Nrf2/HO-1信号通路在右美托咪定减轻小胶质细胞氧糖剥夺-复氧复糖损伤中的作用

Role of Nrf2/HO-1 signaling pathway in dexmedetomidine-induced reduction of oxygen-glucose deprivation and restoration injury to microglia

目的评价核因子-E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路在右美托咪定减轻小胶质细胞氧糖剥夺-复氧复糖损伤中的作用。方法BV-2小胶质细胞加入含10%胎牛血清的高糖DMEM培养基,以1.5×104个/ml的密度接种于96孔培养板(200μl/孔)或以2×105个/ml密度接种于6孔培养板(每孔2 ml),置于37℃、含5%CO_(2)-21%O_(2)-74%N_(2)的正常培养箱中培养。采用随机数字表法分为5组(n=30):正常对照组(C组)、右美托咪定组(D组)、氧糖剥夺/复氧复糖组(OGD/R组)、氧糖剥夺/复氧复糖+右美托咪定组(OGD/R+D组)和氧糖剥夺/复氧复糖+右美托咪定+ML385组(OGD/R+D+ML组)。C组在正常培养箱中继续培养26 h;D组加入终浓度为10μmol/L的右美托咪定孵育2 h,随后在正常培养箱中培养26 h;OGD/R组、OGD/R+D组、OGD/R+D+ML组更换为无糖DMEM培养基,置于37℃、含5%CO_(2)-1%O_(2)-94%N_(2)的培养箱中培养2 h,然后换为含10%胎牛血清的高糖DMEM培养基,在正常培养箱中培养24 h;OGD/R+D组和OGD/R+D+ML组于氧糖剥夺前2 h时加入终浓度为10μmol/L的右美托咪定,OGD/R+D+ML组于加入右美托咪定前30 min时,加入终浓度为4μmol/L的Nrf2抑制剂ML385。采用MTT法检测细胞活力,流式细胞仪检测细胞凋亡率,ELISA检测上清液TNF-α、IL-6和IL-10的浓度,Western blot法检测细胞核Nrf-2、细胞Nrf-2和HO-1表达,RT-PCR法检测细胞HO-1 mRNA表达。结果与C组比较,OGD/R组和OGD/R+D组细胞活力降低,细胞凋亡率和上清液TNF-α、IL-6、IL-10浓度升高,细胞核Nrf2、细胞Nrf-2、HO-1及其mRNA表达上调(P<0.05),D组上述各指标差异无统计学意义(P>0.05);与OGD/R组比较,OGD/R+D组细胞活力和上清液IL-10浓度升高,细胞凋亡率和上清液TNF-α、IL-6浓度降低,细胞核Nrf2、细胞Nrf2、HO-1及其mRNA表达上调(P<0.05),OGD/R+D+ML组上述差异无统计学意义(P>0.05);与OGD/R+D组比较,OGD/R+D+ML组细胞活力和上清液IL-10浓度降低,细胞凋亡率和上清液TNF-α、IL-6浓度升高,细胞核Nrf-2、细胞Nrf2、HO-1及其mRNA表达下调(P<0.05)。结论右美托咪定减轻小胶质细胞氧糖剥夺-复氧复糖损伤的机制与促进Nrf2/HO-1信号通路激活,抑制炎症反应有关。

Objective To evaluate the role of nuclear factor erythroid 2-related factor/heme oxygenase-1(Nrf2/HO-1)signaling pathway in dexmedetomidine-induced reduction of oxygen-glucose deprivation and restoration(OGD/R)injury to microglia.Methods BV-2 microglia were cultured in high-glucose DMEM culture medium supplemented with 10%fetal bovine serum in an normal culture incubator at 37℃(5%CO_(2)-21%O_(2)-74%N_(2)).The cells were seeded in 96-well plates at a density of 1.5×104 cells/ml(200μl/well)or 6-well plates at a density of 2×105 cells/ml(2 ml/well)and divided into 5 groups(n=30 each)using a random number table method:control group(group C),dexmedetomidine group(group D),group OGD/R,OGD/R+dexmedetomidine group(group OGD/R+D)and OGD/R+dexmedetomidine+ML385 group(group OGD/R+D+ML).The cells in group C were continuously cultured in a normal culture incubator for 26 h.In group D,dexmedetomidine at the final concentration of 10μmol/L was added,cells were incubated for 2 h,and then were continuously incubated in a normal culture incubator for 26 h.In OGD/R,OGD/R+D and OGD/R+D+ML groups,the culture medium was replaced with glucose-free DMEM culture medium,cells were cultured for 2 h in an incubator at 37℃(5%CO_(2)-1%O_(2)-94%N_(2)),the culture medium was replaced with high-glucose DMEM culture medium containing 10%fetal bovine serum and then the cells were cultured for 24 h in a normal incubator.Dexmedetomidine at the final concentration of 10μmol/L was added at 2 h before OGD in OGD/R+D and OGD/R+D+ML groups.Nrf-2 inhibitor ML385 at the final concentration of 4μmol/L was added at 30 min before dexmedetomidine was added in group OGD/R+D+ML.Cells in 6 wells in each group were selected randomly for assessment of cell viability(by methyl thiazolyl tetrazolium assay)and apoptosis(using flow cytometry),and for determination of the concentrations of tumor necrosis factor(TNF)-α,interleukin(IL)-6,and IL-10 in the supernatant(using enzyme-linked immunosorbent assay),the expression of Nrf2 in nucleus,Nrf2 and HO-1(by Western blot)and the expression of HO-1 mRNA(by real-time polymerase chain reaction).Results Compared with group C,the cell viability was significantly decreased,cell apoptosis rate and concentrations of TNF-α,IL-6 and IL-10 in the supernatant were increased,and the expression of Nrf2 in nucleus,Nrf2,HO-1 and its mRNA was up-regulated in OGD/R and OGD/R+D groups(P<0.05),and no significant change was found in each parameter mentioned above in group D(P>0.05).Compared with group OGD/R,the cell viability and IL-10 in the supernatant concentration were significantly increased,cell apoptosis rate and concentrations of TNF-αand IL-6 in the supernatant were decreased and the expression of Nrf2 in nucleus,Nrf2,HO-1 and its mRNA was up-regulated in group OGD/R+D(P<0.05),and no significant changes were found in the parameters mentioned above in group OGD/R+D+ML(P>0.05).Compared with group OGD/R+D,the cell viability and concentration of IL-10 in the supernatant were significantly decreased,cell apoptosis rate and concentrations of TNF-αand IL-6 in the supernatant were increased and the expression of Nrf2 in nucleus,Nrf2,HO-1 and its mRNA was down-regulated in group OGD/R+D+ML(P<0.05).Conclusion The mechanism by which dexmedetomidine alleviates OGD/R injury to microglia may be related to promoting the activation of Nrf2/HO-1 signaling pathway and inhibition of inflammatory responses.