Study On Relationship Between Hypoxia-inducible Factor-1a And Dimethyl Fumarate

The fist partDimethyl fumarate inhibits the expression and function of hypoxia-inducible factor-1α (HIF-1α) in osteoblasts.Background Dimethyl fumarate (DMF) was recently approved for first-line treatment of multiple sclerosis, which is an oral tablet, people think it is by decreasing the expression of NF-κB dependent gene functioning. Hypoxia occurs when pro-inflammatory and oxidative stress, however, about the physiological effects of DMF during hypoxia, especially information on HIF-1α expression and functional aspects of the regulation in the past, but few studies have been reported. In this study, we look forward to prove that DMF is a new expression of HIF-1α function inhibitors.Objective To exolore the role of DMF in regulating osteoblastic cells HIF-1α expression and function.Methods Mouse osteoblastic cells MC3T3-E1 (clone 14) were maintained in-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S). Cells were pretreated with DMF at various concentrations from 10 to 100μM for 24 h before hypoxia in the presence or absence of specific proteasome inhibitors MG132 or N-acetyl-leucyl-leucyl-norleucinal. Then media was replaced with α-MEM containing 0.1% FBS and 1% P/S, pre-conditioned in 1% or 21% O2 tension for various time from 1 h to 24 h. For ambient (21%) O2 tension experiments, cells were cultured in a standard humidified incubator at 37 ℃ with a 95% air and 5% CO2 atmosphere. For hypoxia experiments, cells were cultured in humidified incubators at 37℃ with 5% CO2 and with 02 tension reduced to 1% using supplemental N2. Synthesized cDNA by reverse transcriptase polymerase chain reaction (RT-PCR) was used to study the expression of VEGF and IL-8 in MC3T3 E1 cells. Then cellular lysates were subjected to SDS-PAGE followed by immunoblot assays. The supernatants were harvested and assayed for VEGF or IL-8 by ELISA. Radiolabeled HIF-la protein was subjected to SDS-PAGE. Gels were dried using the HydroTech gel drying apparatus (Bio-Rad Laboratories, USA) and autoradiographed. Using everse transcriptase polymerase chain reaction (RT-PCR), immunoprecipitation, western blot assays, enzyme-linked immunosorbent assay (ELISA), HIF-la pulse labeling to pxplore Dimethyl fumarate inhibits the expression and function of hypoxia-inducible factor-la (HIF-la)Results In this study, we found that DMF inhibited hypoxia-induced expression of HIF-la and its target genes such as interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) in MC3T3 El cells. Mechanistically, DMF promoted HIF-la degradation in a proteasome-dependent but von Hippel-Lindau (VHL) protein-independent manner. Importantly, we found that DMF disrupted the interaction between HIF-1α and its chaperone heat shock protein 90 (Hsp90) but promoted the interaction between HIF-1α and the receptor of activated protein kinase C (RACK1). These data suggest that DMF might promote degradation of HIF-1α by affecting its folding and maturation.Conclusions DMF is a novel inhibitor of HIF-1α by changing its functional interaction with chaperone protein HSP90 and RACK1. Our study provides a further insight into the pharmacological function of DMF in the process of hypoxia.The second partExperimental research on the effect of Dimethyl fumarate on the expression and function of hypoxia-inducible factor-la on rat intervertebral discdegenerationBackground Many research results proved spinal degenerative intervertebral disc degeneration is initiating factors, so the intervertebral disc degeneration of pathological and physiological change is our solution is the key of spinal degenerative diseases. However, at present, a lot of research has focused on the biomechanical factors, physiological, biochemical, immune, cytokines and inflammatory mediators aspects of research, but the exact pathogenesis remains unclear. The above factors can reduce the interertebral disc proteoglycan content and nucleus pulposus cells apoptosis, the formation of new blood vessels, and so on, which is widely considered plays an important role in the process of intervertebral disc degeneration. HIF-1 a is a kind of adapt tissue cells to low oxygen environment important molecules, is the body in the condition of low oxygen to adjust the body function of an important oxygen depend on the activation of transcription factors. It can promote vascular endothelial growth factor (VEGF), glucose receptor and erythropoietin (EPO). activate the expression of inflammatory factor, inducing the formation of foam cells, regulation of cell apoptosis and differentiation. Has been proved that hypoxia can increase a few important gene expression, including can promote the formation of new blood vessels and mediated inflammatory response of vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8). HIF-1 alpha protein level is controlled by protein hydrolysis under strict oxygen tension control of the previous studies have shown that the method of immunohistochemical confirmed that the expression of HIF-1 a in outstanding intervertebral disc is significantly higher than normal intervertebral disc, there is a positive correlation between expression levels and cell apoptosis. within the nucleus pulposus intervertebral disc are the expression of HIF-i a corresponding increase with the increase of the nucleus pulposus tissue damage. We according to the first part of research results have confirmed that the DMF is a new type of HIF-1 alpha inhibitors, decision of the experiment to test the new alpha inhibitors HIF-1 dimethyl fumarate on their influence in the process of intervertebral disc degeneration. Therefore explore HIF-1 a in the degeneration of intervertebral disc tissue expression and dimethyl fumarate on its expression, step for revealing and perfect the mechanism of intervertebral disc degeneration, and for the prevention and treatment of intervertebral disc degeneration is of great significance.Objective To get experimental lumbar degenerative intervertebral disc model and evaluate the expression of HIF-1a and the affect of Dimethyl fumarate on HIF-la. by MRI,histology, immumohistochemistry and Western-bloting. Methods 48 Sprague Dawley (SD) rats were enrolled in the study, divided into three groups:normal control group (Group A),degenerative control group(Group B), treatment group of Dimethyl Fumarate (Group C) randomly and equally.First established the animal model of disc degeneration except normal control group A. The rats respectively anesthetized by intraperitoneal injection,prone position, fixed the limbs, skin prepared, steriled, cut skin along the lower back spine,subperiosteal dissection, remove L1-L6 spinous and articular process by rongeur, process, cut of bilateral vertical spine muscles,spraspinous ligament and interspinous ligament,then fascia the skin. Put in a separated cage. Did nothing to the normal control group A.The control group B use placebo equally to group C. Group C use Dimethyl fumarate 50mg-1kg-1d;All the animals were fed 8 weeks. The level of proteoglycan of intervertebral disc tissue and the expression of hypoxia-inducible factor-1α(HIF-1α) were examined by histochemistry,immumohistochemistry and Western-bloting.Results To establish rat model of lumbarintervertebral disc degenerative by operating. MRI scanning, T2 signs had no significant changes in normal control group chang, but it was weaker in treatment group of Dimethyl Fumarate. than that in degenerative control group. The the expression of intervertebral disc were evaluated by histochemical staining, immunohistochemical.The fibrosis of nucleus pulposus and the number of pulposus were obserbved in degenerative control group.Under electron microscopy, Different from normal group nucleus pulposus cells were sparse with long dendrites.The amount of organelles cell and glycogen granules were reduced significantly. Collagenous fibrils appeared fusion, twist and calcification.Through immumohistochemistry,The positive cell ratio of HIF-1α in degenerative control group was much more than that of treatment group of Dimethyl Fumarate and normal control group (P<0.05).Conclusions The high expression of HIF-la in lumbar degenerative disc indicate that HIF-1α must be involved in lumbar disc degenerative closely. Treatment with Dimethyl Fumarate inhibits the expression of HIF-1α and the development of experimental rat lumbar degenerative disc in vivo. It will provide new theory and research evidence occurrance and developmet of lumbar degenerative disc.