The Role Played By Cannabinoid CB2 Receptor Agonist Pretreatment In Microglial Activation And Injury

Microglia is the smallest glial cell in CNS, the number of it accounts for about 5% in all glial cells, and it plays supportive, nutritious, protective, and repairing roles in neurons'physiological activities. Microglia can be activated when CNS is ischemic, injured or infected, so activated microglia is of effectiveness on the functions of CNS. Inflammation can be seen in many CNS diseases, and microglia is of great importance in mammalian CNS inflammation. Lipopolysaccharide(LPS) can be secreted by G- bacteria, and LPS as a kind of proinflammatory substance has been widely used in activating microglia, IFN_-γ can enhance the proinflammatory effect of LPS, and LPS plus IFN_-γ can induce inflammatory injury to microglia. Over-activated microglia can secrete a lot of inflammatory factors, enhance inflammatory reactions and lead to neurons'injury. Pretreatment is a phenomenon in which a prior exposure to a stimulus induces protection against the detrimental effects of a subsequent insult. Multiple stimuli including short episodes of ischemia, electroacupuncture, HBO and hypoxia have been shown to induce pretreatment effects in the brain. As there are CB2 receptors expressing in microglia and we have demonstrated that EA pretreatment can induce neuroprotective effects through activating CB2 receptors in rats'brains, however, whether the effects induced through activating CB2 receptors in glial cells has not been proved. In this study, microglial cells are used to study pretreatment effects caused by CB2 agonist, AM1241, on the activation and injury of microglia induced by LPS plus IFN_-γ, and study the influence caused by AM630, a CB2 selective antagonist, to the pretreatment effects. Part 1 The suitable pretreatment condition of cannabinoid CB2 receptor agonist AM1241Metheds1. To build microglial injury model induced by inflammation. Cells were assigned to 7 groups(n=10): Control and 6 injury groups induced by different concentrations of LPS(0.1mg/ml, 1mg/ml and 10mg/ml) plus different concentrations of IFN_-γ(10U/ml and 50U/ml) separately. Cell number in each well was 3.5⁵×104, and 24 hours after the injury, cell viability was determined by MTT.2. To find a suitable AM1241 concentration for pretreatment. Cells were assigned to 6 groups(n=10) pretreated with or without different concentrations of AM1241(1mM, 2mM, 3mM, 5mM, and 10mM) for 2 hours at the same time. The medium of the 6 groups were changed with normal medium without any drug, 2 hours later, the prior medium was changed with medium containing LPS plus IFN_-γ, 24 hours later, cell viability was determined by MTT. The group with the highest cell viability and lower AM1241 concentration will be the suitable group whose AM1241 concentration will be used in next steps.3. CB2 receptor expression induced by different concentrations of AM1241 pretreatment.Microglia were inoculated in a 6-well plate and separated into 7 groups, after being pretreated with different concentrations of AM1241 for 2 hours, cells were fixed with paraformaldehyde, after being washed with PBS, immunocytochemistry methods were used to investigate CB2 receptor expression in microglia.Results1. The cell viability influenced by LPS plus IFN_-γ for 24 h. Compared with control group, cell viability of the cells exposed to different concentrations of LPS plus IFN_-γ decreased significantly (P<0.05). The combination of LPS (1mg/ml) and IFN_-γ (10U/ml) can decrease cell viability to about 70% compared with the control group, so the concentrations of LPS and IFN_-γ mentioned above were chosen for next steps.2. The influence on cell viability caused by different concentrations of AM1241 pretreatment. Compared with the injury group, AM1241 pretreatment reduced the LPS plus IFN_-γ-induced decrease of cell viability(P<0.05). 5mM of AM1241 was chosen according to the protective effects and drug concentrations. 3. The influence on CB2 expression caused by AM1241. Compared with the control group, AM1241 pretreatment can upregulate the expression of CB2, the higher concentration of AM1241 is, the higher CB2 expression in microglia will be. Part 2 The effects of AM1241 pretreatment on microglial activation and injury induced by LPS plus IFN_-γMetheds1. The influence of AM1241 pretreatment in microglial injury induced by LPS plus IFN_-γ.Cells were assigned to 7 groups(n=10), the mouse N9 microglial cells were pretreated with or without various concentrations of AM1241. They were then exposed to 1 mg/ml LPS plus 10 U/ml IFN_-γ for 24 h at 2 h after the AM1241 pretreatment. The cell viability was assessed by MTT assay; the LDH release was determined by Reagent Kit; the shapes of microglia were investigated by microscope.2. The influence of AM1241 pretreatment in microglial activation induced by LPS plus IFN_-γ.Cells were assigned to 4 groups(n=6): Control group, AM1241 group, LPS/IFN_-γ group and AM1241+LPS/IFN_-γ group, after the AM1241 pretreatment and medium change, the NO and inflammatory factors(TNF- , IL-1βand IL-10) release were determined by Reagent Kit and ELISA separately. 3. The role of PKC in AM1241 pretreatment.Cells were assigned to 6 groups(n=10): Control group, Che group, LPS/IFN_-γ group, Che+LPS/IFN_-γ group, AM1241+LPS/IFN_-γ group and Che+AM1241+LPS/IFN_-γ group. Che+LPS/IFN_-γ group and Che+AM1241+LPS/IFN_-γ group, after the AM1241 pretreatment and medium change, cells were cultured with the medium containing Che, LPS and IFN_-γ for 24 h, then cell viability was determined by MTT.Results1. The influence of AM1241 pretreatment in microglial injury induced by LPS plus IFN_-γ.(1) The influence on cell viability Compared with LPS/IFN_-γ group, cell viability of AM1241+LPS/IFN_-γ group increased significantly(P < 0.05), cell viability of AM1241+AM630+LPS/IFN_-γ group, however, shew no significant difference with it.(2) The LDH release Compared with LPS/IFN_-γ group, LDH release in AM1241+LPS/IFN_-γ group decreased significantly(P < 0.05), cell viability of AM1241+AM630+LPS/IFN_-γ group, however, shew no significant difference with LPS/IFN_-γ group.(3) The shape of microglia Compared with LPS/IFN_-γ group, the injury level of AM1241+LPS/IFN_-γ group decreased markedly, that of AM1241+AM630+LPS/IFN_-γ group, however, shew no marked difference with LPS/IFN_-γ group. 2. The influence of AM1241 pretreatment in microglial activation induced by LPS plus IFN_-γ.(1) Inflammatory factors release Compared with LPS/IFN_-γ group, TNF- , IL-1βand IL-10 release in AM1241+ LPS/IFN_-γ group decreased significantly(P<0.05).(2) The NO release Compared with LPS/IFN_-γ group, NO in AM1241+ LPS/IFN_-γ group decreased significantly(P<0.05).3. The role of PKC in AM1241 pretreatment. Compared with AM1241+LPS/IFN_-γ group and LPS/IFN_-γ group, cell viability of Che+AM1241+LPS/IFN_-γ group decreased significantly(P<0.05).Summary1. AM1241 pretreatment reduces microglial activation and injury induced by LPS plus IFN_-γ; and the pretreatment effects can be reversed by AM630, a CB2 antagonist, the results show that microglial CB2 receptor activation can reduce microglial activation and injury induced by LPS plus IFN_-γ.2. PKC plays an important role in AM1241 pretreatment. As the function of PKC is normal, the pretreatment may be neuroprotective, when it is inhibited, the pretreatment may be harmful to the CNS.