Effect Of CB1 Receptor On LPS-induced Microglial Activation

The endocannabinoid system, comprised by the cannabinoid receptors, endogenous ligands and the enzymes for endocannabinoid biosynthesis and degradation, is crucial for regulating a range of physiological, pathological, pharmacological processes including neuroinflammation. Cannabinoid receptors were composed of at least two subtypes (CB1 receptor and CB2 receptors), belonging to G-protein-coupled receptors superfamily. CB1 receptor is mainly expressed in the central nervous system. Endogenous ligands are a family of lipid messengers, which mainly play a role by activating CB1 of presynaptic membrane to regulate the neurotransmitter release. As pathological process of neurodegenerative diseases related to the inflammatory reaction, eCB system is promised to be an important therapeutic target of various neurological diseases. Microglia is the innate immune cell in brain. Many studies have found activation of CB1 receptor of neurons and glia can regulate immune response involved in disease process. Systemic inflammatory response can induce the central nervous system. LPS, an integral part of the outer membrane of Gram-negative bacteria, is a major pathogenic factor, which can cause brain innate immune response and oxidative stress by multiple means of neural or humoral pathway. Therefore, intraperitoneally (i.p.) administered LPS can be used to establish animal models of infectious neuroinflammation.In current study, a LPS induced model of nueroinflammation was established. Firstly, we explored the effect of neuroinflammation on eCB system by investigating CB1 receptor expression change induced by LPS. Secondly, we investigated the effect of CB1 receptors in the process of modulating neuroinflammation by observing changes of behavior and microglia cell activation after agonist HU210 and antagonist AM281 treatment in mouse model. The results may provide new reference for the research of subsequent mechanism study of CB1 receptor involved in neuroinflammation. This study includes the following sections:(1) Establish of LPS-induced neuroinflammation mice models. C57 mice were randomly divided into three groups:saline group,0.5mg/kg of LPS group and lmg/kg of LPS group. The animals were intraperitoneal injected with different dose of LPS or equal volume of saline for two days. The animal general condition were investigated, including body weight and spleen index. We detected the locomotor activity by open-field test and the activation of microglia by Ibal immunohistochemistry. These indexes were measured after 24 hours of the last injection. The different dose of LPS group both exhibited decreased locomotor activity in open-field test, body weight loss and spleen index increase. And 1mg/kg of LPS showed more prominent body weight loss and spleen index increase. Activation of microglia were induced by 1mg/kg of LPS (P=0.045). The quantitative analysis of Ibal immunohistochemistry showed that 0.5mg/kg of LPS had no significant difference comparing with control group. So intraperitoneal injection of LPS 1mg/kg consecutive two days, was chosen to build neuroinflammation mice model in current study.(2) Exploring CB1 expression change in LPS-induced neuroinflammation models. Through immunohistochemistry staining, we observed expression of CB1 receptors in the cingulate cortex, hippocampus, amygdala and globus pallidus. And results of quantitative analysis showed that the expression of CB1 in the hippocampus exhibited a decreased trend after administration of LPS without significant difference.(3) Intervening eCB system, we investigated the role of the CB1 receptor in activation of microglia and behavior. Mice were injected either agonist HU210 (25?g/kg, 50?g/kg) or antagonist AM281 (3mg/kg) 30 min prior to a second i.p. injection of LPS. The body weight and spleen index of animal were investigated. The locomotor activity and activation of microglia were respectively detected by open-field test and Ibal immunohistochemical staining. These indexes were measured after 24 hours of the last LPS injection. HU210 ameliorate LPS induced increase of mouse spleen index and body weight loss; AM281 had no significant effect. Behavioral results showed that agonist HU210 exaggerated mice locomotor reduction induced by LPS; AM281 have no obvious effect on the locomotor reduction induced by LPS. The morphology results show that, agonist HU210 can inhibit LPS induced microglial activation, antagonist AM281 have no obvious effect. These results show that activation of CB1 receptors by HU210 can play a certain immunosuppressive effect, involved in regulation of LPS-induced microglia activation, consistent with the results of peripheral immune organ.