The Effect Of Expression And Regulation Of Transcription Factors Nf-κB And AP-1in Adenomyosis

Adenomyosis is a common gynecologic disease and the etiology is poorly understood. The prevalence of adenomyosis reported in the literature ranges from5to70%. The common presenting symptoms are dysmenorrheal, enlarged tender uterus, subfertility, and abnormal uterine bleeding. About35%of women with adenomyosis are asymptomatic. Clinical treatment of it is a challenge, with hysterectomy being the treatment of choice. To date, adenomyosis already became one of the most dangerous threats to women health. Nuclear factor-kappa B (NF-kB) and active protein-1(AP-1) are involved in numerous pathologies and known to be a proinflammatory, antiapoptotic and mitogenic factor in many cell types. They are thought to play a pivotal role in pathogenesis of endometriosis. Recent researches indicated their target genes cyclooxygenase-2(COX-2), vascular endothelial growth factor (VEGF) and tissue factor (TF) might be involved in the pathogenesis of endometriosis through stimulating angiogenesis and inflammatory reaction. Published studies on NF-κB and AP-1and their target genes almost focused on the endometriosis, however, correlative studies on the adenomyosis has not been reported yet. To date, we still did not know the status of NF-κB and AP-1in adenomyotic lesions and whether over-expressed molecules were regulated by transcription factors NF-κB and AP-1in adenomyotic stromal cells. According to the researches in the endometriosis, we inferred that during the pathology of adenomyosis, transcription factors NF-κB and AP-1were firstly activated, and then gave rise to over-expresstion of inflammatory mediators and cytokines, and finally these abnormal molecules promoted the development of adenomyosis. Transcription factors NF-κB and AP-1might play a pivotal role in the development of the disease. In addition, it was found out that andrographolide (Andro) could specifically down-regulate NF-kB DNA binding activity and suppress the expression of target genes. Trichostatin A (TSA) could suppress the proliferation and expression of COX-2in the endometrial stromal cells and the growth of implanted lesions in the model mice with endometriosis. According to published results in the endometriosis, we inferred that Andro and TSA might play a role in the adenomyosis through affecting the expression of NF-kB and AP-1.In this study, we would detect the expression of transcription factors NF-kB and AP-1and their target genes COX-2, VEGF and TF in adenomyosis and the effect of TNFα on the transcription factors NF-κB and AP-1in the adenomyotic stromal cells in vitro, and the effect of Andro and TSA on transcription factors NF-κB and AP-1and their downstream target genes COX-2, VEGF and TF.Part I Expression of transcription factors NF-κB and AP-1and downstream molecules in adenomyosisObjective:To detect the expression of transcription factors NF-κB and AP-1and COX-2, VEGF and TF in adenomyotic lesions.Methods:Twenty-three adenomyotic lesion samples and8endometrial tissue samples from women with benign gynecological conditions, but not adenomyosis, were used to extract the nuclear and cytoplasm proteins, and their NF-κB and AP-1DNA binding activities were analyzed by electrophoretic mobility shift analysis (EMSA). Western blot analysis was used to evaluate the expression of NF-κB subunits p50and p65, AP-1subunits c-Jun and c-Fos, COX-2, VEGF and TF.Results:EMSA results showed that NF-κB and AP-1were constitutively activated in adenomyosis, and their activation levels were significantly higher than those of control group (P<0.05). In particular, NF-kB was universally activated constitutively in adenomyosis (87%), and while AP-1was constitutively activated in sizeable portion of adenomyotic lesions (40%). Besides, NF-kB activation level was positively correlated with the severity of dysmenorrheal in patients with adenomyosis (P<0.05). Western Blot results showed that the expressions of p50, p65, c-Jun, c-Fos, COX-2, VEGF and TF were all significantly higher than control endometrium (P<0.05).Conclusions:Transcriptional factors NF-κB and AP-1and COX-2, VEGF and TF may play a critical role in the pathogenesis of adenomyosis.Part â…¡ The effect of TNFa on transcriptional factors NF-â'˜B and AP-1in adenomyotic stromal cellsObjective:To investigate the effect of TNFa on the transcriptional factors NF-κB and AP-1and their downstream target genes COX-2, VEGF and TF in adenomyotic stromal cells and explore whether the over-expressed molecules are regulated by transcription factors NF-κB and AP-1in adenomyosis.Methods:Cultured adenomyotic stromal cells were treated with TNFa and inhibitors of NF-κB and AP-1, and EMSA and Western blot analysis were used to detect the expressions of NF-κB, AP-1and their target genes, COX-2, VEGF and TF.Results:In adenomyotic stromal cells, TNFa significantly induced further activation of NF-κB and AP-1, and increased the protein expression of COX-2, VEGF and TF (P<0.05). When inhibitors of NF-κB and AP-1were added, the expression levels of the three proteins were reduced, and were further reduced by the combined treatment of NF-κB and AP-1inhibitors (P<0.05).Conclusions:In adenomyotic stromal cells, transcriptional factors NF-κB and AP-1are involved in the up-regulation of TNFa-induced COX-2, VEGF and TF. Transcriptional factors NF-κB and AP-1may jointly play a pivotal role in the pathogenesis of adenomyosis Part III Andro and TSA reduce the TNFa-induced expression of NF-κB and AP-1and downstream molecules in adenomyotic stromal cellsObjective:To investigate the effect of Andro and TSA on transcriptional factors NF-κB and AP-1and their downstream target genes COX-2, VEGF and TF in adenomyotic stromal cells. The research may provide some new theoretical guidance for the further application of Andro and TSA.Methods:After adenomyotic stromal cells were treated with Andro and TSA respectively, the nucleoproteins and total proteins were prepared. EMSA and Western Blot analysis were used to detect the expression of TNFa-induced NF-κB and AP-1and their target genes COX-2, VEGF and TF, the three genes known to be involved in adenomyosis.Results:In adenomyotic stromal cells, Andro could suppress the NF-κB DNA binding activity, and significantly reduce the expression of COX-2, VEGF and TF (P<0.05). TSA could consistently reduce the protein products of TNFa-induced COX-2and TF (P<0.05), but long-playing treatment could significantly elevate the TF expression (P<0.05). Effect of TSA on NF-κB DNA binding activity was time-dependent: short-time treatment of TSA up-regulated NF-κB activity, but long-playing treatment down-regulate its activity. TSA could consistently reduce the phosphorylation of P65and c-Jun in adenomyotic stromal cells (P<0.05), and the expression of P50appeared observably down-regulation after24h treatment (P<0.05). Besides, TSA could reduce the phosphorylation of JNK (P<0.05), but the phosphorylation of P38had no change, and the phosphorylation of ERK showed that short-time treatment evidently down-regulated and long-playing treatment significantly up-regulated (P<0.05). When the combination of TSA and Andro was used to treat adenomyotic stromal cells, the protein products of COX-2, VEGF and TF were significantly suppressed (P<0.05), and the effect was superior to TSA treatmentκ(P<0.05). Conclusions:In adenomyotic stromal cells, Andro could be used as the NF-κB inhibitor. TSA could consistently down-regulate the expression of COX-2and VEGF through reducing the transcriptional activities of NF-κB and AP-1.Combined treatment of TSA and Andro was superior to that of TSA.