The Protective Effects And Mechanisms Of Isorhamnetin On Oxidized Low-density Lipoprotein Induced Endothelial Cell Injury In Vitro

Atherosclerosis (AS) is a severe chronic disease caused by many factors, which became more severe recently with the improvement of our life. This phenomenon caused considerable attention.In the pathogenesis of AS, endothelial cells play a critical role. The dysfunction and injury to endothelial cells has believed to be the initiate event in the etiology of AS. Therefore, agents that could protect endothelial cells from injury may be an important way to control and treat with AS.Endothelial dysfunctions, especially elicited by oxidized low-density lipoprotein (ox-LDL), play a critical role in the pathogenesis of AS. LOX-1, a lectin-like receptor for ox-LDL has recently been identified in endothelial cells. Activation of this receptor initiates intracellular signaling pathways, and involves in endothelial NO synthase (eNOS) expression, superoxide generation, endothelium secretory activities alteration, and leads to endothelial activation, dysfunction and apoptosis.In recent years, the clinical importance of herbal drugs has received considerable attention. Flavonoids existing in herbal drugs and diets are known to possess a wide range of biological functions. As reviewed by Middleton, the plant flavonoids exhibited strong antioxidant activity and were applied in inflammation, heart disease, and cancer on mammalian cells. Many flavonoids also showed inhibitory activity toward cyclic nucleotide phosphodiesterases. The consumption of fruits, vegetables and beveragescontaining flavonoids may lower the risk of cardiovascular diseases, including atherosclerosis and hypertension.Since the 1950s, many medicinal preparations of Seabuckthorn, also called Hippophae rhamnoides L, from wild and cultivated materials have been widely used in cardiovascular disorders, radiation damage, and inflammation treatment'' . The antioxidant and free radical scavenging properties, inhibition of LDL oxidation capabilities, anti-tumor activity as well as the activity against ox-LDL induced cell apoptosis of quercetin have already been reported recently. However the intracellular factors and enzymes with which the FST interfered to prevent the cytotoxity of ox-LDL are still unclear.The present study was designed to investigate the protective effects of FST and isorhamnetin on ox-LDL induced endothelial cell line EA.hy926 injuries. And to uncover some of the underlying mechanisms of these effects, thereby may provide part of the pharmacological basis for the clinical application of Ttraditional Chinese Medicines for treatment of AS.Part 1 Screening of extracts from seabuckthorn via estimate the protective effects of them on endothelial cell injuries induced by oxidized low-density lipoprotein in vitro and components identification of the most effective extractAim: The present investigation was undertaken to screen the extracts from seabuckthorn via estimate the protective effects on ox-LDL induced endothelial cell line EA.hy926 injuries in vitro, and to identify the components of the most active extract.Methods: Indices such as nitric oxide (NO) and superoxide dismutase (SOD) were measured. The free radical scavenging activity of the extracts from seabuckthorn was also evaluated. TLC, HPLC, MS was used to separate and identify the components of the most active extract HR20.Results: Cell viability decreased significantly after 24 h treatment with ox-LDL, accompanied with apparent secretion disorders such as NO, SOD reduction. Pretreatment of HR20, one of the extracts from seabuckthorn, could remarkably prevent both cell death and secretion disorders in a concentration-dependent manner. Moreover, HR20 scavenged 52.84% of free radical DPPH, about half of the negative control quercetin. The protective effects of HR20 was stronger than other extracts. There are two major points in the screen of TLC, which were corresponding to quercetin and isorhamnetin. On chromatogram of HPLC, there are two major peaks, with the retention times of 7.58min and 12.23min respectively. They are corresponding to the retention times of standard sample, quercetin and isorhamnetin. In order to verify the major peak, the method of MS was also used, which showed the m/zof 315, corresponding to M-l of isorhamnetin.Part 2 Modulation effects of the active extract containing isorhamnetin (AECI) on oxidized low-density lipoprotein induced alteration of eNOS and LOX-1 expressionAim: To verify the protective effects of the active extract containing isorhamnetin from the level of gene and proteinMethods: RT-PCR and Western-blot were used to evaluate the expression of eNOS (mRNA and protein) and LOX-1 mRNA.Results: It was observed that ox-LDL inhibited eNOS expression and increased LOX -1 expression. Pretreatment with isorhamnetin remarkably inhibited the ox-LDL induced downregulation of eNOS, upregulation of LOX-1, as well as the p38MAPK phosphorylation and superoxide overproduction in endothelial cell line EA.hy926.Part 3 The protective effects and mechanisms of isorhamnetin on oxidized low-density lipoprotein induced endothelial cell injury in vitroAim: The present investigation was undertaken to determine the protective effects of isorhamnetin, the major components of FST, on endothelial cell line EA.hy926 injury induced by oxidized low-density lipoprotein (ox-LDL) and to uncover some of the underlying mechanisms of these effects.Methods: Indices such as cell viability, LDH, NO, SOD and superoxide were measured. RT-PCR was employed to confirm the expression of eNOS mRNA and LOX-1 mRNA. Western blotting was used to evaluate LOX-1, eNOS protein expression, and p38MAPK phosphorylation. Immunocytochemistry was also used to measured the expression of protein eNOS.Results: Cell viability decreased significantly after 24 h treatment with ox-LDL, accompanied with apparent secretion disorders such as NO, SOD reduction and LDH increase. Besides, it was observed that ox-LDL triggered superoxide production, inhibited eNOS expression and increased LOX -1 expression. The data showed that pretreatment with isorhamnetin resulted in remarkable increase of cell viability (.P<0.05) and modulation of secretion disorders mediated by ox-LDL in a concentration-dependant manner. Besides, isorhamnetin pretreatment inhibited the ox-LDL-induced downregulation of eNOS, upregulation of LOX-1, as well as the p38MAPK phosphorylation and superoxide overproduction in endothelial cell line EA.hy926.Conclusions: The data indicate the protective effects of HR20 on endothelial cell line EA.hy926 from injuries induced by ox-LDL. These effects might derive from its antioxidant activity as well as its capability in modulating the expression of eNOS and LOX-1. And isorhamnetin may contribute to these effects of HR20. The effects of isorhamnetin may concern with the receptor (LOX-1), intracellular signaling pathway(p38MAPK activation), eNOS expression and superoxide production.