The Role Of AGEs-RAGE Pathway In POP

The pelvic organ prolapse (POP) is the common disease of middle-aged and the elderly women, which impact their quanlity of life considerably. But the pathogenesis of this condition is still unknown, the prevention and treatment is also difficult yet. So the study about pathogenesis is always the focus of gynecology and obstetrics, which is also the fundermental problem need to be solved promptly.As the main component of pelvic floor surport system, the pelvic connective tissues present the important role for the occurrence and development of POP, which consis of fobroblast and collagen chiefly. The balance of collagen metabolism about synthesis and degradation is regulated by fibroblast in noamal connective tussues. But the structure and function of collagen will change when the balance break, and the pathophsiology will occur accordingly.Nowadays, it is consider that the Advanced Glycated product-ends (AGEs) bind it receptor (RAGE), trigger the cell signaling system, and impact the proliferation or apotosis of fibroblast and metabolism of collagen, which involoved into a lot of pathophysiology, such as skin aging, reconstructure of vessel, Diabetic Kephropathies, gingival hyperplasia, hypertrophic, et al. In skin, AGEs can promote apotosis of fibriblast, surpress the synthesis of collagen, and impact the metoblism of MMP and TIMP, through activating cell signaling pathway. So what is the change of AGEs-RAGE patheway and collagen metabolism in pelvic floor tissues? We designed the following experiment and gain many results to answer this question.The First PartThe expression of collagen Ⅰ, AGEs and RAGE in the vaginal tissue of POP[Obsjective] Detect the content of collagen Ⅰ, AGEs and RAGE from vaginal tissue of POP, and find the relationship of AGEs-RAGE pathway and POP. [Patients and Methods] 44 POP and 46 non-POP in our hospital were detected by immunohistochemistry and Western Blotting, and then the whole gene sequence of RAGE for 24 POP and 25 non-POP out of all above were measured. [Results] It is found that the content of collagen I in vaginal tissue for POP patients was lower than the non-POP, and decreased along with aging (p<0.05); the content of AGEs in vaginal tissues for POP patient was higer than the non-POP (p<0.05); the expression of RAGE was the same between POP and non POP (p>0.05). There were two potential SNP locus after gene sequencing, but they both located in introns far from the extron, which mean the lower correlation with POP. [Conclusion] This study manifested that the increase of AGEs and decrease of collagen Ⅰ in POP patients may contribute to the POP occurrence.The Second PartThe impact of AGEs on fibroblast for the metabolism of collagen Ⅰ in pelvic tissue of POP[Objective] To explore the impact of AGEs on fibroblast for the metabolism of collagen Ⅰ in pelvic tissue of POP. [Patients and method] The fibroblasts in the viginal tissue of 3 POP and 3 non-POP were primarily cultured firstly, then the proliferation and apoptosis of these fibroblasts cultured with AGEs were detected by SRB, and the collagen Ⅰ, MMP-1, TIMP-1, RAGE were tested by Western Blotting and real time PCR after being cultured with different concentration of AGEs and different culture time. [Result] It is found that the fibroblast of POP were apt to apoptosis at lower concentration of AGEs (25mg/L); and the apoptosis of fibroblast for non-POP occurred at higer concentration (75mg/l). The expression of collagen Ⅰ in POP group decreased with the increasing of AGEs; and the expression of MMP-1 increased with the increased of AGEs; the mRNA of TIMP-1 decreased and RAGE presentev small peak with the increase of AGEs, but the protein exprssion of both remain stable. According to the non-POP group, the expression of collagen Ⅰ and MMP-1 change smaller or none. [Conclusion] This study mefested that AGEs could promote apotosis of fibroblast in POP tissues, decreased collagen Ⅰ and increased MMP-1 expression. The Third PartThe activation of signalling pathway of MAPK-p38 and NF-kB-p65 after AGEs bind to RAGE in fibroblast of POP[Objective] To search and test the siginalling pathway of MAPK-p38 and NF-kB-p65 after AGEs bind to RAGE in fibroblast of POP. [Method] we detected the the change of MAPK-p38, NF-kB-p65, collagen I, MMP-1 after the AGEs-RAGE signaling pathway were blocked by SiRNA, SB203580, PDTC, by using WB and RT-PCR. [Results] Firstly, the peak of P-p38 occurred after 16 minutes late for adding AGEs, and the peak of P-p65 occurred after 1hour late. When the RAGE was blocked by SiRNA, the occurrence of P-P38 and P-p65 disappeared; and when the MAPK-p38 was blocked by the by SB203580, the occurrence of NF-kB remain; when NF-kB was blocked by PATC, the product of collagen I increased and mmp-1 decreased. According to the control group, the peak of P-p38 didn’t occur after being stimulated by AGEs. [Conclusion] These results manifest that after AGEs bind to RAGE, the cell siginalling molecule of P-p38 and P-p65 are actived, increase MMP-1 and decrease collagen I, but MAPK-p38 isn’t the only cell siginalling molecule at this level.The Fourth PartThe change of AGEs-RAGE pathway in the repairation of abdominal defect of SD rats[Objective] The aim of this part is to test how the AGEs-RAGE pathway participate the metabolism of collagen I at experimental animal level. [Methods] First, the abdominal defect of 60 SD rats was made and then repaired by three methods:a) polypropylene mesh implantion,20 rats, b) SIS biodesign mesh implantation,20 rats, c) Bilateral rectus suture,20 rats. Then the repair results were observed at 3,9,15,21 months after operation, including the macroscopic view and electron microscope view, the mechanical properties for the tissue of repair location. Thirdly, the expressions of collagen Ⅰ, MMP-1, RAGE in the repair location were detected by immnohistochemistry. [Results] This study showed that the group with SIS mesh had high degree of simulation and soft texture with higher rate of prolapsed (P<0.05), and had lower maximum tensile strength of tissue in the repair location (p<0.05); in the view of electron microscope, the collagen increased slower and appeared thin, the number of fibroblast decreased and the volume of it was smaller, the vascular diameter got smaller; the expression of collagen Ⅰ in the tissue of repair location was lower than two other group, AGEs was higher than other, and RAGE remain stable. The content of AGEs in three groups were all increased in 9 month after operation, and then decreased in 15 month, at 21 month remain lowly. [Conclusion] This study manifested that AGEs inhibited the synthesis of collagen, and decreased to normal 15 month from rapair with result of collagen damage, which might contribute to the occurrence of abdominal prolapsed partly.ConclusionFrom the results above, we can conclude that:1, AGEs-RAGE pathway take part in the metabolism of collagen Ⅰ in vagina tissue for POP patients, and inhibit the synthesis of collagen Ⅰ, and promote the synthesis of MMP-1; 2, AGEs bind to RAGE and activte MAPK-p38 and NF-kB-P65 signaling pathway, but not the only way; 3, AGEs increase at early period of hurt and hamper the repair of damage, then decrease to noamal in following months, so the study about POP patients have the quality of hysteresis.These studies will partly provide explanations for merchanism of POP occurrence; also provide reference for prevention and therapy of POP, for example, enough postpartum recovery and early preventive therapy is suggested. But there are still many questions need to be resolved further.