抗氧化应激转录因子Nrf2在血管钙化发生中的作用及机制研究

发布时间：2018-11-20 21:51

【摘要】：第一部分抗氧化应激转录因子Nrf2在VDN诱导的大鼠血管钙化模型中的作用目的氧化应激是多种疾病合并的血管钙化发生的共同病理因素,但是抗氧化应激机制对血管钙化的研究尚待深入。本研究拟初步探讨机体内源性抗氧化应激转录因子Nrf2及其调控的下游抗氧化应激分子在维生素D3 (Vitamin D3)和尼古丁(Nicotine)诱导的大鼠血管钙化模型(VDN)中的变化,通过在体研究证实其在血管钙化发生中的作用。方法选取20只两月龄的Wistar雄性大鼠,随机分为对照组和VDN钙化组,钙化组大鼠早上9点钟分别给予尼古丁(25mg/kg)灌胃和维生素D3(30万单位/kg)肌肉注射,晚上6点再给予尼古丁25mg/kg灌胃一次,对照组给予生理盐水注射。两个月后取大鼠血管组织：硝酸银染色测定血管钙化的发生程度;试剂盒测定组织钙含量和ALP活性;Westernblot检测成骨标记蛋白Runx2、OPN、βcatenin的表达,同时检测Nrf2及其下游抗氧化酶NQO1的表达。结果与对照组相比,VDN钙化组大鼠血管中层出现明显钙化,血管组织钙含量和ALP活性均显著增加(P0.05),成骨标记蛋白Runx2、OPN、β catenin的表达显著增加：同时,与对照组相比,VDN钙化组大鼠血管组织Nrf2及其诱导的抗氧化酶NQO1的表达也均增加。结论在VDN诱导的大鼠血管钙化中,Nrf2及其下游抗氧化酶NQO1被激活,参与了血管钙化的发生。第二部分抗氧化应激转录因子Nrf2对高磷诱导的血管平滑肌细胞钙化的影响及其分子机制研究目的氧化应激、凋亡和成骨样表型转变是血管平滑肌细胞(vascular smooth muscle cells, VSMCs)参与和调控血管钙化的主要机制,作为机体最重要的内源性抗氧化应激转录因子,Nrf2在氧化应激防御中发挥关键作用,具有显著的抗氧化应激和抗凋亡作用,同时最新的研究也发现,Nrf2是调控骨代谢和稳态的新型作用因子。本实验旨在深入探讨Nrf2对高磷诱导的血管平滑肌细胞钙化的作用及其分子机制。方法酶解法分离Wistar大鼠的原代血管平滑肌细胞,分为对照组,钙化组,Nrf2沉默组和Nrf2激活组。对照组用正常培养基培养,钙化组的细胞培养基中给予10mMβ磷酸甘油(β glycerophosphate, PGP)培养,Nrf2激活组细胞同时选用特异性的Nrf2激活剂DMF和tBHQ干预,Nrf2沉默组细胞应用RNA干扰技术使Nrf2表达沉默。应用茜素红染色检测各组细胞钙化程度；应用试剂盒检测各组细胞ALP活性和钙含量;应用Westernblot和RT-PCR技术检测各组细胞Nrf2及其下游NQO1和HO1的表达,以及各组细胞成骨标记BMP2、Runx2、OPN、β catenin的表达,应用流式细胞术和Westernblot分别检测细胞凋亡比例及cleaved caspase3的表达；应用流式细胞仪及试剂盒分别检测细胞ROS活性和MDA含量。结果①与对照组相比,钙化组细胞培养10天后茜素红染色出现明显的钙化,细胞内钙含量和ALP活性均显著增加,成骨标记蛋白BMP2、Runx2、OPN、β catenin的表达均显著增高,细胞ROS活性及MDA含量显著上升,cleaved caspase3表达及细胞凋亡率也显著增加。②与对照组相比,Nrf2沉默组Nrf2的mRNA水平降低约70%,Nrf2下游抗氧化酶NQO1和HO1表达也显著下降,同时成骨标记蛋白BMP2、Runx2、OPN、β catenin的表达均显著增高,细胞钙含量显著上升。③与钙化组相比,Nrf2激活剂DMF和tBHQ干预之后,Nrf2下游抗氧化酶NQO1被显著激活,同时成骨标记蛋白BMP2、Runx2、OPN、βcatenin的表达均显著下降,细胞ROS活性及MDA含量显著下降,cleaved caspase3表达及细胞凋亡率也显著下降。结论Nrf2通过抑制高磷诱导的血管平滑肌细胞的成骨样表型转变、减轻血管平滑肌细胞的氧化应激和凋亡,进而抑制高磷诱导的血管平滑肌细胞钙化的发生。
[Abstract]:The oxidative stress of the first part of the anti-oxidative stress transcription factor Nrf2 in the vascular calcification model of the rat induced by VDNN is a common pathological factor of the blood vessel calcification combined with various diseases, but the research of the anti-oxidative stress mechanism to the vascular calcification is still to be studied. This study is to explore the changes of endogenous anti-oxidative stress transcription factor Nrf2 and its regulated downstream anti-oxidative stress molecule in the vascular calcification model (VDN) induced by vitamin D3 (Vitamine D3) and nicotine (Nicotine), The role of this in the occurrence of vascular calcification was demonstrated by the in vivo study. Methods Twenty two-month-old Wistar male rats were randomly divided into the control group and the VDN calcified group. The rats were given nicotine (25mg/ kg) and vitamin D3 (30 000 units/ kg) intramuscularly at 9 o 'clock in the calcified group, and the nicotine was given at 6: 00 at night for one time. The control group was given saline injection. After two months, rat blood vessel tissue: silver nitrate staining was used to determine the degree of blood vessel calcification; the content of calcium and ALP activity of the tissue were determined by the kit; Western blot was used to detect the expression of bone marker protein Runx2, OPN, and Catenin, and the expression of Nrf2 and its downstream anti-oxidation enzyme NQO1 was also detected. Results Compared with the control group, there was a significant increase in the levels of calcium and ALP in the vascular middle layer of the VDN-calcified group (P0.05), and the expression of the bone marker protein Runx2, OPN, and Catenin increased significantly (P0.05). The expression of Nrf2 and its induced antioxidant NQO1 in the vascular tissue of the VDN-calcified group was also increased. Conclusion Nrf2 and its downstream anti-oxidation enzyme NQO1 were activated in the vascular calcification induced by VDNN, and were involved in the occurrence of vascular calcification. The effect of the second part of the anti-oxidative stress transcription factor Nrf2 on the calcification of high-phosphorus-induced vascular smooth muscle cells and its molecular mechanism are the main mechanism of vascular smooth muscle cells (VSMCs) to participate in and control the vascular calcification. As the most important endogenous anti-oxidative stress transcription factor, Nrf2 plays a key role in the prevention of oxidative stress, and has significant anti-oxidative stress and anti-apoptotic effects. The purpose of this study was to investigate the role of Nrf2 on the calcification of vascular smooth muscle cells induced by high phosphorus and its molecular mechanism. Methods The primary vascular smooth muscle cells of Wistar rats were divided into control group, calcification group, Nrf2 silencing group and Nrf2 activation group. The control group was cultured with normal medium, and 10 mM triphosphoglycerate (PGP) was given to the cell culture medium of the calcified group, and the specific Nrf2 activator DMF and tBHQ were selected for the Nrf2 activated group cells, and the Nrf2 expression was silenced by the RNA interference technique in the Nrf2 silencing group cells. The expression of Nrf2 and its downstream NQO1 and HO1 in each group was detected by Western blot and RT-PCR, and the expression of BMP2, Runx2, OPN and Catenin in each group was detected by Western blot and RT-PCR. The cell apoptosis and the expression of clear caspase3 were detected by flow cytometry and Western blot. The activity of ROS and the content of MDA were detected by flow cytometry and kit. Results Compared with the control group, the content of the intracellular calcium and the activity of ALP increased significantly after 10 days after the cell culture of the calcified group, and the expression of the bone marker protein BMP2, Runx2, OPN and Catenin increased significantly, and the activity of ROS and the content of MDA increased significantly. The expression of clear caspase3 and the cell apoptosis rate were also increased. Compared with the control group, the mRNA level of Nrf2 in the Nrf2 group was reduced by about 70%, and the expression of NQO1 and HO1 downstream of Nrf2 was also significantly decreased, while the expression of the bone marker protein BMP2, Runx2, OPN, and Catenin increased significantly, and the content of calcium in the cells increased significantly. Compared with the calcified group, the Nrf2 downstream anti-oxidation enzyme NQO1 was significantly activated after the intervention of the Nrf2 activator DMF and tBHQ, while the expression of the bone marker protein BMP2, Runx2, OPN, and Catenin decreased significantly, and the ROS activity and MDA content of the cells decreased significantly, and the clear caspase3 expression and the cell apoptosis rate were also significantly reduced. Conclusion Nrf2 can reduce the oxidative stress and apoptosis of vascular smooth muscle cells by inhibiting the transformation of bone-like phenotype of high-phosphorus-induced vascular smooth muscle cells, and further inhibit the occurrence of high-phosphorus-induced vascular smooth muscle cell calcification.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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