The Molecular Mechanisms Of MAPK/CREF Signaling Pathways Regulated By Estrogen In The Pathogenesis Of Endometriosis-associated Pain

Endometriosis is a common gynecological disease defined by the histologic presence of endometrial-like glands and stroma outside the uterine cavity, which Affects the woman physical and moral integrity seriously. The causes of endometriosis remain unclear although its incidence rate tends to be incrteased. Pain is the the characteristics symptom of endometriosis, but the efficacy of the current treatment including medical and surgical therapy is still not good, the side effect is big, and medicine expense is high, which causes the serious problem. Pain may reduces the patient's quality of life seriously, and affect the patient's family life, and even cause the corresponding social question. On its basic reason is because pain mechanisms until now still not bright, therefore in, only then expounds the pain mechanisms, can the basic solution to pain question obtained.Recent studies have found that endometriotic lesions associated with estrogen appears nerve fibers, which mainly contain the C and Aδ primary sensory neurons that transduct pain signal to the spinal cord dorsal root ganglion (DRG) neurons, where their sympathetic nerve fibers is reduced, leading to the imbalance of sensory and sympathetic nerve fibers. The study also found that estrogen can activate the mitogen-activated protein kinase (MAPK) signaling pathway, and high expression of MAPK is found in endometriotic lesions. Moreover, the intrathecal injection of MAPK inhibitors can reduce the numbr of nerve fibers, the size of the endometriotic lesions, MAPK expression and even make pain disappeared. It has been shown that endometriosis is a visceral referred pain or neuropathic pain, and that MAPK signaling pathway involved in the pathogenesis of neuropathic pain has been recognized. Consquently,we hypothsized that the MAPK signaling pathway mediates the molecular mechanisms of estrogen playing in the pathogenesis of endometriosis-associated pain.Recent studies have also found that participating in the mechanisms of neuropathic pain., estrogen mainly binding its estrogen receptor (ER) on nerve cell membrane activates ER/metabotropic glutamate receptors (mGluRs) signaling pathways, causes intracellular signaling cascade pathways, including phospholipase C (PLC) signaling pathway, MAPK signaling pathway, and makes cyclic AMP response element binding protein (CREB) to bind cAMP response element, leading to the transcription activation of target genes. In this way, the synaptic plasticity of nerve fibers is changed, and the release of neurotransmitters occurs. CREB is ranscription factor that is dependent on the intracellular MAPK signaling pathway. Moreover, the phosphorylation of CREB protein can directly lead to the functional protein synthesis of target genes. It is suggested that estrogen may regulate the molecular mechanisms of MAPK/CREB signaling pathways playing in the pathogenesis of endometriosis-associated pain.Based on our hypothesis, we designed this study. First of all, to establish the different kinds of experimental animal models of endometriosis with a different dose of estrogen, and then to compare the lesion size and severity of pain among the different models of endometriosis and among the different groups of the same kind of endometriosis model establihed with a different dose of estrogen. We aimed to evaluate the associations of estrogen levels with the lesion size and severity of pain. Secondly, we measured the protein and gene expression of MAPK/CREB signaling pathways in spinal cord DRG in the same SD rat model of endometriosis establihed with a different dose of estrogen, and after the spinal intrathecal injection intervention with a variety of inhibitors of MAPK/CREB signaling pathways were performed. We aimed to analysize the effects of different estrogen levels on the protein and gene expression of MAPK/CREB signaling pathways, and the effects of different blockers on the protein and gene expression of MAPK/CREB signaling pathways, and study the regulation role of estrogen on MAPK/CREB signaling pathways. Finally, a culture of DRG cells in vitro was performed, and a comparison was made between in vitro and in vivo, so as to explore and screen the key molecular proteins of neural signal transduction of endometriosis-associated pain, and hope to provide more effective treatments for endometriosis-associated pain by using a molecular targeted therapy. Part1Role of estrogen playing in the pathogenesis of endometriosis and endometriosis-associated painObjective:To understand the relationships of estrogen levels with the size of endometriotic lesions and the severity of endometriosis-associated pain.Methods:After a subcutaneous model of endometriosis by auto-transplantation was established, the models are grouped according to the following:1. Four different kinds of models:Modeling without both ovaries removal was defined as the non-removed group (EEDO group); Modeling with both ovaries removal as the Ovariectomized group (ENDO+OVX group); Modeling with both ovaries removal and with a daily intramuscular injection of estradiol benzoate (0.1ml/kg) application the estrogen group (ENDO+OVX+E2group); Surgery without doing modeling as the sham group (Control group).2. The same kind of model with four doses of estrogen:Estrogen application group (model group);1-fold dose group (IE group),2-fold dose group (2E),4-fold dose group (4E group).Six days after modeling, the size of endometriotic lesions was observed, and measured [volume=1/2a x b2(a maximum diameter, b is perpendicular to the longitudinal diameter)]. Thermal pain threshold was measured by using heat tail-flick test. Serum estrogen levels were measured by using radioimmunoassay.Results:1. Endometriotic lesions were seen in all experimentally-induced SD rate models of endometriosis, and the modeling success rate was100%. 2. Serum estrogen levels were significantly correlated with the size of endometriotic lesions in experimentally-induced SD rat models of endometriosis (P<0.05).3. Serum estrogen levels were significantly correlated with the thermal pain threshold in experimentally-induced SD rat models of endometriosis (P<0.05).Conclusions:1. The successful rate of endometriosis model was100%.2. Serum estrogen levels were related to endometriotic lesions and endometriosis-associated pain Part2Role of estrogen mediated by MAPK/CREB signaling pathways playing in the pathogenesis of endometriosis-associated painObjective:To compare the differences of the protein and gene expressions of the MAPK/CREB signaling pathways following the intervention of different estrogen and inhibitors, so as to clarify the regulation roles of estrogen on MAPK/CREB signaling pathways.Methods:The establishment of SD rat model of endometriosis was previously described as part1. DRG tissue samples were taken from the SD rats model of endometriosis on day6,12and24, and after2hours, the intrathecal injection on day24with0.2mlx5μmol/ml of MEK, ERK, mG1R5, mG1Rl, ER, PKC, PLC inhibitors was performed respectively. The mRNA, and protein expressions and protein phosphorylation of MAPK, CREB, SP, NGF and BDNF in DRG tissue were detected by using PCR and Western Blot techniques respectively.Results:1. Effects of estrogen on the protein and gene expressions of MAPK, CREB, SP, NGF and BDNF in DRG tissueThe gene expressions of MAPK, CREB, SP and BDNF in DRG tissue were increased with increased estrogen levels at the start time. However, a decline trendence to their gene expressions would happen with time going on. The protein expressions of MAPK, CREB, NGF and BDNF, and phosphorylation of MAPK protein in DRG tissue were increased when low doses of estrogen was used. On the contrary, their protein expressions and phosphorylation levels were decreased when high doses of estrogen were used. 2. Effects of the intervention with a variety of inhibitors on the protein expressions of MAPK, CREB, SP, NGF and BDNF in DRG tissueWe found that ERK was through mGluR1signaling pathway, CREB through ERa/mGluR5/MEK1/2signaling pathway and NGF through ERa/mGluR5/PKC/MEK1/2/CREB signaling pathway, but BDNF protein expression was not affected.Conclusions:1. The protein and gene expressions of MAPK/CREB signaling pathways are regulated by estrogen.2. MAPK/CREB signaling pathways may mediate the molecular mechanisms of estrogen playing in the pathogenesis of endometriosis-associated pain. Part3Study of SD rat DRG cells in vitroObjective:A comparative analysis of in vitro and in vivo was performed to further clarify the regulation role of estrogen on MAPK/CREB signaling pathways.Methods:DRG neurons were taken from SD rats with a age of16days of pregnancy. DRG cell culture in vitro was performed routinely. When monolayer adherent cells of DRG neurons were cultured, the cells were treated with2uM MEK,50uM ERK,20uM mG1R5,13uM mG1R1,10uM ER,10uM PKC and5uM PLC, respectively. The cells were collected1h after intervention. Also, the cells were treated with1uM,10uM and100uM of17β-estradiol, respectively, and were collected15min,30min,60min and120min after intervention.The protein expressions of MAPK, CREB, NGF and BDNF, and MAPK phosphorylation levels were detected by using Western Blot.Results:1. Effects of inhibitors on the protein expression of MAPK, CREB, NGF and BDNFWe found that ERK was through ERa/mGluRl/PLC/PKC signaling pathways, CREB through ERα/mGluRl/PKC signaling pathways, BDNF through ERa/mGluR5/PKC/MEK1/2/CREB signaling pathways, but NGF protein expression was not affected.2. Effects of estrogen on MAPK phosphorylation levelsAfter DRG cells were treated with different concentrations of estrogen, ERK proteins were phosphorylated rapidly, and its phosphorylation increased with time at the start time. However, the phosphorylation began to decline gradually with time going on, and reached the peak30minutes after estrogen intervention. Moreover, ERK phosphorylation levels were increased with increased estrogen concentrations.Conclusions:1. MAPK protein phosphorylation in DRG cells was regulated by estrogen.2. MAPK/CREB signaling pathways may mediate the biological function of estrogen.