The Effects Of Arsenite On The Apoptosis Of Human Umbilical Vein Endothelial Cells (HUVECs) And The Underlying Mechanism

The Effects of Arsenite on the Apoptosis of Human Umbilical Vein Endothelial Cells (HUVECs) and the Underlying MechanismArsenic is widely distributed in nature and its occurrence at high levels in drinking water in endemic regions remains a major public health concern because it affects several hundred millions of people worldwide. Chronic exposure to inorganic arsenic is found to cause multiple health disorders.peripheral blood vessels are a major target of arsenic toxicity. Epidemiologic studies carried out over the past decades confirmed the association between arsenic exposure and peripheral vascular disease in a dose-responsive pattern.vascular endothelial cells play a key role in maintaining the integrity and the repair of vascular injury. So the study of the molecular mechanism which arsenic damaged vascular endothelial cells is the premise of arsenic disease control.Which the apoptosis of vascular endothelial cells damaged the integrity of vascular is the initial step of vascular lesions, but the effects of arsenic on the apoptosis of HUVECs have not been elucidated. Human umbilical vein endothelial cells (HUVECs) have been widely used as an in vitro experimental model in studies on blood vessel endothelial cells.Apoptosis plays a crucial role in the development and defense of homeostasis and may have pathological effects that lead to serious health problems.The progression of apoptosis has been determined to be modulated by the redox status of cells. Excessive generation of intracellular ROS may lead to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis. Mitochondria is the main place to the production of ATP, and play an important role in cell survival, metabolism, cell apoptosis. The maintenance of mitochondrial membrane potential is the driving force for ATP generation,80%ROS generated in cells mitochondrial as the byproduct of oxidative phosphorylation. studies show that mitochondria are major contributors to endogenous ROS formation in hepatocytes. Increasing evidence has shown that reactive oxygen species (ROS) are involved in arsenic-induced cytotoxicity.As the important antioxidant enzymes in the body, SOD2 plays a key role in cell redox reactions. The role of SOD2 is also very important in the apoptosis of tumor cells induced by arsenic, but the mechinasm of SOD2 in the aspect of arsenic poisoning has not been elucidated. Studies show that SP1 interacts with SOD2 gene promoter and enhancer, whith regulate the level of SOD2 gene transcription in vivo. However, detailed mechanism of SP1 for protein factors and DNA components involved in regulation of SOD2 gene transcription and the effects of arsenic on its arenot been reported.MicroRNAs (miRNAs) are a class of the 18-25nt single-stranded RNA that do not encode any protein, miRNAs can post-transcriptional regulate gene expression by inhibiting the translation of mRNA or degradating mRNA. At present miRNA chip technology has become an important means of related diseases diagnosis.the role of sodium arsenite on miRNAs expression profile of HUVECs cell research has not been reported.As the main component of ginseng,Ginsenosides Rbl can inhibit tumor cells and anti-aging,but the important role of Ginsenosides Rbl for vascular injury caused by arsenic has not been reported.as one of essential vitamins of humans and animals,Vitamin B12 involved in the synthesis and metabolism in vivo.studies recently report that antiapoptotic efficacy of folic acid and vitamin B(12) against arsenic-induced toxicity. the possible interactions of Ginsenosides Rbl and Vitamin B12 separately or in combination work are not reported, during arsenite injury HUVECs cells.Therefore, Human umbilical vein endothelial cells (HUVECs) had been used as an in vitro experimental model in our studies, analyzed the effects of NaAsO2 on the apoptosis of HUVECs, mitochondrial membrane potential (ΔΨm), ROS, and the expression of the related genes, such as SOD2. molecular mechanism of which arsenic regulates gene transcription is analysed by ChIP. at the same time, to clarify the role of miRNAs in apoptosis induced by arsenite, we analyse the expression profile of miRNAs after arsenite treats HUVECs cells for 24 hours by microarray. miRNAs are analyzed by MEME, and then whose roles are analyzed by the other software. At last, we study the effects of Ginsenosides Rbl and Vitamin B12 during arsenic induced HUVECs cells. Results obtained from this project will provide important theoreties and experimental basis for the mechanism of arsenic poisoning, and the foundation for linical drug research of prevention and treatment of arsenic disease.Part I Arsenic induces HUVECs cell apoptosis by the mitochondrial pathwayObjective:To clarify the molecular mechanisms through which arsenic causes injuries to blood vessels, we analyzed the effects of NaAsO2 on the apoptosis of HUVECs,ATm, ROS, and the expression of the related genes. Methods:apoptotic cells were detected by flow cytometry with Annexin V-FITC/PI dual staining;ΔΨmof HUVECs was detected by flow cytometry with the fluorescent dye Rhodamine123 staining; the levels of intracellular ROS were measured by flow cytometry with a cell based ROS assay kit; the expression of the related genes were detected by quantitative real-time RT-PCR Results:apoptotic cells were significantly increased in a dose-dependent manner following arsenite treatments for 24 hours, and arsenic treatment increase the numbers of the cells with collapsedΔΨm,the addition of N-acetyl-L-cysteine (NAC,5 mM) effectively rescued the cells from apoptosis induced by arsenic (P<0.05 versus the cells only treated with 20μM arsenite), indicating that the oxidative stress was involved in the induced cell apoptosis;the intracellular ROS levels of HUVECs showed a double phase response to arsenic exposure:there was a significant and dose-dependent increase with higher concentrations (≧20μM), and a decrease in ROS with low-concentrations (≦10μM) of arsenic treatments;the mRNA of SOD2 was significantly induced when HUVECs were treated with a low concentration (5μM) of arsenic, which is the reason of decrease in ROS with low-concentrations of arsenic treatments. Conclusion:Arsenic induces HUVECs cell apoptosis by the mitochondrial pathwayPartⅡI Transcription factor SPl mediated the effect of that sodium arsenite promotes gene transcription of SOD2Objective:To study the molecular mechanisms of high expression of SOD2 gene induced by sodium arsenite. Methods:Act-D was added to arsenite-induced HUVECs cells, we analyzed the expression of SOD2; analyzed the effect of sodium arsenite on the transcription factor Spl and the SOD2 promoter cis-element binding ability by ChIP; RT-PCR and immunofluorescence cytochemistry detected SPl expression at the level of mRNA and protein in HUVECs. Results:Act-D completely blocked the expression of SOD2 mRNA induced by sodium arsenite, which indicated sodium arsenite play the role by regulating SOD2 transcription rate; arsenite promoted the binding capacity between the transcription factor Spl and the SOD2 promoter region, and the region was 42bp-172bp; sodium arsenite significantly increased the expression of Spl at the level of mRNA and protein. Conclusion:sodium arsenite promote SOD2 transcription rate through increased the expression of Spl and the binding capacity with the SOD2 promoter region.PartⅢSodium arsenite Changes miRNAs expression profiling in HUVECsObjective:To study the miRNAs expression profiling Changes in HUVECs induced by sodium arsenite. Methods:miRNA microarray dectected the miRNAs expression profiling Changes in HUVECs induced by sodium arsenite; MEME analyzed changed miRNAs; DAVID analyzed target genes of miRNAs and the functions. Results:the expression of 89 miRNAs significantly increased, the expression of 60 miRNAs significantly reduced; there were some common DNA cis-elements in the promoters of miRNAs; target proteins of miRNAs were involved in protein phosphorylation, mRNA splicing and other cellular functions, which provides a new molecular mechanism of arsenite-induced vascular injury. Conlusion:Sodium arsenite Changes miRNAs expression profiling in HUVECs, target proteins of miRNAs were involved in protein phosphorylation, mRNA splicing and other cellular functions.PartⅣGinsenosides and VB12 protected from sodium arsenite on HUVECs damage.Objective:To investigate the protect of Ginsenosides Rbl and Vitamin B12 separately or in combination work,when sodium arsenite induced apoptosis of HUVECs.Methods: Apoptotic cells were detected by flow cytometry with Annexin V-FITC/PI dual staining; the levels of intracellular ROS were measured by flow cytometry with a cell based ROS assay kit. Results:Ginsenosides Rb1 and Vitamin B12 separately or in combination work inhibited apoptosis of HUVECs induced by sodium arsenite,and protected the HUVECs; Ginsenosides Rbl and Vitamin B12 separately inhibited ROS generation in HUVECs induced by sodium arsenite, they had not combination effect. Conlusion:Ginsenosides Rbl and Vitamin B12 separately protected apoptosis of HUVECs induced by sodium arsenite, combinated they had additive effects...