Development of anti-cytokine drugs for treatment of endometriosis

子宫内膜异位症患者的腹膜液（PF）中含有大量分泌多种细胞因子的活化巨噬细胞。我们之前的研究表明，子宫内膜异位症患者的PF中肿瘤坏死因子α (TNFa)和白细胞介素-8 （IL-8）水平显著升高。tnf -8与PF水平有显著相关性。本研究的目的是探讨TNFa在子宫内膜异位症间质细胞增殖中的作用。我们还检测了TNFa诱导IL-8过程中核因子- kb （NF-kB）的激活。39例子宫内膜异位症患者卵巢内的巧克力囊肿是子宫内膜异位症组织的来源。RT-PCR检测IL-8和TNFa受体。采用MTT法研究子宫内膜异位症细胞的增殖。采用Northern blotting和ELISA分别检测IL-8基因和蛋白的表达。采用Western blot和EMSA检测TNFa对NF-kB的激活作用。研究了NF-kB抑制剂TPCK对TNFa作用的影响。在子宫内膜异位症间质细胞中检测到IL-8受体A型、TNF受体I型和II型的转录本。TNFa（10和100 pg/mL）诱导子宫内膜异位症基质细胞IL-8基因和蛋白表达呈剂量依赖性。在培养基中加入IL-8 （25-100 pg/mL）或TNFa （5-100 pg/mL）可促进基质细胞的增殖。通过添加抗il -8抗体，TNFa的刺激作用被消除。这些结果表明，IL-8的作用介导了TNFa对基质细胞增殖的刺激作用。NF-kB的活化通常与IkB的磷酸化有关，随后是蛋白酶体降解和NF-kB核易位。为了检测tnf fa诱导IL-8表达时基质细胞中NF-kB的活化情况，使用磷酸化IkB抗体（p-IkB）进行Western blotting。添加TNFa （100 pg/mL）快速诱导间质细胞p-IkB。EMSA显示，添加TNFa促进活化NF-kB的易位。TPCK预孵育可降低TNFa诱导的IL-8基因和蛋白的表达。IL-8介导的TNFa作用可能通过促进子宫内膜异位症细胞的生长而促进子宫内膜异位症的进展。我们在这里证明了NF-kB激活参与了TNFa诱导子宫内膜异位症组织中的IL-8。因此，TNFa及其信号转导分子NF-kB在子宫内膜异位症的病理生理中起着重要作用。少

Peritoneal fluid (PF) in women with endometriosis contains an increased number of activated macrophages that secrete a variety of cytokines. We have previously shown that the PF levels of tumor necrosis factor alpha (TNFa) and interleukin-8 (IL-8) were significantly higher in patients with endometriosis. There was a significant correlation between PF levels of TNFa and IL-8. The purpose of the study is to investigate the role of TNFa in proliferation of endometriotic stromal cells. We also examined the activation of nulear factor-kB (NF-kB) during the induction of IL-8 by TNFa.Chocolate cyst linings of the ovaries in patients with endometriosis (n=39) were a source of endometriotic tissue. The receptors for IL-8 and TNFa were examined by RT-PCR. Proliferation of endometriotic cells was studied using MTT assay. The expression of IL-8 gene and protein was analyzed by Northern blotting and ELISA, respectively. Western blot analysis and electrophoretic mobility shift assay (EMSA) were used … More to detect NF-kB activation by TNFa. Effects of inhibitor for NF-kB, TPCK, on TNFa action were also examined.Transcripts of IL-8 receptor type A, TNF receptor type I and II were detected in endometriotic stromal cells. TNFa (10 and 100 pg/mL) induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. Addition of IL-8 (25-100 pg/mL) or TNFa (5-100 pg/mL) to the culture medium stimulated the proliferation of stromal cells. The stimulatory effects of TNFa were abolished by adding anti-IL-8 antibody. These results suggest that the action of IL-8 mediates the stimulatory effects of TNFa on stromal cell proliferation. The activation of NF-kB is usually associated with phosphorylation of IkB, followed by its degradation by the proteasome and NF-kB nuclear translocation. In order to detect the activation of NF-kB in stromal cells during inducible expression of IL-8 by TNFa, Western blotting was performed using an antibody for phosphorylated IkB (p-IkB). Addition of TNFa (100 pg/mL) rapidly induced p-IkB in stromal cells. EMSA revealed that adding TNFa promoted translocation of activated NF-kB.Preincubation with TPCK reduced TNFa inducible expression of IL-8 gene and protein. TNFa action mediated by IL-8 may contribute to progression of endometriosis by promoting the growth of endometriotic cells. We demonstrated here that NF-kB activation was involved in the induction of IL-8 by TNFa in endometriotic tissues. Thus, TNFa and its signal transduction molecule, NF-kB, play important roles in the pathophysiology of endometriosis. Less