异氟烷调控NF-κB∕iNOS-COX2信号通路抑制三叉神经节卫星胶质细胞炎症的研究

Inhibition of trigeminal ganglion satellite glial cell inflammation by isohalothane regulating the NF-κB/iNOS-COX2 signalling pathway

目的研究异氟烷(ISO)对离体大鼠三叉神经节卫星胶质细胞炎症的影响,并初步探讨相关机制。方法体外培养大鼠三叉神经节卫星胶质细胞,采用四甲基偶氮唑盐(MTT)比色法和乳酸脱氢酶(LDH)法检测并筛选合适的ISO浓度;将细胞按随机数字表法分为对照组、模型组、ISO组和SN50[核转录因子(NF)-κB抑制剂]组,采用硝酸甘油(GTN)构建卫星胶质细胞炎症模型,其中ISO组用1.5%ISO以2 L/min的速率处理30 min,其余组用相同速率的空气处理30 min,每组设6个复孔;采用酶联免疫吸附测定(ELISA)法和一氧化氮(NO)检测试剂盒检测细胞上清液中白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、前列腺素E2(PGE2)水平和NO的含量,采用Western blotting法测定细胞中NF-κB p65、环氧合酶2(COX2)及诱导型一氧化氮合酶(iNOS)的蛋白表达情况,采用Fluo-3/AM探针负载法检测钙离子浓度。结果ISO组IL-1β、TNF-α、PGE2、NO水平及钙离子浓度分别为(121.49±12.37)ng/L、(88.41±9.53)ng/L、(439.92±28.81)pg/mL、(9.03±1.04)μmol/L、(101.11±8.23)mmol/L,均明显低于模型组(均P<0.05);Western blotting检测发现,ISO组NF-κB p65、COX2及iNOS蛋白表达分别为0.74±0.15、0.59±0.14、0.51±0.11,均明显低于模型组(均P<0.05)。SN50组各指标变化趋势与ISO组一致。结论适当剂量的ISO可以抑制GTN诱导的三叉神经节卫星胶质细胞炎症,其机制可能与减少炎症因子释放、抑制NF-κB/iNOS-COX2信号通路的激活、降低钙离子浓度有关。

Objective To investigate the effect of isohalothane(ISO)on rat trigeminal ganglion satellite glial cell inflammation in vitro and its mechanism.Methods Rat trigeminal ganglion satellite glial cells were cultured in vitro.Methyl thiazolyl tetrazolium and lactate dehydrogenase were used to select the appropriate ISO concentration.Cells were divided into the control group,model group,ISO group and SN50 group[nuclear factor(NF)-κB depressor]group.Nitroglycerin(GTN)was used to construct a satellite glial inflammatory model.The ISO group was treated with 1.5%ISO at a rate of 2 L/min for 30 min,and the other groups were treated with air at the same rate for 30 min.The levels of interleukin-1β(IL-1β),tumour necrosis factor-α(TNF-α)and prostaglandin E2(PGE2)and the content of NO in the cell culture medium were measured by enzyme-linked immunosorbent assay and nitric oxide(NO)assay.The protein expressions of NF-κB p65,cyclooxygenase-2(COX2)and inducible nitric oxide synthase(iNOS)were detected by western blot.The concentration of calcium ions was detected by Fluo-3/AM probe loading method.Results The levels of IL-1β,TNF-α,PGE2 and NO and calcium ion concentration in the ISO group were(121.49±12.37)ng/L,(88.41±9.53)ng/L,(439.92±28.81)pg/mL,(9.03±1.04)μmol/L and(101.11±8.23)mmol/L,respectively,which were significantly lower than those in the model group(all P<0.05).The results of western blotting analysis showed that the protein expression levels of NF-κB p65,COX2 and iNOS in the ISO group were 0.74±0.15,0.59±0.14 and 0.51±0.11,respectively,which were significantly lower than those in the model group(all P<0.05).The changing trend of each index in the SN50 group was consistent with that in the ISO group.Conclusion Appropriate dose of ISO can inhibit GTN induced inflammation of trigeminal ganglion satellite glial cells,and the mechanism may be related to reducing the release of inflammatory factors,inhibiting NF-κB/inos-COX2 signalling pathway and the decreasing the calcium concentration.