miRNA-200b在高压氧综合治疗缺氧缺血脑损伤仔鼠中的作用

Role of microRNA-200b in hyperbaric oxygen therapy for the treatmet of rat offsprings with hypoxic-ischemic brain injury

目的探讨miR-200b在高压氧(hyperbaric oxygen,HBO)联合神经脂苷钠(GM)治疗在脑缺氧缺血损伤仔鼠中的作用.方法挑选30只新生的P7 SD仔鼠,随机选取6只作为正常对照组(A组),其他24只进行左侧颈总动脉的结扎、剪断,造成缺氧,制备缺血缺氧脑损伤模型.将此24只脑损伤模型仔鼠分为4组:模型组(B组)、HBO+ GM+中医康复组(C组)、miR-200b抑制剂+HBO+ GM+中医康复组(D组)、miR-200b激动剂+HBO+ GM+中医康复组(E组).采用HE染色、免疫组化以及高效液相色谱法检测脑皮质层中正常神经细胞的数目、炎性因子的表达水平以及兴奋性氨基酸的表达水平.结果 HBO+ GM+中医康复治疗可有效缓解仔鼠缺血缺氧脑损伤,miR-200b抑制剂对疗效具有协同作用,miR-200b激动剂对疗效具有抑制作用(P<0.05).C、D、E组仔鼠脑梗死体积明显低于B组,D组脑梗死体积明显低于C、E组,C组脑梗死体积明显低于E组,差异均有统计学意义(P<0.05或P<0.01).C、D、E组仔鼠脑皮质正常神经细胞数目较B组明显增多,D组明显高于C、E组,C组仔鼠脑皮质正常神经细胞数目明显高于E组,差异均有统计学意义(P<0.05或P<0.01).C、D、E组仔鼠脑皮质TNF-α和IL-6蛋白相对表达量较B组明显降低,D组仔鼠脑皮质TNF-α和IL-6蛋白相对表达量明显低于C、E组,C组正常神经细胞数目明显低于E组,差异均有统计学意义(P<0.05或P<0.01).C、D、E组仔鼠脑皮质Glu和Asp水平较B组明显降低,D组仔鼠脑皮质Glu和Asp水平明显低于C、E组,C组Glu和Asp水平明显低于E组,差异均有统计学意义(P<0.05或P<0.01).结论 HBO+ GM+中医康复治疗可有效缓解仔鼠缺血缺氧脑损伤,miR-200b抑制剂对疗效具有协同作用,miR-200b激动剂对疗效具有抑制作用.

Objective To investigate the role of miR-200b in the treatment of cerebral hypoxia-ischemia with ganglioside sodium (GM) and hyperbaric oxygen (HBO).Methods Thirty newborn P7 SD rats were randomly selected for the study,6 rats were designated as the control group (or group A) and the remaining 24 received left carotid artery ligation to induce ischemia,and the model of cerebral ischemia/ hypoxia was thus established.Then,the 24 brain injury model rats were divided into 4 groups:the model group (or group B),the HBO combined with GM and TCM rehabilitation group (or group C),the miR-200b inhibitor plus HBO combined with GM and TCM rehabilitation Group (or group D) and the miR-200b agonist plus HBO combined with GM and TCM rehabilitation group (or group E).HE staining,immunohistochemistry and high performance liquid chromatography were used to detect the number of normal nerve cells in the cortex,the expression levels of inflammatory factors and excitatory amino acids.Results The combined treatment with HBO,GM and traditional Chinese medicine rehabilitation therapy could effectively alleviate brain damage induced by hypoxic ischemia,and miR-200b could produce certain synergetic effect,while miR-200b activator could produce certain inhibitive effect(P <0.05).The infarct volumes of the offsprings in group C,D and E were significantly lower than that in group B,and that in group D was significantly lower than that in group C and E,and that in group D was significantly lower than that in group C and group E.Statistical significance could be seen,when comparisons were made between them (P < 0.05 or P < 0.01).The number of normal neurons in the cerebral cortex of the offsprings in groups C,D and E was significantly higher than that in group B,and the number of normal neurons in groups C,D and E was significantly higher than that in group B,the number of normal neurons in group D was significantly higher than that in groups C and E,and the number of normal neurons in group C was significantly higher than that in group E.There was also statistical significance,when comparisons were made between them (P <0.05 or P < 0.01).The relative expression levels of TNF-a and IL-6 protein in the cerebral cortex of the offsprings in group C,D and E were significantly lower than that in group B,the relative expression levels of TNF-a and IL-6 protein in the cerebral cortex of the offsprings in group D were significantly lower than that in group C and E,and the number of normal nerve cells in group C was significantly lower than that in group E,with statistical significance (P <0.05 or P <0.01).The levels of Glu and Asp in the cortex of the offsprings in group C,D and E were significantly lower than those in group B,while the levels of Glu and Asp in the cortex of the offsprings in group D were significantly lower than those in group C and E,and the levels of Glu and Asp in group C were significantly lower than those in group E,also with statistical significance (P < 0.05 or P < 0.01).Conclusion HBO + GM + TCM rehabilitation therapy could effectively alleviate cerebral ischemia and hypoxic injury in offspring rats,and the inhibitor of miRNA-200b could produce certain synergistic effect.