Cyclosporin A Attenuates The Oxidative Injury Of Human Trophoblast Cells

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 pathwayObjectives: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-3cellsObjective: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-3cellsObjective: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 pathwayObjective: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.