Molecular Mechanism Of Tramadol Induced Defectsin Embryo Development

Objective Drug abuse during pregnancy is one of the most important causes of miscarriage. Tramadol is a widely used analgesic, for treatment of pain, inhibition of appetite and relief of depression symptoms, etc. It is also becoming more common for addiction and abuse in younger population. Most of the people addictive to Tramadol are between the ages of 18-30, which is the optimal fertile period for female. Clinical research demonstrated that exposure to Tramadol during pregnancy significantly increases the chance of miscarriage. However, the mechanism remains elusive. Preliminary results of this project showed that embryonic exposure to Tramadol could lead to series of developmental defects in Xenopus laevis, including pigment cell developmental abnormalities, small fetal size, and microcephaly. Since pigment cells and craniofacial region is closely related to neural crest development, we suppose that the effect of Tramadol on embryonic development is most likely intefere embryonic neural crest formation. To test this hypothesis, we investigate the effect of Tramadol on neural crest induction and migration and preliminary possible mechanism. This research will shed light on the addictive mechanism and the root cause in embryonic developmental defects underlying Tramadol exposure.Methods Xenopus laevis embryos at cleavage stage were exposed to different concentration of tramadol(1:200, 1:400 and 1:500 in MBS medium) and the development of the embryos was observed with a dissecting microscope, pictures of the embryonic malformation were taken with a stereomicroscope. Head cartilages from 45-hour embryos were stained with alcian blue to evaluate the development of vraniofacial cartilage. Based on these observations, the teratogenic concentration of tramadol was determined. High Performance Liquid Chromatography method was hired to detect the concentration of tramadol in the embryos, which is comparable to the possible blood concentration in human body. Tramadol-exposed embryos were cultured to neural plate stage and the end teeth stage, by when the embryos are stared to migration. To assess the effect of tramadol on the induction and migration of neural crest, embryos were harvested at a forementioned stages and subjected to the detection of the expression of genes that evolved in the induction of neural crest with gene probe, the transcription levels of Wnt, Fgf, BMP, and Notch/Delta in embryos were also detected with in situ hybridization. The apoptosis of embryos was detected with TUNEL assay and the mRNA levels adhesion molecules in neural crest were also examined by q PCR. Further, we have tested the effect of tramadol on the cell migration by a transwell experiment with U251 cell in vitro.Results 1、 Early embryonic exposure to tramadol leads to the developmental defects including dysplasia of cell pigment, small embryos and small head. 2、 The tramadol-induced dysplasia of embryos are mainly caused by the inhibition of migration of neural crest. 3、 Dyregulated expression of dadherin-dependent adhesion molecules is responsible for the tramadol-induced defect of migration of neural crest.Conclusion Early embryonic exposure to tramadol affected the migration of neural crest and followed by developmental defects. Dyregulated expression of dadherin-dependent adhesion molecules is responsible for the tramadol-induced defect of migration of neural crest. Clinical studies suggested that abuse of tramadol leads to an increased chance of miscarry, development of neural crest is the key procedure of embryo development, therefore, our data indicated that tramadol-induced inhibition of neural crest migration may be the reason for the miscarry under this condition.Objective Atopic dermatitis(AD) is a common chronic inflammatory skin disorder, The prevalence rate of AD is rising dramatically, especially in developed countries, and it now affects 20% people. Meanwhile, a growing body of evidence has shown that patients with AD suffer from a severe psychological stress, which markedly increases the prevalence rate of depression and anxiety disorders. Fluoxetine, a selective serotonin reuptake inhibitor(SSRI), has been widely used as an antidepressant agent in clinic, and it has also shown anti-inflammatory and direct immunosuppressive effects such as suppression of T cell activation, cytokine secretion and proliferation and induction of apoptosis in vitro and in vivo. Thus, it is reasonable to to propose that fluoxetine may ameliorate AD through reducing psychological stress and inflammatory response.we except inhibitory effect of fluoxetine on the development of dermatitis and to obtain basic information about the usefulness of fluoxetine in the treatment of AD.Methods Firstly, a BALB/c mouse model of AD was induced by application of 2,4-d initrochlorobenzene(DNCB) onto hairless dorsal skin. We investigate the effect of 5-HT reuptake inhibitor fluoxetine on this AD model, and its probablly molecular mechanism. We observed the SCORAD score, the scratching bouts, as well as the anxiety- and depressive-like behaviors as a reflection of clinical efficacy of Chronic fluoxetine treatment on AD. We use HE stain and toluidine blue ostain to reflect the epidermal thickness and the number of mast cells in skin tissue. we also detected the mRNA levels of Cytokynesis(IL-2、IL-4、IL-13、IFN-r) in the splen, as well as serum immunoglobulin E(IgE) in diffrent treated mice group to explore the molecular mechanism of 5-HT reuptake inhibitor fluoxetine on AD.Results 1、 Chronic fluoxetine treatment alleviates DNCB-induced AD symptoms 2、 Chronic fluoxetine treatment reduces depressive- and anxiety-like behaviors in AD mice 3、 Chronic fluoxetine treatment suppresses tissue inflammation and the accumulation of mast cells in AD-like skin lesions 4、 Chronic fluoxetine treatment inhibits DNCB-induced serum IgE elevation 5、 Chronic fluoxetine treatment reduces spleen cytokines in AD miceConclusion fluoxetine treatment significantly attenuated AD, Furthermore,it can decrease the levels of interleukin-4(IL-4) and IL-13 in the spleen, inhibit the number of mast cells in skin tissue, Improve the immune imbalance. fluoxetine decrease the psychological stress induced anxiety- and depressive-like behaviors,relieve the clinical symptoms of AD.