TPO对阿霉素作用下心肌细胞自噬和凋亡的影响及机制研究
发布时间：2019-03-22 06:43
【摘要】：阿霉素(doxorubicin,DOX)是一种广泛应用的非常有效的抗肿瘤药物,其心肌毒性是肿瘤治疗中主要关注的问题。心肌细胞自噬在维持心肌功能中发挥重要作用,但自噬是把“双刃剑”,关于自噬在DOX诱导心肌损伤中的作用以及一些心脏保护剂到底是通过促进自噬还是抑制自噬发挥心肌保护作用还存在争议。近几年,促血小板生成素(thrombopoietin,TPO)被发现具有心肌保护作用,有望成为新的心脏保护药物,但TPO是否通过调控自噬来保护心肌细胞尚未见报道。最新研究发现与TPO同源的促红细胞生成素(erythropoietin,EPO)能够调控上皮细胞的自噬,我们提出TPO也可能调控心肌细胞自噬。因此,本研究旨在(1)研究DOX和(或)TPO对心肌自噬的影响;(2)研究TPO调控心肌自噬的分子机制;(3)研究TPO作用下心肌自噬与凋亡的关系。我们将心肌细胞系H9C2分为Control、DOX、DOX+BFA、DOX+TPO四个组。以自噬抑制剂巴佛洛霉素A1(bafilomycin A,BFA)作为实验的阳性对照。各组细胞主要作如下检测:(1)CCK-8染色法检测细胞存活率;(2)Western-blot和实时定量PCR检测自噬和凋亡相关分子的表达;(3)自噬小体检测;(4)共聚焦显微镜检测LC3与LAMP1共定位。研究结果显示,DOX处理使H9C2细胞的存活率明显下降(p0.05),而加入TPO组相较于单加DOX组,细胞存活率升高(p0.05)。加入DOX后,H9C2细胞自噬小体数量明显增多,而同时孵育TPO则使自噬小体数量减少(p0.05)。进一步检测自噬相关基因的表达,我们发现与单DOX处理组相比,TPO使Beclin-1和LC3-Ⅱ的表达降低,而使p62表达升高,提示TPO能够抑制DOX诱导的心肌细胞自噬。为了探讨TPO对心肌自噬调控的机制,我们检测了 GATA-4、ATG14L、Rubicon的表达以及LC3和LAMP1共定位。结果发现,TPO可以增加H9C2细胞中GATA-4的表达。DOX使ATG14L、Rubicon的表达水平下降。与对照组相比,DOX可诱导LAMP1的蛋白水平增加,但加入TPO对LAMP1的蛋白水平无明显影响(p0.001)。此外,TPO对LC3和LAMP1的共定位无影响。最后,我们研究TPO作用下心肌细胞自噬与凋亡的关系。与DOX组相比,TPO使H9C2细胞中caspase-3的mRNA和蛋白水平都有所减少。TPO还可上调Bcl-2蛋白水平,对Bcl-2 mRNA水平则没有明显的影响(p0.05)。综上所述,TPO能够减少DOX诱导的心肌细胞自噬小体的数量,抑制自噬相关分子的表达,提示TPO能够抑制DOX诱导的心肌细胞自噬;对TPO抑制自噬的分子机制进行研究,发现TPO能够促进GATA-4的表达,提示TPO有可能通过促进GATA-4的表达抑制心肌细胞自噬;TPO和BFA都能够使凋亡减少,提示DOX处理的心肌细胞中,抑制自噬能够减少凋亡的发生。
[Abstract]:Doxorubicin (doxorubicin,DOX) is a widely used anti-tumor drug, and its myocardial toxicity is a major concern in the treatment of cancer. Autophagy plays an important role in maintaining myocardial function, but autophagy is a "double-edged sword". The role of autophagy in myocardial injury induced by DOX and whether some cardioprotective agents can promote autophagy or inhibit autophagy are still controversial. In recent years, thrombopoietin (thrombopoietin,TPO) has been found to have cardioprotective effects and is expected to become a new cardioprotective drug. However, it has not been reported whether TPO can protect cardiomyocytes by regulating autophagy. Recent studies have found that erythropoietin (erythropoietin,EPO) homologous with TPO can regulate autophagy in epithelial cells. We suggest that TPO may also regulate autophagy in cardiomyocytes. Therefore, the aim of this study was to (1) study the effects of DOX and / or TPO on myocardial autophagy, (2) study the molecular mechanism of TPO regulating myocardial autophagy, and (3) study the relationship between myocardial autophagy and apoptosis induced by TPO. We divided the myocardial cell line H9C2 into four groups of Control,DOX,DOX BFA,DOX TPO. The autophagy inhibitor bafilomycin A 1 (BFA) was used as the positive control. The main results were as follows: (1) CCK-8 staining was used to detect cell viability; (2) Western-blot and real-time quantitative PCR were used to detect the expression of autophagy and apoptosis-related molecules; (3) autophagy bodies were detected; (4) the co-localization of LC3 and LAMP1 was detected by confocal microscope. The results showed that the survival rate of H9C2 cells treated with DOX was significantly decreased (p0.05), while the survival rate of H9C2 cells treated with TPO was significantly higher than that of DOX alone (p0.05). After adding DOX, the number of autophagy bodies in H9C2 cells increased significantly, while the number of autophagy bodies decreased when TPO was incubated at the same time (p0.05). By further detecting the expression of autophagy-related genes, we found that TPO decreased the expression of Beclin-1 and LC3- 鈪，
本文编号：2445358
[Abstract]:Doxorubicin (doxorubicin,DOX) is a widely used anti-tumor drug, and its myocardial toxicity is a major concern in the treatment of cancer. Autophagy plays an important role in maintaining myocardial function, but autophagy is a "double-edged sword". The role of autophagy in myocardial injury induced by DOX and whether some cardioprotective agents can promote autophagy or inhibit autophagy are still controversial. In recent years, thrombopoietin (thrombopoietin,TPO) has been found to have cardioprotective effects and is expected to become a new cardioprotective drug. However, it has not been reported whether TPO can protect cardiomyocytes by regulating autophagy. Recent studies have found that erythropoietin (erythropoietin,EPO) homologous with TPO can regulate autophagy in epithelial cells. We suggest that TPO may also regulate autophagy in cardiomyocytes. Therefore, the aim of this study was to (1) study the effects of DOX and / or TPO on myocardial autophagy, (2) study the molecular mechanism of TPO regulating myocardial autophagy, and (3) study the relationship between myocardial autophagy and apoptosis induced by TPO. We divided the myocardial cell line H9C2 into four groups of Control,DOX,DOX BFA,DOX TPO. The autophagy inhibitor bafilomycin A 1 (BFA) was used as the positive control. The main results were as follows: (1) CCK-8 staining was used to detect cell viability; (2) Western-blot and real-time quantitative PCR were used to detect the expression of autophagy and apoptosis-related molecules; (3) autophagy bodies were detected; (4) the co-localization of LC3 and LAMP1 was detected by confocal microscope. The results showed that the survival rate of H9C2 cells treated with DOX was significantly decreased (p0.05), while the survival rate of H9C2 cells treated with TPO was significantly higher than that of DOX alone (p0.05). After adding DOX, the number of autophagy bodies in H9C2 cells increased significantly, while the number of autophagy bodies decreased when TPO was incubated at the same time (p0.05). By further detecting the expression of autophagy-related genes, we found that TPO decreased the expression of Beclin-1 and LC3- 鈪，
本文编号：2445358
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