羟基法舒地尔对实验性自身免疫性脑脊髓炎小鼠氧化应激的影响

Effect of Hydroxyfasudil on oxidative stress in experimental autoimmune encephalomyelitis mice

目的:探究羟基法舒地尔(HF)对实验性自身免疫性脑脊髓炎(EAE)小鼠氧化应激的影响及可能机制。方法:将BV2细胞随机分为正常对照组、LPS组和LPS+HF组。HF预处理2 h后给予LPS刺激24 h,收取细胞上清液。将16只C57BL/6小鼠随机平均分为EAE组和HF组。HF组小鼠于免疫后第3天起以HF腹腔注射干预,剂量40 mg/(kg·d)。EAE组小鼠注射等量生理盐水。从免疫当天开始,每隔1 d记录小鼠的临床评分。DPPH法和ABTS法检测不同浓度HF对自由基的清除作用。MTT法检测BV2细胞在不同浓度HF的干预下细胞活力的改变。HE和LFB染色检测脊髓炎症和髓鞘脱失。ELISA检测细胞上清液和小鼠脊髓中RNS、ROS、MDA、SOD、CAT和GSH-Px含量。免疫荧光染色和Western blot检测小鼠脊髓中Nrf2、HO-1表达。结果:HF在3.75~60μg/ml浓度范围内对DPPH和ABTS自由基几乎没有清除作用,在0~15μg/ml浓度范围内对BV2细胞活力没有影响,HF可抑制EAE小鼠的临床症状,改善脊髓炎症和髓鞘脱失,减少氧化产物ROS、RNS和MDA产生,增加抗氧化酶SOD、CAT和GSH-Px表达,同时促进小鼠脊髓Nrf2和HO-1表达。结论:HF抑制EAE小鼠的临床症状,减少中枢神经系统内炎症浸润和髓鞘脱失,其机制可能与HF激活Nrf2/HO-1信号通路,抑制EAE小鼠的氧化应激有关。

Objective:To investigate the effect of Hydroxyfasudil(HF)on oxidative stress in experimental autoimmune encephalomyelitis(EAE)mice and its probable mechanisms.Methods:BV2 cells were randomly separated into normal control group,LPS group and LPS+HF group.After pretreatment with HF for 2 h,LPS was given to stimulate for 24 h,and the cell supernatant was collected.Sixteen C57BL/6 mice were randomly separated into EAE group and HF group.Mice in HF group were intraperitoneally injected with HF[40 mg(/kg·d)]from day 3 after immunization,and mice in EAE group were injected with the same amount of normal saline.From the day of immunization,clinical scores of mice were observed every two days.Scavenging effect of different concentrations of HF on free radicals was tested by DPPH and ABTS.Changes in cell viability of BV2 cells under the intervention of different concentrations of HF were detected by MTT.Inflammation and demyelination of spinal cords were detected by HE and LFB staining.Contents of RNS,ROS,MDA,SOD,CAT and GSH-Px in cell supernatant and mice spinal cords were detected by ELISA,expressions of Nrf2 and HO-1 were detected by immunofluorescence staining and Western blot.Results:HF had hardly scavenging effect on DPPH and ABTS free radicals in concentration range of 3.75~60μg/ml,and no changes in BV2 cells viability were observed when the concentration of HF was in 0~15μg/ml.HF inhibited clinical scores of EAE mice,improved inflammation and demyelination of spinal cords,reduced oxidation products ROS,RNS and MDA,increased antioxidant enzymes SOD,CAT and GSH-Px,and increased expressions of Nrf2 and HO-1 in spinal cords of mice.Conclusion:HF inhibited clinical symptoms of EAE mice and reduced central nervous system inflammatory infiltration and demyelination,which might be related to activation of Nrf2/HO-1 signaling pathway by HF to inhibit oxidative stress in EAE mice.