The Effect Of Nerve Growth Factor In Adenomyosis Producing Pain And Its Mechanism

Adenomyosis is defined as the presence of ectopic endometrial glands and stroma within the myometrium. This disorder mainly affects women more than 40 years old. The prevalence of adenomyosis reported in the literature ranges from 5 to 70%, and largely varies in relation to the number of tissue sections analysed. It was found that adenomysis in 30-60% of hysterectomy specimens. Pain, which could be divided into dysmenorrhoea, dyspareunia, and chronic pelvic pain, is one of the common and severe complaints in symptomatic women with adenomyosis. The prevalence of adenmyosis patients complained dysmenorrhoea was about 64.8%-77.8%. The patho-genic mechanism responsible for adenomyosis and its pain has not been well known as yet. nerve growth factor (NGF) is a key factor in pain producing and transfer. Previous studies indicated that the level of NGF was higher in ectopic endometnrium. It may take part in the important part of the mechanism of adenomyosis, especially for the pain.PartⅠThe expression of NGF in the ectopic endometrium of adenomyosis and its relationship with pain scale and innervationObjective:To investigate the expression of NGF in the ectopic endometrium from adenomyosis patients, and explore the relationship between NGF expression and innervation or pain scale.Methods:45 patients who had adenomyosis and underwent hysterectomy were chosen as experimental group,26 patients who had leiomyoma or Cervical intraepithelial neoplasiaⅢand underwent hysterectomy were chosen as control group. Ectopic endometrium from experimental group and eutopic endometrium from control group were collected in the surgery. The expression of NGF was examined by immunohistochemistry. The density of PGP9.5 positive nerve fibers was detected by immuno-fluorescence. The degree of dysmenoreal, chronic pelvic pain and dyspareunia was detected by Visual rating scale (VAS).Results:The expression of NGF and the density of PGP9.5 positive nerve fibers were higer in adenomyosis pain group than adenomyosis painless and control group. The NGF level and density of PGP9.5 positive nerve fibers in severe dysmenorrheal group were higher than light dysmenorrheal group and painless group in adenmyosis patients. There was no difference of NGF expression in chronic pelvic pain group and no chronic pelvic pain group, so was dyspareunia group and no dyspareunia group. The expression of NGF was correlated with the density of PGP9.5 positive nerve fibers in adenomyosis nodules (r=0.31, p<0.05)Conclusions:the enhanced expression of NGF in ectopic endometrium of adenomyosis might take part in the mechanism of pian, and improving innervation may be one of its ways.PartⅡThe expression of NGF and its receptors in uteri and dorsal root ganglias in mice model with adenomyosisObjective:first, to establish ICR mouse model of adenomyosis with tamoxifen and characterize the adenomyosis nodules, angiogenesis and uterine inflammation in this animal model. Then, examine the expression of NGF and its receptors in uteri or dorsal root ganglias (DRGs) at different stages of estrous cycle and course of adenomyosis.Methods:ICR mice with adenomyosis were induced by orally administration of tamoxifen on days 2-5 after birth. Adenomyosis modle mice and control mice were killed on day 42,85-95,135-145,185-195 and 235-245, respectively. Hematoxylin and eosin (HE) staining was performed to observe the adenomyosis nodules. Microvessel density, diameter and the ratio of area in the uterus were assessed by immunohistochemistrial staining with CD31. The gene expression of bradykinin receptor-1 (BKR-1) and neurokinin1 receptor (NK1-R) was detected by RT-PCR. The expression of NGF and its receptors, tyrosine kinase receptor (trkA) and p75 neurotrophin receptor (p75NTR), in the uteri and dorsal root ganglias (DRGs) of adenomyosis and control mice in 4 age groups of 85-95,135-145,185-195 and 235-245 by western bolt, immunochemistry and real time reverse transcription PCR.Results:All mice dosed with tamoxifen developed adenomyosis with a incidence rate of 100%. The microvessel density and the ratio of area in myometrium of experimental mice were significantly higher than those of the controls (P<0.05). In the uterus of 135-135 d dosage mice, the mRNA expression of BKR-1 and NK1-R was significantly higher than that of controls (P<0.05). In uteri of adenomyosis and control mice, NGF immunoreactivity was detected predominantly in the luminal, glandular epithelial cells and stromal cells of endometrium. Meanwhile, immunolocalization of p75NTR was detected mainly in stromal cells of endometrium and trkA in nerve fibers as well as luminal and glandular epithelial cells. The protein level of NGF, trkA and p75NTR in uteri and mRNA expression of trkA in DRGs were higher in aggravated adenomyosis mice than controls. Based on the data of western blot, the expression of NGF and its receptors in uteri were fluctuated as the estrous cycle changed, and increased gradually while age growing for adenomyosis mice but changed little in control mice. Results of real time reverse transcription PCR also revealed that the mRNA level of trkA in DRGs was increasing as age growing in adenomyosis mice.Conclusions:Adenomyosis induced in mice by tamoxifen has abnormal angiogenesis, inflammation and pain related receptor expression in the uterus. The reinforced expression of NGF and its receptors in uteri and DRGs of adenomyosis mice and the gradual increasing in aggravated adenomyosis might hint their participation in the mechanism of adenomyosis, especially for the pain.PartⅢthe impact factors of NGF expression by adenomyosise endometrium stromal cell and the effect of NGF on adenomyosis endometrium stromal cellObjective:To investigate the factors which may impact the NGF expression of adenomyosis endometrial stromal cell (ESC), and the effect of NGF on ESC from the way of cell proliferation, apoptosis and estrogen synthesisMethods:Patients who had adenomyosis and underwent hysterectomy were chosen as experimental group. Patients who had leiomyoma or cervical intraepithelial neoplasiaⅢand underwent hysterectomy were chosen as control group. Ectopic endometrium from experimental group and eutopic endometrium from control group were collected in the surgery. ESC was separated and cultured. The expression of NGF and its two receptors(p75NTR and trkA) were displayed by Immuno-fluorescence.17β-estradiol, TNF and CoCl2 were added in the culture system, respectively. The expression of NGF by adenomyosis and control ESC was examined by western-bolt. NGF was added in the culture system and the proliferation of ESC was detected by MTT. Then, the mRNA and protein level of aromatase were detected by real time RT-PCR and western-bolt, respectively. H2O2 or CoCl2, as well as NGF were added in the culture system, and than the apoptosis rate of ESC was examined by MTTResults:Both adenomyosis ESC and control ESC expressed NGF and its receptor p75NTR. Neither of them expressed trkA.17β-estradiol could promote the NGF expression by adenomyosis ESC but not control ESC. TNF could promote the NGF synthesis by both adenomyosis and control ESC. CoCl2 could inhibit the control ESC to synthesize NGF, but have no effect on NGF synthesis by adenomyosis ESC. NGF could promote the proliferation and the synthesis of aromatase of ESC from adenomyosis patients. However, NGF had no effect on the protection of adenomyosis and control ESC from H2O2 and CoCl2 induced apoptosis.Conclusions 17β-estradiol and TNF added in the culture system of ESC from patients with adenomyosis could stimulate the expression of NGF; and NGF could promote the proliferation and the expression of key enzyme in estrogen synthesis of ESC from in patients with adenomyosis.