The Neuropeptide CRH Regulates Rat Primary Microglin Activation

Background and ObjectiveMicroglia were firstly described by Rio-Hortega in1919, which has been considered as macrophage in the CNS. Microglia were derived from circulating monocytes. Microglia constitute10-15%of total glial cells in the CNS. As the CNS immune cells, microglia timely screen brain damaged neuron, infection and plaque. When the brain are in pathological or insults, microglia are exquisitely sensitive and easily activated in response to pathophysiological brain insults from ramified state into amoeboid state. Activated microglia promote phagocytosis and antigen presenting function, up-regulate cell antigen markers(MHC, CD14, CD40, CD80, CD86et al) and produce pro-inflammatory cytokines NO, IL-1, IL-6and TNF-a. The periphery immune cells are simultaneously attracted into the central nervous system(CNS). Then activated microglia and immune cells cooperate together in innate and adaptive immune response in the brain. Activated microglia and inflammatory neurotoxity play a critical role in neurodegenerative disease, such as Alzheimer’s disease (AD), Multiple sclerosis(MS) and Parkinson’s disease (PD). CRH and CRHR are the main regulator of the hypothalamus-pituitary-adrenal (HPA) axis, which are involved in neuron activity in cortex and hippocampus. CRH participated in several physiology activities via binding CRHR, including CRHR1and CRHR2. CRHR are7transmenbrane G protein-coupled membrane receptors, which are widely distributed in the brain. CRHR1is mainly distributed in cortex, hippocampus and cerebellum and sensory relay structures. While CRHR2is expressed in a number of subcortical structure. CRH elicits its biological effects by binding CRHRl and CRHR2with signaling pathways such as cAMP, protein kinase C and mito-activated protein kinase(MAPK). Plenty of researchers have identified that CRH evoked by stress response involved in neurodegenerative disease process, such as Alzheimer’s disease and Parkinson’s disease. Researchers found that CRH dramatically decreases, whereas CRHR up-regulate and abnormal CRH neurons appears in AD patients brain. Recent a study identified that CRH augments neuropathlogical process and memory decline. CRH also directly modulates the immune inflammatory response. Now it has no doubt that CRF influences immune cell function and activity via paracrine effect or autocrine and plays a pro-inflammatory role. Also CRH augments the synthesis of pro-inflammatory cytokines TNF-a and IL-6at the mRNA level when macrophage response to lipopolysaccharide (LPS). The related peptide Urocortin decreases LPS induced microglia activation. CRH regulate NF-κB activity via the phosphatephorylation of CREB and MAPK in leukocytes, while NF-κB regulate inflammatory cytokines synthesis and release, such as IL-6and TNF-a. These inflammatory cytokines participate in inflammatory response, immune response and neural cell death/survival. In addition, CRH has been implicated in pathological and pathophysiological responses in various neurodegenerative disorders, including brain traumatic, ischemic and excitotoxic injuries. Several lines of evidence suggest that CRH contributes to neuron loss in vitro and in vivo. But it has not been elucidated that CRH whether directly regulates microglia activation and proinflammatory cytokines release or not.Our laboratory used primary microglia cell culture from neonatal SD rats in vitro to investigate CRH regulate microglia activation, proinflammatory cytokines release and signaling pathways whether or not via phagocytosis, NO production, FACS, ELISA and RT-PCR.Design and MethodsMicroglia were prepared from lor2-day-old neonatal Sprague Daeley rats cerebral. After seven days, microglia were isolated and culture for16-24hours, then add various concentration CRF to stimulate microglia and check the phagocytosis and production of NO, TNF-a and IL-6. LPS as a positive control.Results1. CRH(10-10mol/l,10-12mol/l,10-15mol/l) incubated primary microglia for6hours, CRH(10-15mol/l,10-12mol/l,10-10mol/l) phagocytic activities are slightly higher than control, but have no significant effect. CRH(10-10mol/l,10-12mol/l,10-15mol/l) incubated primary microglia for30min followed by LPS10ng/ml for6hours, microglial phagocytic activities are slightly higher than control, but have no significant effect.2. CRH(10-8mol/l,10-9mol/l,10-10mol/l,10-12mol/l,10-15mol/l) incubated primary microglia for24hours,10-15mol/l CRH could significantly improve primary microglia to produce NO. CRH(10-8mol/l,10-9mol/l,10-10mol/l,10-12mol/l,10-15mol/l) incubated cells for30min followed by LPS10ng/ml for24hours.10-15mol/l CRH could significantly improve LPS induced NO production in primary microglia.3. CRH(10-8mol/l,10-9mol/l,10-10mol/l,10-12mol/l,10-15mol/l) stimulate primary microglia for24hours,10-15mol/l CRH could significantly improve primary microglia to produce proinflammatory cytokines TNF-a and IL-6. CRH(10-8mol/l,10-9mol/l,10-10mol/l,10-12mol/l,10-15mol/l) incubated cells for30min followed by LPS10ng/ml for24hours.10-15mol/l CRH and10-12mol/l CRH could significantly improve LPS induced proinflammatory cytokines TNF-a and IL-6in primary microglia.ConclusionsCRH influenced microglia activation and produced NO, IL-6and TNF-a. CRH incubated microglia for30min,then added lOng/ml LPS for24hours, we found that CRH increased LPS induced microglia activation. All these results suggested that CRH could act as physiological regulator to improve excessive inflammatory microglial responses.