环孢素A缓解人滋养细胞氧化应激损伤的分子机制

发布时间：2018-05-03 18:03

本文选题：环孢素A + 氧化应激　；参考：《复旦大学》2012年博士论文

【摘要】：妊娠初期,滋养细胞具有独特的类似肿瘤细胞的生物学行为,即高增殖、低凋亡和高迁移及侵袭力；同时滋养细胞的生物学行为受到蜕膜微环境的严格调控,这对囊胚植入、胚胎发育和正常妊娠的维持起关键作用。近年来研究发现,氧化应激参与多种病理性妊娠的发病,与流产、先兆子痫、胎儿生长受限(FGR)、早产及死胎等妊娠并发症密切关联。过量的活性氧自由基(ROS)可使磷脂中的不饱和脂肪酸生成过氧化脂质,损伤生物膜；可以抑制蛋白质功能,破坏核酸及染色质,从而伤害机体的各种组织和细胞,包括导致滋养细胞凋亡,降低细胞侵袭力。因此,控制氧化损伤对于成功妊娠和正常妊娠的维持具有重要意义。环孢素A(CsA)是具有划时代意义的免疫抑制剂,其临床应用可显著改善实体器官移植的近期存活率,是器官移植后免疫抑制和抗排斥反应的首选药物。我们课题组首次发现,低浓度CsA可显著提高滋养细胞的增殖与侵袭力,抑制低血清培养诱导的滋养细胞凋亡,并改善小鼠流产模型的妊娠结局,提示CsA可能为滋养细胞功能障碍导致的妊娠疾病的潜在治疗药物。与CsA经典的细胞内信号转导通路不同,我们课题组的研究结果显示,CsA可通过活化MAPK/ERK通路提高滋养细胞的增殖及侵袭力。本研究在前期工作基础上,进一步研究在细胞生物学功能调控中起重要作用的粘着斑激酶(FAK)信号通路是否参与CsA调节滋养细胞的迁移与侵袭；并通过用H202处理人绒毛膜上皮癌细胞系JEG-3细胞,建立滋养细胞氧化损伤的体外模型,在氧化应激条件下进一步解析CsA减轻滋养细胞损伤的分子机制,以及相应的细胞内信号转导通路,为拓展CsA的临床应用提供科学依据。第一部分环孢素A通过FAK信号通路促进滋养细胞迁移与侵袭目的探讨FAK信号通路是否参与介导CsA促进滋养细胞迁移与侵袭,以及FAK信号通路与ERK信号通路间的相互作用。方法用CsA(1gM)处理滋养细胞,或用FAK抑制剂Y15、Src抑制剂PP2、MEK抑制剂U0126分别预处理滋养细胞后再用CsA处理,采用Transwell迁移试验和Matrigel侵袭试验分析滋养细胞的迁移及侵袭力,Western blot分析滋养细胞FAK、Src及ERK的磷酸化水平及E-钙粘蛋白(E-cadherin)和金属基质蛋白酶MMP2、MMP9的表达水平,明胶酶谱实验分析滋养细胞培养上清MMP2、MMP9的活性。结果CsA可显著提高原代滋养细胞及JEG-3细胞FAK及其接头分子Src的磷酸化水平。Y15和PP2均能阻断CsA升调节滋养细胞的迁移及侵袭。Y15和PP2可抑制CsA增高的FAK、Src及ERK的磷酸化水平,U0126可抑制CsA增高的ERK磷酸化水平,但对FAK及Src的磷酸化水平无显著影响。Y15、PP2和U0126均可消除CsA对E-cadherin表达的下调作用和对MMP2、MMP9表达及活性的上调作用。结论CsA通过FAK-Src信号通路促进ERK信号通路活化,下调E-cadherin表达,上调MMP2、MMP9的表达及分泌,进而促进滋养细胞的迁移和侵袭。第二部分氧化应激损伤滋养细胞的生物学行为目的分析H202对JEG-3细胞生物学行为的影响,建立滋养细胞氧化应激损伤模型。方法用不同浓度的H202处理JEG-3细胞24h,采用MTT法分析滋养细胞活力,倒置相差显微镜及荧光显微镜观察细胞形态,DHE荧光探针检测细胞内ROS水平,JC-1荧光探针检测细胞线粒体膜电位,Annexin V/PI双标记检测细胞早期凋亡,Matrigel侵袭试验检测细胞的侵袭力,以建立滋养细胞氧化损伤模型。结果500μM H2O2处理JEG-3细胞24h后,细胞活力显著下降,形态发生明显改变,细胞皱缩,间隙增大,体积减小,细胞核缩小,染色质凝集,呈颗粒团块状分布；细胞ROS产生增加,线粒体膜电位下降,凋亡比例升高,细胞侵袭力显著下降。更高浓度的H202(从7501μM开始)则诱发细胞大量脱落坏死,侵袭力丧失。结论JEG-3细胞经500gM H2O2处理24h呈现明显的氧化应激损伤,用此条件建立氧化应激模型,为研究滋养细胞氧化损伤及抗氧化应激药物的作用机制奠定了基础。第三部分环孢素A缓解氧化应激诱导的滋养细胞损伤目的解析CsA对H2O2诱导滋养细胞生物学行为损伤的保护作用及其分子机制。方法用低浓度CsA(1μM)预处理JEG-3细胞24h,再经H2O2(500μM)刺激诱导氧化损伤,采用MTT比色法分析滋养细胞活力,倒置相差显微镜及荧光显微镜观察细胞形态,Annexin V/PI双标记检测细胞早期凋亡,Matrigel侵袭试验检测细胞的侵袭力,DHE荧光探针检测细胞ROS水平,化学比色法检测细胞丙二醛(MDA)的含量、超氧化物歧化酶(SOD)及过氧化氢酶(CAT)的活性,JC-1荧光探针检测细胞线粒体膜电位,Western blot检测凋亡相关蛋白的表达水平。结果与H202单独处理组比较,JEG-3细胞经低浓度CsA干预后,细胞活力明显升高,形态得以改善；细胞凋亡比例下降,侵袭力增加；细胞内ROS、MDA含量明显下降,SOD.CAT的活性显著升高；线粒体膜电位水平恢复；p53的表达和磷酸化水平下降,Bax表达减少,Bcl-2表达增加,caspase-3前体增多,裂解的PARP大片段减少。结论CsA干预可明显改善氧化应激状态下JEG-3细胞的生物学行为；减轻细胞氧化损伤程度,增强细胞的抗氧化损伤能力；抑制线粒体相关的凋亡信号通路及caspase-3活化,减少滋养细胞凋亡。第四部分环孢素A通过调节FAK-Src和MAPK信号通路缓解滋养细胞氧化损伤目的解析CsA缓解滋养细胞氧化损伤的信号转导通路。方法用低浓度CsA(1μM)处理JEG-3细胞24h,再经H2O2(500μM)刺激诱导氧化损伤,采用Western blot分析CsA对氧化应激的滋养细胞FAK.Src及MAPKs磷酸化水平的影响；在此基础上,用FAK抑制剂Y15、Src抑制剂PP2分别预处理处理JEG-3细胞,MTT法测定细胞活力,DHE荧光探针检测细胞内ROS水平,JC-1荧光探针检测细胞线粒体膜电位,Annexin V/PI双标记检测细胞早期凋亡,Matrigel侵袭实验检测细胞的侵袭力；用MAPKs信号通路抑制剂(SB203580、SP600125、U0126)分别预处理JEG-3细胞,MTT法分析细胞活力。结果H202刺激可引起JEG-3细胞FAK、Src磷酸化水平下降,p38MAPK、JNK和ERK的磷酸化水平升高。CsA预处理JEG-3细胞后再经H202刺激,FAK、Src磷酸化水平明显升高,JNK磷酸化水平明显下降,ERK磷酸化水平无显著性变化。Y15和PP2处理细胞后,再经CsA和H202联合处理,与CsA和H202联合处理组比较,滋养细胞活力下降,ROS产生增加,线粒体膜电位下降,凋亡比例升高,侵袭力下降。p38MAPK抑制剂SB203580或JNK抑制剂SP600125处理细胞后,再经H202处理,滋养细胞活力较H202处理组升高；而MEK抑制剂U0126处理后,细胞活力进一步下降。结论低浓度CsA通过促进FAK-Src信号通路活化,抑制p38MAPK和JNK信号通路活化,发挥对氧化损伤的滋养细胞的保护作用。综上所述,本研究发现低浓度CsA对人滋养细胞的生物学行为具有多重调节作用：(1)通过FAK-Src信号通路促进ERK信号通路活化,下调E-cadherin表达,上调MMP2、MMP9的表达及分泌,促进正常滋养细胞的迁移和侵袭；(2)通过降调节ROS及MDA的生成,升调节SOD和CAT的活性,缓解滋养细胞氧化应激损伤；(3)通过抑制线粒体相关的凋亡通路,降低caspase-3活化水平,抑制氧化应激诱导的滋养细胞凋亡；(4)通过促进FAK-Src信号通路活化,抑制p38MAPK和JNK信号通路活化,发挥对氧化损伤的滋养细胞的保护作用。这些研究结果显示,CsA的药理作用及其细胞内信号转导通路远超过人们迄今为止的认识,有望成为一种新型保胎制剂,为临床防治妊娠失败、子痫前期、胎儿生长受限等病理妊娠提供了一种新的策略。
[Abstract]:In the early stages of pregnancy, the trophoblast has a unique biological behavior similar to the tumor cell, that is, high proliferation, low apoptosis, high migration and invasion. At the same time, the biological behavior of trophoblast is regulated by the decidual microenvironment, which plays a key role in the implantation of blastocyst, embryo development and the maintenance of normal pregnancy. Stress participation in the pathogenesis of multiple pathological pregnancies is closely related to abortion, preeclampsia, fetal growth restriction (FGR), premature and stillbirth complications. Excessive active oxygen free radicals (ROS) can make the unsaturated fatty acids in phospholipid produce lipid peroxidation, damage the membrane of raw materials, inhibit protein function, destroy nucleic acid and chromatin. Therefore, it is important for the maintenance of successful pregnancy and normal pregnancy to control the apoptosis of the trophoblastic cells and reduce the cell invasiveness.
Cyclosporin A (CsA) is an epoch-making immunosuppressant. Its clinical application can significantly improve the survival rate of solid organ transplantation. It is the first choice for immunosuppression and rejection after organ transplantation. We first found that low concentration of CsA can increase the proliferation and invasiveness of trophoblastic cells and inhibit low serum culture for the first time. The induction of trophoblast apoptosis and the improvement of the pregnancy outcome of the mouse abortion model suggest that CsA may be a potential therapeutic drug for gestational disease caused by trophoblastic dysfunction. Unlike the classic CsA intracellular signal transduction pathway, our research group shows that CsA can increase the increase of trophoblastic cells through the activation of the MAPK/ERK pathway. Colonization and invasiveness.
On the basis of earlier work, this study further studies whether the FAK signaling pathway plays an important role in the regulation of cellular biological function, whether CsA regulates the migration and invasion of trophoblastic cells, and establishes an in vitro model of the oxidative damage of trophoblastic cells by using H202 to treat human chorionic epithelial cancer cell line JEG-3 cells. Under the condition of oxidative stress, the molecular mechanism of CsA to reduce the damage of trophoblast and the corresponding intracellular signal transduction pathway are further analyzed to provide a scientific basis for the development of the clinical application of CsA.
Part 1 cyclosporine A promotes the migration and invasion of trophoblast cells through FAK signaling pathway.
Objective to investigate whether FAK signaling pathway mediates CsA promoting the migration and invasion of trophoblasts, and the interaction between FAK signaling pathway and ERK signaling pathway.
Methods the trophoblastic cells were treated with CsA (1gM), or FAK inhibitor Y15, Src inhibitor PP2, and MEK inhibitor U0126 were pretreated with CsA treatment. The migration and invasiveness of trophoblastic cells were analyzed by Transwell migration test and Matrigel invasion test. The expression level of protein (E-cadherin) and matrix metalloproteinase MMP2 and MMP9, and the activity of MMP2 and MMP9 in trophoblast cell culture supernatant were analyzed by gelatinase zymogram.
Results CsA can significantly increase the phosphorylation level of the primary trophoblastic and JEG-3 cell FAK and its joint molecule Src.Y15 and PP2, which can block the CsA liters to regulate the migration of trophoblastic cells and invade the FAK of.Y15 and PP2 to inhibit the increase of CsA. The level of.Y15, PP2 and U0126 could not eliminate the effect of CsA on the expression of E-cadherin and the up regulation of MMP2, MMP9 expression and activity.
Conclusion CsA promotes the activation of ERK signaling pathway through FAK-Src signaling pathway, down regulation of E-cadherin expression, up regulation of the expression and secretion of MMP2, MMP9, and thus promoting the migration and invasion of trophoblast.
The second part is the biological behavior of trophoblast injured by oxidative stress.
Objective to analyze the effect of H202 on the biological behavior of JEG-3 cells and establish a model of oxidative stress injury in trophoblastic cells.
Methods the JEG-3 cell 24h was treated with different concentration of H202. The cell viability was analyzed by MTT method. The cell morphology was observed by inverted phase contrast microscope and fluorescence microscope. The ROS level of the cell was detected by DHE fluorescence probe. The mitochondrial membrane potential was detected by JC-1 fluorescence probe. The early apoptosis of the cell was detected by the Annexin V/PI double labeling, and the Matrigel invasion test was tested. The invasive ability of cells was detected to establish a model of oxidative damage in trophoblastic cells.
Results after 500 M H2O2 treatment of JEG-3 cell 24h, the cell vitality decreased significantly, the morphology changed obviously, the cell crinkle, the gap enlargement, the volume decrease, the nucleus shrinking, the chromatin agglutination, the granular mass distribution, the cell ROS production increased, the mitochondrial membrane potential decreased, the apoptosis ratio increased, the cell invasiveness decreased significantly. A higher concentration of H 202 (starting from 7501 M) induced a large number of cells to shed and necrotic and lose their invasiveness.
Conclusion the 500gM H2O2 treatment of JEG-3 cells showed obvious oxidative stress damage. The oxidative stress model was established by this condition, which laid the foundation for the study of the oxidative damage of trophoblast and the mechanism of antioxidation stress drugs.
The third part cyclosporine A alleviates oxidative stress induced trophoblastic injury.
Objective to analyze the protective effect and molecular mechanism of CsA on H2O2 induced biological behavior damage in trophoblastic cells.
Methods JEG-3 cell 24h was pretreated with low concentration of CsA (1 mu M), and then induced oxidative damage induced by H2O2 (500 M). MTT colorimetric assay was used to analyze the viability of trophoblast. Cell morphology was observed by inverted phase contrast microscope and fluorescence microscope. Annexin V/PI double labeling was used to detect cell early death. Matrigel invasion test was used to detect cell invasiveness and DHE fluorescence detection. The level of cell ROS, the content of MDA, the activity of superoxide dismutase (SOD) and catalase (CAT) were detected by chemical colorimetry. The mitochondrial membrane potential of cell mitochondria was detected by JC-1 fluorescence probe, and the expression level of apoptosis related protein was detected by Western blot.
Results compared with the H202 alone group, the cell viability was significantly higher in the JEG-3 cells after the low concentration of CsA, the cell viability was significantly increased, the cell apoptosis ratio decreased, the invasion force increased, the intracellular ROS, MDA content decreased significantly, the activity of SOD.CAT increased significantly, the mitochondrial membrane potential level was restored, the expression of p53 and the level of phosphorylation decreased, Bax Decreased expression, increased Bcl-2 expression, increased caspase-3 precursors, and reduced PARP fragmentation.
Conclusion CsA intervention can obviously improve the biological behavior of JEG-3 cells under oxidative stress, reduce the degree of oxidative damage and enhance the ability of cell oxidation damage, inhibit mitochondrial related apoptosis signaling pathway and caspase-3 activation, and reduce the apoptosis of trophoblast.
The fourth part of cyclosporine A alleviates trophoblast oxidative damage by regulating FAK-Src and MAPK signaling pathways.
Objective to analyze the signal transduction pathway of CsA in relieving oxidative damage in trophoblastic cells.
Methods the JEG-3 cell 24h was treated with low concentration of CsA (1 mu M), and the oxidative damage induced by H2O2 (500 M) was induced. Western blot was used to analyze the effect of CsA on the level of FAK.Src and MAPKs phosphorylation of the trophoblast cells of oxidative stress. HE fluorescence probe was used to detect intracellular ROS level, JC-1 fluorescence probe was used to detect cell mitochondrial membrane potential, Annexin V/PI was used to detect cell early apoptosis, Matrigel invasion test was used to detect cell invasiveness, and JEG-3 cells were pretreated with MAPKs signal pathway inhibitor (SB203580, SP600125, U0126), and cell viability was analyzed by MTT method.
Results H202 stimulation could cause JEG-3 cells FAK, Src phosphorylation level decreased, p38MAPK, JNK and ERK phosphorylation level increased after.CsA pretreatment JEG-3 cells, FAK, Src phosphorylation level was significantly increased, phosphorylation level decreased obviously, phosphorylation level without significant changes Compared with the combined treatment group of CsA and H202, the vitality of trophoblast, the increase of ROS production, the decrease of the mitochondrial membrane potential, the increase of apoptosis ratio, the decrease of the invasive power of the.P38MAPK inhibitor, SB203580 or the JNK inhibitor SP600125, were followed by the H202 treatment, and the activity of the trophoblastic cells was higher than that of the H202 treatment group, while MEK inhibitor U0126 treated, Cell viability decreased further.
Conclusion low concentration of CsA can promote the activation of FAK-Src signaling pathway, inhibit the activation of p38MAPK and JNK signaling pathways, and play a protective role in the trophoblast cells with oxidative damage.
To sum up, the present study found that low concentration of CsA has multiple regulatory effects on the biological behavior of human trophoblastic cells: (1) promoting activation of ERK signaling pathway through FAK-Src signaling pathway, down regulation of E-cadherin expression, up regulation of MMP2, MMP9 expression and secretion, promoting the migration and invasion of normal trophoblastic cells, and (2) regulating the formation of ROS and MDA by lowering the expression of ROS and MDA, Increase the activity of SOD and CAT to alleviate oxidative stress injury of trophoblast; (3) by inhibiting mitochondrial related apoptosis pathway, reducing caspase-3 activation level and inhibiting the apoptosis of trophoblast induced by oxidative stress; (4) activating the FAK-Src signaling pathway, inhibiting the activation of p38MAPK and JNK signaling pathways and giving full play to the nourishment of oxidative damage The results of these studies show that the pharmacological action of CsA and its intracellular signal transduction pathway are far more than people's understanding so far. It is expected to be a new type of fetal preparation, which provides a new strategy for the clinical prevention and treatment of pregnancy failure, preeclampsia, fetal growth restriction and so on.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321

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