A Study On The Levels Of Human Corticotrophin-releasing Hormone, C-Fos/AP-1 And Connexin-43 In Mechanical Of Onset Of Labor

Preterm labor and post-term birth are two of the major causes of neonatal morbidity and mortality. However, the onset of labor in human is a complicate process and is not fully clarified to date. Understanding the complex physiological process and the mechanism of onset of labor are very important to prevent and treat either immature labor or post-term birth.The researches have demonstrated that corticotrophin-releasing hormone (CRH), which is considered as a promoter of labor, plays an important role on the uterine contraction and onset of labor. But the specific mechanism is unclear so far. The increased expression of connexin-43 (Cx43) in uterine muscle is the important material foundation of uterine contraction and onset of labor. Nuclei transcription factor activator protein-1( AP-1) can combine AP-1 sites on many genes, and participate in regulating target gene transcription. There are AP-1 combine sites in the Cx43 gene promoter region, and AP-1 could promote the Cx43 transcription and protein expression. The research has proved that c-Fos, the AP-1 subunit, plays an important regulatory function on Cx43 expression. So, we imagine to specifically block the AP-1 activation in myometrial cells on the gene level, and competitively inhibit Cx43 expression, thus inhibit onset of labor and provide a new idea for prevention and treatment of preterm labor. As a new gene therapy strategy, Decoy nucleotide technique could specifically block the nuclei transcription factor's activation to inhibit the downstream gene's expression. To prove our imagination and provide the theory basis and technique foundation for prevention and treatment of preterm labor by using AP-1 decoy oligodeoxynucleotide (ODN) drugs, this research firstly use AP-1 decoy ODN to competitively inhibit the AP-1 activation in myometrial cells by Cationic liposomes mediated gene transfection technique, and has done the following two parts study.Part I: A clinical study on the correlation among the levels of human corticotrophin-releasing hormone, c-Fos and connexin-43 in onset of laborObjective To investigate the variation of CRH in plasma, the mRNA and protein expression of c-Fos and gap junction gene connexin-43(Cx43) in the myometrium of term pregnancy women, and to study the correlations among CRH, Cx43 and c-Fos at the onset of labor.Methods 30 cases in labor (L group), 30 cases not in labor (NL group) in term pregnancy, and 30 cases of non-pregnant (NP group) women undergoing hysterectomy due to cervical intraepithelial neoplasia were recruited in this clinical study. Radioimmunity was employed to measure the concentration of CRH in plasma; in situ hybridization assay and SP immunohistochemical techniques were respectively employed to detect the expression levels of c-Fos mRNA, Cx43 mRNA and the corresponding proteins.Results 1) The concentration of CRH in L group (371.79±54.13 pg/ml) was significantly higher than that in NL group (157.23±47.71 pg/ml), P<0.05 , which was significantly higher than that in NP group (5.68±3.18 pg/ml), P<0.05. 2) Most of the positive signal of c-Fos mRNA and protein in human myometrium was located in cell nuclear and a little was in cytoplasm; and the positive index of c-Fos mRNA and protein was 5.44±1.66 and 5.36±1.52 in L group, 4.14±0.88 and 3.86±0.90 in NL group, 1.04±0.38 and 1.24±0.35 in NP group, respectively. There was a significant difference among different groups (p<0.05). 3) The positive signal of Cx43 mRNA and protein in human myometrium was located in the cytoplasm; and the expression of Cx43 mRNA and protein was 5.48±1.43 and 5.58±1.28 in L group, 4.08±0.58 and 4.10±0.52 in NL group, 1.54±0.64 and 1.14±0.59 in NP group, respectively. There was a significant difference among different groups (p<0.05). 4) In each experimental group, there was a positive correlation between human plasma CRH concentration and the expression of c-Fos mRNA and protein in myometrium (r₁=0.61, r₂=0.50), P<0.05; between human plasma CRH concentration and the expression of Cx43 mRNA and protein in myometrium (r₃=0.52, r₄=0.68), P<0.05. A positive correlation was also found with respect to the expression of mRNA and protein between c-Fos and Cx43 (r₅=0.51, r₆=0.60), P<0.05. Conclusion Human plasma CRH, the expression of c-Fos and Cx43 in myometrium is closely correlated to the onset of labor. There is a positive correlations among plasma CRH level, myometrial c-Fos expression and Cx43 expression.Part II: Corticotrophin-releasing hormone activates connexin43 via activator protein-1 transcription factor in human myometrial smooth muscle cellsObjective The aim of this study is to investigate the regulation effects of CRH on Cx43 expression in human myometrial cells and its potential signaling pathway of c-Fos/ activator protein-1 (AP-1) activation.Methods Human myometrial cells collected from nonpregnant women were treated with CRH at different concentration. A transient transfection of AP-1 decoy oligodeoxynucleotide (ODN) was employed to block AP-1 binding sites of Cx43. The transcriptional activity of AP-1 was detected by luciferase assay. Cx43 protein expression was visualized by immunoflorescence staining. mRNA and protein expression of c-Fos and Cx43 were demonstrated by real-time RT-PCR quantitation and Western blot, respectively.Results CRH facilitated Cx43 expression and enhanced AP-1 promoter activity in human uterine smooth muscle cells. CRH significantly increased mRNA and protein expression of c-Fos and Cx43 in a dose-dependent manner (p<0.01, respectively). A transient transfection of AP-1 decoy ODN did not affect the regulation of CRH on c-Fos (p>0.05). However, AP-1 decoy ODN almost completely abolished the enhancement of CRH on Cx43 expression (p<0.01).Conclusions CRH enhances Cx43 mRNA and protein expression through an up-regulation of c-Fos expression in human primary myometrial cells. A blocking of c-Fos/AP-1 binding to Cx43 is supposed to neutralize the up-regulation effects of CRH on Cx43, so as to put off the onset of labor.