PE, a characteristic hypertensive disorder in pregnancy, can lead to maternal placental abruption, pulmonary edema, DIC, HEPLL syndrome, postpartum hemorrhage and other acute complications, and causes fetal distress, stillbirth, neonatal death, etc, and additionally, further gives rise to increased risk of long-term hypertension, which poses a huge threat to pregnant women and the fetus. Although previous studies generally recognized that trophoblast infiltration abnormalities, immune regulation dysfunction, endothelial cell damage, oxidative stress, genetic and nutritional factors can induce PE, its pathogenesis is still obscure at present.
A survey of a family history for PE indicated that apart from ethnic, geographic and socioeconomic factors, genetic factor contributed more than 50% of PE development(23). According to this survey statistics, women who are first-degree biological relatives of the PE suffered were five times more likely to be ill, and those second-degree kinships of the probands twice likely than the health groups as well(23, 24). In the wake of the continual in-depth study of the hereditary basis of PE, some different studies regarding the same susceptibility genes had contradictory results(23, 25, 26). Therefore, these candidate genes for PE are still indistinct and far from understood, needing further investigation.
Many pre-existing researches(27) have demonstrated that oxidative stress is an crucial metabolic pathway and plays more and more important role in PE(28). Experiments executed by Wang YP and Zalba G.(28, 29) indicated that during normal pregnancy, ROS activity and antioxidant activity were always maintained a balance, while during PE, high levels of superoxide (OS) and the main precursor of ROS(30) were present in the placenta, which can cause endothelial dysfunction. NADPH oxidase is the main source of the OS generation and its activity is determined and regulated by genetic variants(31) such as CYBA A640G, C242T, -930A/G), which conditioned the stability of the b558 cytochrome and alter the NADPH oxidase activity. The excessive production of ROS in pregnant women caused by NADPH oxidase in pathological conditions may damage the individual’s vascular endothelium, and ulteriorly, influence the emergence of PE.
NADPH oxidase is distributed in both trophoblast and vascular endothelial cells and is consisted of five subunits: gp91phox, p22phox, p47phox, p67phox, and p40phox(32). As a core group, p22phox subunit, along with the gp91phox subunit according to a ratio of 1:1, constitutes the cytochrome b 558 adhered to the membranes of particular granules, forming a compound, while P47phox, p67phox and p40phox subunits are suspended in cytoplasm and move to cytomembrane to combine with the b558 to generate an active oxidase once the p47phox subunit is phosphorylated stimulated by the cell’s exposure to oxidative stimulation. Since the NOX family are homologues of NADPH oxidase and the catalytic domain include the cytochrome b558, the p22phox subunit is concerned in the activation of NOX1-4 that are also key components in the oxidative stress(33). Encoded by CYBA, the p22phox subunit is located on the long arm of chromosome 16 and contains six exons and five introns, encoding a 195 amino acid fragment(11). The TT genotype at the C242T site of CYBA can make histidine 72 shift to tyrosin of the p22phox subunit and may significantly reduce the NOX2 expression in vascular endothelial cells, TNF-α induced NOX2 activation, and the stability between NOX2 and p22phox, which could further reduce the activity of NADPH oxidase and the production of reactive oxygen, lessen vascular endothelial dysfunction and protect cardiovascular system from related diseases such as hypertension and atherosclerosis(34). In addition, the genome-wide association study (GWAS) of Trifonova EA demonstrated that there was a correlationship between CYBA and PE(12). Thus, our study performed the mRNA and protein expression both in vivo and vitro, and simultaneously, genotypic and allelic frequency distribution analyses of CYBA were manipulated in order to initially explore the possible mechanism consigned to CYBA in PE pathogenesis in Chinese population.
The expressions of CYBA in placental tissues measured by the real-time PCR and Western Blot testified that CYBA displayed a significant increase in mRNA and protein expression levels in PE patients, suggesting that CYBA was very likely to play a role in the related pathways associated with the development of PE. For the sake of further investigating the possible interplay of CYBA, CoCl2 and TNF-α were separately employed to create a hypoxic and inflammatory environment(27, 35) to stimulate the HTR-8/Svneo cell line, a human chorionic trophoblast cell in vitro, to detect whether CYBA mRNA expression is affected by hypoxic environment and cytokines such as inflammatory factors. The results revealed that the expressions of CYBA were indeed remarkably increased after CoCl2 and TNF-α stimulation, implying that the change of the gene in vivo may be contributed to the same factors. Synthesizing the vivo and the vitro experiments, the specific mechanism of CYBA involved in the pathogenesis of PE should be inferred that the CYBA would produce excessive ROS in the placenta once provoked by hypoxia or cytokines such as inflammatory factors, which could break the oxidation and antioxidation balance, and this disequilibrium would cause the endothelial dysfunction, engendering the development of PE. However, this speculation requires in-depth functional studies to prove.
Because of the significant differences in the expressions of CYBA between the PE patients and the control ones, the polymorphism distributions of C242T locus in CYBA were examined among them. However, the statistical results showed that there were no difference in the allelic and genotype frequency of the C242T locus in 1184 PE women and 1421 healthy pregnant women, or even in early-onset, late-onset PE. This consequence indicated that the C242T polymorphism of CYBA may not be associated with PE in Shandong Province, consistent with the results of the Caucasian population(36) and Portugal population(37). In addition, we demonstrated that the genetic variants of rs9932581 and rs1049255 in CYBA might not be associated with PE in Chinese population in our previous study(38). However, the exploration does not exclude the effects including the incomplete coverage, insufficient sample size and diagnose elements such as threshold criteria.
In conclusion, CYBA may play a role in the pathogenesis of oxidative stress in PE, in which it may function via cooperating with TNF-α related inflammatory pathway. Although there was no discrepant distribution of the CYBA C242T polymorphic loci in Chinese population, it is still to perform the genetic studies on more CYBA SNPs for larger-scale and multiple regions to gain further insights into its pathogenesis.