Decabrominated Diphenyl Ether-induced Neurotoxicity In Dorsal Root Ganglion Neurons And The Protective Effect Of IGF-1

Polybrominated diphenyl ether (PBDE-209) as one of most common types of bromide flame retardants has become a persistent environmental pollutant. PBDE-209 is difficult to degrade and can accumulate in the food chains. Decabrorainated diphenyl ether (BDE-209) is the most common PBDE congener currently and can affect human health in many ways. BDE-209 can induce toxicity in nervous system, but the mechanisms are still need to be clarified. Insulin-like growth factor-1 (1GF-1) is a growth-promoting peptide hormone that has been shown to have neurotrophic properties. The capacity of IGF-1 on neuronal survival, neurite outgrowth, and differentiation plays an important role in neuronal development and function maintenance. In the present study, primary cultured dorsal root ganglion (DRG) neurons were used for determining the neurotoxicity caused by BDE-209 in DRG neurons and the protective effects and mechanisms of IGF-1.Part I BDE-209-induced neurotoxicity in dorsal root ganglion neuronsThe neuronal bodies of periphery sensory neurons are located in DRG. The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons are two major neurochemical phenotypical classes in DRG CGRP-IR neurons and NF-200-IR neurons represent neuropeptide-IR and neurofilament-IR neurons in DRG respectively. The growth-associated protein 43 (GAP-43) is expressed in all subpopulations of DRG neurons and is used as a marker to reflect neuronal regenerating status.To determine the neurotoxicity of BDE-209 in DRG neurons, the rat DRG neuronal cultures at 48 h post-culture were exposed to different doses of BDE-209 (10 μmol/L,20 μmol/L, and 40 μmol/L, respectively) for an additional 48 h. The DRG neurons were continuously exposed to culture media as a control. Dimethyl sulfoxide (DMSO) was used as vehicle control. After that, the neurite length was determined by immunofluorescent labeling of microtubule-associated protein 2 (MAP2). The cell activities of the DRG neurons were detected by WST-1 kit. Hoechst 33342 staining was used to observe apoptotic neuronal cell. GSH detection kit and ROS test kit were used to detect the GSH level and ROS level respectively. The mRNA and protein levels of GAP-43, CGRP, and NF-200 were analyzed with real-time PCR and Western blot, respectively. The alterations of GAP-43-IR, CGRP-IR, and NF-200-IR were determined by double immunofluorescent labeling of MAP2 and GAP-43, CGRP, or NF-200.The results showed that BDE-209 exposure caused neurite retraction, decrease of cell viability, and increase of ratio to apoptotic cells. The GSH level decreased and the ROS level increased. The mRNA, protein levels of GAP-43, CGRP, and NF-200 and the percentage of GAP-43-IR, CGRP-IR, and NF-200-IR neurons decreased significantly after treatment with BDE-209. These results showed that BDE-209 can affect the outgrowth of DRG neurons and has neurotoxic effect on different subtypes of DRG neurons.Part II The effects of IGF-1 on dorsal root ganglion neurons with BDE-209-induced neurotoxicityIGF-1 is a polypeptide growth factor that has a variety of functions in neurons. IGF-1, binding with its receptor IGF-1R, may activate extracellular signal- regulated protein kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K) /serine-threonine kinase (Akt) signaling pathways. To assess the neuroprotective effects of IGF-1 on DRG neurons with BDE-209-induced neurotoxicity, the primary cultured rat DRG neurons were exposed to BDE-209 (40 μmol/L), BDE-209 (40 μmol/L) plus IGF-1 (20 nmol/L), ERK1/2 inhibitor PD98059 30 min before treatment with BDE-209 (40 μmol/L) plus IGF-1 (20 nmol/L), PI3K inhibitor LY294002 30 min before treatment with BDE-209 (40 μmol/L) plus IGF-1 (20 nmol/L) for an additional 48 h, respectively. The DRG neurons were continuously exposed to culture media as a control. Then the alterations of neurite length, cell viability, apoptotic status, GSH and ROS levels, GAP-43 expression, and the neurochemical phenotypes were detected.The results showed that IGF-1 promoted neurite outgrowth and neuronal cell viability of DRG neurons with BDE-209-induced neurotoxicity. IGF-1 rescued oxidative stress and apoptotic neuronal cell death caused by BDE-209 exposure. IGF-1 could reverse the decreases of GAP-43 and CGRP, but not NF-200 expression caused by BDE-209 exposure. The effects of IGF-1 could be blocked by pretreatment with ERK1/2 inhibitor PD98059 and the PI3K/Akt inhibitor LY294002, either alone or in combination. IGF-1 may play an important role in neuroprotective effects on DRG neurons especially neuropeptide-IR neurons with BDE-209-induced neurotoxicity through inhibiting oxidative stress and apoptosis and regulating GAP-43 and CGRP expression of DRG neurons. Both ERK1/2 and PI3K/Akt signaling pathways were involved in the effects of IGF-1. These data of the present study provide novel experimental evidence for relieving BDE-209-induced neuropathy.