionsLCLAs and lobeline can protect HVECs against the injury effect of ET-1 by decreasing the PAI-1 mRNA expression and PAI-1 release.Part Three: Influence of Lobelia Chinensis Lour alkaloids and lobeline on NO system of HVECs treated with endothelinMethods.Nitrate reductase assay to determine the concentrations of NO. ECV304 cells cultured, groups divided and medicine administered were the same as PAI-1 concentrations measurement. Concentrations of NO in culture supernatants were measured by nitrate reductase assay according to instruction of NO Kit.GeneChip microarray analysis of eNOS gene expression. ECV304 cells were plated in 7 flasks of 25ml volume at the density of 4X 104 cells /ml, 4ml per flask. When cells attached fully in flasks, change medium without FBS for 6 hours to stopped cells at Gl stage and changed medium containing 10% FBS. There were 7 groups, including control group, ET-1 6-h group, LCLAs 6-h group, lobeline 6-h group, ET-1 12-h group, LCLAs 12-h group, lobeline 12-h group. Gene Super Array analysis just the same as in part one.ResultsNO concentrations in culture supernatants of ECV304 cells NO concentrations in ECV304 cells culture supernatants significantly decreased in ET-1 group compare with control group. LCLAs and lobeline reversed the decrease of NO secretion induced by endothelin (PO.05). Their effects were similar to ET antagonist, BQ788 and BQ123.Gene expression of eNOS in EC V3 04 cells. The eNOS gene expression in ET-1 6-h group was downregulated compared with control group. It suggested that ET-1 could lessen NO release and induce injury of HVECs through downregulating eNOSgene expression pathway. The eNOS gene expression was higher in LCLAs group compared with ET-1 group, but there was no statistical significance. The eNOS gene expression in lobeline 6-h group was significantly upregulated compared with ET-1 6-h group, and it also significantly upregulated in lobeline 12-h group compared with ET-1 12-h group. These results suggested that lobeline could work as ET antagonist to upregulate the eNOS gene expression. The NO release resulted from it and play a protective role in HVECs.ConclusionsET-1 induced eNOS gene downregulated and NO release significantly lessened, which could bring about injury effect in HEVCs. Lobeline might be a novel medicine to protect vascular endothelial cell against the injury by ET-1, the mechanism could be upregulating eNOS gene and increasing NO release of HEVCs. LCLAs had the similar effects.Part Four: Influence of Lobelia Chinensis Lour and lobeline on apoptosis of human vascular cells treated with endothelinMethodsFlow cytometry to determine apoptosis rates Apoptosis rates were explored by flow cytometry technique. ECV304 cells were plated in 8 flasks and cultured in DMEM medium without FBS for 24 hours. After changed DMEM medium containing 2% FBS, Cells were divided into 8 groups, including control group, ET-1 group, 50mg/L LCLAs group, 25mg/L LCLAs group, 12.5mg/L LCLAs group, 50mg/L lobeline group, 25 mg/L lobeline group and 12.5 mg/L lobeline group. After administered medicine for 24 hours, discard the culture supernatants. Cells were trypsinized by 0.25% trypsin and detached from flasks, 1X106 cells suspended in DMEM medium. Cells suspension was centrifuged, washed three times with PBS. Supernatants were discarded. When quivering gently, cells were added to 75% ethanol to fix at 4°C for 24 hours. Centrifuged before discarded ethanol. To determine the apoptosis rates of ECV304, after resuspended in 500 y 1 PBS, the cells were stainedusing propidine iodide (PI), filtered with 300 holes boult and analyzed by flow cytometry.Immunohistochemistry to measure expression of Fas and Bcl-2 protein. When ECV304 cells grown and adhered to glass slides in 24-well plates, they were divided into 10 groups just the same as NO measurement. After administered medicine for 24 hours, supernatants were discarded. Cells were washed 2 times with PBS, and fixed for 30 minutes using 95% ethanol. The expression of Fas and Bcl-2 protein was measured according to the instruction of Kit. Immunocytochemistry staining images were obtained and analyzed the density of yellow color.Statistical analysis The results are presented as mean + SEM. Statistical analyses were conducted using Statistical Analysis Systems SPSS 11.0. One-way ANOVA and q test was used. The differences were considered to be significant at PO.05.ResultsApoptosis rates ofECV304 cell. Apoptosis rates of ECV304 cells in all groups were very low and the differences between them were not obvious. Apoptosis peaks could not be seen in figures. The results suggested that the apoptosis rates of ECV304 couldn't be changed by ET-1. The anti-ET-1 effect of LCLAs and lobeline did not show at apoptosis rates.Expression of Fas. Fas expression in ET-1 group was lower than in control groupCPO.Ol). It suggested that ET-1 inhibited HEVCs apoptosis through Fas pathway. The expression of Fas in BQ788 group and BQ123 group was higher than in ET group(P<0.01). Fas expression in LCLAs groups and lobeline groups was also higher than in ET-1 group (PO.05). However, The level of Fas expression was very low in ECV304 cells. Whether ET-1 has anti-apoptosis effect or not and the anti-ET-1 effect of LCLAs and lobeline needs more research to be verified.Expression of Bcl-2. Bcl-2 expression of ECV304 cells was not different between ET-1 group and control group (/*>0.05). Differences of Bcl-2 expression were not shown among BQ788group, BQ123 group, LCLAs groups, lobeline groups and ET-1 group (7>>0.05). It suggested that ET-1 couldn't affect HEVCs apoptosisthrough Bcl-2 pathway. LCLAs and lobeline couldn't play a protective role through the pathway too.ConclusionsET-1 could not induce HVECs apoptosis. It might inhibit HVECs apoptosis through Fas pathway. LCLAs and Lobeline could work as ET-1 antagonist to increase Fas expression in this experiment. Their effects still need to be further proved. Therefore, cells apoptosis was not a good marker for LCLAS and lobeline to show their effect of anti-ET and endothelial protection.Part Five: Influence of Lobelia Chinensis Lour alkaloids and lobeline on adhesion molecules expression of human vascular endothelial cells treated with endothelinMethodsImmunocytochemistry. The expression of intercellular adhesion molecule -1 (ICAM-1) and vascular cell adhesion molecule -1 (VCAM-1) in ECV304 cells was determined using immunocytochemistry staining. Cells culture, groups divided and immunocytochemistry staining were similar to FAS and Bcl-2 measurement. Data was presented as Mean±SEM, One way ANAVA and q test was used by SYSTAT statistical package. The significance was set at P<0.05.RT-PCR analysis RT-PCR analysis was used to determine the expression of ICAM-1 mRNA and VCAM-1 mRNA in ECV304 cells. ECV304 cells were divided into 10 groups as described in measurement of t-PA and PAI concentration. Total RNA was extracted from ECV304 cells, 5 n g RNA was reversibly transcribed and the PCR was performed. After thermocycling and electrophoresis of the PCR products on 1% agarose gel containing ethidium bromide, the bands were visualized using a UV tansilluminator and gel photographs were obtained. Ratio between mRNA of PAI-1 and beta-actin was calculated.ResultsICAM-1 and VCAV-1 protein expression. ICAM-1 expression of ECV304 cellsin ET-1 group was higher than in control group (PO.01). The expression of ICAM-1 in BQ788 group, BQ123 group, LCLAs groups and lobeline groups was lower than in ET-1 group (PO.01). VCAM-1 expression was weak in control group, but icreased in ET-1 group. The expression of VCAM-1 in other groups was also very low. It suggested that ET-1 induced ICAM-1 and VCAM-1 expression in HVECs. This effect could be inhibited by LCLAs and lobeline, which had similar effects with ET antagonist BQ788 and BQ123.ICAM-1 and VCAV-1 mRNA expression. The expression of ICAM-1 mRNA and VCAM-1 mRNA in ET-1 group was higher than that in control group. The effect could be blocked by BQ788, BQ123, LCLAs and lobeline. The expression of ICAM-1 mRNA and VCAM-1 mRNA in these groups was lower than ET-1 group. It indicated that ET-1 induced the expression of ICAM-1 mRNA and VCAM-1 mRNA in HVECs, LCLAs and lobeline could inhibit the effect of ET-1 and protect HVECs.ConclusionsET-1 could induce ICAM-1 and VCAM-1 expression on both mRNA and protein level. LCLAs and lobeline could block the effect of ET-1, lessening the expression of ICAM-1 and VCAM-1 and play a protective role to HVECs. Lobeline might be the most effective component in Lobelia Chinensis Lour.
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