Effect Of SiRNA Against β-NGF On Nerve Fibers Of A Rat Model With Endometriosis

BackgroundEndometriosis (EMs) is a chronic, benign, oestrogen-dependent multifactorial, gynaecological disease and commonly associated with a range of pelvic pain symptoms such as chronic dysmenorrhoea, premenstrual abdominal and pelvic pain, back pain, dysuria, dyschezia and dyspareunia. Endometriosis-associated pain can be caused by peritoneal inflammation, adhesion formation and specific innervation of endometriotic lesions and is correlated with the presence of deep infiltrating disease. Some researchers have identified nerve fibres in endometriotic lesions in women with endometriosis. Meanwhile, some studies showed that there was increased expression of Nerve Growth Factor (NGF) in eutopic and ectopic endometrium in EMs. NGF was originally discovered as a polypeptide to stimulate the survival and differentiation of peripheral sympathetic and sensory neurons, but over the last decades, this growth factor has been shown to be expressed in a large variety of nonneuronal cell types and to exhibit other biological activities unrelated to neural system development. NGF is well known to play a role in the occurrence of hyperalgesia. First, NGF itself can serve as a pain mediator and it can induce the expression of neuropeptides including Substance P (SP) and Calcitonin gene-related peptide (CGRP), which are involved in modulating central pain transmission. Secondly, NGF can increase the number of sensory neurons and is selectively trophic for small fibre sensory neurons and sympathetic ganglion neurons which participate in mediating pain sensation. Transgenic animals lacking the gene for NGF or its specific Tyrosine kinase receptor-A (TrkA) are born with no small diameter nociceptive sensory nueurons and are hypoalgesic. In contrast, animals that over-express NGF are behaviourally hyperalgesic.EMs is believed to be a chronic inflammatory state, with disturbances of both cell-mediated and humoral immunity. The high concentration of cytokines and prostaglandins found in the peritoneal fluid of patients with EMs may contribute to an increase in nociceptive stimuli on nerve fibres. Proinflammatory cytokines have been shown to contribute to the development of inflammatory pain and hyperalgesia and may be involved in endometriosis pathophysiology. In turn, endometriotic lesions can produce both inflammatory and pain mediators, including Prostaglandin F, Bradykinin and transforming growth factor bl(TGFbl), which may induce local algesic stimulation to nerve fibres. The levels of NGF have been shown to increase substantially in inflamed tissue. In the present study, we applied specific β-nerve growth factor small interfering RNA (β-NGF siRNA) to a rat model with surgically induced EMs to investigate further the influence of siRNA on nerve fibers and pathogenesis of EMs.ObjectiveTo examine whether silencing specific β-NGF siRNA can affect the growth of ectopic endometriotic implants, generalized hyperalgesia, and nerve fibre density in endometriosis.MethodsAdult female Wistar rats, weighing180-210g, were surgically induced endometriosis. Thereafter the EMs rat models were randomly divided into two groups (12rats each), namely the treatment group and the control group. Four specific β-NGF siRNAs were detected by Western blot analysis, and the most efficient specific siRNA was transferred into rats in the treatment group through gene transfer. Meanwhile, the microparticles of mock plasmid were performed for the control group in the same way. The length×width×height of each ectopic transplant that survived from two groups was measured at pre-and post-bombardment. The transplants were collected two weeks after bombardment. Warm-water tail flick test was performed before the rats were sacrificed. The specimens were sectioned and stained immunohistochemically with antibodies against types of nerve fibers. The serums and supernatants of the peritoneal washings in the treatment and control groups were collected for enzyme-linked immunosorbent assay (ELISA) analysis.Results1. The maximum interfering effectiveness of the specific siRNA coated plasmid was screened out by Western blot from the four specific groups. The most efficient specific siRNA was Group3(F=26.12; P<0.001).2. The spherical volumes in the treatment group were much smaller than those in the control group at post-bombardment (P<0.05). A significant difference existed in the tail-flick latencies of the two groups (P<0.05).3. ELISA analysis showed that the concentrations of β-NGF in the serums and supernatants of the peritoneal fluid decreased in the treatment group unlike in the control group (P<0.05).4. Less sympathetic and sensory innervation existed in the treatment group (P<0.05).ConclusionSpecific siRNA-mediated silencing of the β-NGF gene expression after gene transfer suppressed the growth of ectopic endometriotic implants, resulted in a significant improvement in generalized hyperalgesia as well as reduced sympathetic and sensory nerve fiber density in the treatment group.