血必净对抗N-甲基-D-天门冬氨酸受体脑炎小鼠海马神经元的保护作用及机制

Protective effect of Xuebijing on hippocampal neurons in mice with anti-N-methyl-D-aspartate receptor encephalitis and its mechanism

目的探讨血必净对抗N-甲基-D-天门冬氨酸受体(NMDAR)脑炎小鼠的治疗作用及对辅助性T细胞1(Th1)/辅助性T细胞2(Th2)变化的影响。方法实验分组:对照组、模型组、低剂量血必净组、高剂量血必净组,每组10只小鼠,除对照组外,其余3组小鼠采用抗原注射与免疫刺激建立抗NMDAR脑炎模型,低、高剂量血必净组的小鼠分别通过腹腔注射5 ml/kg、10 ml/kg血必净注射液,12 h注射1次,连续3 d;2周后,HE染色观察小鼠脑组织病理学改变,尼氏染色观察小鼠神经细胞形态变化,TUNEL染色检测小鼠神经细胞凋亡情况,ELISA检测小鼠血清和脑脊液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素4(IL-4)、干扰素γ(IFN-γ)含量,免疫组织化学染色与Western blotting检测脑组织IFN-γ、IL-4表达情况,流式细胞术检测外周血Th1、Th2细胞比例分布,并计算Th1/Th2比值。结果与模型组比较,低剂量血必净组和高剂量血必净组的小鼠海马组织损伤得到改善,神经细胞形态变化较小,尼氏小体形态较为完整且数目增加,神经细胞的TUNEL阳性率降低,血清和脑脊液中TNF-α、IL-1β、IFN-γ含量均减少而IL-4含量增加,脑组织内IFN-γ阳性细胞百分比与蛋白相对表达量降低,IL-4阳性细胞百分比与蛋白相对表达量升高,外周血分化簇(CD)4^(+)IFN-γ^(+)标记的Th1细胞比例、Th1/Th2比值降低,且高剂量血必净组小鼠外周血CD4^(+)IL-4^(+)标记的Th2细胞比例升高,差异均具有统计学意义(P<0.05)。与低剂量血必净组比较,高剂量血必净组小鼠的上述各项检测指标改善效果更为明显,且差异也均具有统计学意义(P<0.05)。结论血必净能够改善抗NMDAR脑炎小鼠海马神经元损伤,该作用可能与减少IFN-γ表达、促进IL-4表达进而维持Th1/Th2细胞平衡有关。

Objective To investigate the therapeutic effect of Xuebijing on anti-N-methyl-D-aspartate receptor(NMDAR)encephalitis mice and its effect on the changes of helper T cell 1(Th1)/helper T cell 2(Th2).Methods The experimental groups included control group,model group,low-dose Xuebijing(low-XBJ)group,highdose Xuebijing(high-XBJ)group,with 10 mice in each group,except the control group,the other 3 groups of mice were treated with antigen injection and immune stimulation to establish anti-NMDAR encephalitis models,the mice in the low-XBJ and high-XBJ groups were intraperitoneally injected with 5 ml/kg and 10 ml/kg of Xuebijing injection,respectively,once every 12 hours for 3 consecutive days;After 2 weeks,HE staining was used to observe the pathological changes of mice brain tissue,Nissl staining was used to observe the morphological changes of mice neurons,TUNEL staining was used to detect the apoptosis of mice neurons,ELISA was used to detect the contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-4 and interferon-γ(IFN-γ)in serum and cerebrospinal fluid of mice,immunohistochemical staining and Western blotting were used to detect the expression of IFN-γand IL-4 in brain tissue,flow cytometry was used to detect the distribution of Th1 and Th2 cells in peripheral blood,and the ratio of Th1/Th2 was calculated.Results Compared with the model group,hippocampal tissue damage was improved in low-XBJ group and high-XBJ group,the morphological changes of neurons were small,the morphology of Nissl bodies was more complete and the number of Nissl bodies was increased,the TUNEL positive rate of neurons decreased,the contents of TNF-α,IL-1βand IFN-γin serum and cerebrospinal fluid decreased while the content of IL-4 increased,the percentage of IFN-γpositive cells and the relative expression of protein in brain tissue decreased,the percentage of IL-4 positive cells and the relative expression of protein increased,the proportion of cluster of differentiation(CD)4^(+)IFN-γ^(+)labeled Th1 cells and the ratio of Th1/Th2 in peripheral blood decreased,and the proportion of CD4^(+)IL-4^(+)labeled Th2 cells in peripheral blood of mice in high-XBJ group increased,the differences were statistically significant(P<0.05).In addition,compared with the low-XBJ group,the improvement effect of the above detection indicators in the high-XBJ group was more obvious,and the differences were statistically significant(P<0.05).Conclusion Xuebijing can improve hippocampal neuronal damage in anti-NMDAR encephalitis mice,which may play a therapeutic role by reducing the expression of IFN-γ,promoting the expression of IL-4 and maintaining the balance of Th1/Th2 cells.