ABSTRACT
Objective@#To evaluate the effect of docosahexaenoic acid (DHA) on microglial activation during oxygen-glucose deprivation and restoration (OGD/R) injury.@*Methods@#N9 microglia were inoculated in 96-well culture plates at a density of 104 cells/well for 3-5 days and divided into 3 groups (n=18 each) using a random number table method: control group (group C), group OGD/R and DHA+ OGD/R group. The cells were cultured for 24 h in an incubator at 37 ℃ (95% air-5% CO2) in group C. In OGD/R and DHA groups, the culture medium was replaced by glucose-free culture medium, the cells were cultured for 12 h in an incubator at 37 ℃ (95% N2-5% CO2), and then cells were returned to the normal culture medium and cultured for 24 h in an incubator at 37 ℃ (95% air-5% CO2) to establish the OGD/R injury model. The cells in group DHA were incubated with 25 μmol/L DHA at 12 h before OGD/R. The cell viability was detected using the methyl thiazolyl tetrazolium assay, the activity of lactate dehydrogenase (LDH) was measured by using chemical colorimetric method, activated microglia were counted by immunofluorescence staining, the expression of microglia activation marker iba-1 was detected by Western blot, and the concentrations of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and IL-4 and IL-10 in culture medium were determined using enzyme-linked immunosorbent assay.@*Results@#Compared with the group C, the cell viability was significantly decreased, the LDH activity was increased, the number of activated microglia was increased, and the expression of iba-1 was up-regulated in OGD/R and DHA+ OGD/R groups, the concentrations of TNF-α and IL-6 were significantly increased in group OGD/R, and the concentrations of TNF-α, IL-6, IL-4 and IL-10 were significantly increased in group DHA+ OGD/R (P<0.05). Compared with group OGD/R, the cell viability was significantly increased, the LDH activity was decreased, the number of activated microglia was decreased, the expression of iba-1 was down-regulated, TNF-α and IL-6 concentrations were decreased, and IL-4 and IL-10 concentrations were increased in group DHA (P<0.05).@*Conclusion@#The mechanism by which DHA reduces OGD/R injury may be related to inhibiting activation of microglia and to reducing inflammatory responses.
ABSTRACT
Objective To evaluate the effect of docosahexaenoic acid (DHA) on microglial activation during oxygen-glucose deprivation and restoration (OGD/R) injury.Methods N9 microglia were inoculated in 96-well culture plates at a density of 104 cells/well for 3-5 days and divided into 3 groups (n=18 each) using a random number table method:control group (group C),group OGD/R and DHA+OGD/R group.The cells were cultured for 24 h in an incubator at 37 ℃ (95％ air-5％ CO2) in group C.In OGD/R and DHA groups,the culture medium was replaced by glucose-free culture medium,the cells were cultured for 12 h in an incubator at 37 ℃ (95％ N2-5％ CO2),and then cells were returned to the normal culture medium and cultured for 24 h in an incubator at 37 ℃ (95％ air-5％ CO2) to establish the OGD/R injury model.The cells in group DHA were incubated with 25 μmol/L DHA at 12 h before OGD/R.The cell viability was detected using the methyl thiazolyl tetrazolium assay,the activity of lactate dehydrogenase (LDH)was measured by using chemical colorimetric method,activated microglia were counted by immunofluorescence staining,the expression of microglia activation marker iba-1 was detected by Western blot,and the concentrations of tumor necrosis factor-α (TNF-α),interleukin 6 (IL-6) and IL-4 and IL-10 in culture medium were determined using enzyme-linked immunosorbent assay.Results Compared with the group C,the cell viability was significantly decreased,the LDH activity was increased,the number of activated microglia was increased,and the expression of iba-1 was up-regulated in OGD/R and DHA+OGD/R groups,the concentrations of TNF-α and IL-6 were significantly increased in group OGD/R,and the concentrations of TNF-α,IL-6,IL-4 and IL-10 were significantly increased in group DHA+OGD/R (P＜0.05).Compared with group OGD/R,the cell viability was significantly increased,the LDH activity was decreased,the number of activated microglia was decreased,the expression of iba-1 was down-regulated,TNF-α and IL-6 concentrations were decreased,and IL-4 and IL-10 concentrations were increased in group DHA (P＜0.05).Conclusion The mechanism by which DHA reduces OGD/R injury may be related to inhibiting activation of microglia and to reducing inflammatory responses.
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Objective To evaluate the role of excitatory amino acid transporter 2 ( EAAT2) in can-nabinoid receptor 2 ( CB2 receptor) activation-induced attenuation of microglial injury caused by glutamate. Methods N9 microglial cells were divided into 4 groups ( n=26 each) using a random number table meth-od: control group ( group Con) , glutamate group ( group Glu) , CB2 receptor agonist AM1241 plus gluta-mate group (group AM1241+Glu) and AM1241 plus EAAT inhibitor TBOA plus glutamate group (group AM1241+TBOA+Glu) . The cells were routinely cultured for 30 h in group Con. In group Glu, the cells were routinely cultured for 6 h, and then were incubated for 24 h in the culture medium containing gluta-mate 10 mmol∕L. In group AM1241+Glu, the cells were incubated for 4 h in the culture medium containing AM12412 μmol∕L, and then were routinely cultured for 2 h, and then were incubated for 24 h in the cul-ture medium containing glutamate 10 mmol∕L. In group AM1241+TBOA+Glu, the cells were incubated for 4 h in the culture medium containing AM12412 μmol∕L and TBOA 100 μmol∕L, and then were routinely cultured for 2 h, and then were incubated for 24 h in the culture medium containing glutamate 10 mmol∕L. The cell viability was measured by MTT assay, the activity of lactic dehydrogenase ( LDH) in supernatant was determined using colorimetric method, and the expression of EAAT2 was determined by Western blot. Results Compared with group Con, the cell viability was significantly decreased and LDH activity was in-creased in Glu, AM1241+Glu and AM1241+TBOA+Glu groups, and the expression of EAAT2 was signifi-cantly up-regulated in Glu and AM1241+Glu groups ( P<0. 05) . Compared with group Glu, the cell viabil-ity was significantly increased, LDH activity was decreased, and the expression of EAAT2 was up-regulated in group AM1241+Glu ( P<0. 05) , and no significant change was found in the parameters mentioned above in group AM1241+TBOA+Glu ( P>0. 05) . Compared with group AM1241+Glu, the cell viability was sig-nificantly decreased, LDH activity was increased, and the expression of EAAT2 was down-regulated in group AM1241+TBOA+Glu ( P<0. 05) . Conclusion The mechanism by which the activation of CB2 re-ceptor attenuates microglial injury caused by glutamate is related to up-regulating the expression of EAAT2.
ABSTRACT
ObjectlveTo evaluate the role of cannabinoid receptor 2 (CB2 receptor) in microglial injury induced by glutamate.MethodsMicroglia cells were randomly divided into 4 grups:control group (group C),microglial injury group ( group Ⅰ),specific CB2 receptor agonist AM 1241 group ( group AM1241 ) and specific CB2 receptor antagonist AM630 group (group AM630).In group C,the cells were cultured routinely for 26 h.In group Ⅰ,the cells were incubated in the culture medium containing glutamate 10 mmol/L for 24 h.In group AM1241,the cells were incubated in the culture medium containing AM1241 2 μmol/L for 2 h,and then in the culture medium containing glutamate 10 mmol/L for 24 h.In group AM630,the cells were incubated in the culture medium containing AM630 2 μmol/L for 2 h,and then in the culture medium containing glutamate 10 mmol/L for 24 h.The cell viability and release of LDH were measured.Microglial morphology was observed under microscope.Results Compared with group C,the cell viability was significantly decreased,and the release of LDH was significantly increased in groups Ⅰ,AM1241 and AM630 (P ＜ 0.05).Compared with group Ⅰ,the cell viability was significantly increased,and the release of LDH was significantly decreased in group AM1241 ( P ＜ 0.05).There was no significant difference in the cell viability and the release of LDH between groups 1 and AM630 ( P ＞ 0.05).Conclusion Glutamate induces microglial injury through inhibiting the function of CB2 receptor.