胞磷胆碱钠对大鼠发育期惊厥性脑损伤的保护作用

Protective effects of citicoline sodium on developmental convulsive cerebral injury in rats

目的探究胞磷胆碱钠对大鼠发育期惊厥性脑损伤的保护作用及其机制。方法将SD大鼠随机分为对照组、模型组和实验组,每组10只,用腹腔注射氯化锂+氢溴酸东莨菪碱+毛果芸香碱的方法建立大鼠发育期惊厥模型。造模成功后实验组大鼠腹腔注射胞磷胆碱钠(500 mg·kg^(-1)),每日1次,连续给药1周。对照组和模型组在相同时间点腹腔注射等量0.9%NaCl。用Griess法检测血清中一氧化氮(NO)浓度,用酶联免疫吸附试验(ELISA)法检测脑组织中白细胞介素-6(IL-6)和白细胞介素-1β(IL^(-1)β)含量,用生化法检测脑组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,用实时定量聚合酶链反应(qRT-PCR)法检测脑组织中诱导型一氧化氮合酶(iNOS)mRNA的水平,用蛋白质印迹(Western blot)法检测大鼠脑组织中G蛋白偶联受体39抗体(GPR39)、蛋白激酶B(AKT)和细胞外信号调节激酶(ERK)的蛋白水平。结果对照组、模型组和实验组大鼠脑组织IL-6含量分别为(37.16±6.34)、(119.31±19.26)和(74.52±12.37)pg·mL^(-1),IL^(-1)β含量分别为(7.46±1.25)、(42.73±8.41)和(24.18±4.62)pg·mL^(-1),SOD活性分别为(384.51±34.72)、(229.16±27.42)和(218.47±33.59)U·gp^(-1),MDA含量分别为(1.06±0.15)、(1.82±0.21)和(1.24±0.17)U·gp^(-1),血清NO含量分别为(13.62±2.06)、(29.34±5.37)和(16.55±3.15)μmol·L^(-1),iNOS mRNA相对表达水平分别为1.00±0.15、2.19±0.24和1.62±0.18,GPR39蛋白相对表达水平分别为0.41±0.05、1.03±0.19和0.74±0.08,p-AKT/AKT分别为0.46±0.07、0.13±0.04和0.36±0.05,p-ERK/ERK分别为0.37±0.11、0.11±0.03和0.42±0.05。对照组的上述指标与模型组比较,在统计学上差异均有统计学意义(P<0.01,P<0.001);模型组的上述指标与实验组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01,P<0.001)。结论胞磷胆碱钠能够有效缓解发育期惊厥大鼠脑组织的损伤程度,抑制炎症反应,改善氧化应激,其作用机制可能与GPR39/AKT/ERK信号通路的激活有关。

Objective To explore the protective effect and mechanism of citicoline sodium on developmental convulsive cerebral injury in rats.Method SD rats were randomly divided into control group,model group and experimental group with 10 in each group.The rat developmental convulsive model was established by intraperitoneal injection of lithium chloride,scopolamine hydrobromide and pilocarpine.After successful mo de ling,the rats in experimental group were intraperitoneally injected with cytidine sodium(500 mg·kg^(-1))once a day for one week.The control group and the model group were intraperitoneally injected with the same amount of 0.9%NaCl at the same time point.Griess detection of nitric oxide(NO)concentration in serum;enzyme-linked immunosorbent assay(ELISA)detection of interleukin-6(IL-6)and interleukin-1β(IL^(-1)β)content in cerebrum;biochemical detection detection of superoxide dismutase(SOD)activity and malondialdehyde(MDA)content in cerebrum;real-time quantitative polymerase chain reaction(qRT-PCR)detection of mRNA levels of inducible nitric oxide synthase(iNOS)in cerebrum;Western blot detection of protein levels of G protein-coupled receptors 39 antibody(GPR39),protein kinase B(AKT),and extracellular regulated protein kinases(ERK)in cerebrum.Results In control group,model group and experimental group,the level of IL-6 in rats cerebrum were(37.16±6.34),(119.31±19.26)and(74.52±12.37)pg·mL^(-1),respectively;the contents of IL^(-1)βwere(7.46±1.25),(42.73±8.41)and(24.18±4.62)pg·mL^(-1),respectively;SOD activity were(384.51±34.72),(229.16±27.42)and(218.47±33.59)U·gp-1,respectively;MDA content were(1.06±0.15),(1.82±0.21)and(1.24±0.17)U·gp^(-1),respectively;serum NO levels were(13.62±2.06),(29.34±5.37)and(16.55±3.15)μmol·L^(-1),respectively;mRNA levels of iNOS were 1.00±0.15,2.19±0.24 and 1.62±0.18,respectively;protein levels of GPR39 were 0.41±0.05,1.03±0.19 and 0.74±0.08,respectively;p-AKT/AKT values were 0.46±0.07,0.13±0.04 and 0.36±0.05,respectively;p-ERK/ERK values were 0.37±0.11,0.11±0.03 and 0.42±0.05,respectively.The above indicators showed statistically significant differences between the control group and the model group(P<0.01,P<0.001);the difference between the model group and the experimental group was statistically significant(P<0.05,P<0.01,P<0.001).Conclusion Cytidine sodium can effectively alleviate the degree of developmental convulsive cerebral injury rats,inhibit inflammatory reactions,and improve oxidative stress.Its mechanism may be related to the activation of GPR39/AKT/ERK signaling pathway.