【作者】 李建宽；

【导师】 李青山；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生， 2014， 博士

【摘要】 目的：杜鹃素是存在于植物中的一种主要二氢黄酮化合物,是中药“满山红”(兴安杜鹃Rhododendron dauricum L.)的主要止咳祛痰活性物质,现代药理学研究表明杜鹃素还具有抗菌、抗炎、免疫抑制及血管平滑肌增值抑制作用。杜鹃素与存在于柑橘类水果中的橙皮素、柚皮素同属二氢黄酮化合物,且具有相似的化学结构特征,但是后者在抗氧化及细胞损伤保护方面研究较多,而杜鹃素抗氧化和细胞保护作用研究目前未见报道。本课题第一部分以具有血管内皮细胞特性的EA.hy926细胞株为研究对象研究杜鹃素对过氧化氢(H202)诱导EA.hy926细胞损伤及凋亡的抑制作用；第二部分主要研究杜鹃素对H202诱导的EA.hy926细胞MAPK信号传导通路的调节作用,以阐明其抑制凋亡的分子信号机制；第三部分初步研究杜鹃素对FeC13诱导大鼠颈动脉血栓形成的抑制作用,以体内研究间接探讨其抑制血管内皮氧化损伤作用。本课题主要通过以上三部分研究来揭示杜鹃素抑制氧化应激诱导的血管内皮细胞损伤作用及其可能的分子机制。方法：(1)采用MTT法测定H202对EA.hy926细胞活力影响及杜鹃素对H202诱导的EA.hy926细胞活力下降的抑制作用；(2)采用紫外分光光度法测定杜鹃素对H202诱导EA.hy926细胞内丙二醛(MDA)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性的影响；(3)采用流式细胞术测定杜鹃素对H202诱导EA.hy926细胞内活性氧(ROS)水平的影响；(4)采用Annexin V-FITC/PI双染检测杜鹃素对H202诱导EA.hy926细胞凋亡率的影响；(5)采用RT-PCR检测杜鹃素对H202诱导EA.hy926细胞凋亡相关蛋白Bax mRNA、 Bcl-2mRNA的表达影响；(6)通过Western blot检测杜鹃素对H202诱导EA.hy926细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3的表达影响；(7)通过Westernblot结合激光共聚焦检测杜鹃素对H2O2诱导EA.hy926细胞紧密连接蛋白occludin的表达影响；(8)采用Western blot检测杜鹃素对H202诱导EA.hy926细胞丝裂原活化蛋白激酶(MAPK)信号通路的调控作用；(9)采用紫外分光光度法测定杜鹃素对FeC13诱导的大鼠血清丙二醛(MDA)含量及超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性的影响；(10)采用HE染色检测杜鹃素对FeC13诱导的大鼠颈动脉内血栓形成的病理学变化影响。结果：(1)杜鹃素在浓度小于60μmol/L时对EA.hy926细胞没有检测到细胞毒性作用；(2)杜鹃素在15、30和60μmol/L浓度时具有明显抑制H202诱导的EA.hy926细胞内SOD、GSH-Px活性和细胞活力的降低及MDA和ROS水平的升高(p<0.05),并呈明显剂量关系；(3) Annexin V-FITC/PI双染实验表明杜鹃素在0、15、30和60μmol/L浓度时对H202诱导的EA.hy926细胞凋亡率分别为39.1%、32.35、21.9和15.4%,表明杜鹃素对H202诱导的EA.hy926细胞凋亡具有显著抑制作用(p<0.05);(4) Western blot和RT-PCR检测结果显示杜鹃素在15、30和60μmol/L浓度时具有显著抑制H202诱导EA.hy926细胞内促凋亡蛋白Bax和Bax mRNA表达的升高及抑凋亡蛋白Bcl-2和Bcl-2mRNA表达的降低(p<0.05),且抑制作用呈明显剂量关系;(5) Western blot检测结果表明杜鹃素在15、30和60μmol/L浓度时具有显著抑制H202诱导EA.hy926细胞cleaved caspase-3表达的升高(p<0.05),且抑制作用呈明显剂量关系；(6) Western blot结合激光共聚焦检测结果表明杜鹃素在15、30和60μmol/L浓度时具有显著抑制H202诱导EA.hy926细胞紧密连接蛋白occludin表达的降低(p<0.05),且抑制作用呈明显剂量关系；(7) Western blot检测结果表明杜鹃素对H202诱导的蛋白激酶ERK1/2、p38MAPK、c-Src及蛋白磷酸酶SHP-2磷酸化水平具有明显抑制作用(p<0.05),且抑制作用呈明显剂量关系；(8)大鼠经灌胃给药杜鹃素20、40和80mg/kg,连续14天,FeC13诱导颈主动脉血栓实验结果表明,给药组血清中MDA水平明显低于FeC13模型组,SOD和GSH-Px活性明显高于FeC13模型组(p<0.05),并具有剂量依赖关系;(9)HE染色结果表明高剂量杜鹃素(80mg/kg)具有明显抑制FeCl3诱导的大鼠颈动脉血管内斑块堵塞,表明杜鹃素具有抑制FeC13诱导的血栓形成作用,初步推断可能与其抗氧化应激作用有关。结论：(1)杜鹃素能够抑制H202诱导的EA.hy926细胞凋亡,其作用机制可能是调节细胞内抗氧化酶SOD和GSH-Px活性、调控Bcl-2家族中Bcl-2和Bax基因及蛋白表达以及调节ERK1/2和p38活性；(2)杜鹃素调控ERK1/2和p38活性与其调控上游c-Src及SHP-2信号有关;(3)初步体内实验表明杜鹃素具有抑制FeCl3诱导大鼠颈动脉血栓形成的作用；(4)初步研究表明杜鹃素具有抗氧化应激及预防细胞损伤作用。更多还原

【Abstract】 Objective:Farrerol, a major flavanone present in plants, is the main active substance of "Man-shan-hong"(Rhododendron dauricum L.) for antibechic. Modern pharmacological studies indicate that farrerol also has antibacterial, anti-inflammatory, immune suppression and inhibition of vascular smooth muscle cells. Hesperetin and naringenin are main flavanone present in citrus fruits, which display predominantly antioxidative and cytoprotective activities. However, researches on antioxidative and cytoprotective effects of farrerol are very limited. Therefore this paper was designed to research the antioxidative and cytoprotective effects of farrerol. The first part of this paper demonstrated the antioxidative and cytoprotective effects of farrerol on hydrogen peroxide (H2O2)-induced EA.hy926cell lines. The second part of the paper demonstrated the regulation of farrerol on hydrogen peroxide (H2O2)-induced MAPK activation in EA.hy926cell lines. In the third part, the preliminary antiothrombosis of farrerol in vivo were studied on FeCl3-induce carotid artery thrombosis in rats.Methods:(1) MTT assay was used to detect the inhibition of farrerol on H2O2-induce decrease of EA.hy926cells viability.(2) The intracellular and serum MDA content and SOD, GSH-Px enzymatic activities were detected by UV spectrophotometry to evaluate the regulation of farrerol on H2O2-induced cellular redoxidative level.(3) The regulation of farrerol on H2O2-induced cellular reactive oxygen species (ROS) generation was measured using flow cytometry.(4) PI/Annexin V-FITC double staining assay by flow cytometry was used to detect the effect of farrerol on the cell apoptotic ratio in H2O2-induced EA.hy926cells.(5) The regulation of farrerol on apoptosis-related Bcl-2and Bax genes expressions were obtained by RT-PCR.(6) Western blot method was used to detect the protein expressions of Bcl-2, Bax, cleaved caspase-3, occludin and MAPK activation.(7) Immunofluorescent staining was used to detect the expression of occludin.(8) HE staining was used to detect antithrombosis of farrerol in FeCl3-induce carotid artery thrombosis in rats.Results:(1) Farrerol at the concentration of less60μmol/L had little cytotoxity on EA.hy926cells.(2) Farrerol (15,30, and60μmol/L) inhibited significantly the decreases of cell viability and cellular enzymatic activities of SOD and GSH-Px and the increase of ROS generation in H2O2-induced EA.hy926cells in a dose-dependent manner (p<0.05).(3) PI/Annexin V-FITC double staining assay demonstrated that farrerol at the concentrations of15,30, and60μmol/L attenuated significantly H2O2-induced EA.hy926cells apoptotic ratio from39.1%to32.35%,21.9%, and15.4%, respectively (p<0.05).(4) Western blot and RT-PCR analysis showed that farrerol of15,30, and60μmol/L inhibited significantly H2O2-induced increase of Bax mRNA and protein expression and the decrease of Bcl-2mRNA and protein expression in a dose-dependent manner (p<0.05).(5) Western blot demonstrated that farrerol showed the obvious regulation on the expression of cleaved caspase-3in H2O2-induced EA.hy926cells in a dose-dependent manner (p<0.05).(6) Western blot and Immuno fluorescent staining demonstrated that farrerol showed the obvious regulation on the expression of occludin in H2O2-induced EA.hy926cells in a dose-dependent manner (p<0.05).(7) Western blot demonstrated that farrerol possessed the obvious regulation on the activiation of protein kinases ERK1/2, p38MAPK and c-Src and protein phosphase SHP-2(p<0.05), which were likely associated with the regulation of apoptosis induced by H2O2in EA.hy926cells.(8) In FeCl3-induced rat carotid arterial thrombosis model, compared with the model, farrerol treatment (20,40, and80mg/kg, i.g.) for14days prevented the the increase of MDA content and the decrease of SOD and GSH-Px activity in Spregue Dawey rats serum in a dose-dependent manner (p<0.05).(9) Hematoxylin and eosin (HE) staining indicated that farrerol with high dose (80mg/kg) prevented the thrombosis in FeCl3-induced rat carotid arterial thrombosis model.Conclusions:(1) Farrerol had a inhibitive effect on H2O2-induced apoptosis in EA.hy926cells, which is likely associated with the regulation of intracellular MDA and ROS levels, the activities of SOD and GSH-Px, and modulation of the expression of Bax, Bcl-2, cleavedcaspase-3, as well as the phosphorylation of ERX1/2and p38.(2) The regulation of farrerol on the phosphorylation of ERX1/2was likely associated with the modulation on Src and SHP-2.(3) It was demonstrated preliminarily that farrerol displayed antiathrombosis in FeCl3-induced rat carotid arterial thrombosis model.(4) It was indicated preliminarily that farrerol possessed antioxidative and cytoprotective activities.更多还原