The Study Of The Inhibition Of Lipoxins On The Endometriosis

Objective:1. To investigate the dynamic morphological changing pattern of the ectopic focus, changes of the phagocytosis ability of abdominal cavity macrophage, the expressions and activities of proinflammatory cytokines and matrix metalloproteinases in the mouse endometriosis model.2. To study the effects of IL-1βand 15-epi-LXA4 on the expression inflammatory mediators in human endometriotic glandular epithelial cells and stromal cells.3. To examine the effects of 15-epi-lipoxin A4 (LXA4) on the growth of endometriotic lesions, on the expressions of proinflammatory cytokines and matrix metalloproteinases in endometriosis BALB/c mice.Methods:1. Endometriosis was induced in BALB/c mice. On the 1st,2nd,3rd,4th,5th,6th,9th, 12th,15th,18th and 21st day after the operation, the morphological changing pattern of the ectopic focus was observed. The phagocytosis ability of abdominal cavity macrophage was detected by fluorescent microsphere phagocytosis experiment and flow cytometry. The gene and protein levels of proinflammatory cytokines were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. And the activities of MMP-2 and MMP-9 were measured by gelatin zymography.2. Human endometriotic glandular epithelial cells and stromal cells were isolated and identified by primary cell culture and immunocytochemical method, respectively. Cells were prestimulated in serum-free DMEM with 15-epi-LXA4 (0.1～100 nM) or vehicle for 30 min and further stimulated with 1 ng/ml IL-1βfor different time. The supernatant and the culture cells were used respectively to detect protein and gene expressions of proinflammatory cytokines by ELISA and real-time quantitative PCR.3. BALB/c mice were induced to endometriosis and were intraperitoneally injected 15-epi-LXA4 at a dose of 5μg/kg. The inhibitory effect of 15-epi-LXA4 on the endometriotic lesions was observed. The gene levels of proinflammatory cytokines in the peritoneal fluid cells, eutopic and ectopic endometria were detected by real-time quantitative PCR. The concentrations of inflammatory mediators in the peritoneal fluid were measured by ELISA. The activities of MMP-2 and MMP-9 in the peritoneal fluid were detected by gelatin zymography.Results:1. The morphological change, phagocytosis ability of abdominal cavity macrophage and inflammatory mediators'expression levels were all shown a certain change pattern.2. The macrophage phagocytosis ability, inflammatory mediators (IL-1β, TNF-α, VEGF, MCP-1, MMP-2 and MMP-9) expression levels, activities of MMP-2 and MMP-9 were all significantly higher than the control mice.3. IL-1βcan effectively stimulate the gene and protein expression of IL-6, IL-8, MCP-1, TNF-α, VEGF, MMP-2 and MMP-9 in human endometriotic glandular epithelial cells and stromal cells.4.15-epi-LXA4 can effectively inhibit IL-1β-induced IL-1β, TNF-a, VEGF, MCP-1, MMP-2 and MMP-9 expression in human endometriotic glandular epithelial cells and stromal cells.5.15-epi-LXA4 significantly reduced the weight and size of endometriotic lesions, which did not affect estrous cycling.15-epi-LXA4 can suppress the growth of endometriotic lesions but cannot prevent the development of endometriosis.6.15-epi-LXA4 significantly decreased the expression of IL-1β, TNF-α, MMP-2 and MMP-9 in peritoneal fluid cells of endometriosis mice.15-epi-LXA4 can also inhibit the mRNA level of MMP-2 and MMP-9 in eutopic and ectopic endometria.7.15-epi-LXA4 can strikingly inhibite the activities of MMP-2 and MMP-9.Conclusion:1. The endometrial fragments enter the peritoneal cavity of mice will cause the changes of inflammatory environment, enhancing the phagocytosis ability of macrophage and increasing the expression of inflammatory mediators. And the changes follow a certain pattern, which may bear some relationship with the development and progression of the endometriosis.2.15-epi-LXA4 can effectively suppress the IL-1β-induced inflammatory mediators expression in human endometriotic glandular epithelial cells and stromal cells, which shows well anti-inflammatory and proresolving effects.3.15-epi-LXA4 suppresses the growth of endometriotic lesions possibly by lowering the expression of inflammatory mediators and the activities of MMP-2 and MMP-9. The inflammation self-subside strategy can be a new way to treat endometriosis.