Celastrol通过促进细胞自噬抑制血管平滑肌细胞衰老

发布时间：2018-06-08 00:15

  本文选题：南蛇藤素 + 血管紧张素II　； 参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景血管平滑肌细胞(vascular smooth muscle cells,VSMCs)衰老与心血管疾病的发生发展紧密相关。细胞衰老的特征主要是氧化应激增加,端粒功能障碍和生长停滞,衰老相关蛋白β-半乳糖苷酶(senescence-associatedβ-galactosidase,SAβ-gal)、p53、p21和p16表达增加。血管紧张素II(angiotensin II,Ang II)在动脉粥样硬化的发病机理中起重要作用,可增加活性氧(reactive oxygen species,ROS)的水平并降低细胞的抗氧化能力,最终通过p53/p21依赖机制导致血管衰老,因此,抑制Ang II活性可降低心血管疾病发病率和死亡率。越来越多的证据表明,使用某些药物可以抑制或延缓细胞衰老的发生。已有的研究表明南蛇藤素(celastrol,CeT),这一来源于中药雷公藤的五环三萜类化合物,具有抗氧化和促进细胞自噬的作用。内皮祖细胞经短时间的CeT预处理,即能显著抑制氧化低密度脂蛋白诱导的细胞内ROS水平的升高和细胞衰老。CeT是否影响VSMCs的衰老过程,目前尚未见报道。我们推测,CeT可能通过诱导细胞自噬,而降低Ang II诱导的VSMCs中ROS水平,进而发挥抗衰老作用。研究方法1.采用衰老检测试剂盒对细胞中的SAβ-gal进行染色,Western blot检测细胞内p53和p21的表达水平,流式细胞术检测细胞周期,探讨CeT能否抑制Ang II诱导的VSMCs衰老。2.采用流式细胞技术和ROS检测试剂盒测定VSMCs内ROS水平,探讨Ce T能否抑制Ang II诱导的VSMCs中ROS升高。3.选用免疫荧光技术与激光扫描共聚焦显微镜检测细胞内LC3并计数,Western blot检测LC3 II表达量的变化,探讨CeT是否通过促进细胞自噬,抑制Ang II诱导的VSMCs中ROS升高和衰老的作用。4.Western blot检测PI3K/Akt/m TOR信号通路相关蛋白的表达水平。结果1.Ce T可抑制Ang II诱导的VSMCs衰老:SAβ-gal染色结果可见,VSMCs经100 nmol/L Ang II处理2天后,SAβ-gal染色阳性细胞数目明显增加,而10 nmol/L,50 n M,100 nmol/L CeT预处理使上述衰老比例相对下降,50 nmol/L的作用最为显著;Western blot结果可见,50 nmol/L CeT预处理,可抑制Ang II对p53和p21表达水平的促进作用;流式技术结果可见,采用50 nmol/L CeT预处理后再用Ang II处理,和单纯用Ang II处理相比,G0/G1期的细胞数目相对下降,而S/G2期相对上升。2.50 nmol/L CeT可抑制Ang II诱导的VSMCs中ROS水平的升高。3.CeT可能通过促进细胞自噬,而抑制Ang II诱导的VSMCs中ROS的产生和细胞衰老:免疫荧光技术结果可见,50 nmol/L CeT可显著促进VSMCs的自噬水平;Western blot检测进一步表明,Ce T可促进VSMCs中LC3 II的表达。自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3Ma)可减弱CeT的抗衰老作用,以及Ce T抑制ROS生成的作用。4.Ce T可能通过抑制PI3K/Akt/mTOR信号通路促进VSMCs自噬:Western blot检测表明,Ang II可显著促进PI3K/Akt/m TOR/p70s6k蛋白的表达及其磷酸化水平,而使用CeT预处理VSMCs后,Ang II的上述作用可被显著抑制。结论CeT可通过促进细胞自噬,抑制Ang II诱导的VSMCs内ROS水平的升高,进而抑制Ang II诱导的VSMCs衰老;这可能与CeT抑制PI3K/Akt/m TOR信号转导途径相关。
[Abstract]:Background vascular smooth muscle cells (VSMCs) senescence is closely related to the development of cardiovascular disease. Cell aging is characterized by increased oxidative stress, telomere dysfunction and stagnation of telomere dysfunction and growth, and senescence related protein beta galactosidase (senescence-associated beta -galactosidase, SA beta -gal), p53, p21 and The expression of p16 increases. Angiotensin II (angiotensin II, Ang II) plays an important role in the pathogenesis of atherosclerosis, which can increase the level of reactive oxygen (reactive oxygen species, ROS) and reduce the antioxidant capacity of cells, and eventually lead to vascular senescence through p53/p21 dependence mechanism. Therefore, inhibition of Ang activation can reduce cardiovascular disease. Disease incidence and mortality. More and more evidence suggests that the use of certain drugs can inhibit or delay the occurrence of cell senescence. Studies have shown that celastrol (CeT), which is derived from the five ring three terpenoids of Tripterygium wilfordii, has the effect of antioxidation and promoting cell autophagy. The endothelial progenitor cells (EPCs) have a short time of Ce T preconditioning, which can significantly inhibit the increase of intracellular ROS level induced by oxidized low density lipoprotein and whether cell aging.CeT affects the aging process of VSMCs, has not yet been reported. We speculate that CeT may reduce the ROS level in VSMCs induced by Ang II by inducing autophagy, and then exerts anti-aging effect. Research methods 1. SA beta -gal in cells was stained with the senescence detection kit. The expression level of p53 and p21 in cells was detected by Western blot, cell cycle was detected by flow cytometry, and whether CeT could inhibit VSMCs senescence of Ang II induced VSMCs senescence was determined by flow cytometry and ROS detection kit. The increase of.3. in ROS in VSMCs was determined by immunofluorescence and laser scanning confocal microscopy to detect the intracellular LC3 and to detect the changes in the expression of LC3 II by Western blot. The expression level of 1.Ce T inhibited the VSMCs senescence induced by Ang II: the results of SA beta -gal staining showed that the number of SA beta positive cells increased obviously after 2 days after VSMCs 100 nmol/L Ang II, and 10, 50, and 100 of the pretreatment to reduce the proportion of the senescence. Blot results can be seen that 50 nmol/L CeT preconditioning can inhibit the promotion of Ang II on p53 and p21 expression, and flow technique results can be seen that the number of cells in the period is relatively low compared with Ang II after 50 nmol/L CeT preprocessing. The increase of ROS level in VSMCs may promote autophagy by promoting cell autophagy, which inhibits the production of ROS in VSMCs induced VSMCs and cell senescence. The results of immunofluorescence technique can be seen that 50 nmol/L CeT can significantly promote the autophagy level of VSMCs. 3-methyladenine (3Ma) can weaken the anti-aging effect of CeT and the action of Ce T to inhibit the formation of ROS,.4.Ce T may promote VSMCs autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway. The above effects of Ang II can be significantly inhibited. Conclusion CeT can inhibit the increase of ROS level in VSMCs induced by Ang II by promoting autophagy, which may inhibit Ang II induced VSMCs senescence, which may be related to CeT PI3K/Akt/m signal transduction pathway.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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