Constitutive and tumor necrosis factor-α-induced activation of nuclear factor-κB in adenomyosis and its inhibition by andrographolide  Bin Li, M.S., Ming Chen, Ph.D., Xishi Liu, M.D., Ph.D., Sun-Wei Guo, Ph.D.  Fertility and Sterility  Volume 100, Issue 2, Pages 568-577 (August 2013) DOI: 10.1016/j.fertnstert.2013.04.028 Copyright © 2013 Terms and Conditions

Figure 1 Expression of nuclear factor (NF)-κB proteins in adenomyosis (ADM). (A) Western blotting analysis of NF-κB subunits p65 and p50 in nuclear extracts from adenomyotic lesions (n = 23) and control endometrium (CT = control endometrium; n = 8). (B) Summary data of p65 (P=.000003) and p50 (P=.0008) protein expression. (C) Representative NF-κB electrophoretic mobility shift assay of nuclear extracts from adenomyotic lesions and control endometrium. The specificity of the electrophoretic mobility shift assay was confirmed by adding 200-fold excesses of unlabeled NF-κB oligonucleotides to the binding reaction (CP = cold probe). (D) Comparison with NF-κB bands in adenomyotic lesions and control endometrium (P=.01). (E) Relationship between NF-κB binding activity levels and the severity of dysmenorrhea in patients with adenomyosis (n = 23). P=.0148 by Jonckheere trend test. Fertility and Sterility 2013 100, 568-577DOI: (10.1016/j.fertnstert.2013.04.028) Copyright © 2013 Terms and Conditions

Figure 2 Protein and messenger RNA (mRNA) levels of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and tissue factor (TF) in adenomyosis (ADM) and control endometrium (CT). (A) Expression of COX-2, VEGF, and TF proteins in cytoplasm proteins from adenomyotic lesions (n = 23) and control endometrium (n = 8). (B) Summary data of VEGF (P=.0017), COX-2 (P=.0006), and TF (P=.0013) protein levels. (C) The mRNA levels of three genes in adenomyotic lesion (n = 10) and control endometrium (n = 6). (D) Summary data of COX-2 (P=.0001), TF (P=.0013), and VEGF (P=.0179) mRNA levels. Fertility and Sterility 2013 100, 568-577DOI: (10.1016/j.fertnstert.2013.04.028) Copyright © 2013 Terms and Conditions

Figure 3 Effect of tumor necrosis factor (TNF)-α stimulation on nuclear factor (NF)-κB activation in adenomyotic stromal cells. (A) NF-κB DNA-binding activity after stimulation with TNF-α. Nuclear extracts were prepared adenomyotic stromal cells, and the electrophoretic mobility shift assay was performed with a biotin end-labeled NF-κB probe. For competition experiments, 200-fold excesses of unlabeled NF-κB oligonucleotides were added. (B) Nuclear or total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed with primary specific antibodies specific against p65, p50, and phosph-p65 (p-p65), respectively. (C) Summary data for NF-κB DNA-binding activity after stimulation with TNF-α (n = 6), with the DNA-binding activity in vehicle-treated cells taken as 1 in the Y-axis (P=.003). (D) Summary data for p50 (P=.005), p65 (P=.003), and phosph-p65 (P=.001) protein levels with and without stimulation with TNF-α. Each experiment was done on adenomyotic stromal cells derived from three randomly selected patients. Fertility and Sterility 2013 100, 568-577DOI: (10.1016/j.fertnstert.2013.04.028) Copyright © 2013 Terms and Conditions

Figure 4 Tumor necrosis factor (TNF)-α-induced cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and tissue factor (TF) protein and messenger RNA (mRNA) expression in adenomyotic stromal cells, which were attenuated by pretreatment with andrographolide, a nuclear factor (NF)-κB inhibitor. (A) Effect of 4-hour treatment with or without TNF-α, and NF-κB inhibitor, and then the Western blotting analysis was carried out to detect COX-2, VEGF, and TF expression. (B) Summary data of COX-2, VEGF, and TF protein levels. Each experiment was done on adenomyotic stromal cells derived from three randomly selected patients. (C) Effect of 3-hour treatment of adenomyotic stromal cells with or without TNF-α, and NF-κB inhibitor, and then the real-time reverse transcriptase-polymerase chain reaction (PCR) was used to measure mRNA levels. The relative levels of COX-2, TF, and VEGF were normalized to the β-actin mRNA levels. Each experiment was done on adenomyotic stromal cells derived from 10 randomly selected patients. ∗P<.05, comparison with the vehicle control; and #P<.05, comparison with cells stimulated with TNF-α Fertility and Sterility 2013 100, 568-577DOI: (10.1016/j.fertnstert.2013.04.028) Copyright © 2013 Terms and Conditions