环孢素A缓解人滋养细胞氧化应激损伤的分子机制
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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。
Trophoblast cell exerts a crucial role in implantation and placentation which involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergo a complex process of proliferation, migration and differentiation. A typical feature of placentation in humans is the trophoblasts with high degree invasion to gain access to the maternal circulation in the first trimester pregnancy. The pregnancy physiology depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a disorder of the processes would lead to different types of complications, including pregnancy loss and maternal life-threatening diseases. The inflammation and oxidative stress are associated with the functional disorder of trophoblast cells. The role of oxidative stress in female reproduction is becoming increasingly important, as recent evidence suggests that it plays a part in conditions such as polycystic ovarian syndrome, endometriosis, spontaneous abortion, preeclampsia, hydatidiform mole, embryopathies, preterm labor, and fetal growth restriction (FGR). Reactive oxygen species (ROS) are major species of free radicals, and play an important regulatory role via various signaling transduction pathways in folliculogenesis, oocyte maturation, endometrial cycle, luteolysis, implantation, embryogenesis and pregnancy. Persistent and elevated generation of ROS leads to a disturbance of redox potential that in turn causes oxidative stress. Therefore, keeping proper oxidative balance is needed for successful pregnancy.
Cyclosporin A (CsA), first found in1970s, has made transplantation a breakthrough. Our previous research has found that a low concentration of CsA (10-4～1.0μmol/L) can remarkably promote the proliferation and invasion of human first-trimester trophoblasts while decrease trophoblastic apoptosis induced by serum starvation in vitro. It has been found that CsA promotes trophoblastic invasion through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is different from the classical pathway of CsA. The study in vivo has showed that administration with low dose CsA at the early stage of pregnancy can reduce fetal resorption rate in murine abortion-prone CBA/J×DBA/2matings. These results above suggest that CsA appears to have favorable effect on materno-fetal relationship by regulating behaviors of trophoblast cells.
In the present study, we further investigate the role of FAK signaling pathway in the CsA-promoted migration and invasion of trophoblast cells. In addition, we investigate the molecular mechanism and signaling pathway of CsA protection trophoblast cells against oxidative stress-induced damage with the oxidative stress model established by exposing JEG-3cells to different concentrations of H2O2in vitro. This study will provide more scientific evidence for the expansion of CsA clinical application in the therapeutics for pathological pregnancy.
1. CsA improves migration and invasion of human trophoblast cells via FAK signaling pathway
Objectives:Our previous studies have shown that Cyclosporin A (CsA) promotes the invasiveness of human trophoblast cells via MAPK/ERK signaling pathway. However, the upstream cascades of ERK activation induced by CsA remain poorly defined. Here, we are to investigate whether focal adhesion kinase (FAK) signaling is involved in the CsA-induced trophoblast migration and invasion and ERK activation.
Methods:The migration and invasion of human primary trophoblasts and JEG-3cells were measured by transwell migration and matrigel invasion assays. The CsA-induced activation of FAK, Src or ERK was examined by western blot. The reciprocal regulation between FAK, Src and ERK signaling pathway was investigated with the use of their specific inhibitors Y15, PP2or U0126. The E-cadherin expression and matrix metalloproteinases (MMPs) activity was evaluated by western blot or gelatin zymography, respectively.
Results:CsA increased the phosphorylation of FAK and Src in human primary trophoblasts and JEG-3cells. Both FAK inhibitor Y15and Src inhibitor PP2not only inhibited the activation of FAK and Src, but also suppressed the phosphorylation of ERK in the presence of CsA. They also blocked the CsA-induced migration and invasion of JEG-3cells. U0126inhibited the activation of ERK but had no effect on the activation of FAK or Src induced by CsA. Furthermore, the CsA-promoted expression and activity of MMP2, MMP9were blocked effectively by pretreatment with Y15, PP2or U0126, and these inhibitors also restored the expression of E-cadherin which was down-regulated by CsA.
Conclusions:FAK-Src signaling, the upstream signaling cascade of ERK activation, plays an important role in the CsA-induced trophoblast migration and invasion via down-regulating the expression of E-cadherin and up-regulating the activity of MMP2, MMP9in human trophoblast cells.
2. H2P2-induced oxidative stress damages the biological behaviors of JEG-3cells
Objective:The experimental oxidative stress model of human trophoblast cells in vitro was established to investigate the effect of H2O2-induced oxidative stress on the biological behaviors of JEG-3cells.
Methods:JEG-3cells were treated with H2O2of different concentrations for24h. Cells viability was measured with MTT assay. Cell morphology was observed by inverted microscope and fluorescence microscope with DAPI staining. The ROS production and mitochondrial membrane potential were detected by DHE and JC-1staining, respectively. Cell apoptosis was analyzed by Annexin V/PI staining. Cell invasiveness was evaluated by Matrigel-coated transwell invasion assay.
Results:H2O2decreased the viability and invasiveness, and increased the apoptosis ratio of JEG-3cells in a concentration-dependent manner. H2O2also increased the production of ROS and decreased the mitochondrial membrane potential. JEG-3cells exposed to500μM H2O2showed typical appearance of apoptotic cells, such as cell shrinkage and fragmentation, condensed chromatin. The higher concentration of H2O2induced cell death and cell invasiveness loss.
Conclusion:Treatment with500μM H2O2for24h can induce significant oxidative injury in JEG-3cells, which provides a valuable tool to directly investigate the cellular events of trophoblast oxidative injury and the mechanisms of antioxidants.
3. CsA attenuates the H2O2-induced oxidative injury of JEG-3cells
Objective:To investigate the protective effect and mechanisms of CsA on the H2O2-induced oxidative injury of JEG-3cells.
Methods:JEG-3cells were pretreated with CsA for24h followed by treatment with500μM H2O2for another24h. Cells viability was measured by MTT assay. Cell morphology was observed by inverted microscope and fluorescence microscope with DAPI staining. Cell apoptosis was analyzed by Annexin V/PI staining. Cell invasiveness was evaluated by Matrigel-coated transwell invasion assay. The ROS production and mitochondrial membrane potential were detected by DHE and JC-1staining, respectively. Furthermore, MDA production, SOD and CAT activity were tested with chemical colorimetry assays. The expression of p53, phoph-p53, Bax, Bcl-2, pro caspase-3and cleaved PARP was examined by western blot.
Results:CsA pretreatment dramatically increased JEG-3cell viability, alleviated the morphological injury, reduced the cell apoptosis and improved cell invasion after H2O2treatment. Compared to the H2O2-treated group, CsA pretreatment reduced the production of ROS and MDA, and increased the activity of SOD and CAT. Furthermore, CsA pretreatment restored the mitochondrial membrane potential of JEG-3cells, decreased the expression of p53and phoph-p53, Bax and cleaved PARP, and increased the expression of Bcl-2and pro caspase-3.
Conclusion:CsA displays protective effect of oxidative injured JEG-3cells. CsA attenuates the H2O2-induced oxidative injury by decreasing ROS, MDA production and increasing SOD, CAT activity, and inhibits H2O2-induced apoptosis by suppressing mitochondria related apoptotic signaling pathway and caspase-3activity.
4. CsA attenuates the H2O2-induced oxidative injury of JEG-3cells via regulation of FAK-Src and MAPK pathway
Objective:To explore the signaling pathway by which CsA attenuates the H2O2-induced oxidative injury of JEG-3cells.
Methods:Cells were pretreated with CsA for24h, and then incubated with H2O2for another24h. Y15, PP2, SB203580, SP60015and U0126were used to block the activation of FAK, Src, p38MAPK, JNK and ERK, respectively. Cells viability was measured by MTT assay. Apoptosis was analyzed by Annexin V/PI staining. The ROS production and mitochondrial membrane potential were detected by DHE and JC-1staining, respectively. The invasiveness of JEG-3was investigated with Matrigel-coated transwell invasion assay. The expression and activation of signaling molecules were examined by western blot.
Results:H2O2treatment decreased the activation of FAK and Src, and increased the phosphorylation of ERK, p38MAPK and JNK in JEG-3cells. CsA pretreatment enhanced the activation of FAK, Src and attenuated the phosphorylation of p38, JNK in JEG-3cells after H2O2treatment. However, there was no significant effect on the activation of ERK. Blocking the activation of FAK or Src with Y15or PP2, respectively, reduced the cross activation between FAK and Src, decreased the cell viability, mitochondrial membrane potential and invasiveness, and increased the cell apoptosis ratio and ROS production after the combination treatment of CsA and H2O2. Furthermore, blocking the phosphorylation of p38MAPK or JNK by SB203580or SP60015, respectively, promoted the cell viability reduced by H2O2. However, U0126intensified the inhibitory effect of cell viability by H2O2via inactivating ERK signaling.
Conclusion:CsA attenuates the H2O2-induced oxidative injury of JEG-3cells via promoting the activation of FAK-Src and suppressing the activation of p38MAPK and JNK signaling pathway.
In conclusion, a low concentration of CsA has multiple modulating effect on the biological behaviors of human trophoblast cells:(1) enhances the migration and invasion of trophoblast cells by up-regulating the activity of MMP2and MMP9, and down-regulating the expression of E-cadherin via FAK-Src/ERK signaling pathway;(2) attenuates the H2O2-induced oxidative injury of trophoblast cells by decreasing ROS, MDA production and increasing SOD, CAT activity;(3) inhibits the oxidative stress-induced trophoblast apoptosis via suppressing mitochondria related apoptotic signaling pathway and caspase-3activity;(4) protects trophoblast cells against oxidative damage via promoting the activation of FAK-Src and suppressing the activation of p38MAPK and JNK signaling pathway. Our results suggest that CsA might be used as a unique therapeutics for a series of pregnancy complications, such as miscarriage, preeclampsia, fetal growth restriction, and so on.
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。
Trophoblast cell exerts a crucial role in implantation and placentation which involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergo a complex process of proliferation, migration and differentiation. A typical feature of placentation in humans is the trophoblasts with high degree invasion to gain access to the maternal circulation in the first trimester pregnancy. The pregnancy physiology depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a disorder of the processes would lead to different types of complications, including pregnancy loss and maternal life-threatening diseases. The inflammation and oxidative stress are associated with the functional disorder of trophoblast cells. The role of oxidative stress in female reproduction is becoming increasingly important, as recent evidence suggests that it plays a part in conditions such as polycystic ovarian syndrome, endometriosis, spontaneous abortion, preeclampsia, hydatidiform mole, embryopathies, preterm labor, and fetal growth restriction (FGR). Reactive oxygen species (ROS) are major species of free radicals, and play an important regulatory role via various signaling transduction pathways in folliculogenesis, oocyte maturation, endometrial cycle, luteolysis, implantation, embryogenesis and pregnancy. Persistent and elevated generation of ROS leads to a disturbance of redox potential that in turn causes oxidative stress. Therefore, keeping proper oxidative balance is needed for successful pregnancy.
Cyclosporin A (CsA), first found in1970s, has made transplantation a breakthrough. Our previous research has found that a low concentration of CsA (10-4～1.0μmol/L) can remarkably promote the proliferation and invasion of human first-trimester trophoblasts while decrease trophoblastic apoptosis induced by serum starvation in vitro. It has been found that CsA promotes trophoblastic invasion through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is different from the classical pathway of CsA. The study in vivo has showed that administration with low dose CsA at the early stage of pregnancy can reduce fetal resorption rate in murine abortion-prone CBA/J×DBA/2matings. These results above suggest that CsA appears to have favorable effect on materno-fetal relationship by regulating behaviors of trophoblast cells.
In the present study, we further investigate the role of FAK signaling pathway in the CsA-promoted migration and invasion of trophoblast cells. In addition, we investigate the molecular mechanism and signaling pathway of CsA protection trophoblast cells against oxidative stress-induced damage with the oxidative stress model established by exposing JEG-3cells to different concentrations of H2O2in vitro. This study will provide more scientific evidence for the expansion of CsA clinical application in the therapeutics for pathological pregnancy.
1. CsA improves migration and invasion of human trophoblast cells via FAK signaling pathway
Objectives:Our previous studies have shown that Cyclosporin A (CsA) promotes the invasiveness of human trophoblast cells via MAPK/ERK signaling pathway. However, the upstream cascades of ERK activation induced by CsA remain poorly defined. Here, we are to investigate whether focal adhesion kinase (FAK) signaling is involved in the CsA-induced trophoblast migration and invasion and ERK activation.
Methods:The migration and invasion of human primary trophoblasts and JEG-3cells were measured by transwell migration and matrigel invasion assays. The CsA-induced activation of FAK, Src or ERK was examined by western blot. The reciprocal regulation between FAK, Src and ERK signaling pathway was investigated with the use of their specific inhibitors Y15, PP2or U0126. The E-cadherin expression and matrix metalloproteinases (MMPs) activity was evaluated by western blot or gelatin zymography, respectively.
Results:CsA increased the phosphorylation of FAK and Src in human primary trophoblasts and JEG-3cells. Both FAK inhibitor Y15and Src inhibitor PP2not only inhibited the activation of FAK and Src, but also suppressed the phosphorylation of ERK in the presence of CsA. They also blocked the CsA-induced migration and invasion of JEG-3cells. U0126inhibited the activation of ERK but had no effect on the activation of FAK or Src induced by CsA. Furthermore, the CsA-promoted expression and activity of MMP2, MMP9were blocked effectively by pretreatment with Y15, PP2or U0126, and these inhibitors also restored the expression of E-cadherin which was down-regulated by CsA.
Conclusions:FAK-Src signaling, the upstream signaling cascade of ERK activation, plays an important role in the CsA-induced trophoblast migration and invasion via down-regulating the expression of E-cadherin and up-regulating the activity of MMP2, MMP9in human trophoblast cells.
2. H2P2-induced oxidative stress damages the biological behaviors of JEG-3cells
Objective:The experimental oxidative stress model of human trophoblast cells in vitro was established to investigate the effect of H2O2-induced oxidative stress on the biological behaviors of JEG-3cells.
Methods:JEG-3cells were treated with H2O2of different concentrations for24h. Cells viability was measured with MTT assay. Cell morphology was observed by inverted microscope and fluorescence microscope with DAPI staining. The ROS production and mitochondrial membrane potential were detected by DHE and JC-1staining, respectively. Cell apoptosis was analyzed by Annexin V/PI staining. Cell invasiveness was evaluated by Matrigel-coated transwell invasion assay.
Results:H2O2decreased the viability and invasiveness, and increased the apoptosis ratio of JEG-3cells in a concentration-dependent manner. H2O2also increased the production of ROS and decreased the mitochondrial membrane potential. JEG-3cells exposed to500μM H2O2showed typical appearance of apoptotic cells, such as cell shrinkage and fragmentation, condensed chromatin. The higher concentration of H2O2induced cell death and cell invasiveness loss.
Conclusion:Treatment with500μM H2O2for24h can induce significant oxidative injury in JEG-3cells, which provides a valuable tool to directly investigate the cellular events of trophoblast oxidative injury and the mechanisms of antioxidants.
3. CsA attenuates the H2O2-induced oxidative injury of JEG-3cells
Objective:To investigate the protective effect and mechanisms of CsA on the H2O2-induced oxidative injury of JEG-3cells.
Methods:JEG-3cells were pretreated with CsA for24h followed by treatment with500μM H2O2for another24h. Cells viability was measured by MTT assay. Cell morphology was observed by inverted microscope and fluorescence microscope with DAPI staining. Cell apoptosis was analyzed by Annexin V/PI staining. Cell invasiveness was evaluated by Matrigel-coated transwell invasion assay. The ROS production and mitochondrial membrane potential were detected by DHE and JC-1staining, respectively. Furthermore, MDA production, SOD and CAT activity were tested with chemical colorimetry assays. The expression of p53, phoph-p53, Bax, Bcl-2, pro caspase-3and cleaved PARP was examined by western blot.
Results:CsA pretreatment dramatically increased JEG-3cell viability, alleviated the morphological injury, reduced the cell apoptosis and improved cell invasion after H2O2treatment. Compared to the H2O2-treated group, CsA pretreatment reduced the production of ROS and MDA, and increased the activity of SOD and CAT. Furthermore, CsA pretreatment restored the mitochondrial membrane potential of JEG-3cells, decreased the expression of p53and phoph-p53, Bax and cleaved PARP, and increased the expression of Bcl-2and pro caspase-3.
Conclusion:CsA displays protective effect of oxidative injured JEG-3cells. CsA attenuates the H2O2-induced oxidative injury by decreasing ROS, MDA production and increasing SOD, CAT activity, and inhibits H2O2-induced apoptosis by suppressing mitochondria related apoptotic signaling pathway and caspase-3activity.
4. CsA attenuates the H2O2-induced oxidative injury of JEG-3cells via regulation of FAK-Src and MAPK pathway
Objective:To explore the signaling pathway by which CsA attenuates the H2O2-induced oxidative injury of JEG-3cells.
Methods:Cells were pretreated with CsA for24h, and then incubated with H2O2for another24h. Y15, PP2, SB203580, SP60015and U0126were used to block the activation of FAK, Src, p38MAPK, JNK and ERK, respectively. Cells viability was measured by MTT assay. Apoptosis was analyzed by Annexin V/PI staining. The ROS production and mitochondrial membrane potential were detected by DHE and JC-1staining, respectively. The invasiveness of JEG-3was investigated with Matrigel-coated transwell invasion assay. The expression and activation of signaling molecules were examined by western blot.
Results:H2O2treatment decreased the activation of FAK and Src, and increased the phosphorylation of ERK, p38MAPK and JNK in JEG-3cells. CsA pretreatment enhanced the activation of FAK, Src and attenuated the phosphorylation of p38, JNK in JEG-3cells after H2O2treatment. However, there was no significant effect on the activation of ERK. Blocking the activation of FAK or Src with Y15or PP2, respectively, reduced the cross activation between FAK and Src, decreased the cell viability, mitochondrial membrane potential and invasiveness, and increased the cell apoptosis ratio and ROS production after the combination treatment of CsA and H2O2. Furthermore, blocking the phosphorylation of p38MAPK or JNK by SB203580or SP60015, respectively, promoted the cell viability reduced by H2O2. However, U0126intensified the inhibitory effect of cell viability by H2O2via inactivating ERK signaling.
Conclusion:CsA attenuates the H2O2-induced oxidative injury of JEG-3cells via promoting the activation of FAK-Src and suppressing the activation of p38MAPK and JNK signaling pathway.
In conclusion, a low concentration of CsA has multiple modulating effect on the biological behaviors of human trophoblast cells:(1) enhances the migration and invasion of trophoblast cells by up-regulating the activity of MMP2and MMP9, and down-regulating the expression of E-cadherin via FAK-Src/ERK signaling pathway;(2) attenuates the H2O2-induced oxidative injury of trophoblast cells by decreasing ROS, MDA production and increasing SOD, CAT activity;(3) inhibits the oxidative stress-induced trophoblast apoptosis via suppressing mitochondria related apoptotic signaling pathway and caspase-3activity;(4) protects trophoblast cells against oxidative damage via promoting the activation of FAK-Src and suppressing the activation of p38MAPK and JNK signaling pathway. Our results suggest that CsA might be used as a unique therapeutics for a series of pregnancy complications, such as miscarriage, preeclampsia, fetal growth restriction, and so on.
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