Dissertation > Excellent graduate degree dissertation topics show

Novel Acetylene Phenolic Compound Selaginellin: Anti-aging of Endothelial Cells and Neurobiology Research

Author: WangChenJing
Tutor: LiYuanJian
School: Central South University
Course: Pharmacology
Keywords: Homocysteine selaginellin Cellular senescence Reactive oxygen species Telomerase Sirt1 Glutamate Oxidative Stress PC12 cells Klotho Oxidized low-density lipoprotein LOX-1 NADPH oxidase Alzheimer's disease toll -like receptor 4 NF-κB Lipopolysaccharide
CLC: R965
Type: PhD thesis
Year: 2010
Downloads: 275
Quote: 1
Read: Download Dissertation

Abstract


Background artery atherosclerosis (atherosclerosis, AS) is a chronic, progressive, multiple intimal disease, serious harm to human health. AS etiology and mechanisms are very complex lipids, smoking, hypertension, diabetes, obesity, infection, and other risk factors are closely related. Numerous studies have demonstrated that homocysteine ??(homocysteine, Hcy) are also important factors to promote the formation of AS. Hcy can promote the formation of atherosclerotic plaque, increased mortality of patients with coronary heart disease, is considered to be an independent risk factor of AS. Aging is one of the risk factors for development of AS. Consistent with the many changes of the age-related vascular diseases and the aging vascular cells of characteristic changes. Endothelial dysfunction caused by vasoconstriction abnormal platelet adhesion and aggregation, as well as medial smooth muscle hyperplasia, AS pathogenesis of initiating events. Therefore, the protection of the vascular endothelial cells, antagonize the endothelial cell senescence is an important research direction for prevention and treatment of cardiovascular disease, especially atherosclerosis. Numerous studies show that aging is closely related to the Sirt1 gene play an important role in the maintenance of vascular endothelial function. Sirt1 in recent years found that the anti-aging and longevity genes involved in oxidative stress-induced endothelial cell senescence. The study, Hcy induced the AS role in its induced endothelial cell senescence-related. The Selaginella belonging the the ferns Selaginellaceae Selaginella plants have lower blood pressure, lowering blood sugar, enhance immune function and suppress the role of oxidative stress. Selaginellin a new structure of monomer compounds extracted from Selaginella vitro antioxidant Experimental results show that, selaginellin has strong antioxidant effects. Based on the above research background, the present study intends to observe selaginellin of Hcy induced cell senescence in cultured human umbilical vein endothelial cells and to explore the possible mechanisms involved. Primary cultured human umbilical vein endothelial cells (HUVECs). MTS assay cell viability; β-galactosidase activity (staining), telomerase activity (Real time PCR method) and cell cycle distribution (flow) to detect cell senescence; kit detection of intracellular reactive oxygen fluorescence ROS generation; quantitative real-time PCR to detect cell Sirt1 mRNA expression. The results (1) of Hcy (0.5 M) processing endothelial cells 48 h significantly reduced cell activity; selaginellin (10-7,3 × 10-7 and 10-6 M) for 1 h significantly inhibited Hcy-induced cell activity decline, and a dose-dependent manner; (2) Hcy (0.5 M) treatment of endothelial cells 48 h significantly increased β-galactosidase activity, lower telomerase activity, increase the proportion of Gl phase cells, suggesting that Hcy can induce endothelial cell senescence; Selaginellin (10-7,3 × 10-7 and 10-6 M) pretreatment 1 h can significantly inhibit Hcy-induced β-galactosidase activity was increased telomerase activity reduction cells in G1 phase The increase in the proportion and concentration-dependent manner; (3) of Hcy (0.5 M) processing endothelial cells 48 h significantly increased the level of intracellular ROS; Selaginellin (10-7,3 × 10-7 and 10-6 M) pre- treatment 1 h in a concentration-dependent manner inhibit Hcy induction of intracellular ROS level increase; (4) of Hcy (0.5 M) treatment of endothelial cells 48 h, can significantly with reducing the Sirt1 mRNA in expression; while selaginellin itself can significantly with raised the Sirt1 mRNA in expression, and Sirt1 mRNA can reverse Hcy-induced downregulation. The conclusion Selaginellin inhibited Hcy-induced endothelial cell senescence, which may be related to the inhibition of oxidative stress and upregulation of Sirt1 expression. Background glutamate (Glutamate, Glu) is an excitatory amino acid, plays an important role in the maintenance of the normal function of the nervous system activity. However, in certain pathological cases, such as Alzheimer's disease, Huntington's disease and Parkinson's disease, glutamate large accumulation of release can be induced nerve cell damage and missing. Glutamate-induced neuronal death mainly through two ways: excitotoxicity and oxidative stress. Former by excessive activation of glutamate ionotropic receptors, resulting in intracellular calcium overload trigger cell death; latter glutamate / cystine transporter-mediated cell death pathway. Selaginella have lower blood pressure, lower blood sugar, enhance immune function and suppress the role of oxidative stress, the crude extract can accelerate free radical scavenging, activate superoxide dismutase. Selaginellin in our hospital from Selaginella extract a monomer, has antioxidant properties. The experiments in differentiated PC12 cells to study the protective effect of selaginellin Glu-induced cell damage and apoptosis. Basis Klotho is a new anti-aging gene, can delay a variety of stress-induced apoptosis process, its mechanism and inhibition of oxidative stress, therefore, we further investigate the selaginellin the anti-apoptotic role in whether the up-regulation of Klotho gene. Methods The experiment is divided into normal control group, Glu injury group, Glu concentration selaginellin (10-7,3 × 10-7 and 10-6 M) group and the vehicle control group. Each drug treatment group pre-incubated for 1 h with the differentiation of PC12 cells, and then Glu (10 mM) treatment 24 h. Changes in cell morphology, cell activity (MTS Act) and LDH leakage (colorimetric method) to detect cell damage; Hoechst staining and caspase-3 activity to detect cell apoptosis; ROS generation of reactive oxygen fluorescence detection kit cells; Real-time quantitative PCR detection of intracellular expression of Klotho mRNA. Results (1) PC12 cell lines in culture medium containing 100 ng / mL NGF after 7 d of incubation, the cells differentiate into the anaphase neuron-like cell lines, generating a large number of dendrites; (2) MTS results show :5-20 mM Glu concentration-dependent manner to reduce the activity of PC12 cells, wherein, 10 mM Glu, a 45% reduction in cell activity; (3) morphology results: control group clear edge of the PC12 cells, membrane integrity, dendritic integrity; 10 mM Glu incubated After 24 h, cell body shrinkage, dendritic fracture, reticular structure disappeared; Selaginellin pretreatment in a concentration-dependent manner relieve Glu injury of PC12 cells. Consistent with the changes in cell morphology, Glu can significantly increase LDH leakage and reduce cell activity selaginellin a concentration-dependent reversal of the above-mentioned effect of Glu; (4) Hoechst 33342 staining showed that: Glu for 24 h significantly increased cell shrinkage nucleus fragmentation and chromatin gathered by Hoechst staining positive cells was significantly increased. Consistent with it a significant increase in caspase-3 activity. A concentration-dependently inhibited Glu pro-apoptotic effects in a concentration-dependent manner selaginellin inhibited; (5) Glu (10 mM) treated cells 24 h can significantly enhance the level of reactive oxygen species; pre-treated for 1 h with selaginellin Glu-induced increase in reactive oxygen species generation; (6) Glu significantly cut PC12 cells Klotho mRNA expression selaginellin pretreatment significantly inhibited Glu due to the down-regulation of Klotho mRNA. The conclusion Selaginellin inhibition of glutamate-induced PC12 cell injury and apoptosis by reducing oxidative stress and its anti-apoptotic effects may be associated with up-regulation of Klotho gene expression. Background Alzheimer's disease (Alzheimer's disease, AD) is a progressive degenerative disease of the nervous system caused by a variety of causes, the main pathology is characterized by lack of extracellular amyloid deposition, neurofibrillary tangles and neurons. The pathogenesis of AD is still not very clear. Recent studies suggest that AD incidence may be related to abnormal lipid metabolism and cell. LDL oxidative modification of brain tissue the generated oxidized LDL (oxidized low-density lipoprotein, ox-LDL), causing oxidative stress-induced neuronal apoptosis. Much evidence that neuronal apoptosis is involved in the occurrence of brain aging and AD. In vitro experiments confirmed, ox-LDL can induce a variety of cell apoptosis. Ox-LDL by binding to its receptor exerts its biological effects. Plant lectin-like oxidized low-density lipoprotein receptor-the -1 (lectin-like oxidized low density lipoprotein receptor-1, LOX-1) is the first time in 1997 by Japanese scholars in bovine aortic endothelial cells found new type of ox-LDL receptors. Ox-LDL induced intracellular ROS production, and ROS in a variety of biological processes play an important role in the LOX-1 is activated. We speculate that LOX-1 may be involved in neuronal apoptosis induced by ox-LDL. NADPH oxidase is an important catalytic enzymes superoxide anion production. The study showed that AD pathogenesis, NADPH oxidase is involved in the neuronal damage caused by reactive oxygen species. Selaginella have lower blood pressure, lower blood sugar, enhance immune function and suppress the role of oxidative stress, the crude extract can accelerate free radical scavenging, activate superoxide dismutase. Selaginellin is an extract from Selaginella monomer, The study found selaginellin significant antioxidant role. In this study, abnormal lipid metabolism, selaginellin neuron NADPH oxidase mediated ROS generation, further proven mechanism of its neuroprotective effects. Methods The experiment is divided into normal control group, ox-LDL injury group, three concentrations selaginellin (10-7,3 × 10-7 and 10-6 M) ox-LDL group, anti-LOX-1 of ox-LDL group, selaginellin (10-6 M) group and the vehicle control group. Ox-LDL (100μg/mL) treatment for 24 h, and then the drug treatment group pre-incubated with differentiated PC12 cells lh. MTS assay cell activity; In Hoechst staining, AnnexinV-FITC/PI the double staining and caspase-3 activity detection of apoptosis; kits for detecting intracellular ROS generation reactive oxygen fluorescence; RT-PCR was used to detect the expression of LOX-1 mRNA ; the colorimetric detection NADPH oxidase activity; quantitative real-time PCR detection of intracellular NOX-1, gp91phox, and p47phox mRNA expression. Results (1) MTS :20-100μg / mL ox-LDL concentration dependent decrease in the activity of PC12 cells, which, 100μg/mL ox-LDL and a 40% reduction in cell activity; (2) Ox-LDL treatment of PC12 cells 24 h, can significantly reduce cell activity; Selaginellin (10-3,3 × 10-7 and 10-6M) for 1 h significantly inhibited ox-LDL-induced cell activity and dose-dependent manner; (3) Hoechst 33342 results: ox-LDL for 24 h significantly increased cell shrinkage, nucleus fragmentation and chromatin aggregation, Hoechst staining positive cells was significantly increased; flow cytometry analysis showed, compared with the normal control group, ox- the LDL group PC12 cell apoptosis was significantly higher; apoptosis proportion of Selaginellin pretreatment group decreased significantly compared with ox-LDL group, respectively, and a dose-dependent manner; consistent with it a significant increase in caspase-3 activity; Ox- dose-dependent, pro-apoptotic effects of LDL concentration-dependent manner selaginellin inhibited; (4) Ox-LDL (100μg/mL) cells were treated for 24 h significantly increased the level of reactive oxygen species; pre-treated for 1 h with selaginellin inhibits ox-LDL-induced reactive oxygen species generated increase; Anti-of LOX-1 has a similar role; (5) Ox-LDL can significantly with raised PC12 cells LOX-1 mRNA in the expression; Selaginellin pretreatment can significantly with the inhibition of ox-LDL due to LOX-1 mRNA upregulation; (6) of NADPH oxidase activity test results, a 100μg/mL the role of ox-LDL can increase the activity of NADPH oxidase in differentiated PC12 cells 24 h; different the concentration selaginellin can dose-dependent antagonistic ox-LDL induced NADPH oxidase activity increased; Real time PCR results, 100μg/mL ox-LDL significantly increased NOX-1, gp91phox and p47phox mRNA expression; Selaginellin pretreatment significantly inhibited the ox-LDL due NOX-1, gp91phox, and p47phox mRNA expression down, and in a concentration-dependent manner; Anti-LOX-1 group has a similar role. The conclusion Selaginellin inhibition of oxidized LDL-induced apoptosis in PC12 cells, and its anti-apoptotic effects may be associated with the passage of suppression LOX-1/NADPH oxidase / R0S. Background Alzheimer's disease (Alzheimer's disease, AD) is a common degenerative lesions in the central nervous system. AD etiology, pathogenesis is not entirely clear that genetic factors, apoptosis, free radical damage, aluminum poisoning. The role of inflammatory response in AD has drawn wide attention recently. AD occurrence and development, along with the pathological changes of inflammation. Visible limitations and diffuse astrocytosis in the brain tissue of patients with AD. Some stimulation, astrocytes can release large amounts of inflammatory mediators involved in the substance of the central nervous system inflammatory response triggered and accelerated AD process. More and more evidence shows that the immune response play a decisive role in the occurrence and development process of AD. Toll-like receptors (toll-like receptor, TLR) mediated innate immune receptors, mainly involved in of pathogenic microorganisms product recognition and inflammatory signaling, mediated innate and acquired immunity. Closely related to the occurrence and development of TLR4 with Alzheimer's disease. TLR4 can activate nuclear factor-κB (nuclear factor-kappa B, NF-κB) signaling pathway mediated by a variety of proinflammatory role. Selaginellin extracted from Selaginella a new structure of the monomer compounds with strong antioxidant effect. The study shows that the selaginellin inhibit glutamate and oxidized low-density lipoprotein-induced apoptosis in PC12 cells. Therefore, we speculated the, selaginellin with strong neuroprotective effect. The purpose of this experiment is to observe impact of selaginellin inflammatory response of astrocytes and explore its role in whether the suppression of TLR4-NF-κB signaling pathway. Methods Primary cultured astrocytes, the experiment is divided into normal control group, the LPS injury group, the three concentrations selaginellin (10-7,3 × 10-7 and 10-6 M) LPS group, TLR4 antagonist LPS group selaginellin (10-6 M) group and the vehicle control group. Each drug treatment group astrocytes preincubated for 1 h, and then LPS (1μg/ml) for 24 h. MTS assay cell activity; quantitative real-time PCR detection cells of TLR4, TNF-α, MCP-1, IL-6 and NF-κB p65 mRNA expression; ELISA assay cell culture medium, TNF-α, MCP-1 and IL- 6 content; electrophoretic mobility change analysis of NF-κB activity. Results (1) MTS, the astrocytes exposed in 1μg/mL LPS for 24 h, cell viability decreased by 40%; the different concentrations selaginellin can dose-dependently antagonized LPS cytotoxicity; (2) Real time RCR results Show a 1μg/mL the LPS-treated astrocytes 24 h, significant upregulation of TNF-α, MCP-1 and IL-6 mRNA expression; Selaginellin pretreatment concentration-dependently inhibited the LPS-induced TNF-α MCP-1 and IL-6 mRNA upregulation; similar role of TLR4 antagonist; (3) ELISA results show 1μg/mL the LPS incubation star of glial cells 24 h, the culture supernatant of TNF-alpha, MCP-1 and IL-6 levels were significantly increased; pre-incubated with selaginellin after 1h, and then exposed to LPS were incubated for 24 h in a concentration-dependent manner down the culture supernatant of TNF-alpha, MCP-1 and IL-6 the level; (4) LPS treatment of astrocytes 24 h, significantly increased TLR4 mRNA expression; while selaginellin can reverse the LPS-induced TLR4 mRNA upregulation; (5) Real time PCR results show, 1μg/mL of LPS treatment the astrocytes 24h, significant upregulation of NF-kappa B p65 mRNA expression; Selaginellin pretreatment concentration-dependently inhibited the LPS-induced NF-κB p65 mRNA upregulation; EMSA results show a clear signal bands, LPS can a significant increase in the activity of NF-κB; Selaginellin preincubation astrocytes 1 h in a concentration-dependent decrease in the activity of NF-κB, TLR4 antagonist has a similar role. The Conclusion Selaginellin by reducing inflammatory cytokines TNF-α, MCP-1 and IL-6 synthesis and release of inhibition of LPS-induced astrocyte inflammatory response, and its anti-inflammatory effects may be related to the inhibition of TLR4-NF-κB pathway.

Related Dissertations

  1. Studies on Dynamic Regularity of Antibody Against G. Anatis and Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against G. Anatis Lps,S858.32
  2. Noble Dendrobium Polysaccharides Attenuate Learning and Memory Deficits Induced by Lipopolysaccharide and Its Mechanisms in Rats,R285.5
  3. 3-year changes in lipopolysaccharide newly diagnosed type 2 diabetes and atherosclerosis in patients with the correlation between the evolution of research,R543.5
  4. The Effect of Melatonin on Calcium Overload of Fetal Mouse Brain Cells Due to Lipopolysaccharide,R722.1
  5. Aspirin-triggered Lipoxin Inhibit the Inflammatory Response Induced by Lipopolysaccharide in Primary Astrocytes,R965
  6. Effects of Achyranthis Bidentatae Polysaccharides (ABPS) on Growth Performance, Immune Function and Intestinal Function in Piglets Challenged with Lipopolysaccharide,S828.5
  7. Immunofluorescence Study of LPS-induced NF-κB Expression in Human Decidual Cells,R965
  8. Lipopolysaccharide in aged rat alveolar macrophages secrete cytokines and apoptosis,R965
  9. Toll-like receptor 4 and its ligand lipopolysaccharide on bone marrow mesenchymal stem cell biology function,R329
  10. The Detection of Interleukin-6 in Vitro Human Whole Blood with Exogenous Pyrogen,R927
  11. Therapeutic Effect of Pulmonary Hemocoagulas Delivery on Neonatal Rats with Acute Lung Hemorrhage Induced by Lipopolysaccharide,R96
  12. The Effects of Posttreatment with Isoflurane, Sevoflurane and Desflurane on the Expression of ICAM-1 and VCAM-1 Induced by LPS in RLMVECs,R96
  13. Effect of Penehyclidine Hydrochloride on the Inflammatory Factor and Oxidative Stress in Acute Lung Injury Rats,R965
  14. LPS on carboxylesterase 1 and 2 (CES1, CES2) Expression,R96
  15. Explore antioxidants on human vascular endothelial cells inflammation in rats,R965
  16. Effect and Influencing Factors of FADD on Hepatic Apoptosis Signal Pathway in Experimental Fulminant Hepatic Failure,R575.3
  17. Protective Effects of Keratinocyte Growth Factor-2 on Lipopolysacchride-induced Acute Lung Injury and Its Potential Mechanisms,R563
  18. Change of Albumin in Septic Rats of the Early Phase,R459.7
  19. Expression of Angiotensin Ⅱ Type1 Receptor in RAW264.7 Macrophages and Its Role in the Production of Mediators of Inflammation,R363
  20. The Molecule Mechanism Study of Human Endometrium with Gonghuan Zhixue Tablet,R285.5
  21. Experimental Studies in Antiinflammatory Effects of Licorice Flavonoids,R285

CLC: > Medicine, health > Pharmacy > Pharmacology > Experimental Pharmacology
© 2012 www.DissertationTopic.Net  Mobile